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Sample records for bacterial phosphotransferase system

  1. Changes in the cellular energy state affect the activity of the bacterial phosphotransferase system

    DEFF Research Database (Denmark)

    Rohwer, J.M.; Jensen, Peter Ruhdal; Shinohara, Y.

    1996-01-01

    The effect of different cellular free-energy states on the uptake of methyl alfa-D-glucopyranoside, an analoque of glucose, by Escherichia coli phosphoenolpyruvate:carbohydrate phosphotransferase system was investigated. The intracellular ATP/ADP ratio was varied by changing the expression...... that the initial uptake rate was decreased under conditions of lowered intracellular ATP/ADP ratios, irrespective of which method was used to change the cellular energy state.. When either the expression of the atp operon was changed or 2,4-dinitrophenol was added to wild-type cells, the relationship between...... initial phosphotransferase uptake rate and the logaritm of the ATP/ADP ratio was approxomately linear. These results suggest that the cellular free-energy state, as reflected in the intracellular ATP/ADP ratio, plays an important role in regulating the activity of the phosphotransferase ssystem....

  2. [Transformation of phosphotransferase system in Escherichia coli].

    Science.gov (United States)

    Xiao, Mengrong; Zhang, Liang; Liu, Shuangping; Shi, Guiyang

    2014-10-01

    We constructed several recombinant Escherichia coli strains to transform phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS system) and compared the characteristics of growth and metabolism of the mutants. We knocked-out the key genes ptsI and ptsG in PTS system by using Red homologous recombination in E. coli and meanwhile we also knocked-in the glucose facilitator gene glf from Zymomonas mobilis in the E. coli chromosome. Recombinant E. coli strains were constructed and the effects of cell growth, glucose consumption and acetic acid accumulation were also evaluated in all recombinant strains. The deletion of gene ptsG and ptsI inactivated some PTS system functions and inhibited the growth ability of the cell. Expressing the gene glf can help recombinant E. coli strains re-absorb the glucose through Glf-Glk (glucose facilitator-glucokinase) pathway as it can use ATP to phosphorylate glucose and transport into cell. This pathway can improve the availability of glucose and also reduce the accumulation of acetic acid; it can also broaden the carbon flux in the metabolism pathway.

  3. Phosphoenolpyruvate-dependent fructose phosphotransferase system in Rhodopseudomonas sphaeroides : The coupling between transport and phosphorylation in inside-out vesicles

    NARCIS (Netherlands)

    Lolkema, Juke S.; Robillard, George T.

    The bacterial phosphotransferase systems are believed to catalyze the concomitant transport and phosphorylation of hexoses and hexitols. The transport is from the outside to the inside of the cell. An absolute coupling between transport and phosphorylation has however been questioned in the

  4. Steady State Kinetics of Mannitol Phosphorylation Catalyzed by Enzyme IImtl of the Escherichia coli Phosphoenolpyruvate-dependent Phosphotransferase System

    NARCIS (Netherlands)

    Lolkema, Juke S.; Hoeve-Duurkens, Ria H. ten; Robillard, George T.

    1993-01-01

    The kinetics of mannitol phosphorylation catalyzed by enzyme IImtl of the bacterial P-enolpyruvate-dependent phosphotransferase system are described for three different physical conditions of the enzyme, (i) embedded in the membrane of inside-out (ISO) oriented vesicles, (ii) solubilized and assayed

  5. Phosphoenolpyruvate phosphotransferase system components positively regulate Klebsiella biofilm formation

    Directory of Open Access Journals (Sweden)

    Yu-Tze Horng

    2018-04-01

    Full Text Available Background/Purpose: Klebsiella pneumoniae is one of the leading causes of device-related infections (DRIs, which are associated with attachment of bacteria to these devices to form a biofilm. The latter is composed of not only bacteria but also extracellular polymeric substances (EPSes consisting of extracellular DNAs, polysaccharides, and other macromolecules. The phosphoenolpyruvate (PEP:carbohydrate phosphotransferase system (PTS regulates diverse processes of bacterial physiology. In the genome of K. pneumoniae MGH 78578, we found an uncharacterized enzyme II complex homolog of PTS: KPN00353 (EIIA homolog, KPN00352 (EIIB homolog, and KPN00351 (EIIC homolog. The aim of this study was to characterize the potential physiological role of KPN00353, KPN00352, and KPN00351 in biofilm formation by K. pneumoniae. Methods/Results: We constructed the PTS mutants and recombinant strains carrying the gene(s of PTS. The recombinant K. pneumoniae strain overexpressing KPN00353–KPN00352–KPN00351 produced more extracellular matrix than did the vector control according to transmission and scanning electron microscopy. Judging by quantification of biofilm formation, of extracellular DNA (eDNA, and of capsular polysaccharide, the recombinant strain overexpressing KPN00353-KPN00352-KPN00351 produced more biofilm and capsular polysaccharide after overnight culture and more eDNA in the log phase as compared to the vector control. Conclusion: The genes, KPN00353–KPN00352–KPN00351, encode a putative enzyme II complex in PTS and positively regulate biofilm formation by enhancing production of eDNA and capsular polysaccharide in K. pneumoniae. Five proteins related to chaperones, to the citric acid cycle, and to quorum sensing are upregulated by the KPN00353–KPN00352–KPN00351 system. Keywords: Klebsiella, PTS, Biofilm, eDNA, Polysaccharide

  6. STEADY-STATE KINETICS OF MANNITOL PHOSPHORYLATION CATALYZED BY ENZYME-II(MTL) OF THE ESCHERICHIA-COLI PHOSPHOENOLPYRUVATE-DEPENDENT PHOSPHOTRANSFERASE SYSTEM

    NARCIS (Netherlands)

    LOLKEMA, JS; TENHOEVEDUURKENS, RH; ROBILLARD, GT

    1993-01-01

    The kinetics of mannitol phosphorylation catalyzed by enzyme II(mtl) of the bacterial P-enolpyruvate-dependent phosphotransferase system are described for three different physical conditions of the enzyme, (i) embedded in the membrane of inside-out (ISO) oriented vesicles, (ii) solubilized and

  7. Escherichia coli Phosphoenolpyruvate Dependent Phosphotransferase System. Copurification of HPr and α1-6 Glucan

    NARCIS (Netherlands)

    Dooijewaard, G.; Roossien, F.F.; Robillard, G.T.

    1979-01-01

    A rapid, high-yield procedure has been developed for the purification of HPr from the Escherichia coli phosphoenolpyruvate dependent phosphotransferase system. During this procedure, the protein copurifies with a 2500-dalton homopolysaccharide which we have identified as α1-6 glucan. The results of

  8. Escherichia coli Phosphoenolpyruvate-Dependent Phosphotransferase System : Equilibrium Kinetics and Mechanism of Enzyme I Phosphorylation

    NARCIS (Netherlands)

    Hoving, H; Lolkema, Juke S.; Robillard, George T.

    1981-01-01

    The phosphorylation of enzyme I from the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system was studied by means of isotope exchange between phosphoenolpyruvate and pyruvate. Experiments monitoring 1H-2H exchange showed that enzyme I phosphorylation is accompanied by the

  9. Clusters of reaction rates and concentrations in protein networks such as the phosphotransferase system

    NARCIS (Netherlands)

    Härdin, Hanna M.; Zagaris, Antonios; Willms, Allan R.; Westerhoff, Hans V.

    To understand the functioning of living cells, it is often helpful or even necessary to exploit inherent timescale disparities and focus on long-term dynamic behaviour. In the present study, we explore this type of behaviour for the biochemical network of the phosphotransferase system. We show that,

  10. From the phosphoenolpyruvate phosphotransferase system to selfish metabolism: a story retraced in Pseudomonas putida.

    Science.gov (United States)

    Pflüger-Grau, Katharina; de Lorenzo, Víctor

    2014-07-01

    Although DNA is the ultimate repository of biological information, deployment of its instructions is constrained by the metabolic and physiological status of the cell. To this end, bacteria have evolved intricate devices that connect exogenous signals (e.g. nutrients, physicochemical conditions) with endogenous conditions (metabolic fluxes, biochemical networks) that coordinately influence expression or performance of a large number of cellular functions. The phosphoenolpyruvate:carbohydrate-phosphotransferase system (PTS) is a bacterial multi-protein phosphorylation chain which computes extracellular (e.g. sugars) and intracellular (e.g. phosphoenolpyruvate, nitrogen) signals and translates them into post-translational regulation of target activities through protein-protein interactions. The PTS of Pseudomonas putida KT2440 encompasses one complete sugar (fructose)-related system and the three enzymes that form the so-called nitrogen-related PTS (PTS(N) (tr) ), which lacks connection to transport of substrates. These two PTS branches cross-talk to each other, as the product of the fruB gene (a polyprotein EI-HPr-EIIA) can phosphorylate PtsN (EIIA(N) (tr) ) in vivo. This gives rise to a complex actuator device where diverse physiological inputs are ultimately translated into phosphorylation or not of PtsN (EIIA(N) (tr) ) which, in turn, checks the activity of key metabolic and regulatory proteins. Such a control of bacterial physiology highlights the prominence of biochemical homeostasis over genetic ruling -and not vice versa. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  11. Regulation and characterization of the galactose-phosphoenolpyruvate-dependent phosphotransferase system in Lactobacillus casei.

    OpenAIRE

    Chassy, B M; Thompson, J

    1983-01-01

    Cells of Lactobacillus casei grown in media containing galactose or a metabolizable beta-galactoside (lactose, lactulose, or arabinosyl-beta-D-galactoside) were induced for a galactose-phosphoenolpyruvate-dependent phosphotransferase system (gal-PTS). This high-affinity system (Km for galactose, 11 microM) was inducible in eight strains examined, which were representative of all five subspecies of L. casei. The gal-PTS was also induced in strains defective in glucose- and lactose-phosphoenolp...

  12. Clusters of reaction rates and concentrations in protein networks such as the phosphotransferase system.

    Science.gov (United States)

    Härdin, Hanna M; Zagaris, Antonios; Willms, Allan R; Westerhoff, Hans V

    2014-01-01

    To understand the functioning of living cells, it is often helpful or even necessary to exploit inherent timescale disparities and focus on long-term dynamic behaviour. In the present study, we explore this type of behaviour for the biochemical network of the phosphotransferase system. We show that, during the slow phase that follows a fast initial transient, the network reaction rates are partitioned into clusters corresponding to connected parts of the reaction network. Rates within any of these clusters assume essentially the same value: differences within each cluster are vastly smaller than that from one cluster to another. This rate clustering induces an analogous clustering of the reactive compounds: only the molecular concentrations on the interface between these clusters are produced and consumed at substantially different rates and hence change considerably during the slow phase. The remaining concentrations essentially assume their steady-state values already by the end of the transient phase. Further, we find that this clustering phenomenon occurs for a large number of parameter values and also for models with different topologies; to each of these models, there corresponds a particular network partitioning. Our results show that, in spite of its complexity, the phosphotransferase system tends to behave in a rather simple (yet versatile) way. The persistence of clustering for the perturbed models we examined suggests that it is likely to be encountered in various environmental conditions, as well as in other signal transduction pathways with network structures similar to that of the phosphotransferase system. © 2013 FEBS.

  13. Regulation of lactose-phosphoenolpyruvate-dependent phosphotransferase system and beta-D-phosphogalactoside galactohydrolase activities in Lactobacillus casei.

    Science.gov (United States)

    Chassy, B M; Thompson, J

    1983-01-01

    The lactose-phosphoenolpyruvate-dependent phosphotransferase system (lac-PTS) and beta-D-phosphogalactoside galactohydrolase (P-beta-gal) mediate the metabolism of lactose by Lactobacillus casei. Starved cells of L. casei contained a high intracellular concentration of phosphoenolpyruvate, and this endogenous energy reserve facilitated characterization of phosphotransferase system activities in physiologically intact cells. Data obtained from transport studies with whole cells and from in vitro phosphotransferase system assays with permeabilized cells revealed that the lac-PTS had a high affinity for beta-galactosides (e.g., lactose, lactulose, lactobionic acid, and arabinosyl-beta-D-galactoside). lac-PTS and P-beta-gal activities were determined in wild-type strains and strains defective in the glucose-phosphoenolpyruvate-dependent phosphotransferase system after growth on various sugars and in the presence of potential inducers. We found that (i) the lac genes (i.e., the genes coding for the lac-PTS proteins and P-beta-gal) were induced by metabolizable and non-metabolizable beta-galactosides (presumably acting as their phosphorylated derivatives), (ii) galactose 6-phosphate was not an inducer in most strains, (iii) the ratio of lac-PTS activity to P-beta-gal activity in a given strain was not constant, and (iv) inhibition of lac gene expression during growth on glucose was a consequence of glucose-phosphoenolpyruvate-dependent phosphotransferase system-mediated inducer exclusion, repressive effects of a functional glucose-phosphoenolpyruvate-dependent phosphotransferase system and glucose-derived metabolites. The expression of the lac-PTS structural genes and the expression of the P-beta-gal gene are independently regulated and may be subject to both positive control and negative control. Images PMID:6406426

  14. The global regulatory system Csr senses glucose through the phosphoenolpyruvate: carbohydrate phosphotransferase system.

    Science.gov (United States)

    Pérez-Morales, Deyanira; Bustamante, Víctor H

    2016-02-01

    A novel connection between two regulatory systems controlling crucial biological processes in bacteria, the carbon storage regulator (Csr) system and the glucose-specific phosphotransferase system (PTS), is reported by Leng et al. in this issue. This involves the interaction of unphosphorylated EIIA(Glc), a component of the glucose-specific PTS, with the CsrD protein, which accelerates the decay of the CsrB and CsrC small RNAs via RNase E in Escherichia coli. As unphosphorylated EIIA(G) (lc) is generated in the presence of glucose, the PTS thus acts as a sensor of glucose for the Csr system. Interestingly, another pathway can operate for communication between the Csr system and the glucose-specific PTS. The absence of glucose generates phosphorylated EIIA(Glc) , which activates the enzyme adenylate cyclase to produce cyclic adenosine monophosphate (cAMP) that, in turn, binds to the regulator cAMP receptor protein (CRP). Leng et al. show that the complex cAMP-CRP modestly reduces CsrB decay independently of CsrD. On the other hand, a previous study indicates that the complex cAMP-CRP positively regulates the transcription of CsrB and CsrC in Salmonella enterica. Therefore, EIIA(G) (lc) could work as a molecular switch that regulates the activity of the Csr system, in response to its phosphorylation state determined by the presence or absence of glucose, in order to control gene expression. © 2015 John Wiley & Sons Ltd.

  15. Escherichia coli Phosphoenolpyruvate-Dependent Phosphotransferase System : Mechanism of Phosphoryl-Group Transfer from Phosphoenolpyruvate to HPr

    NARCIS (Netherlands)

    Misset, Onno; Robillard, George T.

    1982-01-01

    The mechanism of phosphoryl-group transfer from phosphoenolpyruvate (PEP) to HPr, catalyzed by enzyme I of the Escherichia coli PEP-dependent phosphotransferase system, has been studied in vitro. Steady-state kinetics and isotope exchange measurements revealed that this reaction cannot be described

  16. 31PHOSPHO-NMR DEMONSTRATION OF PHOSPHOCYSTEINE AS A CATALYTIC INTERMEDIATE ON THE ESCHERICHIA-COLI PHOSPHOTRANSFERASE SYSTEM EIIMTL

    NARCIS (Netherlands)

    MEYER, GH; KRUIZINGA, WH; TAMMINGA, KS; VANWEEGHEL, RP; ROBILLARD, GT; Pas, Hendrikus

    1991-01-01

    The mannitol-specific phosphotransferase system transport protein, Enzyme II(Mtl), contains two catalytically important phosphorylated amino acid residues, both present on the cytoplasmic part of the enzyme. Recently, this portion has been subcloned, purified, and shown to be an enzymatically active

  17. Identification of a phosphoenolpyruvate:fructose phosphotransferase system (fructose-1-phosphate forming) in Listeria monocytogenes.

    Science.gov (United States)

    Mitchell, W J; Reizer, J; Herring, C; Hoischen, C; Saier, M H

    1993-05-01

    Listeria monocytogenes is a gram-positive bacterium whose carbohydrate metabolic pathways are poorly understood. We provide evidence for an inducible phosphoenolpyruvate (PEP):fructose phosphotransferase system (PTS) in this pathogen. The system consists of enzyme I, HPr, and a fructose-specific enzyme II complex which generates fructose-1-phosphate as the cytoplasmic product of the PTS-catalyzed vectorial phosphorylation reaction. Fructose-1-phosphate kinase then converts the product of the PTS reaction to fructose-1,6-bisphosphate. HPr was shown to be phosphorylated by [32P]PEP and enzyme I as well as by [32P]ATP and a fructose-1,6-bisphosphate-activated HPr kinase like those found in other gram-positive bacteria. Enzyme I, HPr, and the enzyme II complex of the Listeria PTS exhibit enzymatic cross-reactivity with PTS enzyme constituents from Bacillus subtilis and Staphylococcus aureus.

  18. Modeling and analysis of flux distributions in the two branches of the phosphotransferase system in Pseudomonas putida

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    Kremling Andreas

    2012-12-01

    Full Text Available Abstract Background Signal transduction plays a fundamental role in the understanding of cellular physiology. The bacterial phosphotransferase system (PTS together with the PEP/pyruvate node in central metabolism represents a signaling unit that acts as a sensory element and measures the activity of the central metabolism. Pseudomonas putida possesses two PTS branches, the C-branch (PTSFru and a second branch (PTSNtr, which communicate with each other by phosphate exchange. Recent experimental results showed a cross talk between the two branches. However, the functional role of the crosstalk remains open. Results A mathematical model was set up to describe the available data of the state of phosphorylation of PtsN, one of the PTS proteins, for different environmental conditions and different strain variants. Additionally, data from flux balance analysis was used to determine some of the kinetic parameters of the involved reactions. Based on the calculated and estimated parameters, the flux distribution during growth of the wild type strain on fructose could be determined. Conclusion Our calculations show that during growth of the wild type strain on the PTS substrate fructose, the major part of the phosphoryl groups is provided by the second branch of the PTS. This theoretical finding indicates a new role of the second branch of the PTS and will serve as a basis for further experimental studies.

  19. EI of the Phosphotransferase System of Escherichia coli: Mathematical Modeling Approach to Analysis of Its Kinetic Properties

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    T. A. Karelina

    2011-01-01

    Full Text Available The mathematical model of the operation of the first enzyme of the Escherichia coli phosphotransferase system, EI, is proposed. Parameters of the kinetic model describing the operation of EI under different conditions are identified on the basis of a large amount of known experimental data. The verified model is employed to predict modes of operation of EI under both in vivo physiological conditions and in vitro nonphysiological conditions. The model predicts that under in vivo physiological conditions, the rate of phosphotransfer from EI to the second protein of the phosphotransferase system HPr by the dimer is much higher than by the monomer. A hypothesis is proposed on the basis of calculations that the transfer by a monomer plays a role in the regulation of chemotaxis. At submicromolar pyruvate concentration, the model predicts nonmonotonic dependence of the phosphotransfer rate on the substrate (PEP concentration.

  20. Improvement of Escherichia coli production strains by modification of the phosphoenolpyruvate:sugar phosphotransferase system

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    Gosset Guillermo

    2005-05-01

    Full Text Available Abstract The application of metabolic engineering in Escherichia coli has resulted in the generation of strains with the capacity to produce metabolites of commercial interest. Biotechnological processes with these engineered strains frequently employ culture media containing glucose as the carbon and energy source. In E. coli, the phosphoenolpyruvate:sugar phosphotransferase system (PTS transports glucose when this sugar is present at concentrations like those used in production fermentations. This protein system is involved in phosphoenolpyruvate-dependent sugar transport, therefore, its activity has an important impact on carbon flux distribution in the phosphoenolpyruvate and pyruvate nodes. Furthermore, PTS has a very important role in carbon catabolite repression. The properties of PTS impose metabolic and regulatory constraints that can hinder strain productivity. For this reason, PTS has been a target for modification with the purpose of strain improvement. In this review, PTS characteristics most relevant to strain performance and the different strategies of PTS modification for strain improvement are discussed. Functional replacement of PTS by alternative phosphoenolpyruvate-independent uptake and phosphorylation activities has resulted in significant improvements in product yield from glucose and productivity for several classes of metabolites. In addition, inactivation of PTS components has been applied successfully as a strategy to abolish carbon catabolite repression, resulting in E. coli strains that use more efficiently sugar mixtures, such as those obtained from lignocellulosic hydrolysates.

  1. Streptococcal phosphoenolpyruvate-sugar phosphotransferase system: amino acid sequence and site of ATP-dependent phosphorylation of HPr

    International Nuclear Information System (INIS)

    Deutscher, J.; Pevec, B.; Beyreuther, K.; Kiltz, H.H.; Hengstenberg, W.

    1986-01-01

    The amino acid sequence of histidine-containing protein (HPr) from Streptococcus faecalis has been determined by direct Edman degradation of intact HPr and by amino acid sequence analysis of tryptic peptides, V8 proteolyptic peptides, thermolytic peptides, and cyanogen bromide cleavage products. HPr from S. faecalis was found to contain 89 amino acid residues, corresponding to a molecular weight of 9438. The amino acid sequence of HPr from S. faecalis shows extended homology to the primary structure of HPr proteins from other bacteria. Besides the phosphoenolpyruvate-dependent phosphorylation of a histidyl residue in HPr, catalyzed by enzyme I of the bacterial phosphotransferase system, HPr was also found to be phosphorylated at a seryl residue in an ATP-dependent protein kinase catalyzed reaction. The site of ATP-dependent phosphorylation in HPr of S faecalis has now been determined. [ 32 P]P-Ser-HPr was digested with three different proteases, and in each case, a single labeled peptide was isolated. Following digestion with subtilisin, they obtained a peptide with the sequence -(P)Ser-Ile-Met-. Using chymotrypsin, they isolated a peptide with the sequence -Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-Gly-Val-Met-. The longest labeled peptide was obtained with V8 staphylococcal protease. According to amino acid analysis, this peptide contained 36 out of the 89 amino acid residues of HPr. The following sequence of 12 amino acid residues of the V8 peptide was determined: -Tyr-Lys-Gly-Lys-Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-. Thus, the site of ATP-dependent phosphorylation was determined to be Ser-46 within the primary structure of HPr

  2. Streptococcal phosphoenolpyruvate-sugar phosphotransferase system: amino acid sequence and site of ATP-dependent phosphorylation of HPr

    Energy Technology Data Exchange (ETDEWEB)

    Deutscher, J.; Pevec, B.; Beyreuther, K.; Kiltz, H.H.; Hengstenberg, W.

    1986-10-21

    The amino acid sequence of histidine-containing protein (HPr) from Streptococcus faecalis has been determined by direct Edman degradation of intact HPr and by amino acid sequence analysis of tryptic peptides, V8 proteolyptic peptides, thermolytic peptides, and cyanogen bromide cleavage products. HPr from S. faecalis was found to contain 89 amino acid residues, corresponding to a molecular weight of 9438. The amino acid sequence of HPr from S. faecalis shows extended homology to the primary structure of HPr proteins from other bacteria. Besides the phosphoenolpyruvate-dependent phosphorylation of a histidyl residue in HPr, catalyzed by enzyme I of the bacterial phosphotransferase system, HPr was also found to be phosphorylated at a seryl residue in an ATP-dependent protein kinase catalyzed reaction. The site of ATP-dependent phosphorylation in HPr of S faecalis has now been determined. (/sup 32/P)P-Ser-HPr was digested with three different proteases, and in each case, a single labeled peptide was isolated. Following digestion with subtilisin, they obtained a peptide with the sequence -(P)Ser-Ile-Met-. Using chymotrypsin, they isolated a peptide with the sequence -Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-Gly-Val-Met-. The longest labeled peptide was obtained with V8 staphylococcal protease. According to amino acid analysis, this peptide contained 36 out of the 89 amino acid residues of HPr. The following sequence of 12 amino acid residues of the V8 peptide was determined: -Tyr-Lys-Gly-Lys-Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-. Thus, the site of ATP-dependent phosphorylation was determined to be Ser-46 within the primary structure of HPr.

  3. Regulatory tasks of the phosphoenolpyruvate-phosphotransferase system of Pseudomonas putida in central carbon metabolism.

    Science.gov (United States)

    Chavarría, Max; Kleijn, Roelco J; Sauer, Uwe; Pflüger-Grau, Katharina; de Lorenzo, Víctor

    2012-01-01

    Two branches of the phosphoenolpyruvate-phosphotransferase system (PTS) operate in the soil bacterium Pseudomonas putida KT2440. One branch encompasses a complete set of enzymes for fructose intake (PTS(Fru)), while the other (N-related PTS, or PTS(Ntr)) controls various cellular functions unrelated to the transport of carbohydrates. The potential of these two systems for regulating central carbon catabolism has been investigated by measuring the metabolic fluxes of isogenic strains bearing nonpolar mutations in PTS(Fru) or PTS(Ntr) genes and grown on either fructose (a PTS substrate) or glucose, the transport of which is not governed by the PTS in this bacterium. The flow of carbon from each sugar was distinctly split between the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways in a ratio that was maintained in each of the PTS mutants examined. However, strains lacking PtsN (EIIA(Ntr)) displayed significantly higher fluxes in the reactions of the pyruvate shunt, which bypasses malate dehydrogenase in the TCA cycle. This was consistent with the increased activity of the malic enzyme and the pyruvate carboxylase found in the corresponding PTS mutants. Genetic evidence suggested that such a metabolic effect of PtsN required the transfer of high-energy phosphate through the system. The EIIA(Ntr) protein of the PTS(Ntr) thus helps adjust central metabolic fluxes to satisfy the anabolic and energetic demands of the overall cell physiology. This study demonstrates that EIIA(Ntr) influences the biochemical reactions that deliver carbon between the upper and lower central metabolic domains for the consumption of sugars by P. putida. These findings indicate that the EIIA(Ntr) protein is a key player for orchestrating the fate of carbon in various physiological destinations in this bacterium. Additionally, these results highlight the importance of the posttranslational regulation of extant enzymatic complexes for increasing the robustness of the

  4. Identification of lactose phosphotransferase systems in Lactobacillus gasseri ATCC 33323 required for lactose utilization.

    Science.gov (United States)

    Francl, Alyssa L; Hoeflinger, Jennifer L; Miller, Michael J

    2012-04-01

    Improving the annotation of sugar catabolism-related genes requires functional characterization. Our objective was to identify the genes necessary for lactose utilization by Lactobacillus gasseri ATCC 33323 (NCK334). The mechanism of lactose transport in many lactobacilli is a lactose/galactose-specific permease, yet no orthologue was found in NCK334. Characterization of an EI knockout strain [EI (enzyme I) is required for phosphotransferase system transporter (PTS) function] demonstrated that L. gasseri requires PTS(s) to utilize lactose. In order to determine which PTS(s) were necessary for lactose utilization, we compared transcript expression profiles in response to lactose for the 15 complete PTSs identified in the NCK334 genome. PTS 6CB (LGAS_343) and PTS 8C (LGAS_497) were induced in the presence of lactose 107- and 53-fold, respectively. However, L. gasseri ATCC 33323 PTS 6CB, PTS 8C had a growth rate similar to that of the wild-type on semisynthetic deMan, Rogosa, Sharpe (MRS) medium with lactose. Expression profiles of L. gasseri ATCC 33323 PTS 6CB, PTS 8C in response to lactose identified PTS 9BC (LGAS_501) as 373-fold induced, whereas PTS 9BC was not induced in NCK334. Elimination of growth on lactose required the inactivation of both PTS 6CB and PTS 9BC. Among the six candidate phospho-β-galactosidase genes present in the NCK334 genome, LGAS_344 was found to be induced 156-fold in the presence of lactose. In conclusion, we have determined that: (1) NCK334 uses a PTS to import lactose; (2) PTS 6CB and PTS 8C gene expression is strongly induced by lactose; and (3) elimination of PTS 6CB and PTS 9BC is required to prevent growth on lactose.

  5. Both IIC and IID Components of Mannose Phosphotransferase System Are Involved in the Specific Recognition between Immunity Protein PedB and Bacteriocin-Receptor Complex.

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    Wanli Zhou

    Full Text Available Upon exposure to exogenous pediocin-like bacteriocins, immunity proteins specifically bind to the target receptor of the mannose phosphotransferase system components (man-PTS IIC and IID, therefore preventing bacterial cell death. However, the specific recognition of immunity proteins and its associated target receptors remains poorly understood. In this study, we constructed hybrid receptors to identify the domains of IIC and/or IID recognized by the immunity protein PedB, which confers immunity to pediocin PA-1. Using Lactobacillus plantarum man-PTS EII mutant W903, the IICD components of four pediocin PA-1-sensitive strains (L. plantarum WQ0815, Leuconostoc mesenteroides 05-43, Lactobacillus salivarius REN and Lactobacillus acidophilus 05-172 were respectively co-expressed with the immunity protein PedB. Well-diffusions assays showed that only the complex formed by LpIICD from L. plantarum WQ0815 with pediocin PA-1 could be recognized by PedB. In addition, a two-step PCR approach was used to construct hybrid receptors by combining LpIIC or LpIID recognized by PedB with the other three heterologous IID or IIC compounds unrecognized by PedB, respectively. The results showed that all six hybrid receptors were recognized by pediocin PA-1. However, when IIC or IID of L. plantarum WQ0815 was replaced with any corresponding IIC or IID component from L. mesenteroides 05-43, L. salivarius REN and L. acidophilus 05-172, all the hybrid receptors could not be recognized by PedB. Taken altogether, we concluded that both IIC and IID components of the mannose phosphotransferase system play an important role in the specific recognition between the bacteriocin-receptor complex and the immunity protein PedB.

  6. Escherichia coli Phosphoenolpyruvate Dependent Phosphotransferase System. NMR Studies of the Conformation of HPr and P-HPr and the Mechanism of Energy Coupling

    NARCIS (Netherlands)

    Dooijewaard, G.; Roossien, F.F.; Robillard, G.T.

    1979-01-01

    1H and 31P nuclear magnetic resonance investigations of the phosphoprotein intermediate P-HPr and the parent molecule HPr of the E. coli phosphoenolpyruvate dependent phosphotransferase system (PTS) show that HPr can exist in two conformations. These conformations influence the protonation state of

  7. Phosphoenolpyruvate-Dependent Fructose Phosphotransferase System of Rhodopseudomonas sphaeroides : Purification and Physicochemical and Immunochemical Characterization of a Membrane-Associated Enzyme I

    NARCIS (Netherlands)

    Brouwer, Marius; Elferink, Marieke G.L.; Robillard, George T.

    1982-01-01

    The phosphotransferase system (PTS) of the phototrophic bacterium Rhodopseudomonas sphaeroides consists of a component located in the cytoplasmic membrane and a membrane-associated enzyme called “soluble factor” (SF). SF has been partially purified by a combination of hydrophobic interaction and

  8. The Small Protein SgrT Controls Transport Activity of the Glucose-Specific Phosphotransferase System.

    Science.gov (United States)

    Lloyd, Chelsea R; Park, Seongjin; Fei, Jingyi; Vanderpool, Carin K

    2017-06-01

    stress to help bacterial cells maintain balanced metabolism and continue growing. Our results indicate that this protein acts on the glucose transport system, inhibiting its activity under stress conditions in order to allow cells to utilize alternative carbon sources. This work sheds new light on a key biological problem: how cells coordinate carbohydrate transport and metabolism. The study also expands our understanding of the functional capacities of small proteins. Copyright © 2017 American Society for Microbiology.

  9. Isolation, characterization, and nucleotide sequence of the Streptococcus mutans mannitol-phosphate dehydrogenase gene and the mannitol-specific factor III gene of the phosphoenolpyruvate phosphotransferase system.

    Science.gov (United States)

    Honeyman, A L; Curtiss, R

    1992-08-01

    Streptococcus mutans, the causative agent of dental caries, utilizes carbohydrates by means of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The PTS facilitates vectorial translocation of metabolizable carbohydrates to form the corresponding sugar-phosphates, which are subsequently converted to glycolytic intermediates. The PTS consists of both sugar-specific and sugar-independent components. Complementation of an Escherichia coli mtlD mutation with a streptococcal recombinant DNA library allowed isolation of the mannitol-1-phosphate dehydrogenase gene (mtlD) and the adjacent sugar-specific mannitol factor III gene (mtlF) from S. mutans. Subsequent transposon mutagenesis of the complementing DNA fragment with Tn5seq1 defined the region that encodes the mtlD-complementing activity, the streptococcal mtlD gene. Nucleotide sequence analysis of this region revealed two complete open reading frames (ORFs) from within the streptococcal mannitol PTS operon. One ORF encodes the mtlD gene product, a 43.0-kDa protein which exhibits similarity to the E. coli and Enterococcus faecalis mannitol-1-phosphate dehydrogenases. The second ORF encodes a 15.8-kDa protein which exhibits similarity to mannitol factor III proteins from several bacterial species. In vitro transcription-translation assays were used to produce proteins of the sizes predicted by the streptococcal ORFs. These data indicate that the S. mutans mannitol PTS utilizes an enzyme II-factor III complex similar to the mannitol system found in other gram-positive organisms, as opposed to that of E. coli, which utilizes an independent enzyme II system.

  10. The histidine-phosphocarrier protein of the phosphoenolpyruvate: sugar phosphotransferase system of Bacillus sphaericus self-associates.

    Directory of Open Access Journals (Sweden)

    Rosa Doménech

    Full Text Available The phosphotransferase system (PTS is involved in the use of carbon sources in bacteria. Bacillus sphaericus, a bacterium with the ability to produce insecticidal proteins, is unable to use hexoses and pentoses as the sole carbon source, but it has ptsHI genes encoding the two general proteins of the PTS: enzyme I (EI and the histidine phosphocarrier (HPr. In this work, we describe the biophysical and structural properties of HPr from B. sphaericus, HPr(bs, and its affinity towards EI of other species to find out whether there is inter-species binding. Conversely to what happens to other members of the HPr family, HPr(bs forms several self-associated species. The conformational stability of the protein is low, and it unfolds irreversibly during heating. The protein binds to the N-terminal domain of EI from Streptomyces coelicolor, EIN(sc, with a higher affinity than that of the natural partner of EIN(sc, HPr(sc. Modelling of the complex between EIN(sc and HPr(bs suggests that binding occurs similarly to that observed in other HPr species. We discuss the functional implications of the oligomeric states of HPr(bs for the glycolytic activity of B. sphaericus, as well as a strategy to inhibit binding between HPr(sc and EIN(sc.

  11. Plasticity of regulation of mannitol phosphotransferase system operon by CRP-cAMP complex in Vibrio cholerae.

    Science.gov (United States)

    Zhou, Yan Yan; Zhang, Hong Zhi; Liang, Wei Li; Zhang, Li Juan; Zhu, Jun; Kan, Biao

    2013-10-01

    The complex of the cyclic AMP receptor protein (CRP) and cAMP is an important transcriptional regulator of numerous genes in prokaryotes. The transport of mannitol through the phosphotransferase systems (PTS) is regulated by the CRP-cAMP complex. The aim of the study is to investigate how the CRP-cAMP complex acting on the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype. The crp mutant strain was generated by homologous recombination to assess the need of CRP to activate the mannitol PTS operon of V. cholerae El Tor. Electrophoretic mobility shift assays (EMSA) and the reporter plasmid pBBRlux were used to confirm the role that the CRP-cAMP complex playing on the mannitol PTS operon mtl. In this study, we confirmed that CRP is strictly needed for the activation of the mtl operon. We further experimentally identified five CRP binding sites within the promoter region upstream of the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype and found that these sites display different affinities for CRP and provide different contributions to the activation of the operon. The five binding sites collectively confer the strong activation of mannitol transfer by CRP in V. cholerae, indicating an elaborate and subtle CRP activation mechanism. Copyright © 2013 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  12. Nitrogen regulator GlnR controls uptake and utilization of non-phosphotransferase-system carbon sources in actinomycetes.

    Science.gov (United States)

    Liao, Cheng-Heng; Yao, Lili; Xu, Ya; Liu, Wei-Bing; Zhou, Ying; Ye, Bang-Ce

    2015-12-22

    The regulatory mechanisms underlying the uptake and utilization of multiple types of carbohydrates in actinomycetes remain poorly understood. In this study, we show that GlnR (central regulator of nitrogen metabolism) serves as a universal regulator of nitrogen metabolism and plays an important, previously unknown role in controlling the transport of non-phosphotransferase-system (PTS) carbon sources in actinomycetes. It was observed that GlnR can directly interact with the promoters of most (13 of 20) carbohydrate ATP-binding cassette (ABC) transporter loci and can activate the transcription of these genes in response to nitrogen availability in industrial, erythromycin-producing Saccharopolyspora erythraea. Deletion of the glnR gene resulted in severe growth retardation under the culture conditions used, with select ABC-transported carbohydrates (maltose, sorbitol, mannitol, cellobiose, trehalose, or mannose) used as the sole carbon source. Furthermore, we found that GlnR-mediated regulation of carbohydrate transport was highly conserved in actinomycetes. These results demonstrate that GlnR serves a role beyond nitrogen metabolism, mediating critical functions in carbon metabolism and crosstalk of nitrogen- and carbon-metabolism pathways in response to the nutritional states of cells. These findings provide insights into the molecular regulation of transport and metabolism of non-PTS carbohydrates and reveal potential applications for the cofermentation of biomass-derived sugars in the production of biofuels and bio-based chemicals.

  13. Electrotransformation and Expression of Bacterial Genes Encoding Hygromycin Phosphotransferase and β-Galactosidase in the Pathogenic Fungus Histoplasma capsulatum

    OpenAIRE

    Woods, Jon P.; Heinecke, Elizabeth L.; Goldman, William E.

    1998-01-01

    We developed an efficient electrotransformation system for the pathogenic fungus Histoplasma capsulatum and used it to examine the effects of features of the transforming DNA on transformation efficiency and fate of the transforming DNA and to demonstrate fungal expression of two recombinant Escherichia coli genes, hph and lacZ. Linearized DNA and plasmids containing Histoplasma telomeric sequences showed the greatest transformation efficiencies, while the plasmid vector had no significant ef...

  14. Synbiotic impact of tagatose on viability of Lactobacillus rhamnosus strain GG mediated by the phosphotransferase system (PTS).

    Science.gov (United States)

    Koh, Ji Hoon; Choi, Seung Hye; Park, Seung Won; Choi, Nag-Jin; Kim, Younghoon; Kim, Sae Hun

    2013-10-01

    Synbiotics, the combination of prebiotics and probiotics, has been shown to produce synergistic effects that promote gastrointestinal well-being of host. Tagatose is a low calorie food ingredient with putative health-promoting benefits. Herein, we investigated its synbiotic impact on the viability of Lactobacillus casei 01 and Lactobacillus rhamnosus strain GG and the potential mechanism involved. Tagatose, as a synbiotic substrate, enhanced the growth of L. casei 01 and L. rhamnosus strain GG compared to other prebiotics. Other gut-indigenous such as Clostridium spp. readily utilized fructooligosaccharide (FOS), the most widely used functional prebiotics, but not tagatose. Additionally, tagatose enhanced probiotic functions of L. casei 01 and L. rhamnosus strain GG by reinforcing their attachment on HT-29 intestine epithelial cells and enhancing their cholesterol-lowering activities. Whole transcriptome study and quantitative real-time polymerase chain reaction (qRT-PCR) test showed that the presence of tagatose in L. rhamnosus strain GG caused induction of a large number of genes associated with carbohydrate metabolism including the phosphotransferase system (PTS). Collectively, these results indicate the tagatose enhanced the growth of L. casei 01 and L. rhamnosus strain GG and their probiotic activities by activating tagatose-associated PTS networks. Importantly, this study highlights the potential application of tagatose and L. casei 01 and/or L. rhamnosus strain GG as a synbiotic partner in functional dairy foods (i.e. yogurt and cheese) and therapeutic dietary supplements. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Enterococcus faecalis Uses a Phosphotransferase System Permease and a Host Colonization-Related ABC Transporter for Maltodextrin Uptake.

    Science.gov (United States)

    Sauvageot, Nicolas; Mokhtari, Abdelhamid; Joyet, Philippe; Budin-Verneuil, Aurélie; Blancato, Víctor S; Repizo, Guillermo D; Henry, Céline; Pikis, Andreas; Thompson, John; Magni, Christian; Hartke, Axel; Deutscher, Josef

    2017-05-01

    Maltodextrin is a mixture of maltooligosaccharides, which are produced by the degradation of starch or glycogen. They are mostly composed of α-1,4- and some α-1,6-linked glucose residues. Genes presumed to code for the Enterococcus faecalis maltodextrin transporter were induced during enterococcal infection. We therefore carried out a detailed study of maltodextrin transport in this organism. Depending on their length (3 to 7 glucose residues), E. faecalis takes up maltodextrins either via MalT, a maltose-specific permease of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), or the ATP binding cassette (ABC) transporter MdxEFG-MsmX. Maltotriose, the smallest maltodextrin, is primarily transported by the PTS permease. A malT mutant therefore exhibits significantly reduced growth on maltose and maltotriose. The residual uptake of the trisaccharide is catalyzed by the ABC transporter, because a malT mdxF double mutant no longer grows on maltotriose. The trisaccharide arrives as maltotriose-6″-P in the cell. MapP, which dephosphorylates maltose-6'-P, also releases P i from maltotriose-6″-P. Maltotetraose and longer maltodextrins are mainly (or exclusively) taken up via the ABC transporter, because inactivation of the membrane protein MdxF prevents growth on maltotetraose and longer maltodextrins up to at least maltoheptaose. E. faecalis also utilizes panose and isopanose, and we show for the first time, to our knowledge, that in contrast to maltotriose, its two isomers are primarily transported via the ABC transporter. We confirm that maltodextrin utilization via MdxEFG-MsmX affects the colonization capacity of E. faecalis , because inactivation of mdxF significantly reduced enterococcal colonization and/or survival in kidneys and liver of mice after intraperitoneal infection. IMPORTANCE Infections by enterococci, which are major health care-associated pathogens, are difficult to treat due to their increasing resistance to clinically

  16. Metabolic engineering for the production of shikimic acid in an evolved Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system.

    Science.gov (United States)

    Escalante, Adelfo; Calderón, Rocío; Valdivia, Araceli; de Anda, Ramón; Hernández, Georgina; Ramírez, Octavio T; Gosset, Guillermo; Bolívar, Francisco

    2010-04-12

    Shikimic acid (SA) is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this work, metabolic engineering approaches were employed to produce SA in Escherichia coli strains derived from an evolved strain (PB12) lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS-) but with capacity to grow on glucose. Derivatives of PB12 strain were constructed to determine the effects of inactivating aroK, aroL, pykF or pykA and the expression of plasmid-coded genes aroGfbr, tktA, aroB and aroE, on SA synthesis. Batch cultures were performed to evaluate the effects of genetic modifications on growth, glucose consumption, and aromatic intermediate production. All derivatives showed a two-phase growth behavior with initial high specific growth rate (mu) and specific glucose consumption rate (qs), but low level production of aromatic intermediates. During the second growth phase the mu decreased, whereas aromatic intermediate production reached its maximum. The double aroK- aroL- mutant expressing plasmid-coded genes (strain PB12.SA22) accumulated SA up to 7 g/L with a yield of SA on glucose of 0.29 mol/mol and a total aromatic compound yield (TACY) of 0.38 mol/mol. Single inactivation of pykF or pykA was performed in PB12.SA22 strain. Inactivation of pykF caused a decrease in mu, qs, SA production, and yield; whereas TACY increased by 33% (0.5 mol/mol). The effect of increased availability of carbon metabolites, their channeling into the synthesis of aromatic intermediates, and disruption of the SA pathway on SA production was studied. Inactivation of both aroK and aroL, and transformation with plasmid-coded genes resulted in the accumulation of SA up to 7 g/L with a yield on glucose of 0.29 mol/mol PB12.SA22, which represents the highest reported yield. The pykF and pykA genes were inactivated in strain PB12.SA22 to increase the production of aromatic compounds in the PTS- background. Results indicate differential roles of Pyk

  17. Identification and functional analysis of the L-ascorbate-specific enzyme II complex of the phosphotransferase system in Streptococcus mutans.

    Science.gov (United States)

    Wu, Xinyu; Hou, Jin; Chen, Xiaodan; Chen, Xuan; Zhao, Wanghong

    2016-03-22

    Streptococcus mutans is the primary etiological agent of human dental caries. It can metabolize a wide variety of carbohydrates and produce large amounts of organic acids that cause enamel demineralization. Phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) plays an important role in carbohydrates uptake of S. mutans. The ptxA and ptxB genes in S. mutans encode putative enzyme IIA and enzyme IIB of the L-ascorbate-specific PTS. The aim of this study was to analyze the function of these proteins and understand the transcriptional regulatory mechanism. ptxA (-), ptxB (-), as well as ptxA (-) , ptxB (-) double-deletion mutants all had more extended lag phase and lower growth yield than wild-type strain UA159 when grown in the medium using L-ascorbate as the sole carbon source. Acid production and acid killing assays showed that the absence of the ptxA and ptxB genes resulted in a reduction in the capacity for acidogenesis, and all three mutant strains did not survive an acid shock. According to biofilm and extracellular polysaccharides (EPS) formation analysis, all the mutant strains formed much less prolific biofilms with small amounts of EPS than wild-type UA159 when using L-ascorbate as the sole carbon source. Moreover, PCR analysis and quantitative real-time PCR revealed that sgaT, ptxA, ptxB, SMU.273, SMU.274 and SMU.275 appear to be parts of the same operon. The transcription levels of these genes were all elevated in the presence of L-ascorbate, and the expression of ptxA gene decreased significantly once ptxB gene was knockout. The ptxA and ptxB genes are involved in the growth, aciduricity, acidogenesis, and formation of biofilms and EPS of S. mutans when L-ascorbate is the sole carbon source. In addition, the expression of ptxA is regulated by ptxB. ptxA, ptxB, and the upstream gene sgaT, the downstream genes SMU.273, SMU.274 and SMU.275 appear to be parts of the same operon, and L-ascorbate is a potential inducer of the operon.

  18. Metabolic engineering for the production of shikimic acid in an evolved Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system

    Directory of Open Access Journals (Sweden)

    Bolívar Francisco

    2010-04-01

    Full Text Available Abstract Background Shikimic acid (SA is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this work, metabolic engineering approaches were employed to produce SA in Escherichia coli strains derived from an evolved strain (PB12 lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS- but with capacity to grow on glucose. Derivatives of PB12 strain were constructed to determine the effects of inactivating aroK, aroL, pykF or pykA and the expression of plasmid-coded genes aroGfbr, tktA, aroB and aroE, on SA synthesis. Results Batch cultures were performed to evaluate the effects of genetic modifications on growth, glucose consumption, and aromatic intermediate production. All derivatives showed a two-phase growth behavior with initial high specific growth rate (μ and specific glucose consumption rate (qs, but low level production of aromatic intermediates. During the second growth phase the μ decreased, whereas aromatic intermediate production reached its maximum. The double aroK- aroL- mutant expressing plasmid-coded genes (strain PB12.SA22 accumulated SA up to 7 g/L with a yield of SA on glucose of 0.29 mol/mol and a total aromatic compound yield (TACY of 0.38 mol/mol. Single inactivation of pykF or pykA was performed in PB12.SA22 strain. Inactivation of pykF caused a decrease in μ, qs, SA production, and yield; whereas TACY increased by 33% (0.5 mol/mol. Conclusions The effect of increased availability of carbon metabolites, their channeling into the synthesis of aromatic intermediates, and disruption of the SA pathway on SA production was studied. Inactivation of both aroK and aroL, and transformation with plasmid-coded genes resulted in the accumulation of SA up to 7 g/L with a yield on glucose of 0.29 mol/mol PB12.SA22, which represents the highest reported yield. The pykF and pykA genes were inactivated in strain PB12.SA22 to increase the production of aromatic compounds in the PTS

  19. Glucose-Specific Enzyme IIA of the Phosphoenolpyruvate:Carbohydrate Phosphotransferase System Modulates Chitin Signaling Pathways in Vibrio cholerae.

    Science.gov (United States)

    Yamamoto, Shouji; Ohnishi, Makoto

    2017-09-15

    In Vibrio cholerae , the genes required for chitin utilization and natural competence are governed by the chitin-responsive two-component system (TCS) sensor kinase ChiS. In the classical TCS paradigm, a sensor kinase specifically phosphorylates a cognate response regulator to activate gene expression. However, our previous genetic study suggested that ChiS stimulates the non-TCS transcriptional regulator TfoS by using mechanisms distinct from classical phosphorylation reactions (S. Yamamoto, J. Mitobe, T. Ishikawa, S. N. Wai, M. Ohnishi, H. Watanabe, and H. Izumiya, Mol Microbiol 91:326-347, 2014, https://doi.org/10.1111/mmi.12462). TfoS specifically activates the transcription of tfoR , encoding a small regulatory RNA essential for competence gene expression. Whether ChiS and TfoS interact directly remains unknown. To determine if other factors mediate the communication between ChiS and TfoS, we isolated transposon mutants that turned off tfoR :: lacZ expression but possessed intact chiS and tfoS genes. We demonstrated an unexpected association of chitin-induced signaling pathways with the glucose-specific enzyme IIA (EIIA glc ) of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) for carbohydrate uptake and catabolite control of gene expression. Genetic and physiological analyses revealed that dephosphorylated EIIA glc inactivated natural competence and tfoR transcription. Chitin-induced expression of the chb operon, which is required for chitin transport and catabolism, was also repressed by dephosphorylated EIIA glc Furthermore, the regulation of tfoR and chb expression by EIIA glc was dependent on ChiS and intracellular levels of ChiS were not affected by disruption of the gene encoding EIIA glc These results define a previously unknown connection between the PTS and chitin signaling pathways in V. cholerae and suggest a strategy whereby this bacterium can physiologically adapt to the existing nutrient status. IMPORTANCE The EIIA glc

  20. Control of Lactose Transport, β-Galactosidase Activity, and Glycolysis by CcpA in Streptococcus thermophilus : Evidence for Carbon Catabolite Repression by a Non-Phosphoenolpyruvate-Dependent Phosphotransferase System Sugar

    NARCIS (Netherlands)

    Bogaard, Patrick T.C. van den; Kleerebezem, Michiel; Kuipers, Oscar P.; Vos, Willem M. de

    2000-01-01

    Streptococcus thermophilus, unlike many other gram-positive bacteria, prefers lactose over glucose as the primary carbon and energy source. Moreover, lactose is not taken up by a phosphoenolpyruvate-dependent phosphotransferase system (PTS) but by the dedicated transporter LacS. In this paper we

  1. Flux control of the bacterial phosphoenolpyruvate: glucose phosphotransferase system and the effect of diffusion..

    NARCIS (Netherlands)

    Francke, C.; Westerhoff, H.V.; Blom, J.G.; Peletier, M.A.

    2002-01-01

    We analyzed the role of diffusion and cell size on the flux control properties of the glucose-PTS of Escherichia coli, in silicon cells under various metabolic conditions. To our surprise, the influence of the concentration of phosphoryl-donor PEP on the distribution of control was small. We found

  2. Enzyme II/sup Mtl/ of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: identification of the activity-linked cysteine on the mannitol carrier

    International Nuclear Information System (INIS)

    Pas, H.H.; Robillard, G.T.

    1988-01-01

    The cysteine of the membrane-bound mannitol-specific enzyme II (EII/sup Mtl/) of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system have been labeled with 4-vinylpyridine. After proteolytic breakdown and reversed-phase HPLC, the peptides containing cysteines 110, 384, and 571 could be identified. N-Ethylmaleimide (NEM) treatment of the native unphosphorylated enzyme results in incorporation of one NEM label per molecule and loss of enzymatic activity. NEM treatment and inactivation prevented 4-vinylpyridine incorporation into the Cys-384-containing peptide, identifying this residue as the activity-linked cysteine. Both oxidation and phosphorylation of the native enzyme protected the enzyme against NEM labeling of Cys-384. Positive identification of the activity-linked cysteine was accomplished by inactivation with [ 14 C]iodoacetamide, proteolytic fragmentation, isolation of the peptide, and amino acid sequencing

  3. Method for construction of bacterial strains with increased succinic acid production

    Science.gov (United States)

    Donnelly, Mark I.; Sanville-Millard, Cynthia; Chatterjee, Ranjini

    2000-01-01

    A fermentation process for producing succinic acid is provided comprising selecting a bacterial strain that does not produce succinic acid in high yield, disrupting the normal regulation of sugar metabolism of said bacterial strain, and combining the mutant bacterial strain and selected sugar in anaerobic conditions to facilitate production of succinic acid. Also provided is a method for changing low yield succinic acid producing bacteria to high yield succinic acid producing bacteria comprising selecting a bacterial strain having a phosphotransferase system and altering the phosphotransferase system so as to allow the bacterial strain to simultaneously metabolize different sugars.

  4. The phosphotransferase system gene ptsI in the endophytic bacterium Bacillus cereus is required for biofilm formation, colonization, and biocontrol against wheat sharp eyespot.

    Science.gov (United States)

    Xu, Yu-Bin; Chen, Mai; Zhang, Ying; Wang, Miao; Wang, Ying; Huang, Qiu-bin; Wang, Xue; Wang, Gang

    2014-05-01

    Natural resistance of wheat plants to wheat sharp eyespot is inadequate, and new strategies for controlling the disease are required. Biological control is an alternative and attractive way of reducing the use of chemicals in agriculture. In this study, we investigated the biocontrol properties of endophytic bacterium Bacillus cereus strain 0-9, which was isolated from the root systems of healthy wheat varieties. The phosphotransferase system is a major regulator of carbohydrate metabolism in bacteria. Enzyme I is one of the protein components of this system. Specific disruption and complementation of the enzyme I-coding gene ptsI from B. cereus was achieved through homologous recombination. Disruption of ptsI in B. cereus caused a 70% reduction in biofilm formation, a 30.4% decrease in biocontrol efficacy, and a 1000-fold reduction in colonization. The growth of ΔptsI mutant strain on G-tris synthetic medium containing glucose as the exclusive carbon source was also reduced. Wild-type properties could be restored to the ΔptsI mutant strain by ptsI complementation. These results suggested that ptsI may be one of the key genes involved in biofilm formation, colonization, and biocontrol of B. cereus and that B. cereus wild-type strain 0-9 may be an ideal biocontrol agent for controlling wheat sharp eyespot. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  5. Lactobacillus casei 64H Contains a Phosphoenolpyruvate-Dependent Phosphotransferase System for Uptake of Galactose, as Confirmed by Analysis of ptsH and Different gal Mutants

    Science.gov (United States)

    Bettenbrock, Katja; Siebers, Ulrike; Ehrenreich, Petra; Alpert, Carl-Alfred

    1999-01-01

    Galactose metabolism in Lactobacillus casei 64H was analyzed by genetic and biochemical methods. Mutants with defects in ptsH, galK, or the tagatose 6-phosphate pathway were isolated either by positive selection using 2-deoxyglucose or 2-deoxygalactose or by an enrichment procedure with streptozotocin. ptsH mutations abolish growth on lactose, cellobiose, N-acetylglucosamine, mannose, fructose, mannitol, glucitol, and ribitol, while growth on galactose continues at a reduced rate. Growth on galactose is also reduced, but not abolished, in galK mutants. A mutation in galK in combination with a mutation in the tagatose 6-phosphate pathway results in sensitivity to galactose and lactose, while a galK mutation in combination with a mutation in ptsH completely abolishes galactose metabolism. Transport assays, in vitro phosphorylation assays, and thin-layer chromatography of intermediates of galactose metabolism also indicate the functioning of a permease/Leloir pathway and a phosphoenolpyruvate-dependent phosphotransferase system (PTS)/tagatose 6-phosphate pathway. The galactose-PTS is induced by growth on either galactose or lactose, but the induction kinetics for the two substrates are different. PMID:9864334

  6. Stimulation of dihydroxyacetone and glycerol kinase activity in Streptococcus faecalis by phosphoenolpyruvate-dependent phosphorylation catalyzed by enzyme I and HPr of the phosphotransferase systems

    International Nuclear Information System (INIS)

    Deutscher, J.; Sauerwald, H.

    1986-01-01

    Recently a report was given of the phosphoenolpyruvate (PEP)-dependent phosphorylation of a 55-kilodalton protein of Streptococus faecalis catalyzed by enzyme I and histidine-containing protein (HPr) of the phosphotransferase system. The purified 55-kilodalton protein was found to exhibit dihydroxyacetone kinase activity. Glycerol was six times more slowly phosphorylated than dihydroxyacetone. The K/sub m/s were found to 0.7 mM for ATP, 0.45 mM for dihydroxyacetone, and 0.9 MM for glycerol. PEP-dependent phosphorylation of dihydroxyacetone kinase stimulated phosphorylation of both substrates about 10-fold. Fructose 1,6-diphosphate at concentrations higher than 2 mM inhibited the activity of phosphorylated and unphosphorylated dihydroxyacetone kinase in a noncompetitive manner. The rate of PEP-dependent phosphorylation of dihydroxyacetone kinase was about 200-fold slower than the phosphorylation rate of III proteins (also called enzyme III or factor III), which so far have been considered the only phosphoryl acceptors of histidyl-phosphorylated HPr. P-Dihydroxyacetone kinase was found to be able to transfer its phosphoryl group in a backward reaction to HPr. Following [ 32 P]PEP-dependent phosphorylation and tryptic digestion of dihydroxyacetone kinase, the authors isolated a labeled peptide composed of 37 amino acids, as determined by amino acid analysis. The single histidyl residue of this peptide most likely carries the phosphoryl group in phosphorylated dihydroxyacetone kinase

  7. Stimulation of dihydroxyacetone and glycerol kinase activity in Streptococcus faecalis by phosphoenolpyruvate-dependent phosphorylation catalyzed by enzyme I and HPr of the phosphotransferase systems

    Energy Technology Data Exchange (ETDEWEB)

    Deutscher, J.; Sauerwald, H.

    1986-06-01

    Recently a report was given of the phosphoenolpyruvate (PEP)-dependent phosphorylation of a 55-kilodalton protein of Streptococus faecalis catalyzed by enzyme I and histidine-containing protein (HPr) of the phosphotransferase system. The purified 55-kilodalton protein was found to exhibit dihydroxyacetone kinase activity. Glycerol was six times more slowly phosphorylated than dihydroxyacetone. The K/sub m/s were found to 0.7 mM for ATP, 0.45 mM for dihydroxyacetone, and 0.9 MM for glycerol. PEP-dependent phosphorylation of dihydroxyacetone kinase stimulated phosphorylation of both substrates about 10-fold. Fructose 1,6-diphosphate at concentrations higher than 2 mM inhibited the activity of phosphorylated and unphosphorylated dihydroxyacetone kinase in a noncompetitive manner. The rate of PEP-dependent phosphorylation of dihydroxyacetone kinase was about 200-fold slower than the phosphorylation rate of III proteins (also called enzyme III or factor III), which so far have been considered the only phosphoryl acceptors of histidyl-phosphorylated HPr. P-Dihydroxyacetone kinase was found to be able to transfer its phosphoryl group in a backward reaction to HPr. Following (/sup 32/P)PEP-dependent phosphorylation and tryptic digestion of dihydroxyacetone kinase, the authors isolated a labeled peptide composed of 37 amino acids, as determined by amino acid analysis. The single histidyl residue of this peptide most likely carries the phosphoryl group in phosphorylated dihydroxyacetone kinase.

  8. Genome-wide Screening Identifies Phosphotransferase System Permease BepA to Be Involved in Enterococcus faecium Endocarditis and Biofilm Formation.

    Science.gov (United States)

    Paganelli, Fernanda L; Huebner, Johannes; Singh, Kavindra V; Zhang, Xinglin; van Schaik, Willem; Wobser, Dominique; Braat, Johanna C; Murray, Barbara E; Bonten, Marc J M; Willems, Rob J L; Leavis, Helen L

    2016-07-15

    Enterococcus faecium is a common cause of nosocomial infections, of which infective endocarditis is associated with substantial mortality. In this study, we used a microarray-based transposon mapping (M-TraM) approach to evaluate a rat endocarditis model and identified a gene, originally annotated as "fruA" and renamed "bepA," putatively encoding a carbohydrate phosphotransferase system (PTS) permease (biofilm and endocarditis-associated permease A [BepA]), as important in infective endocarditis. This gene is highly enriched in E. faecium clinical isolates and absent in commensal isolates that are not associated with infection. Confirmation of the phenotype was established in a competition experiment of wild-type and a markerless bepA mutant in a rat endocarditis model. In addition, deletion of bepA impaired biofilm formation in vitro in the presence of 100% human serum and metabolism of β-methyl-D-glucoside. β-glucoside metabolism has been linked to the metabolism of glycosaminoglycans that are exposed on injured heart valves, where bacteria attach and form vegetations. Therefore, we propose that the PTS permease BepA is directly implicated in E. faecium pathogenesis. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  9. Coutilization of glucose and glycerol enhances the production of aromatic compounds in an Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system

    Directory of Open Access Journals (Sweden)

    Bolívar Francisco G

    2008-01-01

    Full Text Available Abstract Background Escherichia coli strains lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS are capable of coutilizing glucose and other carbon sources due to the absence of catabolite repression by glucose. In these strains, the lack of this important regulatory and transport system allows the coexistence of glycolytic and gluconeogenic pathways. Strains lacking PTS have been constructed with the goal of canalizing part of the phosphoenolpyruvate (PEP not consumed in glucose transport to the aromatic pathway. The deletion of the ptsHIcrr operon inactivates PTS causing poor growth on this sugar; nonetheless, fast growing mutants on glucose have been isolated (PB12 strain. However, there are no reported studies concerning the growth potential of a PTS- strain in mixtures of different carbon sources to enhance the production of aromatics compounds. Results PB12 strain is capable of coutilizing mixtures of glucose-arabinose, glucose-gluconate and glucose-glycerol. This capacity increases its specific growth rate (μ given that this strain metabolizes more moles of carbon source per unit time. The presence of plasmids pRW300aroGfbr and pCLtktA reduces the μ of strain PB12 in all mixtures of carbon sources, but enhances the productivity and yield of aromatic compounds, especially in the glucose-glycerol mixture, as compared to glucose or glycerol cultures. No acetate was detected in the glycerol and the glucose-glycerol batch fermentations. Conclusion Due to the lack of catabolite repression, PB12 strain carrying multicopy plasmids containing tktA and aroGfbr genes is capable of coutilizing glucose and other carbon sources; this capacity, reduces its μ but increases the production of aromatic compounds.

  10. Uptake of N,N'-diacetylchitobiose [(GlcNAc)2] via the phosphotransferase system is essential for chitinase production by Serratia marcescens 2170.

    Science.gov (United States)

    Uchiyama, Taku; Kaneko, Ryousuke; Yamaguchi, Junko; Inoue, Akane; Yanagida, Takahiro; Nikaidou, Naoki; Regue, Miguel; Watanabe, Takeshi

    2003-03-01

    The chiR gene of Serratia marcescens 2170, encoding a LysR-type transcriptional activator, was identified previously as an essential factor for expression of chitinases and a chitin-binding protein, CBP21. To identify other genes that are essential for chitinase production, transposon mutagenesis with mini-Tn5Km1 was carried out, and 25 mutants that were unable to produce chitinases and CBP21 were obtained. Analysis of the mutated gene of one of the mutants, N22, revealed the presence of a pts operon in this bacterium, and a mutation was found in ptsI in the operon. In addition to its inability to produce chitinase, N22 did not grow well on N-acetyl-D-glucosamine (GlcNAc), (GlcNAc)(2), and some other carbon sources, most of which were phosphotransferase system (PTS) sugars. Thus, the inability to produce chitinase was assumed to be caused by the defect in uptake of (GlcNAc)(2) via the PTS, considering that (GlcNAc)(2) is the minimal substrate for chitinase induction and the major product of chitin hydrolysis by chitinases of this bacterium. To confirm this assumption, the chb operon, encoding the (GlcNAc)(2)-specific enzyme II permease, was cloned by reference to its Escherichia coli counterpart, and the Serratia chb operon was shown to comprise chbB, chbC, bglA, chbR, and chbG. Disruption of chbC drastically reduced production of chitinases and CBP21 and impaired growth on colloidal chitin. These results indicate that uptake of (GlcNAc)(2) is mediated by the PTS and that the (GlcNAc)(2)-specific enzyme II permease constitutes its major pathway. Since (GlcNAc)(2) uptake is essential for induction of chitinases and CBP21 production, (GlcNAc)(2) appears to be the key molecule in recognition and utilization of chitin by S. marcescens.

  11. Cloning and biochemical analysis of β-glucoside utilization (bgl) operon without phosphotransferase system in Pectobacterium carotovorum subsp. carotovorum LY34.

    Science.gov (United States)

    An, Chang Long; Kim, Min Keun; Kang, Tae Ho; Kim, Jungho; Kim, Hoon; Yun, Han Dae

    2012-09-06

    β-Glucosidases are widespread in bacteria and involved in the metabolism of various carbohydrate substrates. Studying of β-glucoside utilization (bgl) operons on operon of Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34) will help us understanding how β-glucoside utilization (bgl) operon can cooperate with other systems in bacterium caused soft-rot disease. Pcc LY34 causes soft-rot disease in plants and expresses multiple enzymatic forms of β-glucosidases. To fully explore the β-glucoside utilization system in Pcc LY34, we have isolated a bgl operon from a genomic library for screening of β-glucosidase activities. Sequence analysis of a 3050bp cloned DNA fragment (accession number AY870655) shows two open reading frames (bglY and bglK) that are predicted to encode proteins of 474 and 278 amino acid residues, respectively. Pair wise similarity analysis suggests BglY is a beta-glucosidase (a member of glycosyl hydrolase family 1) and BglK is a transcriptional antiterminator protein. bglY promoter region contains an inverted repeat sequence similar to transcriptional terminator. Different from other four β-glucoside utilization operons of Pcc LY34 strain, BglY contains signal peptide sequences as extracellular β-glucosidase. Comparisons of five β-glucoside utilization operons of Pcc LY34 strain showed that bglYK operon does not have phosphotransferase system domain which are responsible for sugar transportation. BglY shares 33-44% identity with other four β-glucosidases of Pcc LY34 strain. Enzyme assay showed that purified BglY enzyme hydrolyzed salicin, arbutin, pNPG, and MUG, and exhibited maximal activity at pH 7.0 and 40°C. This activity was enhanced Mg(2+). Site-directed mutagenesis revealed E166 and E371 are critical of BglY's β-glucosidase activity. Copyright © 2012 Elsevier GmbH. All rights reserved.

  12. sigma(54)-mediated control of the mannose phosphotransferase sytem in Lactobacillus plantarum impacts on carbohydrate metabolism

    NARCIS (Netherlands)

    Stevens, Marc J. A.; Molenaar, Douwe; de Jong, Anne; De Vos, Willem M.; Kleerebezem, Michiel

    Sigma factors direct specific binding of the bacterial RNA polymerase to the promoter. Here we present the elucidation of the sigma(54) regulon in Lactobacillus plantarum. A sequence-based regulon prediction of sigma(54)-dependent promoters revealed an operon encoding a mannose phosphotransferase

  13. Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: purification and characterization of the mannitol-specific enzyme III/sup mtl/ of Staphylococcus aureus and Staphylococcus carnosus and homology with the enzyme II/sup mtl/ of Escherichia coli

    International Nuclear Information System (INIS)

    Reiche, B.; Frank, R.; Deutscher, J.; Meyer, N.; Hengstenberg, W.

    1988-01-01

    Enzyme III/sup mtl/ is part of the mannitol phosphotransferase system of Staphylococcus aureus and Staphylococcus carnosus and is phosphorylated by phosphoenolpyruvate in a reaction sequence requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase) and the histidine-containing protein HPr. In this paper, the authors report the isolation of III/sup mtl/ from both S. aureus and S. carnosus and the characterization of the active center. After phosphorylation of III/sup mtl/ with [ 32 P]PEP, enzyme I, and HPr, the phosphorylated protein was cleaved with endoproteinase GLu(C). The amino acid sequence of the S. aureus peptide carrying the phosphoryl group was found to be Gln-Val-Val-Ser-Thr-Phe-Met-Gly-Asn-Gly-Leu-Ala-Ile-Pro-His-Gly-Thr-Asp-Asp. The corresponding peptide from S. carnosus shows an equal sequence except that the first residue is Ala instead of Gln. These peptides both contain a single histidyl residue which they assume to carry the phosphoryl group. All proteins of the PTS so far investigated indeed carry the phosphoryl group attached to a histidyl residue. According to sodium dodecyl sulfate gels, the molecular weight of the III/sup mtl/ proteins was found to be 15,000. They have also determined the N-terminal sequence of both proteins. Comparison of the III/sup mtl/ peptide sequences and the C-terminal part of the enzyme II/sup mtl/ of Escherichia coli reveals considerable sequence homology, which supports the suggestion that II/sup mtl/ of E. coli is a fusion protein of a soluble III protein with a membrane-bound enzyme II

  14. Purification and Characterization of Aminoglycoside Phosphotransferase APH(6)-Id, a Streptomycin Inactivating Enzyme

    OpenAIRE

    Ashenafi, Meseret; Ammosova, Tatiana; Nekhai, Sergei; Byrnes, W. Malcolm

    2013-01-01

    As part of an overall project to characterize the streptomycin phosphotransferase enzyme APH(6)-Id, which confers bacterial resistance to streptomycin, we cloned, expressed, purified and characterized the enzyme. When expressed in E. coli, the recombinant enzyme increased by up to 70-fold the minimum inhibitory concentration (MIC) needed to inhibit cell growth. Size exclusion chromatography gave a molecular mass of 31.4 ± 1.3 kDa for the enzyme, showing that it functions as a monomer. Activit...

  15. Synthesis and Physicochemical Characterization of D-Tagatose-1-Phosphate: The Substrate of the Tagatose-1-Phosphate Kinase in the Phosphotransferase System-Mediated D-Tagatose Catabolic Pathway of Bacillus licheniformis.

    Science.gov (United States)

    Van der Heiden, Edwige; Delmarcelle, Michaël; Simon, Patricia; Counson, Melody; Galleni, Moreno; Freedberg, Darón I; Thompson, John; Joris, Bernard; Battistel, Marcos D

    2015-01-01

    We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multicomponent phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by (31)P and (1)H nuclear magnetic resonance spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-tagatose catabolic pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TF(His6)) of Escherichia coli. The active fusion enzyme was named TagK-TF(His6). Tag-1P and D-fructose-1-phosphate are substrates for the TagK-TF(His6) enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate and D-fructose-6-phosphate are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as the substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific Enzyme II in E. coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and Enzyme I to restore the phosphate transfer is demonstrated. © 2015 S. Karger AG, Basel.

  16. Control of lactose transport, beta-galactosidase activity, and glycolysis by CcpA in Streptococcus thermophilus: evidence for carbon catabolite repression by a non-phosphoenolpyruvate-dependent phosphotransferase system sugar.

    Science.gov (United States)

    van den Bogaard, P T; Kleerebezem, M; Kuipers, O P; de Vos, W M

    2000-11-01

    Streptococcus thermophilus, unlike many other gram-positive bacteria, prefers lactose over glucose as the primary carbon and energy source. Moreover, lactose is not taken up by a phosphoenolpyruvate-dependent phosphotransferase system (PTS) but by the dedicated transporter LacS. In this paper we show that CcpA plays a crucial role in the fine-tuning of lactose transport, beta-galactosidase (LacZ) activity, and glycolysis to yield optimal glycolytic flux and growth rate. A catabolite-responsive element (cre) was identified in the promoter of the lacSZ operon, indicating a possible role for regulation by CcpA. Transcriptional analysis showed a sevenfold relief of repression in the absence of a functional CcpA when cells were grown on lactose. This CcpA-mediated repression of lacSZ transcription did not occur in wild-type cells during growth on galactose, taken up by the same LacS transport system. Lactose transport during fermentation was increased significantly in strains carrying a disrupted ccpA gene. Moreover, a ccpA disruption strain was found to release substantial amounts of glucose into the medium when grown on lactose. Transcriptional analysis of the ldh gene showed that expression was induced twofold during growth on lactose compared to glucose or galactose, in a CcpA-dependent manner. A reduced rate of glycolysis concomitant with an increased lactose transport rate could explain the observed expulsion of glucose in a ccpA disruption mutant. We propose that CcpA in S. thermophilus acts as a catabolic regulator during growth on the preferred non-PTS sugar lactose. In contrast to other bacteria, S. thermophilus possesses an overcapacity for lactose uptake that is repressed by CcpA to match the rate-limiting glycolytic flux.

  17. Control of Lactose Transport, β-Galactosidase Activity, and Glycolysis by CcpA in Streptococcus thermophilus: Evidence for Carbon Catabolite Repression by a Non-Phosphoenolpyruvate-Dependent Phosphotransferase System Sugar

    Science.gov (United States)

    van den Bogaard, Patrick T. C.; Kleerebezem, Michiel; Kuipers, Oscar P.; de Vos, Willem M.

    2000-01-01

    Streptococcus thermophilus, unlike many other gram-positive bacteria, prefers lactose over glucose as the primary carbon and energy source. Moreover, lactose is not taken up by a phosphoenolpyruvate-dependent phosphotransferase system (PTS) but by the dedicated transporter LacS. In this paper we show that CcpA plays a crucial role in the fine-tuning of lactose transport, β-galactosidase (LacZ) activity, and glycolysis to yield optimal glycolytic flux and growth rate. A catabolite-responsive element (cre) was identified in the promoter of the lacSZ operon, indicating a possible role for regulation by CcpA. Transcriptional analysis showed a sevenfold relief of repression in the absence of a functional CcpA when cells were grown on lactose. This CcpA-mediated repression of lacSZ transcription did not occur in wild-type cells during growth on galactose, taken up by the same LacS transport system. Lactose transport during fermentation was increased significantly in strains carrying a disrupted ccpA gene. Moreover, a ccpA disruption strain was found to release substantial amounts of glucose into the medium when grown on lactose. Transcriptional analysis of the ldh gene showed that expression was induced twofold during growth on lactose compared to glucose or galactose, in a CcpA-dependent manner. A reduced rate of glycolysis concomitant with an increased lactose transport rate could explain the observed expulsion of glucose in a ccpA disruption mutant. We propose that CcpA in S. thermophilus acts as a catabolic regulator during growth on the preferred non-PTS sugar lactose. In contrast to other bacteria, S. thermophilus possesses an overcapacity for lactose uptake that is repressed by CcpA to match the rate-limiting glycolytic flux. PMID:11029416

  18. Purification and Characterization of Aminoglycoside Phosphotransferase APH(6)-Id, a Streptomycin Inactivating Enzyme

    Science.gov (United States)

    Ashenafi, Meseret; Ammosova, Tatiana; Nekhai, Sergei; Byrnes, W. Malcolm

    2014-01-01

    As part of an overall project to characterize the streptomycin phosphotransferase enzyme APH(6)-Id, which confers bacterial resistance to streptomycin, we cloned, expressed, purified and characterized the enzyme. When expressed in E. coli, the recombinant enzyme increased by up to 70-fold the minimum inhibitory concentration (MIC) needed to inhibit cell growth. Size exclusion chromatography gave a molecular mass of 31.4 ± 1.3 kDa for the enzyme, showing that it functions as a monomer. Activity was assayed using three methods: (1) an HPLC-based method that measures the consumption of streptomycin over time; (2) a spectrophotometric method that utilizes a coupled assay; and (3) a radioenzymatic method that detects production of 32P-labeled streptomycin phosphate. Altogether, the three methods demonstrated that streptomycin was consumed in the APH(6)-Id-catalyzed reaction, ATP was hydrolyzed, and streptomycin phosphate was produced in a substrate-dependent manner, demonstrating that APH(6)-Id is a streptomycin phosphotransferase. Steady state kinetic analysis gave the following results: Km(streptomycin) of 0.38 ± 0.13 mM, Km(ATP) of 1.03 ± 0.1 mM, Vmax of 3.2 ± 1.1 μmol/min/mg and kcat of 1.7 ± 0.6 s−1. Our study demonstrates that APH(6)-Id is a bona fide streptomycin phosphotransferase, functions as a monomer, and confers resistance to streptomycin. PMID:24248535

  19. Purification and characterization of aminoglycoside phosphotransferase APH(6)-Id, a streptomycin-inactivating enzyme.

    Science.gov (United States)

    Ashenafi, Meseret; Ammosova, Tatiana; Nekhai, Sergei; Byrnes, W Malcolm

    2014-02-01

    As part of an overall project to characterize the streptomycin phosphotransferase enzyme APH(6)-Id, which confers bacterial resistance to streptomycin, we cloned, expressed, purified, and characterized the enzyme. When expressed in Escherichia coli, the recombinant enzyme increased by up to 70-fold the minimum inhibitory concentration needed to inhibit cell growth. Size-exclusion chromatography gave a molecular mass of 31.4 ± 1.3 kDa for the enzyme, showing that it functions as a monomer. Activity was assayed using three methods: (1) an HPLC-based method that measures the consumption of streptomycin over time; (2) a spectrophotometric method that utilizes a coupled assay; and (3) a radioenzymatic method that detects production of (32)P-labeled streptomycin phosphate. Altogether, the three methods demonstrated that streptomycin was consumed in the APH(6)-Id-catalyzed reaction, ATP was hydrolyzed, and streptomycin phosphate was produced in a substrate-dependent manner, demonstrating that APH(6)-Id is a streptomycin phosphotransferase. Steady-state kinetic analysis gave the following results: K(m)(streptomycin) of 0.38 ± 0.13 mM, K(m)(ATP) of 1.03 ± 0.1 mM, V(max) of 3.2 ± 1.1 μmol/min/mg, and k(cat) of 1.7 ± 0.6 s(-1). Our study demonstrates that APH(6)-Id is a bona fide streptomycin phosphotransferase, functions as a monomer, and confers resistance to streptomycin.

  20. Structural characterization of the novel aminoglycoside phosphotransferase AphVIII from Streptomyces rimosus with enzymatic activity modulated by phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Boyko, Konstantin M., E-mail: kmb@inbi.ras.ru [Bach Institute of Biochemistry, Federal Research Centre of Biotechnology of the Russian Academy of Sciences, Leninsky Prospekt. 33, Bld. 2, 119071, Moscow (Russian Federation); National Research Center “Kurchatov Institute”, Kurchatov Complex of NBICS-technologies, Akad. Kurchatova sqr., 1, Moscow, 123182 (Russian Federation); Gorbacheva, Marina A.; Korzhenevskiy, Dmitry A. [National Research Center “Kurchatov Institute”, Kurchatov Complex of NBICS-technologies, Akad. Kurchatova sqr., 1, Moscow, 123182 (Russian Federation); Alekseeva, Maria G.; Mavletova, Dilara A.; Zakharevich, Natalia V.; Elizarov, Sergey M.; Rudakova, Natalia N.; Danilenko, Valery N. [Vavilov Institute of General Genetics, Russian Academy of Sciences, Gubkina str. 3, Moscow, 119333 (Russian Federation); Popov, Vladimir O. [Bach Institute of Biochemistry, Federal Research Centre of Biotechnology of the Russian Academy of Sciences, Leninsky Prospekt. 33, Bld. 2, 119071, Moscow (Russian Federation); National Research Center “Kurchatov Institute”, Kurchatov Complex of NBICS-technologies, Akad. Kurchatova sqr., 1, Moscow, 123182 (Russian Federation)

    2016-09-02

    Aminoglycoside phosphotransferases represent a broad class of enzymes that promote bacterial resistance to aminoglycoside antibiotics via the phosphorylation of hydroxyl groups in the latter. Here we report the spatial structure of the 3′-aminoglycoside phosphotransferase of novel VIII class (AphVIII) solved by X-ray diffraction method with a resolution of 2.15 Å. Deep analysis of APHVIII structure and its comparison with known structures of aminoglycoside phosphotransferases of various types reveals that AphVIII has a typical two-domain fold and, however, possesses some unique characteristics that distinguish the enzyme from its known homologues. The most important difference is the presence of the activation loop with unique Ser146 residue. We demonstrate that in the apo-state of the enzyme the activation loop does not interact with other parts of the enzyme and seems to adopt catalytically competent state only after substrate binding. - Highlights: • 3D structure of the novel aminoglycoside phosphotransferase AphVIII was obtained. • AphVIII activation loop is clearly identified in the electron density. • AphVIII has some unique structural features in its substrate C-ring binding pocket.

  1. Bacterial ferrous iron transport: the Feo system.

    Science.gov (United States)

    Lau, Cheryl K Y; Krewulak, Karla D; Vogel, Hans J

    2016-03-01

    To maintain iron homeostasis within the cell, bacteria have evolved various types of iron acquisition systems. Ferric iron (Fe(3+)) is the dominant species in an oxygenated environment, while ferrous iron (Fe(2+)) is more abundant under anaerobic conditions or at low pH. For organisms that must combat oxygen limitation for their everyday survival, pathways for the uptake of ferrous iron are essential. Several bacterial ferrous iron transport systems have been described; however, only the Feo system appears to be widely distributed and is exclusively dedicated to the transport of iron. In recent years, many studies have explored the role of the FeoB and FeoA proteins in ferrous iron transport and their contribution toward bacterial virulence. The three-dimensional structures for the Feo proteins have recently been determined and provide insight into the molecular details of the transport system. A highly select group of bacteria also express the FeoC protein from the same operon. This review will provide a comprehensive look at the structural and functional aspects of the Feo system. In addition, bioinformatics analyses of the feo operon and the Feo proteins have been performed to complement our understanding of this ubiquitous bacterial uptake system, providing a new outlook for future studies. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. The redox state and the phosphorylation state of the mannitol-specific carrier of the E. coli phosphoenolpyruvate-dependent phosphotransferase system

    NARCIS (Netherlands)

    Robillard, G.T.; Pas, H.H.; Gage, D.; Elferink, M.G.L.

    1988-01-01

    This review summarizes the recent developments in identifying the activity-linked cysteine as one of the phosphorylation sites on the mannitol-specific EII of the E. coli phosphoenolpyruvate-dependent mannitol transport system. Two phosphorylation sites have been identified, one being the HPr/P-HPr

  3. Analytical sedimentation of the IIAChb and IIBChb proteins of the Escherichia coli N,N'-diacetylchitobiose phosphotransferase system. Demonstration of a model phosphotransfer transition state complex.

    Science.gov (United States)

    Keyhani, N; Rodgers, M E; Demeler, B; Hansen, J C; Roseman, S

    2000-10-20

    The phosphoenolpyruvate:glycose transferase system (PTS) is a prototypic signaling system responsible for the vectorial uptake and phosphorylation of carbohydrate substrates. The accompanying papers describe the proteins and product of the Escherichia coli N, N-diacetylchitobiose ((GlcNAc)(2)) PTS-mediated permease. Unlike most PTS transporters, the Chb system is composed of two soluble proteins, IIA(Chb) and IIB(Chb), and one transmembrane receptor (IIC(Chb)). The oligomeric states of PTS permease proteins and phosphoproteins have been difficult to determine. Using analytical ultracentrifugation, both dephospho and phosphorylated IIA(Chb) are shown to exist as stable dimers, whereas IIB(Chb), phospho-IIB(Chb) and the mutant Cys10SerIIB(Chb) are monomers. The mutant protein Cys10SerIIB(Chb) is unable to accept phosphate from phospho-IIA(Chb) but forms a stable higher order complex with phospho-IIA(Chb) (but not with dephospho-IIA(Chb)). The stoichiometry of proteins in the purified complex was determined to be 1:1, indicating that two molecules of Cys10SerIIB(Chb) are associated with one phospho-IIA(Chb) dimer in the complex. The complex appears to be a transition state analogue in the phosphotransfer reaction between the proteins. A model is presented that describes the concerted assembly and disassembly of IIA(Chb)-IIB(Chb) complexes contingent on phosphorylation-dependent conformational changes, especially of IIA(Chb).

  4. Autoinducer 2 Signaling via the Phosphotransferase FruA Drives Galactose Utilization by Streptococcus pneumoniae, Resulting in Hypervirulence

    Directory of Open Access Journals (Sweden)

    Claudia Trappetti

    2017-01-01

    Full Text Available Communication between bacterial cells is crucial for the coordination of diverse cellular processes that facilitate environmental adaptation and, in the case of pathogenic species, virulence. This is achieved by the secretion and detection of small signaling molecules called autoinducers, a process termed quorum sensing. To date, the only signaling molecule recognized by both Gram-positive and Gram-negative bacteria is autoinducer 2 (AI-2, synthesized by the metabolic enzyme LuxS (S-ribosylhomocysteine lyase as a by-product of the activated methyl cycle. Homologues of LuxS are ubiquitous in bacteria, suggesting a key role in interspecies, as well as intraspecies, communication. Gram-negative bacteria sense and respond to AI-2 via the Lsr ABC transporter system or by the LuxP/LuxQ phosphorelay system. However, homologues of these systems are absent from Gram-positive bacteria and the AI-2 receptor is unknown. Here we show that in the major human pathogen Streptococcus pneumoniae, sensing of exogenous AI-2 is dependent on FruA, a fructose-specific phosphoenolpyruvate-phosphotransferase system that is highly conserved in Gram-positive pathogens. Importantly, AI-2 signaling via FruA enables the bacterium to utilize galactose as a carbon source and upregulates the Leloir pathway, thereby leading to increased production of capsular polysaccharide and a hypervirulent phenotype.

  5. Bacterial systems for production of heterologous proteins.

    Science.gov (United States)

    Zerbs, Sarah; Frank, Ashley M; Collart, Frank R

    2009-01-01

    Proteins are the working molecules of all biological systems and participate in a majority of cellular chemical reactions and biological processes. Knowledge of the properties and function of these molecules is central to an understanding of chemical and biological processes. In this context, purified proteins are a starting point for biophysical and biochemical characterization methods that can assist in the elucidation of function. The challenge for production of proteins at the scale and quality required for experimental, therapeutic and commercial applications has led to the development of a diverse set of methods for heterologous protein production. Bacterial expression systems are commonly used for protein production as these systems provide an economical route for protein production and require minimal technical expertise to establish a laboratory protein production system.

  6. Crystallization and preliminary crystallographic analysis of hygromycin B phosphotransferase from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Iino, Daisuke [Department of Bioscience, Tokyo University of Agriculture, Tokyo 156-8502 (Japan); Takakura, Yasuaki [Division of Integrative Environmental Sciences, Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki 305-8572 (Japan); Kuroiwa, Mika; Kawakami, Ryouta; Sasaki, Yasuyuki [Department of Bioscience, Tokyo University of Agriculture, Tokyo 156-8502 (Japan); Hoshino, Takayuki [Division of Integrative Environmental Sciences, Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki 305-8572 (Japan); Ohsawa, Kanju [Department of Bioscience, Tokyo University of Agriculture, Tokyo 156-8502 (Japan); Nakamura, Akira [Division of Integrative Environmental Sciences, Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki 305-8572 (Japan); Yajima, Shunsuke, E-mail: yshun@nodai.ac.jp [Department of Bioscience, Tokyo University of Agriculture, Tokyo 156-8502 (Japan)

    2007-08-01

    The crystallization and preliminary X-ray studies of the aminoglycoside antibiotic-modifying enzyme hygromycin B phosphotransferase from E. coli are reported. Aminoglycoside antibiotics, such as hygromycin, kanamycin, neomycin, spectinomycin and streptomycin, inhibit protein synthesis by acting on bacterial and eukaryotic ribosomes. Hygromycin B phosphotransferase (Hph; EC 2.7.1.119) converts hygromycin B to 7′′-O-phosphohygromycin using a phosphate moiety from ATP, resulting in the loss of its cell-killing activity. The Hph protein has been crystallized for the first time using a thermostable mutant and the hanging-drop vapour-diffusion method. The crystal provided diffraction data to a resolution of 2.1 Å and belongs to space group P3{sub 2}21, with unit-cell parameters a = b = 71.0, c = 125.0 Å. Crystals of complexes of Hph with hygromycin B and AMP-PNP or ADP have also been obtained in the same crystal form as that of the apoprotein.

  7. Crystallization and preliminary crystallographic analysis of hygromycin B phosphotransferase from Escherichia coli

    International Nuclear Information System (INIS)

    Iino, Daisuke; Takakura, Yasuaki; Kuroiwa, Mika; Kawakami, Ryouta; Sasaki, Yasuyuki; Hoshino, Takayuki; Ohsawa, Kanju; Nakamura, Akira; Yajima, Shunsuke

    2007-01-01

    The crystallization and preliminary X-ray studies of the aminoglycoside antibiotic-modifying enzyme hygromycin B phosphotransferase from E. coli are reported. Aminoglycoside antibiotics, such as hygromycin, kanamycin, neomycin, spectinomycin and streptomycin, inhibit protein synthesis by acting on bacterial and eukaryotic ribosomes. Hygromycin B phosphotransferase (Hph; EC 2.7.1.119) converts hygromycin B to 7′′-O-phosphohygromycin using a phosphate moiety from ATP, resulting in the loss of its cell-killing activity. The Hph protein has been crystallized for the first time using a thermostable mutant and the hanging-drop vapour-diffusion method. The crystal provided diffraction data to a resolution of 2.1 Å and belongs to space group P3 2 21, with unit-cell parameters a = b = 71.0, c = 125.0 Å. Crystals of complexes of Hph with hygromycin B and AMP-PNP or ADP have also been obtained in the same crystal form as that of the apoprotein

  8. Particle surface area and bacterial activity in recirculating aquaculture systems

    DEFF Research Database (Denmark)

    Pedersen, Per Bovbjerg; von Ahnen, Mathis; Fernandes, Paulo

    2017-01-01

    Suspended particles in recirculating aquaculture systems (RAS) provide surface area that can be colonized by bacteria. More particles accumulate as the intensity of recirculation increases thus potentially increasing the bacterial carrying capacity of the systems. Applying a recent, rapid, cultur...... for determining bacterial activity might provide a means for future monitoring and assessment of microbial water quality in aquaculture farming systems......Suspended particles in recirculating aquaculture systems (RAS) provide surface area that can be colonized by bacteria. More particles accumulate as the intensity of recirculation increases thus potentially increasing the bacterial carrying capacity of the systems. Applying a recent, rapid, culture......-independent fluorometric detection method (Bactiquant®) for measuring bacterial activity, the current study explored the relationship between total particle surface area (TSA, derived from the size distribution of particles >5 μm) and bacterial activity in freshwater RAS operated at increasing intensity of recirculation...

  9. Soil bacterial diversity changes in different broomcorn millet intercropping systems.

    Science.gov (United States)

    Cao, Xiaoning; Liu, Sichen; Wang, Junjie; Wang, Haigang; Chen, Ling; Tian, Xiang; Zhang, Lijun; Chang, Jianwu; Wang, Lun; Mu, Zhixin; Qiao, Zhijun

    2017-12-01

    Plants growing in soil and the diverse microorganisms with which they are in direct contact have adapted to exploit their close association for mutual benefit. Various intercropping systems have been used to control plant disease and improve productivity in fields. Although high-throughput sequencing approaches have provided new insights into the soil bacterial community, current knowledge of intercropping of broomcorn millet with different leguminous plants is limited. In this study, characterization of different bacterial communities of monoculture and intercropping systems was achieved by deep sequencing. A total of 4684 operational taxonomic units were classified to the species level with good sampling depth and sequencing coverage. The abundance of Actinobacteria, Bacteroidetes, Proteobacteria, Acidobacteria, and Gemmatimonadetes varied at different growth stages and was related to growth of the intercropped plant. According to diversity analyses, Glycomyces, Aeromicrobium, Adhaeribacter, and Streptomyces were the dominant genera. In addition, we predicted functional gene composition based on bacterial OTUs present. Functional results showed that membrane transport and nutrient metabolism was highly abundant in all samples, although abundance varied at different growth stages, which indicated these pathways might be affected by the dominant categories of bacterial community. The dynamic changes observed during intercropping of broomcorn millet with different leguminous plants suggest that soil bacterial community structure exhibits a crop species-specific pattern. Further, agronomic trait data from different broomcorn millet intercropping systems were consistent with functional results and suggest that agronomic traits may be influenced by soil bacterial communities. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Development of bacterial cell-based system for intracellular ...

    African Journals Online (AJOL)

    Development of bacterial cell-based system for intracellular antioxidant activity screening assay using green fluorescence protein (GFP) reporter. ... Both strains demonstrated that quercetin and α- tocopherol exhibited the most potent and significant antioxidant activity with more than 60% reduction of intracellular superoxide ...

  11. CRISPR technologies for bacterial systems: Current achievements and future directions.

    Science.gov (United States)

    Choi, Kyeong Rok; Lee, Sang Yup

    2016-11-15

    Throughout the decades of its history, the advances in bacteria-based bio-industries have coincided with great leaps in strain engineering technologies. Recently unveiled clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) systems are now revolutionizing biotechnology as well as biology. Diverse technologies have been derived from CRISPR/Cas systems in bacteria, yet the applications unfortunately have not been actively employed in bacteria as extensively as in eukaryotic organisms. A recent trend of engineering less explored strains in industrial microbiology-metabolic engineering, synthetic biology, and other related disciplines-is demanding facile yet robust tools, and various CRISPR technologies have potential to cater to the demands. Here, we briefly review the science in CRISPR/Cas systems and the milestone inventions that enabled numerous CRISPR technologies. Next, we describe CRISPR/Cas-derived technologies for bacterial strain development, including genome editing and gene expression regulation applications. Then, other CRISPR technologies possessing great potential for industrial applications are described, including typing and tracking of bacterial strains, virome identification, vaccination of bacteria, and advanced antimicrobial approaches. For each application, we note our suggestions for additional improvements as well. In the same context, replication of CRISPR/Cas-based chromosome imaging technologies developed originally in eukaryotic systems is introduced with its potential impact on studying bacterial chromosomal dynamics. Also, the current patent status of CRISPR technologies is reviewed. Finally, we provide some insights to the future of CRISPR technologies for bacterial systems by proposing complementary techniques to be developed for the use of CRISPR technologies in even wider range of applications. Copyright © 2016. Published by Elsevier Inc.

  12. Compensatory expression of human -Acetylglucosaminyl-1-phosphotransferase subunits in mucolipidosis type III gamma

    OpenAIRE

    Pohl , Sandra; Tiede , Stephan; Castrichini , Monica; Cantz , Michael; Gieselmann , Volkmar; Braulke , Thomas

    2009-01-01

    Abstract The N-Acetylglucosaminyl-1-phosphotransferase plays a key role in the generation of mannose 6-phosphate (M6P) recognition markers essential for efficient transport of lysosomal hydrolases to lysosomes. The phosphotransferase is composed of six subunits (?2, ?2, ?2). The ?- and ?-subunits are catalytically active and encoded by a single gene, GNPTAB, whereas the ?-subunit encoded by GNPTG is proposed to recognize conformational structures common to lysosomal enzymes. Defects in GN...

  13. Harnessing CRISPR-Cas systems for bacterial genome editing.

    Science.gov (United States)

    Selle, Kurt; Barrangou, Rodolphe

    2015-04-01

    Manipulation of genomic sequences facilitates the identification and characterization of key genetic determinants in the investigation of biological processes. Genome editing via clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) constitutes a next-generation method for programmable and high-throughput functional genomics. CRISPR-Cas systems are readily reprogrammed to induce sequence-specific DNA breaks at target loci, resulting in fixed mutations via host-dependent DNA repair mechanisms. Although bacterial genome editing is a relatively unexplored and underrepresented application of CRISPR-Cas systems, recent studies provide valuable insights for the widespread future implementation of this technology. This review summarizes recent progress in bacterial genome editing and identifies fundamental genetic and phenotypic outcomes of CRISPR targeting in bacteria, in the context of tool development, genome homeostasis, and DNA repair. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Small intestinal bacterial overgrowth in patients with systemic sclerosis

    Directory of Open Access Journals (Sweden)

    Saara Rawn

    2017-01-01

    Full Text Available Small intestinal bacterial overgrowth (SIBO is common in patients with systemic sclerosis (SSc yet often goes underrecognized in clinical practice. In patients with SSc, untreated SIBO may result in marked morbidity and possible mortality. The pathogenesis of SIBO is multifactorial and relates to immune dysregulation, vasculopathy, and dysmotility. This article reviews various diagnostic approaches and therapeutic options for SIBO. Treatment modalities mainly include prokinetics, probiotics, and antibiotics.

  15. Revisiting the Nucleotide and Aminoglycoside Substrate Specificity of the Bifunctional Aminoglycoside Acetyltransferase(6′)-Ie/Aminoglycoside Phosphotransferase(2″)-Ia Enzyme*

    Science.gov (United States)

    Frase, Hilary; Toth, Marta; Vakulenko, Sergei B.

    2012-01-01

    The bifunctional aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6′)-Ie/aminoglycoside phosphotransferase(2″)-Ia, or AAC(6′)-Ie/APH(2″)-Ia, is the major source of aminoglycoside resistance in Gram-positive bacterial pathogens. In previous studies, using ATP as the cosubstrate, it was reported that the APH(2″)-Ia domain of this enzyme is unique among aminoglycoside phosphotransferases, having the ability to inactivate an unusually broad spectrum of aminoglycosides, including 4,6- and 4,5-disubstituted and atypical. We recently demonstrated that GTP, and not ATP, is the preferred cosubstrate of this enzyme. We now show, using competition assays between ATP and GTP, that GTP is the exclusive phosphate donor at intracellular nucleotide levels. In light of these findings, we reevaluated the substrate profile of the phosphotransferase domain of this clinically important enzyme. Steady-state kinetic characterization using the phosphate donor GTP demonstrates that AAC(6′)-Ie/APH(2″)-Ia phosphorylates 4,6-disubstituted aminoglycosides with high efficiency (kcat/Km = 105-107 m−1 s−1). Despite this proficiency, no resistance is conferred to some of these antibiotics by the enzyme in vivo. We now show that phosphorylation of 4,5-disubstituted and atypical aminoglycosides are negligible and thus these antibiotics are not substrates. Instead, these aminoglycosides tend to stimulate an intrinsic GTPase activity of the enzyme. Taken together, our data show that the bifunctional enzyme efficiently phosphorylates only 4,6-disubstituted antibiotics; however, phosphorylation does not necessarily result in bacterial resistance. Hence, the APH(2″)-Ia domain of the bifunctional AAC(6′)-Ie/APH(2″)-Ia enzyme is a bona fide GTP-dependent kinase with a narrow substrate profile, including only 4,6-disubstituted aminoglycosides. PMID:23115238

  16. Bacterial biodegradation of neonicotinoid pesticides in soil and water systems.

    Science.gov (United States)

    Hussain, Sarfraz; Hartley, Carol J; Shettigar, Madhura; Pandey, Gunjan

    2016-12-01

    Neonicotinoids are neurotoxic systemic insecticides used in plant protection worldwide. Unfortunately, application of neonicotinoids affects both beneficial and target insects indiscriminately. Being water soluble and persistent, these pesticides are capable of disrupting both food chains and biogeochemical cycles. This review focuses on the biodegradation of neonicotinoids in soil and water systems by the bacterial community. Several bacterial strains have been isolated and identified as capable of transforming neonicotinoids in the presence of an additional carbon source. Environmental parameters have been established for accelerated transformation in some of these strains. Studies have also indicated that enhanced biotransformation of these pesticides can be accomplished by mixed microbial populations under optimised environmental conditions. Substantial research into the identification of neonicotinoid-mineralising bacterial strains and identification of the genes and enzymes responsible for neonicotinoid degradation is still required to complete the understanding of microbial biodegradation pathways, and advance bioremediation efforts. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. Applications of CRISPR/Cas System to Bacterial Metabolic Engineering

    Directory of Open Access Journals (Sweden)

    Suhyung Cho

    2018-04-01

    Full Text Available The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas adaptive immune system has been extensively used for gene editing, including gene deletion, insertion, and replacement in bacterial and eukaryotic cells owing to its simple, rapid, and efficient activities in unprecedented resolution. Furthermore, the CRISPR interference (CRISPRi system including deactivated Cas9 (dCas9 with inactivated endonuclease activity has been further investigated for regulation of the target gene transiently or constitutively, avoiding cell death by disruption of genome. This review discusses the applications of CRISPR/Cas for genome editing in various bacterial systems and their applications. In particular, CRISPR technology has been used for the production of metabolites of high industrial significance, including biochemical, biofuel, and pharmaceutical products/precursors in bacteria. Here, we focus on methods to increase the productivity and yield/titer scan by controlling metabolic flux through individual or combinatorial use of CRISPR/Cas and CRISPRi systems with introduction of synthetic pathway in industrially common bacteria including Escherichia coli. Further, we discuss additional useful applications of the CRISPR/Cas system, including its use in functional genomics.

  18. Role of Honey in Topical and Systemic Bacterial Infections.

    Science.gov (United States)

    Hussain, Muhammad Barkaat

    2018-01-01

    The development of bacterial resistance to antibiotics has made it more difficult and expensive to treat infections. Honey is getting worldwide attention as a topical therapeutic agent for wound infections and potential future candidate for systemic infections. The purpose of this review was to summarise different antibacterial bio-active compounds in honey, their synergistic interaction and their clinical implications in topical and systemic infections. In addition, contemporary testing methods for evaluating peroxide and non-peroxide antibacterial activity of honey were also critically appraised. MEDLINE, EMBASE, Cochrane Library, Pub Med, reference lists and databases were used to review the literature. Honey contains several unique antibacterial components. These components are believed to act on diverse bacterial targets, are broad spectrum, operate synergistically, prevent biofilm formation, and decrease production of virulence factors. Moreover, honey has the ability to block bacterial communication (quorum sensing), and therefore, it is unlikely that bacteria develop resistance against honey. Bacterial resistance against honey has not been documented so far. Unlike conventional antibiotics, honey only targets pathogenic bacteria without disturbing the growth of normal gastrointestinal flora when taken orally. It also contains prebiotics, probiotics, and zinc and enhances the growth of beneficial gut flora. The presence of such plethora of antibacterial properties in one product makes it a promising candidate not only in wound infections but also in systemic and particularly for gastrointestinal infections. Agar diffusion assay, being used for evaluating antibacterial activity of honey, is not the most appropriate and sensitive assay as it only detects non-peroxide activity when present at a higher level. Therefore, there is a need to develop more sensitive techniques that may be capable of detecting and evaluating different important components in honey as

  19. Bacterial ghosts (BGs)--advanced antigen and drug delivery system.

    Science.gov (United States)

    Kudela, Pavol; Koller, Verena Juliana; Lubitz, Werner

    2010-08-16

    Bacterial ghosts (BGs) are empty bacterial envelopes of Gram-negative bacteria produced by controlled expression of cloned gene E, forming a lysis tunnel structure within the envelope of the living bacteria. BGs are devoid of cytoplasmic content and possess all bacterial bio-adhesive surface properties in their original state while not posing any infectious threat. BGs are ideally suited as an advanced drug delivery system (ADDS) for toxic substances in tumor therapy. The inner space of BGs can be loaded with either single components or combinations of peptides, drugs or DNA which provides an opportunity to design new types of (polyvalent) drug delivery vehicles. Uptake of BGs loaded with Doxorubicin (Dox) by CaCo2 cells led to effective Dox release from endo-lysosomal compartments and accumulation in the nucleus. Viability and proliferative capacity of the cells were significantly decreased (2-3 orders of magnitude) after internalization of Dox loaded BGs as compared to cells incubated with free Dox. The same effect was observed with leukemia cells. Melanoma cells also revealed a high capability to internalize BGs. These results indicate that BGs are able to target a range of types of cancer. BGs have also been investigated as DNA delivery vectors. Studies show DNA loaded BGs are efficiently phagocytosed and internalized by both professional APCs and tumor cells with up to 82% of cells expressing the plasmid-encoded reporter gene. Our studies with BGs as an ADDS system contribute (i) to optimize drug delivery for the treatment of cancer; (ii) define specific conditions for selection and preparation of BG formulations; (iii) and provide a background for the clinical application of BGs in cancer therapy.

  20. Type III secretion systems: the bacterial flagellum and the injectisome

    Science.gov (United States)

    Diepold, Andreas; Armitage, Judith P.

    2015-01-01

    The flagellum and the injectisome are two of the most complex and fascinating bacterial nanomachines. At their core, they share a type III secretion system (T3SS), a transmembrane export complex that forms the extracellular appendages, the flagellar filament and the injectisome needle. Recent advances, combining structural biology, cryo-electron tomography, molecular genetics, in vivo imaging, bioinformatics and biophysics, have greatly increased our understanding of the T3SS, especially the structure of its transmembrane and cytosolic components, the transcriptional, post-transcriptional and functional regulation and the remarkable adaptivity of the system. This review aims to integrate these new findings into our current knowledge of the evolution, function, regulation and dynamics of the T3SS, and to highlight commonalities and differences between the two systems, as well as their potential applications. PMID:26370933

  1. Purification, Crystallization And Preliminary X-Ray Analysis of Aminoglycoside-2 ''-Phosphotransferase-Ic [APH(2 '')-Ic] From Enterococcus Gallinarum

    Energy Technology Data Exchange (ETDEWEB)

    Byrnes, L.J.; /SLAC, SSRL; Badarau, A.; Vakulenko, S.B.; /Notre Dame U.; Smith, C.A.; /SLAC, SSRL

    2009-04-30

    Bacterial resistance to aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, aminoglycoside-2{double_prime}-phosphotransferase-Ic [APH(2{double_prime})-Ic] from Enterococcus gallinarum, has been cloned and the wild-type protein (comprising 308 amino-acid residues) and three mutants that showed elevated minimum inhibitory concentrations towards gentamicin (F108L, H258L and a double mutant F108L/H258L) were expressed in Escherichia coli and subsequently purified. All APH(2{double_prime})-Ic variants were crystallized in the presence of 14-20%(w/v) PEG 4000, 0.25 M MgCl{sub 2}, 0.1 M Tris-HCl pH 8.5 and 1 mM Mg{sub 2}GTP. The crystals belong to the monoclinic space group C2, with one molecule in the asymmetric unit. The approximate unit-cell parameters are a = 82.4, b = 54.2, c = 77.0 {angstrom}, {beta} = 108.8{sup o}. X-ray diffraction data were collected to approximately 2.15 {angstrom} resolution from an F108L crystal at beamline BL9-2 at SSRL, Stanford, California, USA.

  2. Bacterial burden in the operating room: impact of airflow systems.

    Science.gov (United States)

    Hirsch, Tobias; Hubert, Helmine; Fischer, Sebastian; Lahmer, Armin; Lehnhardt, Marcus; Steinau, Hans-Ulrich; Steinstraesser, Lars; Seipp, Hans-Martin

    2012-09-01

    Wound infections present one of the most prevalent and frequent complications associated with surgical procedures. This study analyzes the impact of currently used ventilation systems in the operating room to reduce bacterial contamination during surgical procedures. Four ventilation systems (window-based ventilation, supported air nozzle canopy, low-turbulence displacement airflow, and low-turbulence displacement airflow with flow stabilizer) were analyzed. Two hundred seventy-seven surgical procedures in 6 operating rooms of 5 different hospitals were analyzed for this study. Window-based ventilation showed the highest intraoperative contamination (13.3 colony-forming units [CFU]/h) followed by supported air nozzle canopy (6.4 CFU/h; P = .001 vs window-based ventilation) and low-turbulence displacement airflow (3.4 and 0.8 CFU/h; P system showed no increase of contamination in prolonged durations of surgical procedures. This study shows that intraoperative contamination can be significantly reduced by the use of adequate ventilation systems. Copyright © 2012 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.

  3. The Bacterial Ghost platform system: production and applications.

    Science.gov (United States)

    Langemann, Timo; Koller, Verena Juliana; Muhammad, Abbas; Kudela, Pavol; Mayr, Ulrike Beate; Lubitz, Werner

    2010-01-01

    The Bacterial Ghost (BG) platform technology is an innovative system for vaccine, drug or active substance delivery and for technical applications in white biotechnology. BGs are cell envelopes derived from Gram-negative bacteria. BGs are devoid of all cytoplasmic content but have a preserved cellular morphology including all cell surface structures. Using BGs as delivery vehicles for subunit or DNA-vaccines the particle structure and surface properties of BGs are targeting the carrier itself to primary antigen-presenting cells. Furthermore, BGs exhibit intrinsic adjuvant properties and trigger an enhanced humoral and cellular immune response to the target antigen. Multiple antigens of the native BG envelope and recombinant protein or DNA antigens can be combined in a single type of BG. Antigens can be presented on the inner or outer membrane of the BG as well as in the periplasm that is sealed during BG formation. Drugs or supplements can also be loaded to the internal lumen or periplasmic space of the carrier. BGs are produced by batch fermentation with subsequent product recovery and purification via tangential flow filtration. For safety reasons all residual bacterial DNA is inactivated during the BG production process by the use of staphylococcal nuclease A and/or the treatment with β-propiolactone. After purification BGs can be stored long-term at ambient room temperature as lyophilized product. The production cycle from the inoculation of the pre-culture to the purified BG concentrate ready for lyophilization does not take longer than a day and thus meets modern criteria of rapid vaccine production rather than keeping large stocks of vaccines. The broad spectrum of possible applications in combination with the comparably low production costs make the BG platform technology a safe and sophisticated product for the targeted delivery of vaccines and active agents as well as carrier of immobilized enzymes for applications in white biotechnology. © 2010 Landes

  4. Regulation of bacterial virulence by Csr (Rsm) systems.

    Science.gov (United States)

    Vakulskas, Christopher A; Potts, Anastasia H; Babitzke, Paul; Ahmer, Brian M M; Romeo, Tony

    2015-06-01

    Most bacterial pathogens have the remarkable ability to flourish in the external environment and in specialized host niches. This ability requires their metabolism, physiology, and virulence factors to be responsive to changes in their surroundings. It is no surprise that the underlying genetic circuitry that supports this adaptability is multilayered and exceedingly complex. Studies over the past 2 decades have established that the CsrA/RsmA proteins, global regulators of posttranscriptional gene expression, play important roles in the expression of virulence factors of numerous proteobacterial pathogens. To accomplish these tasks, CsrA binds to the 5' untranslated and/or early coding regions of mRNAs and alters translation, mRNA turnover, and/or transcript elongation. CsrA activity is regulated by noncoding small RNAs (sRNAs) that contain multiple CsrA binding sites, which permit them to sequester multiple CsrA homodimers away from mRNA targets. Environmental cues sensed by two-component signal transduction systems and other regulatory factors govern the expression of the CsrA-binding sRNAs and, ultimately, the effects of CsrA on secretion systems, surface molecules and biofilm formation, quorum sensing, motility, pigmentation, siderophore production, and phagocytic avoidance. This review presents the workings of the Csr system, the paradigm shift that it generated for understanding posttranscriptional regulation, and its roles in virulence networks of animal and plant pathogens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  5. Comparative and evolutionary analysis of the bacterial homologous recombination systems.

    Directory of Open Access Journals (Sweden)

    2005-08-01

    Full Text Available Homologous recombination is a housekeeping process involved in the maintenance of chromosome integrity and generation of genetic variability. Although detailed biochemical studies have described the mechanism of action of its components in model organisms, there is no recent extensive assessment of this knowledge, using comparative genomics and taking advantage of available experimental data on recombination. Using comparative genomics, we assessed the diversity of recombination processes among bacteria, and simulations suggest that we missed very few homologs. The work included the identification of orthologs and the analysis of their evolutionary history and genomic context. Some genes, for proteins such as RecA, the resolvases, and RecR, were found to be nearly ubiquitous, suggesting that the large majority of bacterial genomes are capable of homologous recombination. Yet many genomes show incomplete sets of presynaptic systems, with RecFOR being more frequent than RecBCD/AddAB. There is a significant pattern of co-occurrence between these systems and antirecombinant proteins such as the ones of mismatch repair and SbcB, but no significant association with nonhomologous end joining, which seems rare in bacteria. Surprisingly, a large number of genomes in which homologous recombination has been reported lack many of the enzymes involved in the presynaptic systems. The lack of obvious correlation between the presence of characterized presynaptic genes and experimental data on the frequency of recombination suggests the existence of still-unknown presynaptic mechanisms in bacteria. It also indicates that, at the moment, the assessment of the intrinsic stability or recombination isolation of bacteria in most cases cannot be inferred from the identification of known recombination proteins in the genomes.

  6. Portable bacterial identification system based on elastic light scatter patterns

    Directory of Open Access Journals (Sweden)

    Bae Euiwon

    2012-08-01

    Full Text Available Abstract Background Conventional diagnosis and identification of bacteria requires shipment of samples to a laboratory for genetic and biochemical analysis. This process can take days and imposes significant delay to action in situations where timely intervention can save lives and reduce associated costs. To enable faster response to an outbreak, a low-cost, small-footprint, portable microbial-identification instrument using forward scatterometry has been developed. Results This device, weighing 9 lb and measuring 12 × 6 × 10.5 in., utilizes elastic light scatter (ELS patterns to accurately capture bacterial colony characteristics and delivers the classification results via wireless access. The overall system consists of two CCD cameras, one rotational and one translational stage, and a 635-nm laser diode. Various software algorithms such as Hough transform, 2-D geometric moments, and the traveling salesman problem (TSP have been implemented to provide colony count and circularity, centering process, and minimized travel time among colonies. Conclusions Experiments were conducted with four bacteria genera using pure and mixed plate and as proof of principle a field test was conducted in four different locations where the average classification rate ranged between 95 and 100%.

  7. [Cashmere goat bacterial artificial chromosome recombination and cell transfection system].

    Science.gov (United States)

    Huang, Tian; Cao, Zhongyang; Yang, Yaohui; Cao, Gengsheng

    2016-03-01

    The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination.

  8. A robust platform for chemical genomics in bacterial systems.

    Science.gov (United States)

    French, Shawn; Mangat, Chand; Bharat, Amrita; Côté, Jean-Philippe; Mori, Hirotada; Brown, Eric D

    2016-03-15

    While genetic perturbation has been the conventional route to probing bacterial systems, small molecules are showing great promise as probes for cellular complexity. Indeed, systematic investigations of chemical-genetic interactions can provide new insights into cell networks and are often starting points for understanding the mechanism of action of novel chemical probes. We have developed a robust and sensitive platform for chemical-genomic investigations in bacteria. The approach monitors colony volume kinetically using transmissive scanning measurements, enabling acquisition of growth rates and conventional endpoint measurements. We found that chemical-genomic profiles were highly sensitive to concentration, necessitating careful selection of compound concentrations. Roughly 20,000,000 data points were collected for 15 different antibiotics. While 1052 chemical-genetic interactions were identified using the conventional endpoint biomass approach, adding interactions in growth rate resulted in 1564 interactions, a 50-200% increase depending on the drug, with many genes uncharacterized or poorly annotated. The chemical-genetic interaction maps generated from these data reveal common genes likely involved in multidrug resistance. Additionally, the maps identified deletion backgrounds exhibiting class-specific potentiation, revealing conceivable targets for combination approaches to drug discovery. This open platform is highly amenable to kinetic screening of any arrayable strain collection, be it prokaryotic or eukaryotic. © 2016 French et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  9. Live bacterial delivery systems for development of mucosal vaccines

    NARCIS (Netherlands)

    Thole, J.E.R.; Dalen, P.J. van; Havenith, C.E.G.; Pouwels, P.H.; Seegers, J.F.M.L.; Tielen, F.D.; Zee, M.D. van der; Zegers, N.D.; Shaw, M.

    2000-01-01

    By expression of foreign antigens in attenuated strains derived from bacterial pathogens and in non-pathogenic commensal bacteria, recombinant vaccines are being developed that aim to stimulate mucosal immunity. Recent advances in the pathogenesis and molecular biology of these bacteria have allowed

  10. Action of nucleotide phosphotransferase of Escherichia coli on nicotinamide riboside and nicotinamide mononucleotide.

    Science.gov (United States)

    Brunngraber, E F; Chargaff, E

    1977-01-01

    The action of the nucleotide phosphotransferase of Escherichia coli on nicotinamide riboside and on its 5'-phosphate results in the addition of one phosphate moiety to each of the substrates. Although the proof is not conclusive, it is likely that the phosphate group is transferred to the 3'-hydroxyl of the ribose. This is in contrast to the behavior of the enzyme toward NAD in which only the adenylic acid portion is phosphorylated enzymically. PMID:144913

  11. Convergent bacterial microbiotas in the fungal agricultural systems of insects.

    Science.gov (United States)

    Aylward, Frank O; Suen, Garret; Biedermann, Peter H W; Adams, Aaron S; Scott, Jarrod J; Malfatti, Stephanie A; Glavina del Rio, Tijana; Tringe, Susannah G; Poulsen, Michael; Raffa, Kenneth F; Klepzig, Kier D; Currie, Cameron R

    2014-11-18

    The ability to cultivate food is an innovation that has produced some of the most successful ecological strategies on the planet. Although most well recognized in humans, where agriculture represents a defining feature of civilization, species of ants, beetles, and termites have also independently evolved symbioses with fungi that they cultivate for food. Despite occurring across divergent insect and fungal lineages, the fungivorous niches of these insects are remarkably similar, indicating convergent evolution toward this successful ecological strategy. Here, we characterize the microbiota of ants, beetles, and termites engaged in nutritional symbioses with fungi to define the bacterial groups associated with these prominent herbivores and forest pests. Using culture-independent techniques and the in silico reconstruction of 37 composite genomes of dominant community members, we demonstrate that different insect-fungal symbioses that collectively shape ecosystems worldwide have highly similar bacterial microbiotas comprised primarily of the genera Enterobacter, Rahnella, and Pseudomonas. Although these symbioses span three orders of insects and two phyla of fungi, we show that they are associated with bacteria sharing high whole-genome nucleotide identity. Due to the fine-scale correspondence of the bacterial microbiotas of insects engaged in fungal symbioses, our findings indicate that this represents an example of convergence of entire host-microbe complexes. The cultivation of fungi for food is a behavior that has evolved independently in ants, beetles, and termites and has enabled many species of these insects to become ecologically important and widely distributed herbivores and forest pests. Although the primary fungal cultivars of these insects have been studied for decades, comparatively little is known of their bacterial microbiota. In this study, we show that diverse fungus-growing insects are associated with a common bacterial community composed of the

  12. Genetic reporter system for positioning of proteins at the bacterial pole.

    Science.gov (United States)

    Fixen, Kathryn R; Janakiraman, Anuradha; Garrity, Sean; Slade, Daniel J; Gray, Andrew N; Karahan, Nilay; Hochschild, Ann; Goldberg, Marcia B

    2012-01-01

    Spatial organization within bacteria is fundamental to many cellular processes, although the basic mechanisms underlying localization of proteins to specific sites within bacteria are poorly understood. The study of protein positioning has been limited by a paucity of methods that allow rapid large-scale screening for mutants in which protein positioning is altered. We developed a genetic reporter system for protein localization to the pole within the bacterial cytoplasm that allows saturation screening for mutants in Escherichia coli in which protein localization is altered. Utilizing this system, we identify proteins required for proper positioning of the Shigella autotransporter IcsA. Autotransporters, widely distributed bacterial virulence proteins, are secreted at the bacterial pole. We show that the conserved cell division protein FtsQ is required for localization of IcsA and other autotransporters to the pole. We demonstrate further that this system can be applied to the study of proteins other than autotransporters that display polar positioning within bacterial cells. Many proteins localize to specific sites within bacterial cells, and localization to these sites is frequently critical to proper protein function. The mechanisms that underlie protein localization are incompletely understood, in part because of the paucity of methods that allow saturation screening for mutants in which protein localization is altered. We developed a genetic reporter assay that enables screening of bacterial populations for changes in localization of proteins to the bacterial pole, and we demonstrate the utility of the system in identifying factors required for proper localization of the polar Shigella autotransporter protein IcsA. Using this method, we identify the conserved cell division protein FtsQ as being required for positioning of IcsA to the bacterial pole. We demonstrate further that the requirement for FtsQ for polar positioning applies to other autotransporters

  13. Pattern formation and morphology transitions in bacterial systems

    Science.gov (United States)

    Arouh, Scott

    Bacteria grown on a semi-solid agar surface have been observed to form branching, chiral, and ring patterns as the colony envelope propagates outward. We model transitions between the branching and chiral patterns, analyze the effect of directed bacterial motion (chemotaxis) on the branching instability, and analyze a model for ring generation. Our model for transitions between branching and chiral patterns is a variant of Ben-Jacob's Communicating Walkers Models. We demonstrate that arbitrarily small nucleation regions of the new phase may be sufficient for the transformation to proceed. We also illustrate the phase transformations with plots of the colony envelope velocities as a function of environmental parameters. Based on the appearance of simulated colony patterns, we propose that experimentally observed global morphology transitions may be the result of single genetic mutations, and we predict biological values for the corresponding mutation rate. Our analysis of the effect of chemotaxis on a branching instability starts with an existing model for a branching instability. This instability is fundamentally caused by the need for limited nutrient to diffuse towards the colony. We add to this model the effect of bacteria moving chemotactically in response to the nutrient gradient. Our results show that this additional effect has a tendency to suppress the instability. Although we perform our calculations within the context of a simple "cutoff" model of colony dynamics, we expect our results to apply for more complex and hence more realistic approaches. We also analyze a model proposed by Medvedev, Kaper, and Kopell for ring formation. We perform a linear stability calculation for the model equations and find critical spatial decay rates to stability, but we later find that these are not relevant to the ring generation mechanism. By observing numerical bacterial density profiles near the colony edge, we identify a consolidation front distinct from the colony

  14. Microbial Genomics: The Expanding Universe of Bacterial Defense Systems.

    Science.gov (United States)

    Forsberg, Kevin J; Malik, Harmit S

    2018-04-23

    Bacteria protect themselves against infection using multiple defensive systems that move by horizontal gene transfer and accumulate in genomic 'defense islands'. A recent study exploited these features to uncover ten novel defense systems, substantially expanding the catalog of bacterial defense systems and predicting the discovery of many more. Copyright © 2018 Elsevier Ltd. All rights reserved.

  15. Bacterial and eukaryotic systems collide in the three Rs of Methanococcus.

    Science.gov (United States)

    Parker, Richard P; Walters, Alison D; Chong, James P J

    2011-01-01

    Methanococcus maripaludis S2 is a methanogenic archaeon with a well-developed genetic system. Its mesophilic nature offers a simple system in which to perform complementation using bacterial and eukaryotic genes. Although information-processing systems in archaea are generally more similar to those in eukaryotes than those in bacteria, the order Methanococcales has a unique complement of DNA replication proteins, with multiple MCM (minichromosome maintenance) proteins and no obvious originbinding protein. A search for homologues of recombination and repair proteins in M. maripaludis has revealed a mixture of bacterial, eukaryotic and some archaeal-specific homologues. Some repair pathways appear to be completely absent, but it is possible that archaeal-specific proteins could carry out these functions. The replication, recombination and repair systems in M. maripaludis are an interesting mixture of eukaryotic and bacterial homologues and could provide a system for uncovering novel interactions between proteins from different domains of life.

  16. CRISPR/Cas systems: new players in gene regulation and bacterial physiology

    Directory of Open Access Journals (Sweden)

    David eWeiss

    2014-04-01

    Full Text Available CRISPR-Cas systems are bacterial defenses against foreign nucleic acids derived from bacteriophages, plasmids or other sources. These systems are targeted in an RNA-dependent, sequence-specific manner, and are also adaptive, providing protection against previously encountered foreign elements. In addition to their canonical function in defense against foreign nucleic acid, their roles in various aspects of bacterial physiology are now being uncovered. We recently revealed a role for a Cas9-based Type II CRISPR-Cas system in the control of endogenous gene expression, a novel form of prokaryotic gene regulation. Cas9 functions in association with two small RNAs to target and alter the stability of an endogenous transcript encoding a bacterial lipoprotein (BLP. Since BLPs are recognized by the host innate immune protein Toll-like Receptor 2 (TLR2, CRISPR-Cas-mediated repression of BLP expression facilitates evasion of TLR2 by the intracellular bacterial pathogen Francisella novicida, and is essential for its virulence. Here we describe the Cas9 regulatory system in detail, as well as data on its role in controlling virulence traits of Neisseria meningitidis and Campylobacter jejuni. We also discuss potential roles of CRISPR-Cas systems in the response to envelope stress and other aspects of bacterial physiology. Since ~45% of bacteria and ~83% of Archaea encode these machineries, the newly appreciated regulatory functions of CRISPR-Cas systems are likely to play broad roles in controlling the pathogenesis and physiology of diverse prokaryotes.

  17. Small-molecule inhibition of bacterial two-component systems to combat antibiotic resistance and virulence.

    Science.gov (United States)

    Worthington, Roberta J; Blackledge, Meghan S; Melander, Christian

    2013-07-01

    Infections caused by multidrug-resistant bacteria are a considerable and increasing global problem. The development of new antibiotics is not keeping pace with the rapid evolution of resistance to almost all clinically available drugs, and novel strategies are required to fight bacterial infections. One such strategy is the control of pathogenic behaviors, as opposed to simply killing bacteria. Bacterial two-component system (TCS) signal transduction pathways control many pathogenic bacterial behaviors, such as virulence, biofilm formation and antibiotic resistance and are, therefore, an attractive target for the development of new drugs. This review presents an overview of TCS that are potential targets for such a strategy, describes small-molecules inhibitors of TCS identified to date and discusses assays for the identification of novel inhibitors. The future perspective for the identification and use of inhibitors of TCS to potentially provide new therapeutic options for the treatment of drug-resistant bacterial infections is discussed.

  18. Radiometric assay of bacterial growth: analysis of factors determining system performance and optimization of assay technique

    International Nuclear Information System (INIS)

    Boonkitticharoen, V.; Ehrhardt, J.C.; Kirchner, P.T.

    1987-01-01

    A quantitative technique for the measurement of 14 CO 2 released from a bacterial culture was evaluated. The technique uses liquid scintillation counting to record 14 CO 2 accumulation on a fluor-impregnated filter paper within a double-chambered scintillation vial that also houses the bacterial growth medium. We have successfully identified and corrected the major causes for a variably low detection efficiency, and also established the optimum mixture of reagents for the detection system. Incorporation of Triton X-100 into the scintillation fluid used for the detector reduced the variability between identical assays in a single batch from 50% to 5%, and, in conjunction with an increase in the scintillator concentration, raised the counting efficiency from 30% to 70-88%. The response of the improved detector is linear over a wide range of count-rates. Another significant modification was the interchange of growth and detector chambers. Overall, a 40-fold increase in count-rate during the exponential phase of bacterial growth was obtained by improving 14 CO 2 detection efficiency, increasing the rate of 14 CO 2 transfer from liquid to gas phases and enlarging the growth supporting capacity of the detector system. The minimum detection time for bacterial growth was shortened and the exponential phase of bacterial proliferation was lengthened by at least 2 hr. High counting efficiency, precision, and linearity make the improved detector a sensitive and reliable tool for radiometry of bacterial growth and metabolism

  19. Bacterial community composition of a wastewater treatment system reliant on N{sub 2} fixation

    Energy Technology Data Exchange (ETDEWEB)

    Reid, N.M.; Bowers, T.H.; Lloyd-Jones, G. [Scion, Rotorua (New Zealand)

    2008-05-15

    The temporal stability and change of the dominant phylogenetic groups of the domain bacteria were studied in a model plant-based industrial wastewater treatment system showing high levels of organic carbon removal supported by high levels of N{sub 2} fixation. Community profiles were obtained through terminal restriction fragment length polymorphism analysis and cloning of 16S rRNA amplicons followed by sequencing. Bacterial community profiles showed that ten common terminal restriction fragments made up approximately 50% of the measured bacterial community. As much as 42% of the measured bacterial community could be monitored by using quantitative PCR and primers that targeted three dominant operational taxonomic units. Despite changes in wastewater composition and dissolved oxygen levels, the bacterial community composition appeared stable and was dominated by {alpha}-Proteobacteria and {beta}-Proteobacteria, with a lesser amount of the highly diverse bacterial phylum Bacteroidetes. A short period of considerable change in the bacterial community composition did not appear to affect treatment performance indicating functional redundancy in this treatment system. (orig.)

  20. A Fuzzy Expert System for Distinguishing between Bacterial and Aseptic Meningitis

    Directory of Open Access Journals (Sweden)

    Mostafa Langarizadeh

    2015-05-01

    Data were extracted from 106 records of patients with meningitis (42 cases with bacterial meningitis in order to evaluate the proposed system. The system accuracy, specificity, and sensitivity were 89%, 92 %, and 97%, respectively. The area under the ROC curve was 0.93, and Kappa test revealed a good level of agreement (k=0.84, P

  1. A Gateway((R)) -compatible bacterial adenylate cyclase-based two-hybrid system

    Czech Academy of Sciences Publication Activity Database

    Ouellette, S. P.; Gauliard, E.; Antošová, Zuzana; Ladant, D.

    2014-01-01

    Roč. 6, č. 3 (2014), s. 259-267 ISSN 1758-2229 Institutional support: RVO:67985823 Keywords : bacterial two-hybrid system * protein–protein interactions * cell division * Gateway((R))(GW) cloning system Subject RIV: EE - Microbiology, Virology Impact factor: 3.293, year: 2014

  2. The use of hygromycin phosphotransferase gene (hpt) with an ...

    African Journals Online (AJOL)

    Previously, we have developed a marker-off system to truncate a selec marker in transgenic plants by locating one end of the transposon in the intron of the marker gene (glyphosate-tolerant epsps gene). In order to expand this technique to those marker genes without intron in this report, we created an artificial intron ...

  3. Breakdown of the innate immune system by bacterial proteases

    NARCIS (Netherlands)

    Laarman, A.J.

    2011-01-01

    Bacteria have developed many strategies to circumvent our immune system to survive and colonize human tissues. One of these strategies is by secreting proteases that specifically target the innate immune system. Aureolysin is a metalloprotease from Staphylococcus aureus which target the main

  4. Bacterial elicitors and plant signaling in induced systemic resistance

    OpenAIRE

    Bakker, P.A.H.M.; Pelt, J.A. van; Sluis, I. van der; Pieterse, C.M.J.

    2008-01-01

    Plant root colonizing, fluorescent Pseudomonas spp. have been studied for decades for their plant growth promoting properties and their effective suppression of soil borne plant diseases. The modes of action that play a role in disease suppression by these bacteria include siderophore-mediated competition for iron, antibiosis, and induced systemic resistance (ISR). The involvement of ISR is typically studied in systems in which the Pseudomonas bacteria and the pathogen are inoculated and rema...

  5. Changes in bacterial composition of biofilm in a metropolitan drinking water distribution system.

    Science.gov (United States)

    Revetta, R P; Gomez-Alvarez, V; Gerke, T L; Santo Domingo, J W; Ashbolt, N J

    2016-07-01

    This study examined the development of bacterial biofilms within a metropolitan distribution system. The distribution system is fed with different source water (i.e. groundwater, GW and surface water, SW) and undergoes different treatment processes in separate facilities. The biofilm community was characterized using 16S rRNA gene clone libraries and functional potential analysis, generated from total DNA extracted from coupons in biofilm annular reactors fed with onsite drinking water for up to 18 months. Differences in the bacterial community structure were observed between GW and SW. Representatives that explained the dissimilarity were associated with the classes Betaproteobacteria, Alphaproteobacteria, Actinobacteria, Gammaproteobacteria and Firmicutes. After 9 months the biofilm bacterial community from both GW and SW were dominated by Mycobacterium species. The distribution of the dominant operational taxonomic unit (OTU) (Mycobacterium) positively correlated with the drinking water distribution system (DWDS) temperature. In this study, the biofilm community structure observed between GW and SW were dissimilar, while communities from different locations receiving SW did not show significant differences. The results suggest that source water and/or the water quality shaped by their respective treatment processes may play an important role in shaping the bacterial communities in the distribution system. In addition, several bacterial groups were present in all samples, suggesting that they are an integral part of the core microbiota of this DWDS. These results provide an ecological insight into biofilm bacterial structure in chlorine-treated drinking water influenced by different water sources and their respective treatment processes. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  6. Bacterial Autoimmunity Due to a Restriction-Modification System.

    Science.gov (United States)

    Pleška, Maroš; Qian, Long; Okura, Reiko; Bergmiller, Tobias; Wakamoto, Yuichi; Kussell, Edo; Guet, Călin C

    2016-02-08

    Restriction-modification (RM) systems represent a minimal and ubiquitous biological system of self/non-self discrimination in prokaryotes [1], which protects hosts from exogenous DNA [2]. The mechanism is based on the balance between methyltransferase (M) and cognate restriction endonuclease (R). M tags endogenous DNA as self by methylating short specific DNA sequences called restriction sites, whereas R recognizes unmethylated restriction sites as non-self and introduces a double-stranded DNA break [3]. Restriction sites are significantly underrepresented in prokaryotic genomes [4-7], suggesting that the discrimination mechanism is imperfect and occasionally leads to autoimmunity due to self-DNA cleavage (self-restriction) [8]. Furthermore, RM systems can promote DNA recombination [9] and contribute to genetic variation in microbial populations, thus facilitating adaptive evolution [10]. However, cleavage of self-DNA by RM systems as elements shaping prokaryotic genomes has not been directly detected, and its cause, frequency, and outcome are unknown. We quantify self-restriction caused by two RM systems of Escherichia coli and find that, in agreement with levels of restriction site avoidance, EcoRI, but not EcoRV, cleaves self-DNA at a measurable rate. Self-restriction is a stochastic process, which temporarily induces the SOS response, and is followed by DNA repair, maintaining cell viability. We find that RM systems with higher restriction efficiency against bacteriophage infections exhibit a higher rate of self-restriction, and that this rate can be further increased by stochastic imbalance between R and M. Our results identify molecular noise in RM systems as a factor shaping prokaryotic genomes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. UV Radiation Damage and Bacterial DNA Repair Systems

    Science.gov (United States)

    Zion, Michal; Guy, Daniel; Yarom, Ruth; Slesak, Michaela

    2006-01-01

    This paper reports on a simple hands-on laboratory procedure for high school students in studying both radiation damage and DNA repair systems in bacteria. The sensitivity to ultra-violet (UV) radiation of both "Escherichia coli" and "Serratia marcescens" is tested by radiating them for varying time periods. Two growth temperatures are used in…

  8. CRISPR technologies for bacterial systems: Current achievements and future directions

    DEFF Research Database (Denmark)

    Choi, Kyeong Rok; Lee, Sang Yup

    2016-01-01

    Throughout the decades of its history, the advances in bacteria-based bio-industries have coincided with great leaps in strain engineering technologies. Recently unveiled clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) systems are now revolution......Throughout the decades of its history, the advances in bacteria-based bio-industries have coincided with great leaps in strain engineering technologies. Recently unveiled clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) systems are now...... revolutionizing biotechnology as well as biology. Diverse technologies have been derived from CRISPR/Cas systems in bacteria, yet the applications unfortunately have not been actively employed in bacteria as extensively as in eukaryotic organisms. A recent trend of engineering less explored strains in industrial...... microbiology-metabolic engineering, synthetic biology, and other related disciplines-is demanding facile yet robust tools, and various CRISPR technologies have potential to cater to the demands. Here, we briefly review the science in CRISPR/Cas systems and the milestone inventions that enabled numerous CRISPR...

  9. Galectin-3 directs antimicrobial guanylate binding proteins to vacuoles furnished with bacterial secretion systems.

    Science.gov (United States)

    Feeley, Eric M; Pilla-Moffett, Danielle M; Zwack, Erin E; Piro, Anthony S; Finethy, Ryan; Kolb, Joseph P; Martinez, Jennifer; Brodsky, Igor E; Coers, Jörn

    2017-02-28

    Many invasive bacteria establish pathogen-containing vacuoles (PVs) as intracellular niches for microbial growth. Immunity to these infections is dependent on the ability of host cells to recognize PVs as targets for host defense. The delivery of several host defense proteins to PVs is controlled by IFN-inducible guanylate binding proteins (GBPs), which themselves dock to PVs through poorly characterized mechanisms. Here, we demonstrate that GBPs detect the presence of bacterial protein secretion systems as "patterns of pathogenesis" associated with PVs. We report that the delivery of GBP2 to Legionella -containing vacuoles is dependent on the bacterial Dot/Icm secretion system, whereas the delivery of GBP2 to Yersinia- containing vacuoles (YCVs) requires hypersecretion of Yersinia translocon proteins. We show that the presence of bacterial secretion systems directs cytosolic carbohydrate-binding protein Galectin-3 to PVs and that the delivery of GBP1 and GBP2 to Legionella- containing vacuoles or YCVs is substantially diminished in Galectin-3-deficient cells. Our results illustrate that insertion of bacterial secretion systems into PV membranes stimulates Galectin-3-dependent recruitment of antimicrobial GBPs to PVs as part of a coordinated host defense program.

  10. Putative bacterial interactions from metagenomic knowledge with an integrative systems ecology approach

    OpenAIRE

    Bordron, P.; Latorre, M.; Cortés, M.; González, M.; Thiele, S.; Siegel, A.; Maass, A.; Eveillard, D.

    2016-01-01

    Abstract Following the trend of studies that investigate microbial ecosystems using different metagenomic techniques, we propose a new integrative systems ecology approach that aims to decipher functional roles within a consortium through the integration of genomic and metabolic knowledge at genome scale. For the sake of application, using public genomes of five bacterial strains involved in copper bioleaching: Acidiphilium cryptum, Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidan...

  11. A bacterial pathogen uses distinct type III secretion systems to alternate between host kingdoms

    Science.gov (United States)

    Plant and animal-pathogenic bacteria utilize phylogenetically distinct type III secretion systems (T3SS) that produce needle-like injectisomes or pili for the delivery of effector proteins into host cells. Pantoea stewartii subsp. stewartii (Pnss), the causative agent of Stewart’s bacterial wilt and...

  12. HRT and nutrients affect bacterial communities grown on recirculation aquaculture system effluents

    NARCIS (Netherlands)

    Schneider, O.; Chabrillon-Popelka, M.; Smidt, H.; Haenen, O.L.M.; Sereti, V.; Eding, E.H.; Verreth, J.A.J.

    2007-01-01

    In a recirculation aquaculture system the drumfilter effluent can be used as substrate for heterotrophic bacterial production, which can be recycled as feed. Because the bacteria might contain pathogens, which could reduce its suitability as feed, it is important to characterize these communities.

  13. Bacterial contamination of table eggs and the influence of housing systems

    NARCIS (Netherlands)

    Reu, de K.; Messens, W.; Heyndrickx, M.; Rodenburg, T.B.; Uyttendaele, M.; Herman, L.

    2008-01-01

    With the introduction of alternative housing systems for laying hens in the EU, recent research has focussed on the bacterial contamination of table eggs, e.g. eggshell and egg content contamination. Contamination of eggshells with aerobic bacteria is generally higher for nest eggs from non-cage

  14. Role of toll like receptors in bacterial and viral diseases – A systemic ...

    African Journals Online (AJOL)

    Background: Toll like receptors are key-receptors of the innate immune system, but their role against bacterial and viral infections are yet to be understood. Aim: The present study is aimed to investigate the diversity and frequency distribution of 10 TLR genes among typhoid fever and HIV+ patients. In this study, 44 samples ...

  15. A Bacterial-Based Algorithm to Simulate Complex Adaptative Systems

    OpenAIRE

    González Rodríguez, Diego; Hernández Carrión, José Rodolfo

    2014-01-01

    Paper presented at the 13th International Conference on Simulation of Adaptive Behavior which took place at Castellón, Spain in 2014, July 22-25. Bacteria have demonstrated an amazing capacity to overcome envi-ronmental changes by collective adaptation through genetic exchanges. Using a distributed communication system and sharing individual strategies, bacteria propagate mutations as innovations that allow them to survive in different envi-ronments. In this paper we present an agent-based...

  16. Biofilm bacterial communities in urban drinking water distribution systems transporting waters with different purification strategies.

    Science.gov (United States)

    Wu, Huiting; Zhang, Jingxu; Mi, Zilong; Xie, Shuguang; Chen, Chao; Zhang, Xiaojian

    2015-02-01

    Biofilm formation in drinking water distribution systems (DWDS) has many adverse consequences. Knowledge of microbial community structure of DWDS biofilm can aid in the design of an effective control strategy. However, biofilm bacterial community in real DWDS and the impact of drinking water purification strategy remain unclear. The present study investigated the composition and diversity of biofilm bacterial community in real DWDSs transporting waters with different purification strategies (conventional treatment and integrated treatment). High-throughput Illumina MiSeq sequencing analysis illustrated a large shift in the diversity and structure of biofilm bacterial community in real DWDS. Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Nitrospirae, and Cyanobacteria were the major components of biofilm bacterial community. Proteobacteria (mainly Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria) predominated in each DWDS biofilm, but the compositions of the dominant proteobacterial classes and genera and their proportions varied among biofilm samples. Drinking water purification strategy could shape DWDS biofilm bacterial community. Moreover, Pearson's correlation analysis indicated that Actinobacteria was positively correlated with the levels of total alkalinity and dissolved organic carbon in tap water, while Firmicutes had a significant positive correlation with nitrite nitrogen.

  17. Bacterial community characterization of water and intestine of the shrimp Litopenaeus stylirostris in a biofloc system.

    Science.gov (United States)

    Cardona, Emilie; Gueguen, Yannick; Magré, Kevin; Lorgeoux, Bénédicte; Piquemal, David; Pierrat, Fabien; Noguier, Florian; Saulnier, Denis

    2016-07-19

    Biofloc technology (BFT), a rearing method with little or no water exchange, is gaining popularity in aquaculture. In the water column, such systems develop conglomerates of microbes, algae and protozoa, together with detritus and dead organic particles. The intensive microbial community presents in these systems can be used as a pond water quality treatment system, and the microbial protein can serve as a feed additive. The current problem with BFT is the difficulty of controlling its bacterial community composition for both optimal water quality and optimal shrimp health. The main objective of the present study was to investigate microbial diversity of samples obtained from different culture environments (Biofloc technology and clear seawater) as well as from the intestines of shrimp reared in both environments through high-throughput sequencing technology. Analyses of the bacterial community identified in water from BFT and "clear seawater" (CW) systems (control) containing the shrimp Litopenaeus stylirostris revealed large differences in the frequency distribution of operational taxonomic units (OTUs). Four out of the five most dominant bacterial communities were different in both culture methods. Bacteria found in great abundance in BFT have two principal characteristics: the need for an organic substrate or nitrogen sources to grow and the capacity to attach to surfaces and co-aggregate. A correlation was found between bacteria groups and physicochemical and biological parameters measured in rearing tanks. Moreover, rearing-water bacterial communities influenced the microbiota of shrimp. Indeed, the biofloc environment modified the shrimp intestine microbiota, as the low level (27 %) of similarity between intestinal bacterial communities from the two treatments. This study provides the first information describing the complex biofloc microbial community, which can help to understand the environment-microbiota-host relationship in this rearing system.

  18. Significant relationship between soil bacterial community structure and incidence of bacterial wilt disease under continuous cropping system.

    Science.gov (United States)

    She, Siyuan; Niu, Jiaojiao; Zhang, Chao; Xiao, Yunhua; Chen, Wu; Dai, Linjian; Liu, Xueduan; Yin, Huaqun

    2017-03-01

    Soil bacteria are very important in biogeochemical cycles and play significant role in soil-borne disease suppression. Although continuous cropping is responsible for soil-borne disease enrichment, its effect on tobacco plant health and how soil bacterial communities change are yet to be elucidated. In this study, soil bacterial communities across tobacco continuous cropping time-series fields were investigated through high-throughput sequencing of 16S ribosomal RNA genes. The results showed that long-term continuous cropping could significantly alter soil microbial communities. Bacterial diversity indices and evenness indices decreased over the monoculture span and obvious variations for community structures across the three time-scale tobacco fields were detected. Compared with the first year, the abundances of Arthrobacter and Lysobacter showed a significant decrease. Besides, the abundance of the pathogen Ralstonia spp. accumulated over the monoculture span and was significantly correlated with tobacco bacterial wilt disease rate. Moreover, Pearson's correlation demonstrated that the abundance of Arthrobacter and Lysobacter, which are considered to be beneficial bacteria had significant negative correlation with tobacco bacterial wilt disease. Therefore, after long-term continuous cropping, tobacco bacterial wilt disease could be ascribed to the alteration of the composition as well as the structure of the soil microbial community.

  19. Specificity and complexity in bacterial quorum-sensing systems

    Science.gov (United States)

    Hawver, Lisa A.; Jung, Sarah A.; Ng, Wai-Leung

    2016-01-01

    Quorum sensing (QS) is a microbial cell-to-cell communication process that relies on the production and detection of chemical signals called autoinducers (AIs) to monitor cell density and species complexity in the population. QS allows bacteria to behave as a cohesive group and coordinate collective behaviors. While most QS receptors display high specificity to their AI ligands, others are quite promiscuous in signal detection. How do specific QS receptors respond to their cognate signals with high fidelity? Why do some receptors maintain low signal recognition specificity? In addition, many QS systems are composed of multiple intersecting signaling pathways: what are the benefits of preserving such a complex signaling network when a simple linear ‘one-to-one’ regulatory pathway seems sufficient to monitor cell density? Here, we will discuss different molecular mechanisms employed by various QS systems that ensure productive and specific QS responses. Moreover, the network architectures of some well-characterized QS circuits will be reviewed to understand how the wiring of different regulatory components achieves different biological goals. PMID:27354348

  20. The role of the bacterial mismatch repair system in SOS-induced mutagenesis: a theoretical background

    International Nuclear Information System (INIS)

    Belov, O.V.; Kapralov, M.I.; Chuluunbaatar, O.; Sweilam, N.H.

    2012-01-01

    A theoretical study is performed of the possible role of the methyl-directed mismatch repair system in the ultraviolet-induced mutagenesis of Escherichia coli bacterial cells. For this purpose, a mathematical model of the bacterial mismatch repair system is developed. Within this model, the key pathways of this type of repair are simulated on the basis of modern experimental data related to its mechanisms. Here we have modelled in detail five main pathways of DNA misincorporation removal with different DNA exonucleases. Using our calculations, we have tested the hypothesis that the bacterial mismatch repair system is responsible for the removal of the nucleotides misincorporated by DNA polymerase V (the UmuD' 2 C complex) during ultraviolet-induced SOS response. For the theoretical analysis of the mutation frequency, we have combined the proposed mathematical approach with the model of SOS-induced mutagenesis in the E.coli bacterial cell developed earlier. Our calculations support the hypothesis that methyl-directed mismatch repair influences the mutagenic effect of ultraviolet radiation

  1. A Fuzzy Expert System for Distinguishing between Bacterial and Aseptic Meningitis

    Directory of Open Access Journals (Sweden)

    Mostafa Langarizadeh

    2015-05-01

    Full Text Available Introduction Bacterial meningitis is a known infectious disease which occurs at early ages and should be promptly diagnosed and treated. Bacterial and aseptic meningitis are hard to be distinguished. Therefore, physicians should be highly informed and experienced in this area. The main aim of this study was to suggest a system for distinguishing between bacterial and aseptic meningitis, using fuzzy logic.    Materials and Methods In the first step, proper attributes were selected using Weka 3.6.7 software. Six attributes were selected using Attribute Evaluator, InfoGainAttributeEval, and Ranker search method items. Then, a fuzzy inference engine was designed using MATLAB software, based on Mamdani’s fuzzy logic method with max-min composition, prod-probor, and centroid defuzzification. The rule base consisted of eight rules, based on the experience of three specialists and information extracted from textbooks. Results Data were extracted from 106 records of patients with meningitis (42 cases with bacterial meningitis in order to evaluate the proposed system. The system accuracy, specificity, and sensitivity were 89%, 92 %, and 97%, respectively. The area under the ROC curve was 0.93, and Kappa test revealed a good level of agreement (k=0.84, P

  2. Bacterial derived proteoliposome as ideal delivery system and cellular adjuvant.

    Science.gov (United States)

    Rodríguez, Tamara; Pérez, Oliver a; Ugrinovic, Sanja; Bracho, Gustavo; Mastroeni, Pietro

    2006-04-12

    We explored the potential of a proteoliposome (PL) from the outer membrane of N. meningitidis B, as an immunopotentiator and as a vector for antigen delivery to dendritic cells (DC). DC were incubated with PL resulting in up-regulation of MHC-II, CD40, CD80, and CD86 expression and production of TNFalpha and IL12(p70). Ovoalbumin (OVA) was incorporated within PL (PL-OVA). PL-OVA presented OVA-specific peptides to CD4+ and CD8+ OVA-specific T-cell hybridomas. PL exerts an immunomodulatory effect on DC and is a general system to deliver antigens for presentation to CD4+ and CD8+ T-cells possibly implicated in the induction CD8+ cytotoxic T lymphocytes (CTLs) responses.

  3. Intestinal Dysbiosis, Barrier Dysfunction, and Bacterial Translocation Account for CKD-Related Systemic Inflammation.

    Science.gov (United States)

    Andersen, Kirstin; Kesper, Marie Sophie; Marschner, Julian A; Konrad, Lukas; Ryu, Mi; Kumar Vr, Santhosh; Kulkarni, Onkar P; Mulay, Shrikant R; Romoli, Simone; Demleitner, Jana; Schiller, Patrick; Dietrich, Alexander; Müller, Susanna; Gross, Oliver; Ruscheweyh, Hans-Joachim; Huson, Daniel H; Stecher, Bärbel; Anders, Hans-Joachim

    2017-01-01

    CKD associates with systemic inflammation, but the underlying cause is unknown. Here, we investigated the involvement of intestinal microbiota. We report that collagen type 4 α3-deficient mice with Alport syndrome-related progressive CKD displayed systemic inflammation, including increased plasma levels of pentraxin-2 and activated antigen-presenting cells, CD4 and CD8 T cells, and Th17- or IFNγ-producing T cells in the spleen as well as regulatory T cell suppression. CKD-related systemic inflammation in these mice associated with intestinal dysbiosis of proteobacterial blooms, translocation of living bacteria across the intestinal barrier into the liver, and increased serum levels of bacterial endotoxin. Uremia did not affect secretory IgA release into the ileum lumen or mucosal leukocyte subsets. To test for causation between dysbiosis and systemic inflammation in CKD, we eradicated facultative anaerobic microbiota with antibiotics. This eradication prevented bacterial translocation, significantly reduced serum endotoxin levels, and fully reversed all markers of systemic inflammation to the level of nonuremic controls. Therefore, we conclude that uremia associates with intestinal dysbiosis, intestinal barrier dysfunction, and bacterial translocation, which trigger the state of persistent systemic inflammation in CKD. Uremic dysbiosis and intestinal barrier dysfunction may be novel therapeutic targets for intervention to suppress CKD-related systemic inflammation and its consequences. Copyright © 2016 by the American Society of Nephrology.

  4. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications

    Directory of Open Access Journals (Sweden)

    Chew Chieng Yeo

    2016-02-01

    Full Text Available Toxin-antitoxin (TA systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

  5. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications.

    Science.gov (United States)

    Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

    2016-02-19

    Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

  6. Purification, crystallization and preliminary X-ray analysis of Enterococcus faecium aminoglycoside-2′′-phosphotransferase-Ib [APH(2′′)-Ib

    Energy Technology Data Exchange (ETDEWEB)

    Walanj, Rupa; Young, Paul; Baker, Heather M.; Baker, Edward N.; Metcalf, Peter [Laboratory of Structural Biology, School of Biological Sciences, University of Auckland, Auckland (New Zealand); Chow, Joseph W.; Lerner, Stephen [Division of Infectious Diseases, Wayne State University School of Medicine and VA Medical Center, Detroit, Michigan 48201 (United States); Vakulenko, Sergei [Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556 (United States); Smith, Clyde A., E-mail: csmith@slac.stanford.edu [Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CA 94025 (United States); Laboratory of Structural Biology, School of Biological Sciences, University of Auckland, Auckland (New Zealand)

    2005-04-01

    APH(2′′)-Ib is an enzyme responsible for high-level gentamicin resistance in E. faecium isolates. Native crystals of this enzyme have been prepared and preliminary X-ray diffraction experiments have been undertaken. Bacterial resistance to the aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, APH(2′′)-Ib, has been cloned and the protein (comprising 299 amino-acid residues) expressed in Escherichia coli, purified and crystallized in the presence of 16%(w/v) PEG 3350 and gentamicin. The crystals belong to the monoclinic space group P2{sub 1}, with approximate unit-cell parameters a = 79.7, b = 58.8, c = 81.4 Å, β = 98.4°, and preliminary X-ray diffraction analysis is consistent with the presence of two molecules in the asymmetric unit. Synchrotron diffraction data to approximately 2.65 Å resolution were collected from a native APH(2′′)-Ib crystal at beamline BL9-2 at SSRL (Stanford, CA, USA). Selenium-substituted crystals have also been produced and structure determination is proceeding.

  7. Auto-production of biosurfactants reverses the coffee ring effect in a bacterial system

    Science.gov (United States)

    Sempels, Wouter; de Dier, Raf; Mizuno, Hideaki; Hofkens, Johan; Vermant, Jan

    2013-04-01

    The deposition of material at the edge of evaporating droplets, known as the ‘coffee ring effect’, is caused by a radially outward capillary flow. This phenomenon is common to a wide array of systems including colloidal and bacterial systems. The role of surfactants in counteracting these coffee ring depositions is related to the occurrence of local vortices known as Marangoni eddies. Here we show that these swirling flows are universal, and not only lead to a uniform deposition of colloids but also occur in living bacterial systems. Experiments on Pseudomonas aeruginosa suggest that the auto-production of biosurfactants has an essential role in creating a homogeneous deposition of the bacteria upon drying. Moreover, at biologically relevant conditions, intricate time-dependent flows are observed in addition to the vortex regime, which are also effective in reversing the coffee ring effect at even lower surfactant concentrations.

  8. A Bacterial Analysis Platform: An Integrated System for Analysing Bacterial Whole Genome Sequencing Data for Clinical Diagnostics and Surveillance

    DEFF Research Database (Denmark)

    Thomsen, Martin Christen Frølund; Ahrenfeldt, Johanne; Bellod Cisneros, Jose Luis

    2016-01-01

    web-based tools we developed a single pipeline for batch uploading of whole genome sequencing data from multiple bacterial isolates. The pipeline will automatically identify the bacterial species and, if applicable, assemble the genome, identify the multilocus sequence type, plasmids, virulence genes...... and antimicrobial resistance genes. A short printable report for each sample will be provided and an Excel spreadsheet containing all the metadata and a summary of the results for all submitted samples can be downloaded. The pipeline was benchmarked using datasets previously used to test the individual services...... and made publicly available, providing easy-to-use automated analysis of bacterial whole genome sequencing data. The platform may be of immediate relevance as a guide for investigators using whole genome sequencing for clinical diagnostics and surveillance. The platform is freely available at: https...

  9. A Phosphorylation Switch on Lon Protease Regulates Bacterial Type III Secretion System in Host.

    Science.gov (United States)

    Zhou, Xiaofeng; Teper, Doron; Andrade, Maxuel O; Zhang, Tong; Chen, Sixue; Song, Wen-Yuan; Wang, Nian

    2018-01-23

    Most pathogenic bacteria deliver virulence factors into host cytosol through type III secretion systems (T3SS) to perturb host immune responses. The expression of T3SS is often repressed in rich medium but is specifically induced in the host environment. The molecular mechanisms underlying host-specific induction of T3SS expression is not completely understood. Here we demonstrate in Xanthomonas citri that host-induced phosphorylation of the ATP-dependent protease Lon stabilizes HrpG, the master regulator of T3SS, conferring bacterial virulence. Ser/Thr/Tyr phosphoproteome analysis revealed that phosphorylation of Lon at serine 654 occurs in the citrus host. In rich medium, Lon represses T3SS by degradation of HrpG via recognition of its N terminus. Genetic and biochemical data indicate that phosphorylation at serine 654 deactivates Lon proteolytic activity and attenuates HrpG proteolysis. Substitution of alanine for Lon serine 654 resulted in repression of T3SS gene expression in the citrus host through robust degradation of HrpG and reduced bacterial virulence. Our work reveals a novel mechanism for distinct regulation of bacterial T3SS in different environments. Additionally, our data provide new insight into the role of protein posttranslational modification in the regulation of bacterial virulence. IMPORTANCE Type III secretion systems (T3SS) are an essential virulence trait of many bacterial pathogens because of their indispensable role in the delivery of virulence factors. However, expression of T3SS in the noninfection stage is energy consuming. Here, we established a model to explain the differential regulation of T3SS in host and nonhost environments. When Xanthomonas cells are grown in rich medium, the T3SS regulator HrpG is targeted by Lon protease for proteolysis. The degradation of HrpG leads to downregulated expression of HrpX and the hrp / hrc genes. When Xanthomonas cells infect the host, specific plant stimuli can be perceived and induce Lon

  10. Putative bacterial interactions from metagenomic knowledge with an integrative systems ecology approach.

    Science.gov (United States)

    Bordron, Philippe; Latorre, Mauricio; Cortés, Maria-Paz; González, Mauricio; Thiele, Sven; Siegel, Anne; Maass, Alejandro; Eveillard, Damien

    2016-02-01

    Following the trend of studies that investigate microbial ecosystems using different metagenomic techniques, we propose a new integrative systems ecology approach that aims to decipher functional roles within a consortium through the integration of genomic and metabolic knowledge at genome scale. For the sake of application, using public genomes of five bacterial strains involved in copper bioleaching: Acidiphilium cryptum, Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Leptospirillum ferriphilum, and Sulfobacillus thermosulfidooxidans, we first reconstructed a global metabolic network. Next, using a parsimony assumption, we deciphered sets of genes, called Sets from Genome Segments (SGS), that (1) are close on their respective genomes, (2) take an active part in metabolic pathways and (3) whose associated metabolic reactions are also closely connected within metabolic networks. Overall, this SGS paradigm depicts genomic functional units that emphasize respective roles of bacterial strains to catalyze metabolic pathways and environmental processes. Our analysis suggested that only few functional metabolic genes are horizontally transferred within the consortium and that no single bacterial strain can accomplish by itself the whole copper bioleaching. The use of SGS pinpoints a functional compartmentalization among the investigated species and exhibits putative bacterial interactions necessary for promoting these pathways. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  11. System automation for a bacterial colony detection and identification instrument via forward scattering

    International Nuclear Information System (INIS)

    Bae, Euiwon; Hirleman, E Daniel; Aroonnual, Amornrat; Bhunia, Arun K; Robinson, J Paul

    2009-01-01

    A system design and automation of a microbiological instrument that locates bacterial colonies and captures the forward-scattering signatures are presented. The proposed instrument integrates three major components: a colony locator, a forward scatterometer and a motion controller. The colony locator utilizes an off-axis light source to illuminate a Petri dish and an IEEE1394 camera to capture the diffusively scattered light to provide the number of bacterial colonies and two-dimensional coordinate information of the bacterial colonies with the help of a segmentation algorithm with region-growing. Then the Petri dish is automatically aligned with the respective centroid coordinate with a trajectory optimization method, such as the Traveling Salesman Algorithm. The forward scatterometer automatically computes the scattered laser beam from a monochromatic image sensor via quadrant intensity balancing and quantitatively determines the centeredness of the forward-scattering pattern. The final scattering signatures are stored to be analyzed to provide rapid identification and classification of the bacterial samples

  12. A Phosphorylation Switch on Lon Protease Regulates Bacterial Type III Secretion System in Host

    Directory of Open Access Journals (Sweden)

    Xiaofeng Zhou

    2018-01-01

    Full Text Available Most pathogenic bacteria deliver virulence factors into host cytosol through type III secretion systems (T3SS to perturb host immune responses. The expression of T3SS is often repressed in rich medium but is specifically induced in the host environment. The molecular mechanisms underlying host-specific induction of T3SS expression is not completely understood. Here we demonstrate in Xanthomonas citri that host-induced phosphorylation of the ATP-dependent protease Lon stabilizes HrpG, the master regulator of T3SS, conferring bacterial virulence. Ser/Thr/Tyr phosphoproteome analysis revealed that phosphorylation of Lon at serine 654 occurs in the citrus host. In rich medium, Lon represses T3SS by degradation of HrpG via recognition of its N terminus. Genetic and biochemical data indicate that phosphorylation at serine 654 deactivates Lon proteolytic activity and attenuates HrpG proteolysis. Substitution of alanine for Lon serine 654 resulted in repression of T3SS gene expression in the citrus host through robust degradation of HrpG and reduced bacterial virulence. Our work reveals a novel mechanism for distinct regulation of bacterial T3SS in different environments. Additionally, our data provide new insight into the role of protein posttranslational modification in the regulation of bacterial virulence.

  13. Chemical inhibitors of the type three secretion system: disarming bacterial pathogens.

    Science.gov (United States)

    Duncan, Miles C; Linington, Roger G; Auerbuch, Victoria

    2012-11-01

    The recent and dramatic rise of antibiotic resistance among bacterial pathogens underlies the fear that standard treatments for infectious disease will soon be largely ineffective. Resistance has evolved against nearly every clinically used antibiotic, and in the near future, we may be hard-pressed to treat bacterial infections previously conquered by "magic bullet" drugs. While traditional antibiotics kill or slow bacterial growth, an important emerging strategy to combat pathogens seeks to block the ability of bacteria to harm the host by inhibiting bacterial virulence factors. One such virulence factor, the type three secretion system (T3SS), is found in over two dozen Gram-negative pathogens and functions by injecting effector proteins directly into the cytosol of host cells. Without T3SSs, many pathogenic bacteria are unable to cause disease, making the T3SS an attractive target for novel antimicrobial drugs. Interdisciplinary efforts between chemists and microbiologists have yielded several T3SS inhibitors, including the relatively well-studied salicylidene acylhydrazides. This review highlights the discovery and characterization of T3SS inhibitors in the primary literature over the past 10 years and discusses the future of these drugs as both research tools and a new class of therapeutic agents.

  14. BSim: an agent-based tool for modeling bacterial populations in systems and synthetic biology.

    Directory of Open Access Journals (Sweden)

    Thomas E Gorochowski

    Full Text Available Large-scale collective behaviors such as synchronization and coordination spontaneously arise in many bacterial populations. With systems biology attempting to understand these phenomena, and synthetic biology opening up the possibility of engineering them for our own benefit, there is growing interest in how bacterial populations are best modeled. Here we introduce BSim, a highly flexible agent-based computational tool for analyzing the relationships between single-cell dynamics and population level features. BSim includes reference implementations of many bacterial traits to enable the quick development of new models partially built from existing ones. Unlike existing modeling tools, BSim fully considers spatial aspects of a model allowing for the description of intricate micro-scale structures, enabling the modeling of bacterial behavior in more realistic three-dimensional, complex environments. The new opportunities that BSim opens are illustrated through several diverse examples covering: spatial multicellular computing, modeling complex environments, population dynamics of the lac operon, and the synchronization of genetic oscillators. BSim is open source software that is freely available from http://bsim-bccs.sf.net and distributed under the Open Source Initiative (OSI recognized MIT license. Developer documentation and a wide range of example simulations are also available from the website. BSim requires Java version 1.6 or higher.

  15. Speed and Displacement Control System of Bearingless Brushless DC Motor Based on Improved Bacterial Foraging Algorithm

    Directory of Open Access Journals (Sweden)

    Diao Xiaoyan

    2016-01-01

    Full Text Available To solve the deficiencies of long optimization time and poor precision existing in conventional bacterial foraging algorithm (BFA in the process of parameter optimization, an improved bacterial foraging algorithm (IBFA is proposed and applied to speed and displacement control system of bearingless brushless DC (Bearingless BLDC motors. To begin with the fundamental principle of BFA, the proposed method is introduced and the individual intelligence is efficiently used in the process of parameter optimization, and then the working principle of bearingless BLDC motors is expounded. Finally, modeling and simulation of the speed and displacement control system of bearingless BLDC motors based on the IBFA are carried out by taking the software of MATLAB/Simulink as a platform. Simulation results show that, speed overshoot, torque ripple and rotor position oscillation are dramatically reduced, thus the proposed method has good application prospects in the field of bearingless motors.

  16. Bacterial Ghosts as antigen and drug delivery system for ocular surface diseases: Effective internalization of Bacterial Ghosts by human conjunctival epithelial cells.

    Science.gov (United States)

    Kudela, Pavol; Koller, Verena Juliana; Mayr, Ulrike Beate; Nepp, Johannes; Lubitz, Werner; Barisani-Asenbauer, Talin

    2011-05-20

    The purpose of the presented investigation was to examine the efficiency of the novel carrier system Bacterial Ghosts (BGs), which are empty bacterial cell envelopes of Gram-negative bacteria to target human conjunctival epithelial cells, as well as to test the endocytic capacity of conjunctival cells after co-incubation with BGs generated from different bacterial species, and to foreclose potential cytotoxic effects caused by BGs. The efficiency of conjunctival cells to internalize BGs was investigated using the Chang conjunctival epithelial cell line and primary human conjunctiva-derived epithelial cells (HCDECs) as in vitro model. A high capacity of HCDECs to functionally internalize BGs was detected with the level of internalization depending on the type of species used for BGs generation. Detailed analysis showed no cytotoxic effect of BGs on HCDECs independently of the used bacterial species. Moreover, co-incubation with BGs did not enhance expression of both MHC class I and class II molecules by HCDECs, but increased expression of ICAM-1. The high rates of BG's internalization by HCDECs with no BG-mediated cytotoxic impact designate this carrier system to be a promising candidate for an ocular surface drug delivery system. BGs could be useful for future therapeutic ocular surface applications and eye-specific disease vaccine development including DNA transfer. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Bacterial toxin-antitoxin gene system as containment control in yeast cells

    DEFF Research Database (Denmark)

    Kristoffersen, P.; Jensen, G. B.; Gerdes, K.

    2000-01-01

    The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. The Escherichia coli relE and relB genes were expressed in the yeast Saccharomyces cerevisiae, Expression of the relE gene was highly toxic to yeast cells. However, expression...... fermentation processes in which the escape of genetically modified cells would be considered highly risky....

  18. The Bacterial Type III Secretion System as a Target for Developing New Antibiotics

    OpenAIRE

    McShan, Andrew C.; De Guzman, Roberto N.

    2015-01-01

    Antibiotic resistance in pathogens requires new targets for developing novel antibacterials. The bacterial type III secretion system (T3SS) is an attractive target for developing antibacterials as it is essential in the pathogenesis of many Gram-negative bacteria. The T3SS consists of structural proteins, effectors and chaperones. Over 20 different structural proteins assemble into a complex nanoinjector that punctures a hole on the eukaryotic cell membrane to allow the delivery of effectors ...

  19. Chemical interference with iron transport systems to suppress bacterial growth of Streptococcus pneumoniae.

    Directory of Open Access Journals (Sweden)

    Xiao-Yan Yang

    Full Text Available Iron is an essential nutrient for the growth of most bacteria. To obtain iron, bacteria have developed specific iron-transport systems located on the membrane surface to uptake iron and iron complexes such as ferrichrome. Interference with the iron-acquisition systems should be therefore an efficient strategy to suppress bacterial growth and infection. Based on the chemical similarity of iron and ruthenium, we used a Ru(II complex R-825 to compete with ferrichrome for the ferrichrome-transport pathway in Streptococcus pneumoniae. R-825 inhibited the bacterial growth of S. pneumoniae and stimulated the expression of PiuA, the iron-binding protein in the ferrichrome-uptake system on the cell surface. R-825 treatment decreased the cellular content of iron, accompanying with the increase of Ru(II level in the bacterium. When the piuA gene (SPD_0915 was deleted in the bacterium, the mutant strain became resistant to R-825 treatment, with decreased content of Ru(II. Addition of ferrichrome can rescue the bacterial growth that was suppressed by R-825. Fluorescence spectral quenching showed that R-825 can bind with PiuA in a similar pattern to the ferrichrome-PiuA interaction in vitro. These observations demonstrated that Ru(II complex R-825 can compete with ferrichrome for the ferrichrome-transport system to enter S. pneumoniae, reduce the cellular iron supply, and thus suppress the bacterial growth. This finding suggests a novel antimicrobial approach by interfering with iron-uptake pathways, which is different from the mechanisms used by current antibiotics.

  20. A controllable bacterial lysis system to enhance biological safety of live attenuated Vibrio anguillarum vaccine.

    Science.gov (United States)

    Chu, Teng; Guan, Lingyu; Shang, Pengfei; Wang, Qiyao; Xiao, Jingfan; Liu, Qin; Zhang, Yuanxing

    2015-08-01

    Bacterial strains used as backbone for the generation of vaccine prototypes should exhibit an adequate and stable safety profile. Given the fact that live attenuated vaccines often contain some potential risks in virulence recovery and spread infections, new approaches are greatly needed to improve their biological safety. Here, a critically iron-regulated promoter PviuA was screened from Vibrio anguillarum, which was demonstrated to respond to iron-limitation signal both in vitro and in vivo. By using PviuA as a regulatory switch to control the expression of phage P22 lysis cassette 13-19-15, a novel in vivo inducible bacterial lysis system was established in V. anguillarum. This system was proved to be activated by iron-limitation signals and then effectively lyse V. anguillarum both in vitro and in vivo. Further, this controllable bacterial lysis system, after being transformed into a live attenuated V. anguillarum vaccine strain MVAV6203, was confirmed to significantly improve biological safety of the live attenuated vaccine without impairing its immune protection efficacy. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Profiling bacterial communities associated with sediment-based aquaculture bioremediation systems under contrasting redox regimes

    Science.gov (United States)

    Robinson, Georgina; Caldwell, Gary S.; Wade, Matthew J.; Free, Andrew; Jones, Clifford L. W.; Stead, Selina M.

    2016-12-01

    Deposit-feeding invertebrates are proposed bioremediators in microbial-driven sediment-based aquaculture effluent treatment systems. We elucidate the role of the sediment reduction-oxidation (redox) regime in structuring benthic bacterial communities, having direct implications for bioremediation potential and deposit-feeder nutrition. The sea cucumber Holothuria scabra was cultured on sediments under contrasting redox regimes; fully oxygenated (oxic) and redox stratified (oxic-anoxic). Taxonomically, metabolically and functionally distinct bacterial communities developed between the redox treatments with the oxic treatment supporting the greater diversity; redox regime and dissolved oxygen levels were the main environmental drivers. Oxic sediments were colonised by nitrifying bacteria with the potential to remediate nitrogenous wastes. Percolation of oxygenated water prevented the proliferation of anaerobic sulphate-reducing bacteria, which were prevalent in the oxic-anoxic sediments. At the predictive functional level, bacteria within the oxic treatment were enriched with genes associated with xenobiotics metabolism. Oxic sediments showed the greater bioremediation potential; however, the oxic-anoxic sediments supported a greater sea cucumber biomass. Overall, the results indicate that bacterial communities present in fully oxic sediments may enhance the metabolic capacity and bioremediation potential of deposit-feeder microbial systems. This study highlights the benefits of incorporating deposit-feeding invertebrates into effluent treatment systems, particularly when the sediment is oxygenated.

  2. Bacterial Community Structure Shifted by Geosmin in Granular Activated Carbon System of Water Treatment Plants.

    Science.gov (United States)

    Pham, Ngoc Dung; Lee, Eun-Hee; Chae, Seon-Ha; Cho, Yongdeok; Shin, Hyejin; Son, Ahjeong

    2016-01-01

    We investigated the relation between the presence of geosmin in water and the bacterial community structure within the granular activated carbon (GAC) system of water treatment plants in South Korea. GAC samples were collected in May and August of 2014 at three water treatment plants (Sungnam, Koyang, and Yeoncho in Korea). Dissolved organic carbon and geosmin were analyzed before and after GAC treatment. Geosmin was found in raw water from Sungnam and Koyang water treatment plants but not in that from Yeoncho water treatment plant. Interestingly, but not surprisingly, the 16S rRNA clone library indicated that the bacterial communities from the Sungnam and Koyang GAC systems were closely related to geosmin-degrading bacteria. Based on the phylogenetic tree and multidimensional scaling plot, bacterial clones from GAC under the influence of geosmin were clustered with Variovorax paradoxus strain DB 9b and Comamonas sp. DB mg. In other words, the presence of geosmin in water might have inevitably contributed to the growth of geosmin degraders within the respective GAC system.

  3. Defense islands in bacterial and archaeal genomes and prediction of novel defense systems.

    Science.gov (United States)

    Makarova, Kira S; Wolf, Yuri I; Snir, Sagi; Koonin, Eugene V

    2011-11-01

    The arms race between cellular life forms and viruses is a major driving force of evolution. A substantial fraction of bacterial and archaeal genomes is dedicated to antivirus defense. We analyzed the distribution of defense genes and typical mobilome components (such as viral and transposon genes) in bacterial and archaeal genomes and demonstrated statistically significant clustering of antivirus defense systems and mobile genes and elements in genomic islands. The defense islands are enriched in putative operons and contain numerous overrepresented gene families. A detailed sequence analysis of the proteins encoded by genes in these families shows that many of them are diverged variants of known defense system components, whereas others show features, such as characteristic operonic organization, that are suggestive of novel defense systems. Thus, genomic islands provide abundant material for the experimental study of bacterial and archaeal antivirus defense. Except for the CRISPR-Cas systems, different classes of defense systems, in particular toxin-antitoxin and restriction-modification systems, show nonrandom clustering in defense islands. It remains unclear to what extent these associations reflect functional cooperation between different defense systems and to what extent the islands are genomic "sinks" that accumulate diverse nonessential genes, particularly those acquired via horizontal gene transfer. The characteristics of defense islands resemble those of mobilome islands. Defense and mobilome genes are nonrandomly associated in islands, suggesting nonadaptive evolution of the islands via a preferential attachment-like mechanism underpinned by the addictive properties of defense systems such as toxins-antitoxins and an important role of horizontal mobility in the evolution of these islands.

  4. Comparison of bacterial community characteristics between complete and shortcut denitrification systems for quinoline degradation.

    Science.gov (United States)

    Zhang, Xiaomei; Hua, Xiufu; Yue, Xiuping

    2017-02-01

    For quinoline-denitrifying degradation, very few researches focused on shortcut denitrification process and its bacterial community characteristics. In this study, complete and shortcut denitrification systems were constructed simultaneously for quinoline degradation. By calculation, specific quinoline removal rates were 0.905 and 1.123 g/(gVSS d), respectively, in the complete and shortcut systems, and the latter was 1.24 times of the former. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), high-throughput sequencing, and quantitative PCR (qPCR) techniques based on 16S rRNA were jointly applied to compare microbial community structures of two systems. Many denitrifying bacteria phyla, classes, and genera were detected in the two systems. Phylum Proteobacteria, Class Gammaproteobacteria, and Genus Alicycliphilus denitrificans were the dominant contributors for quinoline-denitrifying degradation. In the shortcut denitrification system, main and specific strains playing crucial roles were more; the species richness and the total abundance of functional genes (narG, nirS, nirK, and nosZ) were higher compared with the complete denitrification system. It could be supposed that inorganic-nitrogen reductase activity of bacterial community was stronger in the shortcut denitrification system, which was the intrinsic reason to result in higher denitrification rate.

  5. Engineering of GlcNAc-1-Phosphotransferase for Production of Highly Phosphorylated Lysosomal Enzymes for Enzyme Replacement Therapy.

    Science.gov (United States)

    Liu, Lin; Lee, Wang-Sik; Doray, Balraj; Kornfeld, Stuart

    2017-06-16

    Several lysosomal enzymes currently used for enzyme replacement therapy in patients with lysosomal storage diseases contain very low levels of mannose 6-phosphate, limiting their uptake via mannose 6-phosphate receptors on the surface of the deficient cells. These enzymes are produced at high levels by mammalian cells and depend on endogenous GlcNAc-1-phosphotransferase α/β precursor to phosphorylate the mannose residues on their glycan chains. We show that co-expression of an engineered truncated GlcNAc-1-phosphotransferase α/β precursor and the lysosomal enzyme of interest in the producing cells resulted in markedly increased phosphorylation and cellular uptake of the secreted lysosomal enzyme. This method also results in the production of highly phosphorylated acid β-glucocerebrosidase, a lysosomal enzyme that normally has just trace amounts of this modification.

  6. Application of Biomaterials and Inkjet Printing to Develop Bacterial Culture System

    Directory of Open Access Journals (Sweden)

    Tithimanan Srimongkon

    2015-01-01

    Full Text Available We created an automated bioassay system based on inkjet printing. Compared to conventional manual bacterial culture systems our printing approach improves the quality as well as the processing speed. A hydrophobic/hydrophilic pattern as a container supporting a culture medium was built on filter paper using a toluene solution of polystyrene for hydrophobization, followed by toluene printing to create several hydrophilic areas. As culture media we used a novel poly(vinyl alcohol based hydrogel and a standard calcium alginate hydrogel. The poly(vinyl alcohol hydrogel was formed by physical crosslinking poly(vinyl alcohol with adipic acid dihydrazide solutions. The conditions of poly(vinyl alcohol gelation were optimized for inkjet printability and the optimum mixture ratio was determined. The calcium alginate hydrogel was formed by chemical reaction between sodium alginate and CaCl2 solutions. Together with nutrients both hydrogel solutions were successfully printed on paper by means of the modified inkjet printer. The amount of each solution was demanded simply by outputting CMYK values. In the last step bacterial cells were printed on both hydrogel media. For both media we achieved a stable bacteria growth which was confirmed by microscopical imaging of the developed bacterial colonies.

  7. Dynamics of immune system gene expression upon bacterial challenge and wounding in a social insect (Bombus terrestris.

    Directory of Open Access Journals (Sweden)

    Silvio Erler

    2011-03-01

    Full Text Available The innate immune system which helps individuals to combat pathogens comprises a set of genes representing four immune system pathways (Toll, Imd, JNK and JAK/STAT. There is a lack of immune genes in social insects (e.g. honeybees when compared to Diptera. Potentially, this might be compensated by an advanced system of social immunity (synergistic action of several individuals. The bumble bee, Bombus terrestris, is a primitively eusocial species with an annual life cycle and colonies headed by a single queen. We used this key pollinator to study the temporal dynamics of immune system gene expression in response to wounding and bacterial challenge.Antimicrobial peptides (AMP (abaecin, defensin 1, hymenoptaecin were strongly up-regulated by wounding and bacterial challenge, the latter showing a higher impact on the gene expression level. Sterile wounding down-regulated TEP A, an effector gene of the JAK/STAT pathway, and bacterial infection influenced genes of the Imd (relish and JNK pathway (basket. Relish was up-regulated within the first hour after bacterial challenge, but decreased strongly afterwards. AMP expression following wounding and bacterial challenge correlates with the expression pattern of relish whereas correlated expression with dorsal was absent. Although expression of AMPs was high, continuous bacterial growth was observed throughout the experiment.Here we demonstrate for the first time the temporal dynamics of immune system gene expression in a social insect. Wounding and bacterial challenge affected the innate immune system significantly. Induction of AMP expression due to wounding might comprise a pre-adaptation to accompanying bacterial infections. Compared with solitary species this social insect exhibits reduced immune system efficiency, as bacterial growth could not be inhibited. A negative feedback loop regulating the Imd-pathway is suggested. AMPs, the end product of the Imd-pathway, inhibited the up-regulation of the

  8. Differentiation of Xanthomonas spp. Causing Bacterial Spot in Bulgaria Based on Biolog System

    Directory of Open Access Journals (Sweden)

    Mariya Stoyanova

    2014-01-01

    Full Text Available During the last 20 years, the causative agents of bacterial spot of tomato and pepper have been subjected to many studies and reclassifications. According to the current data, the species are four (X. euvesicatoria, X. vesicatoria, X. gardneri, and X. perforans and cause similar symptoms in plants but possess different phenotypic properties. This work provides the full metabolic characteristics obtained by Biolog system of bacterial spot’s xanthomonads based on a large selection of strains from different vegetable-producing regions of Bulgaria with accent on their major differentiating properties which could be used for species differentiation by metabolic profiles. The results are compared to the data available in the literature in order to clarify the strong features of each species and distinguish the variable ones. Simple characteristics like amylase activity and utilization of cis-aconitate cannot serve alone for differentiation.

  9. Modeling quorum sensing trade-offs between bacterial cell density and system extension from open boundaries

    Science.gov (United States)

    Marenda, Mattia; Zanardo, Marina; Trovato, Antonio; Seno, Flavio; Squartini, Andrea

    2016-12-01

    Bacterial communities undergo collective behavioural switches upon producing and sensing diffusible signal molecules; a mechanism referred to as Quorum Sensing (QS). Exemplarily, biofilm organic matrices are built concertedly by bacteria in several environments. QS scope in bacterial ecology has been debated for over 20 years. Different perspectives counterpose the role of density reporter for populations to that of local environment diffusivity probe for individual cells. Here we devise a model system where tubes of different heights contain matrix-embedded producers and sensors. These tubes allow non-limiting signal diffusion from one open end, thereby showing that population spatial extension away from an open boundary can be a main critical factor in QS. Experimental data, successfully recapitulated by a comprehensive mathematical model, demonstrate how tube height can overtake the role of producer density in triggering sensor activation. The biotic degradation of the signal is found to play a major role and to be species-specific and entirely feedback-independent.

  10. Characterization of the bacterial metagenome in an industrial algae bioenergy production system

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Shi [Chinese Academy of Sciences; Fulbright, Scott P [Colorado State University; Zeng, Xiaowei [Chinese Academy of Sciences; Yates, Tracy [Solix Biofuels; Wardle, Greg [Solix Biofuels; Chisholm, Stephen T [Colorado State University; Xu, Jian [Chinese Academy of Sciences; Lammers, Peter [New Mexico State University

    2011-03-16

    Cultivation of oleaginous microalgae for fuel generally requires growth of the intended species to the maximum extent supported by available light. The presence of undesired competitors, pathogens and grazers in cultivation systems will create competition for nitrate, phosphate, sulfate, iron and other micronutrients in the growth medium and potentially decrease microalgal triglyceride production by limiting microalgal health or cell density. Pathogenic bacteria may also directly impact the metabolism or survival of individual microalgal cells. Conversely, symbiotic bacteria that enhance microalgal growth may also be present in the system. Finally, the use of agricultural and municipal wastes as nutrient inputs for microalgal production systems may lead to the introduction and proliferation of human pathogens or interfere with the growth of bacteria with beneficial effects on system performance. These considerations underscore the need to understand bacterial community dynamics in microalgal production systems in order to assess microbiome effects on microalgal productivity and pathogen risks. Here we focus on the bacterial component of microalgal production systems and describe a pipeline for metagenomic characterization of bacterial diversity in industrial cultures of an oleaginous alga, Nannochloropsis salina. Environmental DNA was isolated from 12 marine algal cultures grown at Solix Biofuels, a region of the 16S rRNA gene was amplified by PCR, and 16S amplicons were sequenced using a 454 automated pyrosequencer. The approximately 70,000 sequences that passed quality control clustered into 53,950 unique sequences. The majority of sequences belonged to thirteen phyla. At the genus level, sequences from all samples represented 169 different genera. About 52.94% of all sequences could not be identified at the genus level and were classified at the next highest possible resolution level. Of all sequences, 79.92% corresponded to 169 genera and 70 other taxa. We

  11. Dynamics of highly polydisperse colloidal suspensions as a model system for bacterial cytoplasm.

    Science.gov (United States)

    Hwang, Jiye; Kim, Jeongmin; Sung, Bong June

    2016-08-01

    There are various kinds of macromolecules in bacterial cell cytoplasm. The size polydispersity of the macromolecules is so significant that the crystallization and the phase separation could be suppressed, thus stabilizing the liquid state of bacterial cytoplasm. On the other hand, recent experiments suggested that the macromolecules in bacterial cytoplasm should exhibit glassy dynamics, which should be also affected significantly by the size polydispersity of the macromolecules. In this work, we investigate the anomalous and slow dynamics of highly polydisperse colloidal suspensions, of which size distribution is chosen to mimic Escherichia coli cytoplasm. We find from our Langevin dynamics simulations that the diffusion coefficient (D_{tot}) and the displacement distribution functions (P(r,t)) averaged over all colloids of different sizes do not show anomalous and glassy dynamic behaviors until the system volume fraction ϕ is increased up to 0.82. This indicates that the intrinsic polydispersity of bacterial cytoplasm should suppress the glass transition and help maintain the liquid state of the cytoplasm. On the other hand, colloids of each kind show totally different dynamic behaviors depending on their size. The dynamics of colloids of different size becomes non-Gaussian at a different range of ϕ, which suggests that a multistep glass transition should occur. The largest colloids undergo the glass transition at ϕ=0.65, while the glass transition does not occur for smaller colloids in our simulations even at the highest value of ϕ. We also investigate the distribution (P(θ,t)) of the relative angles of displacement for macromolecules and find that macromolecules undergo directionally correlated motions in a sufficiently dense system.

  12. Vibrio vulnificus Type 6 Secretion System 1 Contains Anti-Bacterial Properties.

    Directory of Open Access Journals (Sweden)

    Selina R Church

    Full Text Available Vibrio vulnificus is a bacterium responsible for severe gastroenteritis, sepsis and wound infections. Gastroenteritis and sepsis are commonly associated with the consumption of raw oysters, whereas wound infection is often associated with the handling of contaminated fish. Although classical virulence factors of this emerging pathogen are well characterised, there remains a paucity of knowledge regarding the general biology of this species. To investigate the presence of previously unreported virulence factors, we applied whole genome sequencing to a panel of ten V. vulnificus strains with varying virulence potentials. This identified two novel type 6 secretion systems (T6SSs, systems that are known to have a role in bacterial virulence and population dynamics. By utilising a range of molecular techniques and assays we have demonstrated the functionality of one of these T6SSs. Furthermore, we have shown that this system is subject to thermoregulation and is negatively regulated by increasing salinity concentrations. This secretion system was also shown to be involved in the killing of V. vulnificus strains that did not possess this system and a model is proposed as to how this interaction may contribute to population dynamics within V. vulnificus strains. In addition to this intra-species killing, this system also contributes to the killing of inter bacterial species and may have a role in the general composition of Vibrio species in the environment.

  13. Encyclopedia of bacterial gene circuits whose presence or absence correlate with pathogenicity--a large-scale system analysis of decoded bacterial genomes.

    Science.gov (United States)

    Shestov, Maksim; Ontañón, Santiago; Tozeren, Aydin

    2015-10-13

    Bacterial infections comprise a global health challenge as the incidences of antibiotic resistance increase. Pathogenic potential of bacteria has been shown to be context dependent, varying in response to environment and even within the strains of the same genus. We used the KEGG repository and extensive literature searches to identify among the 2527 bacterial genomes in the literature those implicated as pathogenic to the host, including those which show pathogenicity in a context dependent manner. Using data on the gene contents of these genomes, we identified sets of genes highly abundant in pathogenic but relatively absent in commensal strains and vice versa. In addition, we carried out genome comparison within a genus for the seventeen largest genera in our genome collection. We projected the resultant lists of ortholog genes onto KEGG bacterial pathways to identify clusters and circuits, which can be linked to either pathogenicity or synergy. Gene circuits relatively abundant in nonpathogenic bacteria often mediated biosynthesis of antibiotics. Other synergy-linked circuits reduced drug-induced toxicity. Pathogen-abundant gene circuits included modules in one-carbon folate, two-component system, type-3 secretion system, and peptidoglycan biosynthesis. Antibiotics-resistant bacterial strains possessed genes modulating phagocytosis, vesicle trafficking, cytoskeletal reorganization, and regulation of the inflammatory response. Our study also identified bacterial genera containing a circuit, elements of which were previously linked to Alzheimer's disease. Present study produces for the first time, a signature, in the form of a robust list of gene circuitry whose presence or absence could potentially define the pathogenicity of a microbiome. Extensive literature search substantiated a bulk majority of the commensal and pathogenic circuitry in our predicted list. Scanning microbiome libraries for these circuitry motifs will provide further insights into the complex

  14. 1,2-Diacylglycerol choline phosphotransferase catalyzes the final step in the unique Treponema denticola phosphatidylcholine biosynthesis pathway.

    Science.gov (United States)

    Vences-Guzmán, Miguel Ángel; Paula Goetting-Minesky, M; Guan, Ziqiang; Castillo-Ramirez, Santiago; Córdoba-Castro, Luz América; López-Lara, Isabel M; Geiger, Otto; Sohlenkamp, Christian; Christopher Fenno, J

    2017-03-01

    Treponema denticola synthesizes phosphatidylcholine through a licCA-dependent CDP-choline pathway identified only in the genus Treponema. However, the mechanism of conversion of CDP-choline to phosphatidylcholine remained unclear. We report here characterization of TDE0021 (herein designated cpt) encoding a 1,2-diacylglycerol choline phosphotransferase homologous to choline phosphotransferases that catalyze the final step of the highly conserved Kennedy pathway for phosphatidylcholine synthesis in eukaryotes. T. denticola Cpt catalyzed in vitro phosphatidylcholine formation from CDP-choline and diacylglycerol, and full activity required divalent manganese. Allelic replacement mutagenesis of cpt in T. denticola resulted in abrogation of phosphatidylcholine synthesis. T. denticola Cpt complemented a Saccharomyces cerevisiae CPT1 mutant, and expression of the entire T. denticola LicCA-Cpt pathway in E. coli resulted in phosphatidylcholine biosynthesis. Our findings show that T. denticola possesses a unique phosphatidylcholine synthesis pathway combining conserved prokaryotic choline kinase and CTP:phosphocholine cytidylyltransferase activities with a 1,2-diacylglycerol choline phosphotransferase that is common in eukaryotes. Other than in a subset of mammalian host-associated Treponema that includes T. pallidum, this pathway is found in neither bacteria nor Archaea. Molecular dating analysis of the Cpt gene family suggests that a horizontal gene transfer event introduced this gene into an ancestral Treponema well after its divergence from other spirochetes. © 2016 John Wiley & Sons Ltd.

  15. Recent trends of modern bacterial insecticides for pest control practice in integrated crop management system.

    Science.gov (United States)

    Chattopadhyay, Pritam; Banerjee, Goutam; Mukherjee, Sayantan

    2017-05-01

    Food security and safety are the major concern in ever expanding human population on the planet earth. Each and every year insect pests cause a serious damage in agricultural field that cost billions of dollars annually to farmers. The loss in term of productivity and high cost of chemical pesticides enhance the production cost. Irrespective use of chemical pesticides (such as Benzene hexachloride, Endosulfan, Aldicarb, and Fenobucarb) in agricultural field raised several types of environmental issues. Furthermore, continuous use of chemical pesticides creates a selective pressure which helps in emerging of resistance pest. These excess chemical pesticide residues also contaminate the environment including the soil and water. Therefore, the biological control of insect pest in the agricultural field gains more importance due to food safety and environment friendly nature. In this regard, bacterial insecticides offer better alternative to chemical pesticides. It not only helps to establish food security through fighting against insect pests but also ensure the food safety. In this review, we have categorized insect pests and the corresponding bacterial insecticides, and critically analyzed the importance and mode of action of bacterial pesticides. We also have summarized the use of biopesticides in integrated pest management system. We have tried to focus the future research area in this field for the upcoming scientists.

  16. Responses of Bacterial Communities to CuO Nanoparticles in Activated Sludge System.

    Science.gov (United States)

    Wang, Xiaohui; Li, Jing; Liu, Rui; Hai, Reti; Zou, Dexun; Zhu, Xiaobiao; Luo, Nan

    2017-05-16

    The main objectives of this study were to investigate the influence of copper oxide nanoparticles (CuO NPs) on wastewater nutrient removal, bacterial community and molecular ecological network in activated sludge. The results showed that long-term exposure to 1 mg/L CuO NPs induced an increase of effluent concentrations of ammonia and total phosphorus, which was consistent with the inhibition of enzyme activities of ammonia monooxygenase, nitrite oxidoreductase, exopolyphosphatase, and polyphosphate in the presence of CuO NPs. MiSeq sequencing data indicated that CuO NPs significantly decreased the bacterial diversity and altered the overall bacterial community structure in activated sludge. Some genera involved in nitrogen and phosphorus removal, such as Nitrosomonas, Acinetobacter, and Pseudomonas decreased significantly. Molecular ecological network analysis showed that network interactions among different phylogenetic populations were markedly changed by CuO NPs. For example, β-Proteobacteria, playing an important role in nutrients removal, had less complex interactions in the presence of CuO NPs. These shifts of the abundance of related genera, together with the network interactions may be associated with the deterioration of ammonia and phosphorus removal. This study provides insights into our understanding of shifts in the bacteria community and their molecular ecological network under CuO NPs in activated sludge systems.

  17. Peptidomimetic Small Molecules Disrupt Type IV Secretion System Activity in Diverse Bacterial Pathogens

    Directory of Open Access Journals (Sweden)

    Carrie L. Shaffer

    2016-04-01

    Full Text Available Bacteria utilize complex type IV secretion systems (T4SSs to translocate diverse effector proteins or DNA into target cells. Despite the importance of T4SSs in bacterial pathogenesis, the mechanism by which these translocation machineries deliver cargo across the bacterial envelope remains poorly understood, and very few studies have investigated the use of synthetic molecules to disrupt T4SS-mediated transport. Here, we describe two synthetic small molecules (C10 and KSK85 that disrupt T4SS-dependent processes in multiple bacterial pathogens. Helicobacter pylori exploits a pilus appendage associated with the cag T4SS to inject an oncogenic effector protein (CagA and peptidoglycan into gastric epithelial cells. In H. pylori, KSK85 impedes biogenesis of the pilus appendage associated with the cag T4SS, while C10 disrupts cag T4SS activity without perturbing pilus assembly. In addition to the effects in H. pylori, we demonstrate that these compounds disrupt interbacterial DNA transfer by conjugative T4SSs in Escherichia coli and impede vir T4SS-mediated DNA delivery by Agrobacterium tumefaciens in a plant model of infection. Of note, C10 effectively disarmed dissemination of a derepressed IncF plasmid into a recipient bacterial population, thus demonstrating the potential of these compounds in mitigating the spread of antibiotic resistance determinants driven by conjugation. To our knowledge, this study is the first report of synthetic small molecules that impair delivery of both effector protein and DNA cargos by diverse T4SSs.

  18. Innate immune system favors emergency monopoiesis at the expense of DC-differentiation to control systemic bacterial infection in mice.

    Science.gov (United States)

    Pasquevich, Karina A; Bieber, Kristin; Günter, Manina; Grauer, Matthias; Pötz, Oliver; Schleicher, Ulrike; Biedermann, Tilo; Beer-Hammer, Sandra; Bühring, Hans-Jörg; Rammensee, Hans-Georg; Zender, Lars; Autenrieth, Ingo B; Lengerke, Claudia; Autenrieth, Stella E

    2015-10-01

    DCs are professional APCs playing a crucial role in the initiation of T-cell responses to combat infection. However, systemic bacterial infection with various pathogens leads to DC-depletion in humans and mice. The mechanisms of pathogen-induced DC-depletion remain poorly understood. Previously, we showed that mice infected with Yersinia enterocolitica (Ye) had impaired de novo DC-development, one reason for DC-depletion. Here, we extend these studies to gain insight into the molecular mechanisms of DC-depletion and the impact of different bacteria on DC-development. We show that the number of bone marrow (BM) hematopoietic progenitors committed to the DC lineage is reduced following systemic infection with different Gram-positive and Gram-negative bacteria. This is associated with a TLR4- and IFN-γ-signaling dependent increase of committed monocyte progenitors in the BM and mature monocytes in the spleen upon Ye-infection. Adoptive transfer experiments revealed that infection-induced monopoiesis occurs at the expense of DC-development. Our data provide evidence for a general response of hematopoietic progenitors upon systemic bacterial infections to enhance monocyte production, thereby increasing the availability of innate immune cells for pathogen control, whereas impaired DC-development leads to DC-depletion, possibly driving transient immunosuppression in bacterial sepsis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Comparative study of two ATP : L-arginine phosphotransferases of molecular weight 84 000.

    Science.gov (United States)

    Thiem, N V; Lacombe, G; Thoai, N V

    1975-01-23

    Solen ensisensis muscle arginine kinase (ATP : L-arginine phosphotransferase, EC 2.7.3.3) was isolated in an homogeneous state. Its molecular weight was found to be about 80 000. The properties of this enzyme were compared with those of arginine kinase from Sipunculus nudus, an enzyme which also has a molecular weight of about 80 000. Both enzymes have several reactive thiol groups (8 thiol groups in the Solen kinase and 12 in the Sipunculus enzyme were titrateable with 5,5'-dithio-bis-(2-nitrobenzoic) acid and histidine residues (both enzymes have 6 reactive histidine residues). These kinases were, therefore, highly susceptible to oxidation. Both enzymes show the same pH optimum and absolute specificity towards the guanidine substrate, L-arginine. The reaction kinetics of both enzymes are of the sequential type. In the presence of alpha-aminoacids of Mg2+-ADP, similar spectral effects were obtained. The enzymes differ in their enzymic activities and in their rate of recovery following urea denaturation. The most important difference that appeared to be a special feature of the Sipunculus enzyme is that the spectrum of the Mg2+-ADP-enzyme complex is strongly intensified by L-arginine.

  20. [Studies on the repair of damaged DNA in bacteriophage, bacterial and mammalian systems]: Final report

    International Nuclear Information System (INIS)

    Friedberg, E.C.

    1987-08-01

    This study sought to exploit the use of uv radiation as a source of genomic damage. We explored the molecular mechanism of the repair of DNA damage at a number of different levels of biological organization, by investigating bacteriophage, bacterial, yeast and mammalian cells. Not only have observations obtained in one biological system suggested specific experimental approaches in others, but we have also learned that some biochemical pathways for DNA repair are unique to specific organisms. Our studies are summarized in terms of 4 major areas of research activity that span the past 16 years. 86 refs

  1. Evidence for Bacterial Sulfate Reduction in a Fissured-porous Karst System in Southern Germany

    Science.gov (United States)

    Einsiedl, F.; Mayer, B.

    2005-12-01

    Twenty five percent of the world's population uses karst water as drinking water resources. Since karst groundwater systems are highly vulnerable to contamination, groundwater protection and self purification is a major challenge. Up to now research in karst groundwater systems has predominantly concentrated on hydrodynamic processes. Little is known about anoxic processes in oxygen dominated, fracture-matrix diffusion controlled karst aquifers. Isotope measurements comprise a promising tool to identify biogeochemical processes such as bacterial (dissimilatory) sulfate reduction in karstic aquifers. The goal of this study was to determine the sources and the processes affecting sulfate in an oxygen-rich karst aquifer in southern Germany and their dependence on hydrogeological parameters. This was achieved by interpreting tritium data with a simple lumped parameter approach and assessing variations in concentrations and isotopic compositions of sulfate and dissolved inorganic carbon (DIC) with respect to groundwater age. Young groundwater (characterized by comparatively high sulfate concentrations (0.36 mM) and δ34S values similar to those of recent atmospheric deposition (1.5‰). In contrast groundwater with mean residence times >60 years had significantly lower sulfate concentrations (0.08 mM) and markedly higher δ34S values (7.5‰). These results indicate that in karst systems with matrix porosity, bacterial (dissimilatory) sulfate reduction may occur. This process has the potential to contribute to long-term biodegradation of contaminants in the porous rock matrix representing the dominant water reservoir in fissured-porous karst aquifers.

  2. A quorum sensing-based in vivo expression system and its application in multivalent bacterial vaccine.

    Science.gov (United States)

    Chu, Teng; Ni, Chunshan; Zhang, Lingzhi; Wang, Qiyao; Xiao, Jingfan; Zhang, Yuanxing; Liu, Qin

    2015-03-18

    Delivery of antigens by live bacterial carriers can elicit effective humoral and cellular responses and may be an attractive strategy for live bacterial vaccine production through introduction of a vector that expresses an exogenous protective antigen. To overcome the instability and metabolic burden associated with plasmid introduction, alternative strategies, such as the use of in vivo-inducible promoters, have been proposed. However, screening an ideal in vivo-activated promoter with high efficiency and low leak expression in a particular strain poses great challenges to many researchers. In this work, we constructed an in vivo antigen-expressing vector suitable for Edwardsiella tarda, an enteric Gram-negative invasive intracellular pathogen of both animals and humans. By combining quorum sensing genes from Vibrio fischeri with iron uptake regulons, a synthetic binary regulation system (ironQS) for E. tarda was designed. In vitro expression assay demonstrated that the ironQS system is only initiated in the absence of Fe2+ in the medium when the cell density reaches its threshold. The ironQS system was further confirmed in vivo to present an in vivo-triggered and cell density-dependent expression pattern in larvae and adult zebrafish. A recombinant E. tarda vector vaccine candidate WED(ironQS-G) was established by introducing gapA34, which encodes the protective antigen glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the fish pathogen Aeromonas hydrophila LSA34 into ironQS system, and the immune protection afforded by this vaccine was assessed in turbot (Scophtalmus maximus). Most of the vaccinated fish survived under the challenge with A. hydrophila LSA34 (RPS=67.0%) or E. tarda EIB202 (RPS=72.3%). Quorum sensing system has been extensively used in various gene structures in synthetic biology as a well-functioning and population-dependent gene circuit. In this work, the in vivo expression system, ironQS, maintained the high expression efficiency of the

  3. Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions.

    Directory of Open Access Journals (Sweden)

    Sandra Schwarz

    2010-08-01

    Full Text Available Bacteria that live in the environment have evolved pathways specialized to defend against eukaryotic organisms or other bacteria. In this manuscript, we systematically examined the role of the five type VI secretion systems (T6SSs of Burkholderia thailandensis (B. thai in eukaryotic and bacterial cell interactions. Consistent with phylogenetic analyses comparing the distribution of the B. thai T6SSs with well-characterized bacterial and eukaryotic cell-targeting T6SSs, we found that T6SS-5 plays a critical role in the virulence of the organism in a murine melioidosis model, while a strain lacking the other four T6SSs remained as virulent as the wild-type. The function of T6SS-5 appeared to be specialized to the host and not related to an in vivo growth defect, as ΔT6SS-5 was fully virulent in mice lacking MyD88. Next we probed the role of the five systems in interbacterial interactions. From a group of 31 diverse bacteria, we identified several organisms that competed less effectively against wild-type B. thai than a strain lacking T6SS-1 function. Inactivation of T6SS-1 renders B. thai greatly more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas fluorescens and Serratia proteamaculans-leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly displaced in mixed biofilms with P. putida, whereas wild-type cells persisted and overran the competitor. Our data show that T6SSs within a single organism can have distinct functions in eukaryotic versus bacterial cell interactions. These systems are likely to be a decisive factor in the survival of bacterial cells of one species in intimate association with those of another, such as in polymicrobial communities present both in the environment and in many infections.

  4. Bacterial Associations with Diatoms Influence Host Health in a Xenic Model System

    Science.gov (United States)

    Baker, L.; Kemp, P. F.

    2016-02-01

    Diatoms are photosynthetic unicellular eukaryotes found ubiquitously in aquatic systems. Microorganisms such as bacteria are frequently found attached to diatoms and may influence the fitness of their host. The most commonly used model organisms in studies of diatom-bacterial associations are Alteromonas and Marinobacter. Some strains of Alteromonas are capable of parasitism, producing chitinases or having algicidal interactions; some strains of Marinobacter are capable of mutualism, providing its host with vital nutrients. In this study, multiple strains of Alteromonas and Marinobacter were isolated from the centric diatom Chaetoceros sp KBDT20. Isolates were added back in varying concentration to cultures of their original xenic diatom host, and to cultures of a smaller, xenic naïve host, Chaetoceros sp. KBDT32. The growth rate of the diatom host was monitored using flow cytometry to assess the impact of the added bacterial isolates on host health. Our results suggest that all strains of Alteromonas tested have an antagonistic relationship with both the original as well as the naïve host while all strains of Marinobacter tested have a synergistic relationship with both diatom cultures. The functional basis for these relationships is being explored by supplementing xenic diatom cultures with materials essential for diatom growth that may be contributed by bacteria, such as B-vitamins and bioavailable trace metals. The colonization rates and competitive interactions between bacteria are investigated through surface colonization studies. The goal of this study is to better inform our understanding of how bacterial associates of diatom populations may contribute to their health, success, or failure in aquatic systems.

  5. Strain ŽP - the first bacterial conjugation-based "kill"-"anti-kill" antimicrobial system.

    Science.gov (United States)

    Starčič Erjavec, Marjanca; Petkovšek, Živa; Kuznetsova, Marina V; Maslennikova, Irina L; Žgur-Bertok, Darja

    2015-11-01

    As multidrug resistant bacteria pose one of the greatest risks to human health new alternative antibacterial agents are urgently needed. One possible mechanism that can be used as an alternative to traditional antibiotic therapy is transfer of killing agents via conjugation. Our work was aimed at providing a proof of principle that conjugation-based antimicrobial systems are possible. We constructed a bacterial conjugation-based "kill"-"anti-kill" antimicrobial system employing the well known Escherichia coli probiotic strain Nissle 1917 genetically modified to harbor a conjugative plasmid carrying the "kill" gene (colicin ColE7 activity gene) and a chromosomally encoded "anti-kill" gene (ColE7 immunity gene). The constructed strain acts as a donor in conjugal transfer and its efficiency was tested in several types of conjugal assays. Our results clearly demonstrate that conjugation-based antimicrobial systems can be highly efficient. Copyright © 2015. Published by Elsevier Inc.

  6. Novel detection system for biomolecules using nano-sized bacterial magnetic particles and magnetic force microscopy.

    Science.gov (United States)

    Amemiya, Yosuke; Tanaka, Tsuyoshi; Yoza, Brandon; Matsunaga, Tadashi

    2005-11-21

    A system for streptavidin detection using biotin conjugated to nano-sized bacterial magnetic particles (BMPs) has been developed. BMPs, isolated from magnetic bacteria, were used as magnetic markers for magnetic force microscopy (MFM) imaging. The magnetic signal was obtained from a single particle using MFM without application of an external magnetic field. The number of biotin conjugated BMPs (biotin-BMPs) bound to streptavidin immobilized on the glass slides increased with streptavidin concentrations up to 100 pg/ml. The minimum streptavidin detection limit using this technique is 1 pg/ml, which is 100 times more sensitive than a conventional fluorescent detection system. This is the first report using single domain nano-sized magnetic particles as magnetic markers for biosensing. This assay system can be used for immunoassay and DNA detection with high sensitivities.

  7. Defense Islands in Bacterial and Archaeal Genomes and Prediction of Novel Defense Systems ▿†‡

    OpenAIRE

    Makarova, Kira S.; Wolf, Yuri I.; Snir, Sagi; Koonin, Eugene V.

    2011-01-01

    The arms race between cellular life forms and viruses is a major driving force of evolution. A substantial fraction of bacterial and archaeal genomes is dedicated to antivirus defense. We analyzed the distribution of defense genes and typical mobilome components (such as viral and transposon genes) in bacterial and archaeal genomes and demonstrated statistically significant clustering of antivirus defense systems and mobile genes and elements in genomic islands. The defense islands are enrich...

  8. Molecular Epidemiologic Typing Systems of Bacterial Pathogens: Current Issues and Perpectives

    Directory of Open Access Journals (Sweden)

    Struelens Marc J

    1998-01-01

    Full Text Available The epidemiologic typing of bacterial pathogens can be applied to answer a number of different questions: in case of outbreak, what is the extent and mode of transmission of epidemic clone(s ? In case of long-term surveillance, what is the prevalence over time and the geographic spread of epidemic and endemic clones in the population? A number of molecular typing methods can be used to classify bacteria based on genomic diversity into groups of closely-related isolates (presumed to arise from a common ancestor in the same chain of transmission and divergent, epidemiologically-unrelated isolates (arising from independent sources of infection. Ribotyping, IS-RFLP fingerprinting, macrorestriction analysis of chromosomal DNA and PCR-fingerprinting using arbitrary sequence or repeat element primers are useful methods for outbreak investigations and regional surveillance. Library typing systems based on multilocus sequence-based analysis and strain-specific probe hybridization schemes are in development for the international surveillance of major pathogens like Mycobacterium tuberculosis. Accurate epidemiological interpretation of data obtained with molecular typing systems still requires additional research on the evolution rate of polymorphic loci in bacterial pathogens.

  9. Characterization of CRISPR-Cas system in clinical Staphylococcus epidermidis strains revealed its potential association with bacterial infection sites

    DEFF Research Database (Denmark)

    Li, Qiuchun; Xie, Xiaolei; Yin, Kequan

    2016-01-01

    Staphylococcus epidermidis is considered as a major cause of nosocomial infections, bringing an immense burden to healthcare systems. Virulent phages have been confirmed to be efficient in combating the pathogen, but the prensence of CRISPR-Cas system, which is a bacterial immune system eliminating...

  10. Long-Term Bacterial Dynamics in a Full-Scale Drinking Water Distribution System

    Science.gov (United States)

    Prest, E. I.; Weissbrodt, D. G.; Hammes, F.; van Loosdrecht, M. C. M.; Vrouwenvelder, J. S.

    2016-01-01

    Large seasonal variations in microbial drinking water quality can occur in distribution networks, but are often not taken into account when evaluating results from short-term water sampling campaigns. Temporal dynamics in bacterial community characteristics were investigated during a two-year drinking water monitoring campaign in a full-scale distribution system operating without detectable disinfectant residual. A total of 368 water samples were collected on a biweekly basis at the water treatment plant (WTP) effluent and at one fixed location in the drinking water distribution network (NET). The samples were analysed for heterotrophic plate counts (HPC), Aeromonas plate counts, adenosine-tri-phosphate (ATP) concentrations, and flow cytometric (FCM) total and intact cell counts (TCC, ICC), water temperature, pH, conductivity, total organic carbon (TOC) and assimilable organic carbon (AOC). Multivariate analysis of the large dataset was performed to explore correlative trends between microbial and environmental parameters. The WTP effluent displayed considerable seasonal variations in TCC (from 90 × 103 cells mL-1 in winter time up to 455 × 103 cells mL-1 in summer time) and in bacterial ATP concentrations (water temperature variations. These fluctuations were not detected with HPC and Aeromonas counts. The water in the network was predominantly influenced by the characteristics of the WTP effluent. The increase in ICC between the WTP effluent and the network sampling location was small (34 × 103 cells mL-1 on average) compared to seasonal fluctuations in ICC in the WTP effluent. Interestingly, the extent of bacterial growth in the NET was inversely correlated to AOC concentrations in the WTP effluent (Pearson’s correlation factor r = -0.35), and positively correlated with water temperature (r = 0.49). Collecting a large dataset at high frequency over a two year period enabled the characterization of previously undocumented seasonal dynamics in the

  11. Long-Term Bacterial Dynamics in a Full-Scale Drinking Water Distribution System

    KAUST Repository

    Prest, E. I.

    2016-10-28

    Large seasonal variations in microbial drinking water quality can occur in distribution networks, but are often not taken into account when evaluating results from short-term water sampling campaigns. Temporal dynamics in bacterial community characteristics were investigated during a two-year drinking water monitoring campaign in a full-scale distribution system operating without detectable disinfectant residual. A total of 368 water samples were collected on a biweekly basis at the water treatment plant (WTP) effluent and at one fixed location in the drinking water distribution network (NET). The samples were analysed for heterotrophic plate counts (HPC), Aeromonas plate counts, adenosine-tri-phosphate (ATP) concentrations, and flow cytometric (FCM) total and intact cell counts (TCC, ICC), water temperature, pH, conductivity, total organic carbon (TOC) and assimilable organic carbon (AOC). Multivariate analysis of the large dataset was performed to explore correlative trends between microbial and environmental parameters. The WTP effluent displayed considerable seasonal variations in TCC (from 90 × 103 cells mL-1 in winter time up to 455 × 103 cells mL-1 in summer time) and in bacterial ATP concentrations (<1–3.6 ng L-1), which were congruent with water temperature variations. These fluctuations were not detected with HPC and Aeromonas counts. The water in the network was predominantly influenced by the characteristics of the WTP effluent. The increase in ICC between the WTP effluent and the network sampling location was small (34 × 103 cells mL-1 on average) compared to seasonal fluctuations in ICC in the WTP effluent. Interestingly, the extent of bacterial growth in the NET was inversely correlated to AOC concentrations in the WTP effluent (Pearson’s correlation factor r = -0.35), and positively correlated with water temperature (r = 0.49). Collecting a large dataset at high frequency over a two year period enabled the characterization of previously

  12. Long-Term Bacterial Dynamics in a Full-Scale Drinking Water Distribution System.

    Science.gov (United States)

    Prest, E I; Weissbrodt, D G; Hammes, F; van Loosdrecht, M C M; Vrouwenvelder, J S

    2016-01-01

    Large seasonal variations in microbial drinking water quality can occur in distribution networks, but are often not taken into account when evaluating results from short-term water sampling campaigns. Temporal dynamics in bacterial community characteristics were investigated during a two-year drinking water monitoring campaign in a full-scale distribution system operating without detectable disinfectant residual. A total of 368 water samples were collected on a biweekly basis at the water treatment plant (WTP) effluent and at one fixed location in the drinking water distribution network (NET). The samples were analysed for heterotrophic plate counts (HPC), Aeromonas plate counts, adenosine-tri-phosphate (ATP) concentrations, and flow cytometric (FCM) total and intact cell counts (TCC, ICC), water temperature, pH, conductivity, total organic carbon (TOC) and assimilable organic carbon (AOC). Multivariate analysis of the large dataset was performed to explore correlative trends between microbial and environmental parameters. The WTP effluent displayed considerable seasonal variations in TCC (from 90 × 103 cells mL-1 in winter time up to 455 × 103 cells mL-1 in summer time) and in bacterial ATP concentrations (water temperature variations. These fluctuations were not detected with HPC and Aeromonas counts. The water in the network was predominantly influenced by the characteristics of the WTP effluent. The increase in ICC between the WTP effluent and the network sampling location was small (34 × 103 cells mL-1 on average) compared to seasonal fluctuations in ICC in the WTP effluent. Interestingly, the extent of bacterial growth in the NET was inversely correlated to AOC concentrations in the WTP effluent (Pearson's correlation factor r = -0.35), and positively correlated with water temperature (r = 0.49). Collecting a large dataset at high frequency over a two year period enabled the characterization of previously undocumented seasonal dynamics in the distribution

  13. Long-Term Bacterial Dynamics in a Full-Scale Drinking Water Distribution System.

    Directory of Open Access Journals (Sweden)

    E I Prest

    Full Text Available Large seasonal variations in microbial drinking water quality can occur in distribution networks, but are often not taken into account when evaluating results from short-term water sampling campaigns. Temporal dynamics in bacterial community characteristics were investigated during a two-year drinking water monitoring campaign in a full-scale distribution system operating without detectable disinfectant residual. A total of 368 water samples were collected on a biweekly basis at the water treatment plant (WTP effluent and at one fixed location in the drinking water distribution network (NET. The samples were analysed for heterotrophic plate counts (HPC, Aeromonas plate counts, adenosine-tri-phosphate (ATP concentrations, and flow cytometric (FCM total and intact cell counts (TCC, ICC, water temperature, pH, conductivity, total organic carbon (TOC and assimilable organic carbon (AOC. Multivariate analysis of the large dataset was performed to explore correlative trends between microbial and environmental parameters. The WTP effluent displayed considerable seasonal variations in TCC (from 90 × 103 cells mL-1 in winter time up to 455 × 103 cells mL-1 in summer time and in bacterial ATP concentrations (<1-3.6 ng L-1, which were congruent with water temperature variations. These fluctuations were not detected with HPC and Aeromonas counts. The water in the network was predominantly influenced by the characteristics of the WTP effluent. The increase in ICC between the WTP effluent and the network sampling location was small (34 × 103 cells mL-1 on average compared to seasonal fluctuations in ICC in the WTP effluent. Interestingly, the extent of bacterial growth in the NET was inversely correlated to AOC concentrations in the WTP effluent (Pearson's correlation factor r = -0.35, and positively correlated with water temperature (r = 0.49. Collecting a large dataset at high frequency over a two year period enabled the characterization of previously

  14. The DinJ/RelE Toxin-Antitoxin System Suppresses Bacterial Proliferation and Virulence of Xylella fastidiosa in Grapevine.

    Science.gov (United States)

    Burbank, Lindsey P; Stenger, Drake C

    2017-04-01

    Xylella fastidiosa, the causal agent of Pierce's disease of grapes, is a slow-growing, xylem-limited, bacterial pathogen. Disease progression is characterized by systemic spread of the bacterium through xylem vessel networks, causing leaf-scorching symptoms, senescence, and vine decline. It appears to be advantageous to this pathogen to avoid excessive blockage of xylem vessels, because living bacterial cells are generally found in plant tissue with low bacterial cell density and minimal scorching symptoms. The DinJ/RelE toxin-antitoxin system is characterized here for a role in controlling bacterial proliferation and population size during plant colonization. The DinJ/RelE locus is transcribed from two separate promoters, allowing for coexpression of antitoxin DinJ with endoribonuclease toxin RelE, in addition to independent expression of RelE. The ratio of antitoxin/toxin expressed is dependent on bacterial growth conditions, with lower amounts of antitoxin present under conditions designed to mimic grapevine xylem sap. A knockout mutant of DinJ/RelE exhibits a hypervirulent phenotype, with higher bacterial populations and increased symptom development and plant decline. It is likely that DinJ/RelE acts to prevent excessive population growth, contributing to the ability of the pathogen to spread systemically without completely blocking the xylem vessels and increasing probability of acquisition by the insect vector.

  15. The phosphotransferase VanU represses expression of four qrr genes antagonizing VanO-mediated quorum-sensing regulation in Vibrio anguillarum.

    Science.gov (United States)

    Weber, Barbara; Lindell, Kristoffer; El Qaidi, Samir; Hjerde, Erik; Willassen, Nils-Peder; Milton, Debra L

    2011-12-01

    Vibrio anguillarum utilizes quorum sensing to regulate stress responses required for survival in the aquatic environment. Like other Vibrio species, V. anguillarum contains the gene qrr1, which encodes the ancestral quorum regulatory RNA Qrr1, and phosphorelay quorum-sensing systems that modulate the expression of small regulatory RNAs (sRNAs) that destabilize mRNA encoding the transcriptional regulator VanT. In this study, three additional Qrr sRNAs were identified. All four sRNAs were positively regulated by σ(54) and the σ(54)-dependent response regulator VanO, and showed a redundant activity. The Qrr sRNAs, together with the RNA chaperone Hfq, destabilized vanT mRNA and modulated expression of VanT-regulated genes. Unexpectedly, expression of all four qrr genes peaked at high cell density, and exogenously added N-acylhomoserine lactone molecules induced expression of the qrr genes at low cell density. The phosphotransferase VanU, which phosphorylates and activates VanO, repressed expression of the Qrr sRNAs and stabilized vanT mRNA. A model is presented proposing that VanU acts as a branch point, aiding cross-regulation between two independent phosphorelay systems that activate or repress expression of the Qrr sRNAs, giving flexibility and precision in modulating VanT expression and inducing a quorum-sensing response to stresses found in a constantly changing aquatic environment.

  16. Systems biology of bacterial persistence, a metabolism-driven strategy for survival

    NARCIS (Netherlands)

    Radzikowski, Jakub

    2017-01-01

    Bacteriële persisten zijn bacteriën die antibiotica tolereren en kunnen opnieuw vermenigvuldigen na antibiotische behandeling. Ze veroorzaken infecties, bijvoorbeeld tuberculose. Ze zijn verschillend van antibiotica resistente bacteriën, omdat het mechanisme van antibiotische tolerantie niet

  17. A slow-release system of bacterial cellulose gel and nanoparticles for hydrophobic active ingredients.

    Science.gov (United States)

    Numata, Yukari; Mazzarino, Leticia; Borsali, Redouane

    2015-01-01

    A combination of bacterial cellulose (BC) gel and amphiphilic block copolymer nanoparticles was investigated as a drug delivery system (DDS) for hydrophobic active ingredients. Poly(ethylene oxide)-b-poly(caprolactone) (PEO-b-PCL) and retinol were used as the block copolymer and hydrophobic active ingredient, respectively. The BC gel was capable of incorporating copolymer nanoparticles and releasing them in an acetic acid-sodium acetate buffer solution (pH 5.2) at 37 °C. The percentage of released copolymer reached a maximum value of approximately 60% after 6h and remained constant after 24h. The percentage of retinol released from the copolymer-containing BC gel reached a maximum value at 4h. These results show that the combination of BC gel and nanoparticles is a slow-release system that may be useful in the cosmetic and biomedical fields for skin treatment and preparation. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Vanadate and phosphotransferases with special emphasis on ouabain/Na, K-ATPase interaction

    International Nuclear Information System (INIS)

    Hansen, O.

    1983-01-01

    A short introduction to the chemistry and possible physiological or toxicological role of vanadium is given. The +5 oxidation state, vanadate, is a very efficient inhibitor of several phosphotransferases and for that reason a prosperous tool in the study of such enzymes. Special emphasis is placed on studies with vanadate on the sodium pump. Vanadate appears to supplement ouabain as high affinity inhibitor of Na,K-ATPase. They are also complementary to one another since vanadate binds to the cytoplasmic aspect and ouabain to the extracellular side of the cell membrane, and moreover, they potentiate the binding of one another. The hypothesis that vanadate may act as a transition state analogue of phosphate seems supported by the observation that vanadate, like phosphate, is able to induce ouabain binding to Na,K-ATPase. The vanadate affinity is much higher than that of phosphate, however, and vanadate remains bound in a rather stable enzyme-vanadate-ouabain complex. Fluorescene studies indicate that different subspecies of an E 2 -conformation of the enzyme is obtained with vanadate and with ouabain. In molecular studies on Na,K-ATPase, e.g. in studies on the protein folding through the plasma-membrane, one can imagine that the simultaneous binding of an extracellular marker, ouabain, and of the intracellular marker, vanadate, may be most helpful tools. A proposed physiological regulatory role of vanadium on the pump activity seems less likely considering the simultaneous acceleration of K + -uptake which is probably due to adenylate cyclase activation. Vanadate seems more likely to have a pharmacological role as a cofactor for digitals binding to Na,K-ATPase. Finally, the vasoconstriction evoked by vanadate could indicate a pathophysiological role of the ion and vanadate could then become useful tool in experimental hypertension and uremia. (author)

  19. Lactose metabolism in Streptococcus lactis: studies with a mutant lacking glucokinase and mannose-phosphotransferase activities

    International Nuclear Information System (INIS)

    Thompson, J.; Chassy, B.M.; Egan, W.

    1985-01-01

    A mutant of Streptococcus lactis 133 has been isolated that lacks both glucokinase and phosphoenolpyruvate-dependent mannose- phosphotransferase (mannose-PTS) activities. The double mutant S. lactis 133 mannose-PTSd GK- is unable to utilize either exogenously supplied or intracellularly generated glucose for growth. Fluorographic analyses of metabolites formed during the metabolism of [ 14 C]lactose labeled specifically in the glucose or galactosyl moiety established that the cells were unable to phosphorylate intracellular glucose. However, cells of S. lactis 133 mannose-PTSd GK- readily metabolized intracellular glucose 6-phosphate, and the growth rates and cell yield of the mutant and parental strains on sucrose were the same. During growth on lactose, S. lactis 133 mannose-PTSd GK- fermented only the galactose moiety of the disaccharide, and 1 mol of glucose was generated per mol of lactose consumed. For an equivalent concentration of lactose, the cell yield of the mutant was 50% that of the wild type. The specific rate of lactose utilization by growing cells of S. lactis 133 mannose-PTSd GK- was ca. 50% greater than that of the wild type, but the cell doubling times were 70 and 47 min, respectively. High-resolution 31 P nuclear magnetic resonance studies of lactose transport by starved cells of S. lactis 133 and S. lactis 133 mannose-PTSd GK- showed that the latter cells contained elevated lactose-PTS activity. Throughout exponential growth on lactose, the mutant maintained an intracellular steady-state glucose concentration of 100 mM

  20. Lactose metabolism in Streptococcus lactis: studies with a mutant lacking glucokinase and mannose-phosphotransferase activities

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, J.; Chassy, B.M.; Egan, W.

    1985-04-01

    A mutant of Streptococcus lactis 133 has been isolated that lacks both glucokinase and phosphoenolpyruvate-dependent mannose- phosphotransferase (mannose-PTS) activities. The double mutant S. lactis 133 mannose-PTSd GK- is unable to utilize either exogenously supplied or intracellularly generated glucose for growth. Fluorographic analyses of metabolites formed during the metabolism of (/sup 14/C)lactose labeled specifically in the glucose or galactosyl moiety established that the cells were unable to phosphorylate intracellular glucose. However, cells of S. lactis 133 mannose-PTSd GK- readily metabolized intracellular glucose 6-phosphate, and the growth rates and cell yield of the mutant and parental strains on sucrose were the same. During growth on lactose, S. lactis 133 mannose-PTSd GK- fermented only the galactose moiety of the disaccharide, and 1 mol of glucose was generated per mol of lactose consumed. For an equivalent concentration of lactose, the cell yield of the mutant was 50% that of the wild type. The specific rate of lactose utilization by growing cells of S. lactis 133 mannose-PTSd GK- was ca. 50% greater than that of the wild type, but the cell doubling times were 70 and 47 min, respectively. High-resolution /sup 31/P nuclear magnetic resonance studies of lactose transport by starved cells of S. lactis 133 and S. lactis 133 mannose-PTSd GK- showed that the latter cells contained elevated lactose-PTS activity. Throughout exponential growth on lactose, the mutant maintained an intracellular steady-state glucose concentration of 100 mM.

  1. Structure of a bacterial type III secretion system in contact with a host membrane in situ

    Science.gov (United States)

    Nans, Andrea; Kudryashev, Mikhail; Saibil, Helen R.; Hayward, Richard D.

    2015-12-01

    Many bacterial pathogens of animals and plants use a conserved type III secretion system (T3SS) to inject virulence effector proteins directly into eukaryotic cells to subvert host functions. Contact with host membranes is critical for T3SS activation, yet little is known about T3SS architecture in this state or the conformational changes that drive effector translocation. Here we use cryo-electron tomography and sub-tomogram averaging to derive the intact structure of the primordial Chlamydia trachomatis T3SS in the presence and absence of host membrane contact. Comparison of the averaged structures demonstrates a marked compaction of the basal body (4 nm) occurs when the needle tip contacts the host cell membrane. This compaction is coupled to a stabilization of the cytosolic sorting platform-ATPase. Our findings reveal the first structure of a bacterial T3SS from a major human pathogen engaged with a eukaryotic host, and reveal striking `pump-action' conformational changes that underpin effector injection.

  2. Bacterial composition in a metropolitan drinking water distribution system utilizing different source waters.

    Science.gov (United States)

    Gomez-Alvarez, Vicente; Humrighouse, Ben W; Revetta, Randy P; Santo Domingo, Jorge W

    2015-03-01

    We investigated the bacterial composition of water samples from two service areas within a drinking water distribution system (DWDS), each associated with a different primary source of water (groundwater, GW; surface water, SW) and different treatment process. Community analysis based on 16S rRNA gene clone libraries indicated that Actinobacteria (Mycobacterium spp.) and α-Proteobacteria represented nearly 43 and 38% of the total sequences, respectively. Sequences closely related to Legionella, Pseudomonas, and Vibrio spp. were also identified. In spite of the high number of sequences (71%) shared in both areas, multivariable analysis revealed significant differences between the GW and SW areas. While the dominant phylotypes where not significantly contributing in the ordination of samples, the populations associated with the core of phylotypes (1-10% in each sample) significantly contributed to the differences between both service areas. Diversity indices indicate that the microbial community inhabiting the SW area is more diverse and contains more distantly related species coexisting with local assemblages as compared with the GW area. The bacterial community structure of SW and GW service areas were dissimilar, suggesting that their respective source water and/or water quality parameters shaped by the treatment processes may contribute to the differences in community structure observed.

  3. Animals devoid of pulmonary system as infection models in the study of lung bacterial pathogens

    Science.gov (United States)

    López Hernández, Yamilé; Yero, Daniel; Pinos-Rodríguez, Juan M.; Gibert, Isidre

    2015-01-01

    Biological disease models can be difficult and costly to develop and use on a routine basis. Particularly, in vivo lung infection models performed to study lung pathologies use to be laborious, demand a great time and commonly are associated with ethical issues. When infections in experimental animals are used, they need to be refined, defined, and validated for their intended purpose. Therefore, alternative and easy to handle models of experimental infections are still needed to test the virulence of bacterial lung pathogens. Because non-mammalian models have less ethical and cost constraints as a subjects for experimentation, in some cases would be appropriated to include these models as valuable tools to explore host–pathogen interactions. Numerous scientific data have been argued to the more extensive use of several kinds of alternative models, such as, the vertebrate zebrafish (Danio rerio), and non-vertebrate insects and nematodes (e.g., Caenorhabditis elegans) in the study of diverse infectious agents that affect humans. Here, we review the use of these vertebrate and non-vertebrate models in the study of bacterial agents, which are considered the principal causes of lung injury. Curiously none of these animals have a respiratory system as in air-breathing vertebrates, where respiration takes place in lungs. Despite this fact, with the present review we sought to provide elements in favor of the use of these alternative animal models of infection to reveal the molecular signatures of host–pathogen interactions. PMID:25699030

  4. Evaluation of a pulsed xenon ultraviolet disinfection system to decrease bacterial contamination in operating rooms.

    Science.gov (United States)

    El Haddad, Lynn; Ghantoji, Shashank S; Stibich, Mark; Fleming, Jason B; Segal, Cindy; Ware, Kathy M; Chemaly, Roy F

    2017-10-10

    Environmental cleanliness is one of the contributing factors for surgical site infections in the operating rooms (ORs). To decrease environmental contamination, pulsed xenon ultraviolet (PX-UV), an easy and safe no-touch disinfection system, is employed in several hospital environments. The positive effect of this technology on environmental decontamination has been observed in patient rooms and ORs during the end-of-day cleaning but so far, no study explored its feasibility between surgical cases in the OR. In this study, 5 high-touch surfaces in 30 ORs were sampled after manual cleaning and after PX-UV intervention mimicking between-case cleaning to avoid the disruption of the ORs' normal flow. The efficacy of a 1-min, 2-min, and 8-min cycle were tested by measuring the surfaces' contaminants by quantitative cultures using Tryptic Soy Agar contact plates. We showed that combining standard between-case manual cleaning of surfaces with a 2-min cycle of disinfection using a portable xenon pulsed ultraviolet light germicidal device eliminated at least 70% more bacterial load after manual cleaning. This study showed the proof of efficacy of a 2-min cycle of PX-UV in ORs in eliminating bacterial contaminants. This method will allow a short time for room turnover and a potential reduction of pathogen transmission to patients and possibly surgical site infections.

  5. A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors

    Directory of Open Access Journals (Sweden)

    Qian Xu

    2017-12-01

    Full Text Available Human noroviruses (HuNoVs are the dominant cause of food-borne outbreaks of acute gastroenteritis. However, fundamental researches on HuNoVs, such as identification of viral receptors have been limited by the currently immature system to culture HuNoVs and the lack of efficient small animal models. Previously, we demonstrated that the recombinant protruding domain (P domain of HuNoVs capsid proteins were successfully anchored on the surface of Escherichia coli BL21 cells after the bacteria were transformed with a plasmid expressing HuNoVs P protein fused with bacterial transmembrane anchor protein. The cell-surface-displayed P proteins could specifically recognize and bind to histo-blood group antigens (HBGAs, receptors of HuNoVs. In this study, an upgraded bacterial surface displayed system was developed as a new platform to discover candidate receptors of HuNoVs. A thrombin-susceptible “linker” sequence was added between the sequences of bacterial transmembrane anchor protein and P domain of HuNoV (GII.4 capsid protein in a plasmid that displays the functional P proteins on the surface of bacteria. In this new system, the surface-displayed HuNoV P proteins could be released by thrombin treatment. The released P proteins self-assembled into small particles, which were visualized by electron microscopy. The bacteria with the surface-displayed P proteins were incubated with pig stomach mucin which contained HBGAs. The bacteria-HuNoV P proteins-HBGAs complex could be collected by low speed centrifugation. The HuNoV P proteins-HBGAs complex was then separated from the recombinant bacterial surface by thrombin treatment. The released viral receptor was confirmed by using the monoclonal antibody against type A HBGA. It demonstrated that the new system was able to capture and easily isolate receptors of HuNoVs. This new strategy provides an alternative, easier approach for isolating unknown receptors/ligands of HuNoVs from different samples

  6. 31P NMR Spectroscopy Revealed Adenylate kinase-like Activity and Phosphotransferase-like Activity from F1-ATPase of Escherichia coli

    International Nuclear Information System (INIS)

    Kim, Hyun Won

    2011-01-01

    Adenylate kinase-like activity and phosphotransferase-like activity from F 1 -ATPase of Escherichia coli was revealed by 31 P NMR spectroscopy. Incubation of F 1 -ATPase with ADP in the presence of Mg 2+ shows the appearance of 31 P resonances from AMP and Pi, suggesting generation of AMP and ATP by adenylate kinase-like activity and the subsequent hydrolysis to Pi. Incubation of F1-ATPase with ADP in the presence of methanol shows additional peak from methyl phosphate, suggesting phosphotransferase-like activity of F 1 -ATPase. Both adenylate kinase-like activity and phosphotransferase-like activity has not been reported from F 1 -ATPase of Escherichia coli. 31 P NMR could be a valuable tool for the investigation of phosphorous related enzyme

  7. Novel Approaches to Manipulating Bacterial Pathogen Biofilms: Whole-Systems Design Philosophy and Steering Microbial Evolution.

    Science.gov (United States)

    Penn, Alexandra S

    2016-01-01

    Understanding and manipulating bacterial biofilms is crucial in medicine, ecology and agriculture and has potential applications in bioproduction, bioremediation and bioenergy. Biofilms often resist standard therapies and the need to develop new means of intervention provides an opportunity to fundamentally rethink our strategies. Conventional approaches to working with biological systems are, for the most part, "brute force", attempting to effect control in an input and effort intensive manner and are often insufficient when dealing with the inherent non-linearity and complexity of living systems. Biological systems, by their very nature, are dynamic, adaptive and resilient and require management tools that interact with dynamic processes rather than inert artefacts. I present an overview of a novel engineering philosophy which aims to exploit rather than fight those properties, and hence provide a more efficient and robust alternative. Based on a combination of evolutionary theory and whole-systems design, its essence is what I will call systems aikido; the basic principle of aikido being to interact with the momentum of an attacker and redirect it with minimal energy expenditure, using the opponent's energy rather than one's own. In more conventional terms, this translates to a philosophy of equilibrium engineering, manipulating systems' own self-organisation and evolution so that the evolutionarily or dynamically stable state corresponds to a function which we require. I illustrate these ideas with a description of a proposed manipulation of environmental conditions to alter the stability of co-operation in the context of Pseudomonas aeruginosa biofilm infection of the cystic fibrosis lung.

  8. Impact of Cropping Systems, Soil Inoculum, and Plant Species Identity on Soil Bacterial Community Structure.

    Science.gov (United States)

    Ishaq, Suzanne L; Johnson, Stephen P; Miller, Zach J; Lehnhoff, Erik A; Olivo, Sarah; Yeoman, Carl J; Menalled, Fabian D

    2017-02-01

    Farming practices affect the soil microbial community, which in turn impacts crop growth and crop-weed interactions. This study assessed the modification of soil bacterial community structure by organic or conventional cropping systems, weed species identity [Amaranthus retroflexus L. (redroot pigweed) or Avena fatua L. (wild oat)], and living or sterilized inoculum. Soil from eight paired USDA-certified organic and conventional farms in north-central Montana was used as living or autoclave-sterilized inoculant into steam-pasteurized potting soil, planted with Am. retroflexus or Av. fatua and grown for two consecutive 8-week periods to condition soil nutrients and biota. Subsequently, the V3-V4 regions of the microbial 16S rRNA gene were sequenced by Illumina MiSeq. Treatments clustered significantly, with living or sterilized inoculum being the strongest delineating factor, followed by organic or conventional cropping system, then individual farm. Living inoculum-treated soil had greater species richness and was more diverse than sterile inoculum-treated soil (observed OTUs, Chao, inverse Simpson, Shannon, P soil contained more Chloroflexi and Acidobacteria, while the sterile inoculum soil had more Bacteroidetes, Firmicutes, Gemmatimonadetes, and Verrucomicrobia. Organically farmed inoculum-treated soil had greater species richness, more diversity (observed OTUs, Chao, Shannon, P soil. Cyanobacteria were higher in pots growing Am. retroflexus, regardless of inoculum type, for three of the four organic farms. Results highlight the potential of cropping systems and species identity to modify soil bacterial communities, subsequently modifying plant growth and crop-weed competition.

  9. Impact of well intake systems on bacterial, algae, and organic carbon reduction in SWRO desalination systems, SAWACO, Jeddah, Saudi Arabia

    KAUST Repository

    Dehwah, Abdullah

    2014-07-18

    The intake system can play a significant role in improving the feed water quality and ultimately influence the performance of downstream components of the seawater reverse osmosis desalination processes. In most cases, open-ocean intakes produce poor feed water quality in terms of the abundance of naturally occurring organic matter, which increases the risk of membrane fouling. An alternative intake is the subsurface system, which is based on the riverbank filtration concept that provides natural filtration and biological treatment of the feed water prior to the entry of the water into the desalination plant. The use of subsurface intakes normally improves the raw water quality by reducing suspended solids, algae, bacterial, and dissolved organic carbon concentrations. Therefore, the risk of biofouling caused by these substances can be reduced by implementing the appropriate type of intake system. The use of well intake systems was investigated along the Red Sea shoreline of Saudi Arabia in the Jeddah region. Data were collected from a seawater reverse osmosis (SWRO) plant with a capacity of 10,000 m3/d. The well system produces feed water from an artificial-fill peninsula that was constructed atop of the seabed. Ten wells have been constructed on the peninsula for extracting raw seawater. Water samples were collected from nearby surface seawater as a reference and from selected individual wells. The percentage of algae and bacterial removal by induced filtration process was evaluated by comparison of the seawater concentrations with the well discharges. Transparent exopolymer particles and organic carbon fractions reduction was also measured. The quality of raw water extracted from the well systems was highly improved compared with the raw seawater source. It was observed that algae were virtually 100% removed and the bacterial concentration was significantly removed by the aquifer matrix. The detailed analysis of organic carbon fraction using liquid

  10. Fungal–bacterial consortia increase diuron degradation in water-unsaturated systems

    DEFF Research Database (Denmark)

    Ellegaard-Jensen, Lea; Knudsen, Berith Elkær; Johansen, Anders

    2014-01-01

    organism. In addition, fungal hyphae may function as transport vectors for bacteria, thereby facilitating a more effective spreading of degrader organisms in the soil. A more rapid mineralization of the phenylurea herbicide diuron was found in sand with added microbial consortia consisting of both...... degrading bacteria and fungi. Facilitated transport of bacteria by fungal hyphae was demonstrated using a system where herbicide-spiked sand was separated from the consortium by a layer of sterile glass beads. Several fungal–bacterial consortia were investigated by combining different diuron......-degrading bacteria (Sphingomonas sp. SRS2, Variovorax sp. SRS16, and Arthrobacter globiformis D47) and fungi (Mortierella sp. LEJ702 and LEJ703). The fastest mineralization of 14C-labeled diuron was seen in the consortium consisting of Mortierella LEJ702, Variovorax SRS16, and A. globiformis D47, as measured...

  11. Evidence for alternative quaternary structure in a bacterial Type III secretion system chaperone

    Energy Technology Data Exchange (ETDEWEB)

    Barta, Michael L.; Zhang, Lingling; Picking, Wendy L.; Geisbrecht, Brian V. (UMKC); (OKLU)

    2010-10-05

    Type III secretion systems are a common virulence mechanism in many Gram-negative bacterial pathogens. These systems use a nanomachine resembling a molecular needle and syringe to provide an energized conduit for the translocation of effector proteins from the bacterial cytoplasm to the host cell cytoplasm for the benefit of the pathogen. Prior to translocation specialized chaperones maintain proper effector protein conformation. The class II chaperone, Invasion plasmid gene (Ipg) C, stabilizes two pore forming translocator proteins. IpgC exists as a functional dimer to facilitate the mutually exclusive binding of both translocators. In this study, we present the 3.3 {angstrom} crystal structure of an amino-terminally truncated form (residues 10-155, denoted IpgC10-155) of the class II chaperone IpgC from Shigella flexneri. Our structure demonstrates an alternative quaternary arrangement to that previously described for a carboxy-terminally truncated variant of IpgC (IpgC{sup 1-151}). Specifically, we observe a rotationally-symmetric 'head-to-head' dimerization interface that is far more similar to that previously described for SycD from Yersinia enterocolitica than to IpgC1-151. The IpgC structure presented here displays major differences in the amino terminal region, where extended coil-like structures are seen, as opposed to the short, ordered alpha helices and asymmetric dimerization interface seen within IpgC{sup 1-151}. Despite these differences, however, both modes of dimerization support chaperone activity, as judged by a copurification assay with a recombinant form of the translocator protein, IpaB. Conclusions: From primary to quaternary structure, these results presented here suggest that a symmetric dimerization interface is conserved across bacterial class II chaperones. In light of previous data which have described the structure and function of asymmetric dimerization, our results raise the possibility that class II chaperones may

  12. Improving water quality using settleable microalga Ettlia sp. and the bacterial community in freshwater recirculating aquaculture system of Danio rerio.

    Science.gov (United States)

    Chun, Seong-Jun; Cui, Yingshun; Ahn, Chi-Yong; Oh, Hee-Mock

    2018-05-15

    A highly settleable microalga, Ettlia sp., was applied to a freshwater recirculating aquaculture system (RAS) of Danio rerio to improve the treatment of nitrogenous compounds. The growth characteristics of the microalgae, water quality parameters, and bacterial communities were monitored for 73 days. In the treatment RAS, the inoculated Ettlia sp. grew up to 1.26 g/L and dominated (>99%) throughout the experiment, whereas naturally occurring microalgae grew to 0.57 g/L in the control RAS. The nitrate, nitrite, and ammonium concentrations in the treatment RAS were reduced by 50.1%, 73.3%, and 24.2%, respectively, compared to the control RAS. A bacterial community analysis showed that Rhodospirillales, Phycisphaerae, Chlorobiales, and Burkholderiales were the major bacterial groups in the later phase of the treatment RAS. Meanwhile, a network analysis among the Ettlia sp., bacterial groups, and environmental parameters, revealed that the bacterial groups played key roles in both water quality improvement and Ettlia sp. growth. In conclusion, the inoculation and growth of the Ettlia sp. and its associated bacteria in the RAS produced beneficial effects on the water quality by reducing the nitrogenous compounds and providing a favorable environment for certain bacterial groups to further improve water quality. Copyright © 2018 Elsevier Ltd. All rights reserved.

  13. Membrane Lipids as Indicators for Viable Bacterial Communities Inhabiting Petroleum Systems.

    Science.gov (United States)

    Gruner, Andrea; Mangelsdorf, Kai; Vieth-Hillebrand, Andrea; Horsfield, Brian; van der Kraan, Geert M; Köhler, Thomas; Janka, Christoph; Morris, Brandon E L; Wilkes, Heinz

    2017-08-01

    Microbial activity in petroleum reservoirs has been implicated in a suite of detrimental effects including deterioration of petroleum quality, increases in oil sulfur content, biofouling of steel pipelines and other infrastructures, and well plugging. Here, we present a biogeochemical approach, using phospholipid fatty acids (PLFAs), for detecting viable bacteria in petroleum systems. Variations within the bacterial community along water flow paths (producing well, topside facilities, and injection well) can be elucidated in the field using the same technique, as shown here within oil production plants in the Molasse Basin of Upper Austria. The abundance of PLFAs is compared to total cellular numbers, as detected by qPCR of the 16S rDNA gene, to give an overall comparison between the resolutions of both methods in a true field setting. Additionally, the influence of biocide applications on lipid- and DNA-based quantification was investigated. The first oil field, Trattnach, showed significant PLFA abundances and cell numbers within the reservoir and topside facilities. In contrast, the second field (Engenfeld) showed very low PLFA levels overall, likely due to continuous treatment of the topside facilities with a glutaraldehyde-based antimicrobial. In comparison, Trattnach is dosed once per week in a batch fashion. Changes within PLFA compositions across the flow path, throughout the petroleum production plants, point to cellular adaptation within the system and may be linked to shifts in the dominance of certain bacterial types in oil reservoirs versus topside facilities. Overall, PLFA-based monitoring provides a useful tool to assess the abundance and high-level taxonomic diversity of viable microbial populations in oil production wells, topside infrastructure, pipelines, and other related facilities.

  14. Frequency of dental caries in active and inactive systemic lupus erythematous patients: salivary and bacterial factors.

    Science.gov (United States)

    Loyola Rodriguez, J P; Galvan Torres, L J; Martinez Martinez, R E; Abud Mendoza, C; Medina Solis, C E; Ramos Coronel, S; Garcia Cortes, J O; Domínguez Pérez, R A

    2016-10-01

    The objective of this study was to determine dental caries frequency and to analyze salivary and bacterial factors associated with active and inactive systemic lupus erythematous (SLE) patients. Also, a proposal to identify dental caries by a surface, teeth, and the patient was developed. A cross-sectional, blinded study that included 60 SLE patients divided into two groups of 30 subjects each, according to the Activity Index for Diagnosis of Systemic Lupus Erythematous (SLEDAI). The decayed, missing, and filled teeth (DMFT) index and Integrative Dental Caries Index (IDCI) were used for analyzing dental caries. The saliva variables recorded were: flow, pH, and buffer capacity. The DNA copies of Streptococcus mutans and Streptococcus sobrinus were estimated by real-time PCR. The caries frequency was 85% for SLE subjects (73.3% for inactive systemic lupus erythematous (ISLE) and 100% for active systemic lupus erythematous (ASLE)); DMFT for the SLE group was 12.6 ± 5.7 and the IDCI was (9.8 ± 5.9). The ASLE group showed a salivary flow of 0.65 compared with 0.97 ml/1 min from the ISLE group; all variables mentioned above showed a statistical difference (p dental caries in epidemiological studies. © The Author(s) 2016.

  15. Multilevel Regulation of Bacterial Gene Expression with the Combined STAR and Antisense RNA System.

    Science.gov (United States)

    Lee, Young Je; Kim, Soo-Jung; Moon, Tae Seok

    2018-02-16

    Synthetic small RNA regulators have emerged as a versatile tool to predictably control bacterial gene expression. Owing to their simple design principles, small size, and highly orthogonal behavior, these engineered genetic parts have been incorporated into genetic circuits. However, efforts to achieve more sophisticated cellular functions using RNA regulators have been hindered by our limited ability to integrate different RNA regulators into complex circuits. Here, we present a combined RNA regulatory system in Escherichia coli that uses small transcription activating RNA (STAR) and antisense RNA (asRNA) to activate or deactivate target gene expression in a programmable manner. Specifically, we demonstrated that the activated target output by the STAR system can be deactivated by expressing two different types of asRNAs: one binds to and sequesters the STAR regulator, affecting the transcription process, while the other binds to the target mRNA, affecting the translation process. We improved deactivation efficiencies (up to 96%) by optimizing each type of asRNA and then integrating the two optimized asRNAs into a single circuit. Furthermore, we demonstrated that the combined STAR and asRNA system can control gene expression in a reversible way and can regulate expression of a gene in the genome. Lastly, we constructed and simultaneously tested two A AND NOT B logic gates in the same cell to show sophisticated multigene regulation by the combined system. Our approach establishes a methodology for integrating multiple RNA regulators to rationally control multiple genes.

  16. Evolution of fracture permeability due to co-colloidal bacterial transport in a coupled fracture-skin-matrix system

    Directory of Open Access Journals (Sweden)

    N. Natarajan

    2012-07-01

    Full Text Available A numerical model is developed for investigating the evolution of fracture permeability in a coupled fracture-matrix system in the presence of fracture-skin with simultaneous colloidal and bacterial transport, by taking into account the effects of thermal stress and silica precipitation/dissolution, which is computed using linear reaction kinetics. The non-linear coupled equations are numerically modeled using the fully implicit finite difference method and a constant continuous source is adopted while modeling thermal, contaminant, colloidal and bacterial transport. Due to co-colloid bacterial transport under non-isothermal conditions, in a coupled fracture-skin-matrix system, the fracture apertures vary spatially, with a corresponding pressure variation for a constant discharge. A series of numerical experiments were conducted for analyzing the spatial variation of fracture aperture in response to the combined effects of thermal stress, silica precipitation/dissolution, and simultaneous colloidal and bacterial transport in the presence of the fracture-skin. The simulation results suggest that temperature and contaminant concentration of the mobile fluid within the fracture increases with reduction in initial fracture aperture. The pattern of variation followed by the fracture aperture is nearly the same in the presence and absence of bacterial transport but the magnitude of the fracture aperture is low under the influence of bacterial transport. The variation in the fracture aperture resulting from precipitation-dissolution and thermoelastic stress is significant when the fracture aperture is very low and reduces with increment in fracture aperture. The variation in fracture aperture and pressure remains the same for both undersaturated and supersaturated fluid entering the fracture due to the influence of bacterial transport at the inlet of the fracture.

  17. Long-term impact of systemic bacterial infection on the cerebral vasculature and microglia

    Directory of Open Access Journals (Sweden)

    Püntener Ursula

    2012-06-01

    Full Text Available Abstract Background Systemic infection leads to generation of inflammatory mediators that result in metabolic and behavioural changes. Repeated or chronic systemic inflammation leads to a state of innate immune tolerance: a protective mechanism against overactivity of the immune system. In this study, we investigated the immune adaptation of microglia and brain vascular endothelial cells in response to systemic inflammation or bacterial infection. Methods Mice were given repeated doses of lipopolysaccharide (LPS or a single injection of live Salmonella typhimurium. Inflammatory cytokines were measured in serum, spleen and brain, and microglial phenotype studied by immunohistochemistry. To assess priming of the innate immune response in the brain, mice were infected with Salmonella typhimurium and subsequently challenged with a focal unilateral intracerebral injection of LPS. Results Repeated systemic LPS challenges resulted in increased brain IL-1β, TNF-α and IL-12 levels, despite attenuated systemic cytokine production. Each LPS challenge induced significant changes in burrowing behaviour. In contrast, brain IL-1β and IL-12 levels in Salmonella typhimurium-infected mice increased over three weeks, with high interferon-γ levels in the circulation. Behavioural changes were only observed during the acute phase of the infection. Microglia and cerebral vasculature display an activated phenotype, and focal intracerebral injection of LPS four weeks after infection results in an exaggerated local inflammatory response when compared to non-infected mice. Conclusions These studies reveal that the innate immune cells in the brain do not become tolerant to systemic infection, but are primed instead. This may lead to prolonged and damaging cytokine production that may have a profound effect on the onset and/or progression of pre-existing neurodegenerative disease.

  18. Inhibition of bacterial oxidation of ferrous iron by lead nitrate in sulfate-rich systems

    Science.gov (United States)

    Wang, Hongmei; Gong, Linfeng; Cravotta,, Charles A.; Yang, Xiaofen; Tuovinen, Olli H.; Dong, Hailiang; Fu, Xiang

    2013-01-01

    Inhibition of bacterial oxidation of ferrous iron (Fe(II)) by Pb(NO3)2 was investigated with a mixed culture of Acidithiobacillus ferrooxidans. The culture was incubated at 30 °C in ferrous-sulfate medium amended with 0–24.2 mM Pb(II) added as Pb(NO3)2. Anglesite (PbSO4) precipitated immediately upon Pb addition and was the only solid phase detected in the abiotic controls. Both anglesite and jarosite (KFe3(SO4)2(OH)6) were detected in inoculated cultures. Precipitation of anglesite maintained dissolved Pb concentrations at 16.9–17.6 μM regardless of the concentrations of Pb(NO3)2 added. Fe(II) oxidation was suppressed by 24.2 mM Pb(NO3)2 addition even when anglesite was removed before inoculation. Experiments with 0–48 mM KNO3 demonstrated that bacterial Fe(II) oxidation decreased as nitrate concentration increased. Therefore, inhibition of Fe(II) oxidation at 24.2 mM Pb(NO3)2 addition resulted from nitrate toxicity instead of Pb addition. Geochemical modeling that considered the initial precipitation of anglesite to equilibrium followed by progressive oxidation of Fe(II) and the precipitation of jarosite and an amorphous iron hydroxide phase, without allowing plumbojarosite to precipitate were consistent with the experimental time-series data on Fe(II) oxidation under biotic conditions. Anglesite precipitation in mine tailings and other sulfate-rich systems maintains dissolved Pb concentrations below the toxicity threshold of A. ferrooxidans.

  19. Modulation of bacterial Type III secretion system by a spermidine transporter dependent signaling pathway.

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    Lian Zhou

    Full Text Available BACKGROUND: Many gram-negative bacterial pathogens employ Type III secretion systems (T3SS to inject effector proteins into host cells in infectious processes. METHODOLOGY/PRINCIPAL FINDINGS: By screening a transposon mutant library of P. aeruginosa, we found that mutation of spuDEFGH, which encode a major spermidine uptake system, abolished the expression of the exsCEBA operon that codes for key T3SS regulators under inducing conditions (low calcium. Whole genome microarray analysis revealed that inactivation of the spermidine uptake system significantly decreased the transcriptional expression of most, if not all, T3SS genes. Consistently, the spermidine uptake mutants showed decreased expression of the T3SS genes in responding to host cell extract and attenuated cytotoxicity. Furthermore, exogenous addition of spermidine to the wild type strain PAO1 enhanced the expression of exsCEBA and also the effector protein genes. CONCLUSION/SIGNIFICANCE: Cumulatively, these data have depicted a novel spermidine transporter-dependent signaling pathway, which appears to play an essential role in modulation of T3SS expression in P. aeruginosa.

  20. Phytoplankton production systems in a shellfish hatchery: variations of the bacterial load and diversity of vibrios.

    Science.gov (United States)

    Dubert, J; Fernández-Pardo, A; Nóvoa, S; Barja, J L; Prado, S

    2015-06-01

    Outbreaks of disease caused by some Vibrio species represent the main production bottleneck in shellfish hatcheries. Although the phytoplankton used as food is one of the main sources of bacteria, studies of the associated bacterial populations, specifically vibrios, are scarce. The aim of the study was the microbiological monitoring of the microalgae as the first step in assessing the risk disease for bivalve cultures. Two phytoplankton production systems were sampled weekly throughout 1-year period in a bivalve hatchery. Quantitative analysis revealed high levels of marine heterotrophic bacteria in both systems throughout the study. Presumptive vibrios were detected occasionally and at low concentrations. In most of the cases, they belonged to the Splendidus and Harveyi clades. The early detection of vibrios in the microalgae may be the key for a successful bivalve culture. Their abundance and diversity were affected by factors related to the hatchery environment. This work represents the first long study where the presence of vibrios was evaluated rigorously in phytoplankton production systems and provides a suitable microbiological protocol to control and guarantee the quality of the algal cultures to avoid the risk of transferring potential pathogens to shellfish larvae and/or broodstock. © 2015 The Society for Applied Microbiology.

  1. Quantification and risks associated with bacterial aerosols near domestic greywater-treatment systems

    Energy Technology Data Exchange (ETDEWEB)

    Benami, Maya; Busgang, Allison; Gillor, Osnat; Gross, Amit, E-mail: amgross@exchange.bgu.ac.il

    2016-08-15

    Greywater (GW) reuse can alleviate water stress by lowering freshwater consumption. However, GW contains pathogens that may compromise public health. During the GW-treatment process, bioaerosols can be produced and may be hazardous to human health if inhaled, ingested, or come in contact with skin. Using air-particle monitoring, BioSampler®, and settle plates we sampled bioaerosols emitted from recirculating vertical flow constructed wetlands (RVFCW) – a domestic GW-treatment system. An array of pathogens and indicators were monitored using settle plates and by culturing the BioSampler® liquid. Further enumeration of viable pathogens in the BioSampler® liquid utilized a newer method combining the benefits of enrichment with molecular detection (MPN-qPCR). Additionally, quantitative microbial risk assessment (QMRA) was applied to assess risks of infection from a representative skin pathogen, Staphylococcus aureus. According to the settle-plate technique, low amounts (0–9.7 × 10{sup 4} CFU m{sup −2} h{sup −1}) of heterotrophic bacteria, Staphylococcus spp., Pseudomonas spp., Klebsiella pneumoniae, Enterococcus spp., and Escherichia coli were found to aerosolize up to 1 m away from the GW systems. At the 5 m distance amounts of these bacteria were not statistically different (p > 0.05) from background concentrations tested over 50 m away from the systems. Using the BioSampler®, no bacteria were detected before enrichment of the GW-aerosols. However, after enrichment, using an MPN-qPCR technique, viable indicators and pathogens were occasionally detected. Consequently, the QMRA results were below the critical disability-adjusted life year (DALY) safety limits, a measure of overall disease burden, for S. aureus under the tested exposure scenarios. Our study suggests that health risks from aerosolizing pathogens near RVFCW GW-treatment systems are likely low. This study also emphasizes the growing need for standardization of bioaerosol-evaluation techniques

  2. The effect of a commercial UV disinfection system on the bacterial load of shell eggs.

    NARCIS (Netherlands)

    Reu, de K.; Grijspeerdt, K.; Hermann, L.M.; Heyndrickx, M.; Uytendaele, M.; Debevere, J.; Putirulan, F.F.; Bolder, N.M.

    2006-01-01

    Aims: To study the effect of UV irradiation on the bacterial load of shell eggs and of a roller conveyor belt. Methods and Results: The natural bacterial load on the eggshell of clean eggs was significantly reduced by a standard UV treatment of 4.7 s; from 4.47 to 3.57 log CFU per eggshell. For very

  3. [Investigation of bacterial and viral etiology in community acquired central nervous system infections with molecular methods].

    Science.gov (United States)

    Kahraman, Hasip; Tünger, Alper; Şenol, Şebnem; Gazi, Hörü; Avcı, Meltem; Örmen, Bahar; Türker, Nesrin; Atalay, Sabri; Köse, Şükran; Ulusoy, Sercan; Işıkgöz Taşbakan, Meltem; Sipahi, Oğuz Reşat; Yamazhan, Tansu; Gülay, Zeynep; Alp Çavuş, Sema; Pullukçu, Hüsnü

    2017-07-01

    In this multicenter prospective cohort study, it was aimed to evaluate the bacterial and viral etiology in community-acquired central nervous system infections by standart bacteriological culture and multiplex polymerase chain reaction (PCR) methods. Patients hospitalized with central nervous system infections between April 2012 and February 2014 were enrolled in the study. Demographic and clinical information of the patients were collected prospectively. Cerebrospinal fluid (CSF) samples of the patients were examined by standart bacteriological culture methods, bacterial multiplex PCR (Seeplex meningitis-B ACE Detection (Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Listeria monocytogenes, Group B streptococci) and viral multiplex PCR (Seeplex meningitis-V1 ACE Detection kits herpes simplex virus-1 (HSV1), herpes simplex virus-2 (HSV2), varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein Barr virus (EBV) and human herpes virus 6 (HHV6)) (Seeplex meningitis-V2 ACE Detection kit (enteroviruses)). Patients were classified as purulent meningitis, aseptic meningitis and encephalitis according to their clinical, CSF (leukocyte level, predominant cell type, protein and glucose (blood/CSF) levels) and cranial imaging results. Patients who were infected with a pathogen other than the detection of the kit or diagnosed as chronic meningitis and other diseases during the follow up, were excluded from the study. A total of 79 patients (28 female, 51 male, aged 42.1 ± 18.5) fulfilled the study inclusion criteria. A total of 46 patients were classified in purulent meningitis group whereas 33 were in aseptic meningitis/encephalitis group. Pathogens were detected by multiplex PCR in 41 patients. CSF cultures were positive in 10 (21.7%) patients (nine S.pneumoniae, one H.influenzae) and PCR were positive for 27 (58.6%) patients in purulent meningitis group. In this group one type of bacteria were detected in 18 patients (14 S.pneumoniae, two N

  4. Modeling of the bacterial mechanism of methicillin-resistance by a systems biology approach.

    Directory of Open Access Journals (Sweden)

    Ida Autiero

    Full Text Available BACKGROUND: A microorganism is a complex biological system able to preserve its functional features against external perturbations and the ability of the living systems to oppose to these external perturbations is defined "robustness". The antibiotic resistance, developed by different bacteria strains, is a clear example of robustness and of ability of the bacterial system to acquire a particular functional behaviour in response to environmental changes. In this work we have modeled the whole mechanism essential to the methicillin-resistance through a systems biology approach. The methicillin is a beta-lactamic antibiotic that act by inhibiting the penicillin-binding proteins (PBPs. These PBPs are involved in the synthesis of peptidoglycans, essential mesh-like polymers that surround cellular enzymes and are crucial for the bacterium survival. METHODOLOGY: The network of genes, mRNA, proteins and metabolites was created using CellDesigner program and the data of molecular interactions are stored in Systems Biology Markup Language (SBML. To simulate the dynamic behaviour of this biochemical network, the kinetic equations were associated with each reaction. CONCLUSIONS: Our model simulates the mechanism of the inactivation of the PBP by methicillin, as well as the expression of PBP2a isoform, the regulation of the SCCmec elements (SCC: staphylococcal cassette chromosome and the synthesis of peptidoglycan by PBP2a. The obtained results by our integrated approach show that the model describes correctly the whole phenomenon of the methicillin resistance and is able to respond to the external perturbations in the same way of the real cell. Therefore, this model can be useful to develop new therapeutic approaches for the methicillin control and to understand the general mechanism regarding the cellular resistance to some antibiotics.

  5. Genome-wide analysis of bacterial determinants of plant growth promotion and induced systemic resistance by Pseudomonas fluorescens

    NARCIS (Netherlands)

    Cheng, Xu; Etalo, Desalegn W.; van de Mortel, Judith E.; Dekkers, Ester; Nguyen, Linh; Medema, Marnix H; Raaijmakers, Jos M.

    2017-01-01

    Pseudomonas fluorescens strain SS101 (Pf.SS101) promotes growth of Arabidopsis thaliana, enhances greening and lateral root formation, and induces systemic resistance (ISR) against the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). Here, targeted and untargeted approaches were adopted to

  6. A novel in vivo inducible expression system in Edwardsiella tarda for potential application in bacterial polyvalence vaccine.

    Science.gov (United States)

    Mu, Wei; Guan, Lingyu; Yan, Yijian; Liu, Qin; Zhang, Yuanxing

    2011-12-01

    Recombinant bacterial vector vaccine is an attractive vaccination strategy to induce the immune response to a carried protective antigen, and the main concern of bacterial vector vaccine is to establish a stable antigen expression system in vector bacteria. Edwardsiella tarda is an important facultative intracellular pathogen of both animals and humans, and its attenuated derivates are excellent bacterial vectors for use in recombinant vaccine design. In this study, we design an in vivo inducible expression system in E. tarda and establish potential recombinant E. tarda vector vaccines. With wild type strain E. tarda EIB202 as a vector, 53 different bacteria-originated promoters were examined for iron-responsive transcription in vitro, and the promoters P(dps) and P(yncE) showed high transcription activity. The transcription profiles in vivo of two promoters were further assayed, and P(dps) revealed an enhanced in vivo inducible transcription in macrophage, larvae and adult zebra fish. The gapA34 gene, encoding the protective antigen GAPDH from the fish pathogen Aeromonas hydrophila LSA34, was introduced into the P(dps)-based protein expression system, and transformed into attenuated E. tarda strains. The resultant recombinant vector vaccine WED/pUTDgap was evaluated in turbot (Scophtalmus maximus). Over 60% of the vaccinated fish survived under the challenge with A. hydrophila LSA34 and E. tarda EIB202, suggesting that the P(dps)-based antigen delivery system had great potential in bacterial vector vaccine application. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Bacterial Communities of Diverse Drosophila Species: Ecological Context of a Host–Microbe Model System

    Science.gov (United States)

    Bhatnagar, Srijak; Eisen, Jonathan A.; Kopp, Artyom

    2011-01-01

    Drosophila melanogaster is emerging as an important model of non-pathogenic host–microbe interactions. The genetic and experimental tractability of Drosophila has led to significant gains in our understanding of animal–microbial symbiosis. However, the full implications of these results cannot be appreciated without the knowledge of the microbial communities associated with natural Drosophila populations. In particular, it is not clear whether laboratory cultures can serve as an accurate model of host–microbe interactions that occur in the wild, or those that have occurred over evolutionary time. To fill this gap, we characterized natural bacterial communities associated with 14 species of Drosophila and related genera collected from distant geographic locations. To represent the ecological diversity of Drosophilids, examined species included fruit-, flower-, mushroom-, and cactus-feeders. In parallel, wild host populations were compared to laboratory strains, and controlled experiments were performed to assess the importance of host species and diet in shaping bacterial microbiome composition. We find that Drosophilid flies have taxonomically restricted bacterial communities, with 85% of the natural bacterial microbiome composed of only four bacterial families. The dominant bacterial taxa are widespread and found in many different host species despite the taxonomic, ecological, and geographic diversity of their hosts. Both natural surveys and laboratory experiments indicate that host diet plays a major role in shaping the Drosophila bacterial microbiome. Despite this, the internal bacterial microbiome represents only a highly reduced subset of the external bacterial communities, suggesting that the host exercises some level of control over the bacteria that inhabit its digestive tract. Finally, we show that laboratory strains provide only a limited model of natural host–microbe interactions. Bacterial taxa used in experimental studies are rare or absent in

  8. Sorting of bacterial lipoproteins to the outer membrane by the Lol system.

    Science.gov (United States)

    Narita, Shin-ichiro; Tokuda, Hajime

    2010-01-01

    Bacterial lipoproteins comprise a subset of membrane proteins with a lipid-modified cysteine residue at their amino termini through which they are anchored to the membrane. In Gram-negative bacteria, lipoproteins are localized on either the inner or the outer membrane. The Lol system is responsible for the transport of lipoproteins to the outer membrane.The Lol system comprises an inner-membrane ABC transporter LolCDE complex, a periplasmic carrier protein, LolA, and an outer membrane receptor protein, LolB. Lipoproteins are synthesized as precursors in the cytosol and then translocated across the inner membrane by the Sec translocon to the outer leaflet of the inner membrane, where lipoprotein precursors are processed to mature lipoproteins. The LolCDE complex then mediates the release of outer membrane-specific lipoproteins from the inner membrane while the inner membrane-specific lipoproteins possessing Asp at position 2 are not released by LolCDE because it functions as a LolCDE avoidance signal, causing the retention of these lipoproteins in the inner membrane. A water-soluble lipoprotein-LolA complex is formed as a result of the release reaction mediated by LolCDE. This complex traverses the hydrophilic periplasm to reach the outer membrane, where LolB accepts a lipoprotein from LolA and then catalyzes its incorporation into the inner leaflet of the outer membrane.

  9. Bacterial Keratitis

    Science.gov (United States)

    ... Español Eye Health / Eye Health A-Z Bacterial Keratitis Sections What Is Bacterial Keratitis? Bacterial Keratitis Symptoms ... Lens Care Bacterial Keratitis Treatment What Is Bacterial Keratitis? Leer en Español: ¿Qué Es la Queratitis Bacteriana? ...

  10. Exploring the complex response to linuron of bacterial communities from biopurification systems by means of cultivation-independent methods

    DEFF Research Database (Denmark)

    Dealtry, Simone; Nour, Eman H.; Holmsgaard, Peter Nikolai

    2016-01-01

    captured from BPS+/BPS-, and in three transconjugants from BPS+ the gene hylA was detected. Our data suggest the existence of a multispecies linuron degrading bacterial food web and an involvement of IncP-1 plasmids in the adaptation of bacterial communities to pesticide pollution in BPSs.......On-farm biopurification systems (BPSs) treat pesticide-contaminated wastewater at farms through biodegradation and sorption processes. However, information on the microbiota involved in pesticide removal in BPSs is scarce. Here we report on the response of BPS bacterial communities to the herbicide...... to taxa that were previously reported from linuron degrading consortia cultivated from soil (Hyphomicrobiaceae, Comamonadaceae, Micrococcaceae). In addition, numerous taxa with increased relative abundance were identified that were previously not associated with linuron degradation. The relative abundance...

  11. Bacterial communities in the collection and chlorinated distribution sections of a drinking water system in Budapest, Hungary.

    Science.gov (United States)

    Homonnay, Zalán G; Török, György; Makk, Judit; Brumbauer, Anikó; Major, Eva; Márialigeti, Károly; Tóth, Erika

    2014-07-01

    Bacterial communities of a bank-filtered drinking water system were investigated by aerobic cultivation and clone library analysis. Moreover, bacterial communities were compared using sequence-aided terminal restriction fragment length polymorphism (T-RFLP) fingerprinting at ten characteristic points located at both the collecting and the distributing part of the water supply system. Chemical characteristics of the samples were similar, except for the presence of chlorine residuals in the distribution system and increased total iron concentration in two of the samples. Assimilable organic carbon (AOC) concentration increased within the collection system, it was reduced by chlorination and it increased again in the distribution system. Neither fecal indicators nor pathogens were detected by standard cultivation techniques. Chlorination reduced bacterial diversity and heterotrophic plate counts. Community structures were found to be significantly different before and after chlorination: the diverse communities in wells and the collection system were dominated by chemolithotrophic (e.g., Gallionella and Nitrospira) and oligocarbophilic-heterotrophic bacteria (e.g., Sphingomonas, Sphingopyxis, and Bradyrhizobium). After chlorination in the distribution system, the most characteristic bacterium was related to the facultative methylotrophic Methylocella spp. Communities changed within the distribution system too, Mycobacterium spp. or Sphingopyxis spp. became predominant in certain samples. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Differences of serum procalcitonin levels between bacterial infection and flare in systemic lupus erythematosus patients

    Science.gov (United States)

    Patrick, J.; Marpaung, B.; Ginting, Y.

    2018-03-01

    Differentiate bacterial infections from flare in SLE patients is difficult to do because clinical symptoms of infection is similar to flare. SLE patients with infection require antibiotic therapy with decreased doses of immunosuppressant while in flare diseases require increased immunosuppressant. Procalcitonin (PCT), a biological marker, increased in serum patients with bacterial infections and expected to be a solution of problem. The aim of this study was to examine the function of PCT serum as marker to differentiate bacterial infection and flare in SLE patients. This cross-sectional study was conducted in Adam Malik Hospital from January-July 2017. We examined 80 patients SLE flare (MEX-SLEDAI>5), screen PCT and culture according to focal infection. Data were statistically analyzed. 80 SLE patients divided into 2 groups: bacterial infection group (31 patients) and non-infection/flare group (49 patients). Median PCT levels of bacterial infection group was 1.66 (0.04-8.45)ng/ml while flare group was 0.12 (0.02-0.81)ng/ml. There was significant difference of serum Procalcitonin level between bacterial infection and flare group in SLE patients (p=0.001). Procalcitonin serum levels can be used as a biomarker to differentiate bacterial infections and flare in SLE patients.

  13. Structure, function, and evolution of bacterial ATP-binding cassette systems

    Energy Technology Data Exchange (ETDEWEB)

    Davidson, A.L.; Dassa, E.; Orelle, C.; Chen, J. (Purdue)

    2010-07-27

    HisP, the proteins suspected to energize these transporters, shared as much as 32% identity in amino acid residues when their sequences were aligned (171). Later, it was found that several bacterial proteins involved in uptake of nutrients, export of toxins, cell division, bacterial nodulation of plants, and DNA repair displayed the same similarity in their sequences (127, 196). This led to the notion that the conserved protein, which had been shown to bind ATP (198, 201), would probably energize the systems mentioned above by coupling the energy of ATP hydrolysis to transport. The latter was demonstrated with the maltose and histidine transporters by use of isolated membrane vesicles (105, 379) and purified transporters reconstituted into proteoliposomes (30, 98). The determination of the sequence of the first eukaryotic protein strongly similar to these bacterial transporters (the P-glycoprotein, involved in resistance of cancer cells to multiple drugs) (169, 179) demonstrated that these proteins were not restricted to prokaryotes. Two names, 'traffic ATPases' (15) and the more accepted name 'ABC transporters' (193, 218), were proposed for members of this new superfamily. ABC systems can be divided into three main functional categories, as follows. Importers mediate the uptake of nutrients in prokaryotes. The nature of the substrates that are transported is very wide, including mono- and oligosaccharides, organic and inorganic ions, amino acids, peptides, ironsiderophores, metals, polyamine cations, opines, and vitamins. Exporters are involved in the secretion of various molecules, such as peptides, lipids, hydrophobic drugs, polysaccharides, and proteins, including toxins such as hemolysin. The third category of systems is apparently not involved in transport, with some members being involved in translation of mRNA and in DNA repair. Despite the large, diverse population of substrates handled and the difference in the polarity of transport

  14. Bacterial Effectors and Their Functions in the Ubiquitin-Proteasome System: Insight from the Modes of Substrate Recognition

    Directory of Open Access Journals (Sweden)

    Minsoo Kim

    2014-08-01

    Full Text Available Protein ubiquitination plays indispensable roles in the regulation of cell homeostasis and pathogenesis of neoplastic, infectious, and neurodegenerative diseases. Given the importance of this modification, it is to be expected that several pathogenic bacteria have developed the ability to utilize the host ubiquitin system for their own benefit. Modulation of the host ubiquitin system by bacterial effector proteins inhibits innate immune responses and hijacks central signaling pathways. Bacterial effectors mimic enzymes of the host ubiquitin system, but may or may not be structurally similar to the mammalian enzymes. Other effectors bind and modify components of the host ubiquitin system, and some are themselves subject to ubiquitination. This review will describe recent findings, based on structural analyses, regarding how pathogens use post-translational modifications of proteins to establish an infection.

  15. Bacterial effectors and their functions in the ubiquitin-proteasome system: insight from the modes of substrate recognition.

    Science.gov (United States)

    Kim, Minsoo; Otsubo, Ryota; Morikawa, Hanako; Nishide, Akira; Takagi, Kenji; Sasakawa, Chihiro; Mizushima, Tsunehiro

    2014-08-18

    Protein ubiquitination plays indispensable roles in the regulation of cell homeostasis and pathogenesis of neoplastic, infectious, and neurodegenerative diseases. Given the importance of this modification, it is to be expected that several pathogenic bacteria have developed the ability to utilize the host ubiquitin system for their own benefit. Modulation of the host ubiquitin system by bacterial effector proteins inhibits innate immune responses and hijacks central signaling pathways. Bacterial effectors mimic enzymes of the host ubiquitin system, but may or may not be structurally similar to the mammalian enzymes. Other effectors bind and modify components of the host ubiquitin system, and some are themselves subject to ubiquitination. This review will describe recent findings, based on structural analyses, regarding how pathogens use post-translational modifications of proteins to establish an infection.

  16. Metagenomics reveals that detoxification systems are underrepresented in marine bacterial communities.

    Science.gov (United States)

    Bengtsson-Palme, Johan; Alm Rosenblad, Magnus; Molin, Mikael; Blomberg, Anders

    2014-09-01

    Environmental shotgun sequencing (metagenomics) provides a new way to study communities in microbial ecology. We here use sequence data from the Global Ocean Sampling (GOS) expedition to investigate toxicant selection pressures revealed by the presence of detoxification genes in marine bacteria. To capture a broad range of potential toxicants we selected detoxification protein families representing systems protecting microorganisms from a variety of stressors, such as metals, organic compounds, antibiotics and oxygen radicals. Using a bioinformatics procedure based on comparative analysis to finished bacterial genomes we found that the amount of detoxification genes present in marine microorganisms seems surprisingly small. The underrepresentation is particularly evident for toxicant transporters and proteins involved in detoxifying metals. Exceptions are enzymes involved in oxidative stress defense where peroxidase enzymes are more abundant in marine bacteria compared to bacteria in general. In contrast, catalases are almost completely absent from the open ocean environment, suggesting that peroxidases and peroxiredoxins constitute a core line of defense against reactive oxygen species (ROS) in the marine milieu. We found no indication that detoxification systems would be generally more abundant close to the coast compared to the open ocean. On the contrary, for several of the protein families that displayed a significant geographical distribution, like peroxidase, penicillin binding transpeptidase and divalent ion transport protein, the open ocean samples showed the highest abundance. Along the same lines, the abundance of most detoxification proteins did not increase with estimated pollution. The low level of detoxification systems in marine bacteria indicate that the majority of marine bacteria have a low capacity to adapt to increased pollution. Our study exemplifies the use of metagenomics data in ecotoxicology, and in particular how anthropogenic

  17. Control of corrosive bacterial community by bronopol in industrial water system.

    Science.gov (United States)

    Narenkumar, Jayaraman; Ramesh, Nachimuthu; Rajasekar, Aruliah

    2018-01-01

    Ten aerobic corrosive bacterial strains were isolated from a cooling tower water system (CWS) which were identified based on the biochemical characterization and 16S rRNA gene sequencing. Out of them, dominant corrosion-causing bacteria, namely, Bacillus thuringiensis EN2, Terribacillus aidingensis EN3, and Bacillus oleronius EN9, were selected for biocorrosion studies on mild steel 1010 (MS) in a CWS. The biocorrosion behaviour of EN2, EN3, and EN9 strains was studied using immersion test (weight loss method), electrochemical analysis, and surface analysis. To address the corrosion problems, an anti-corrosive study using a biocide, bronopol was also demonstrated. Scanning electron microscopy and Fourier-transform infrared spectroscopy analyses of the MS coupons with biofilm developed after exposure to CWS confirmed the accumulation of extracellular polymeric substances and revealed that biofilms was formed as microcolonies, which subsequently cause pitting corrosion. In contrast, the biocide system, no pitting type of corrosion, was observed and weight loss was reduced about 32 ± 2 mg over biotic system (286 ± 2 mg). FTIR results confirmed the adsorption of bronopol on the MS metal surface as protective layer (co-ordination of NH 2 -Fe 3+ ) to prevent the biofilm formation and inhibit the corrosive chemical compounds and thus led to reduction of corrosion rate (10 ± 1 mm/year). Overall, the results from WL, EIS, SEM, XRD, and FTIR concluded that bronopol was identified as effective biocide and corrosion inhibitor which controls the both chemical and biocorrosion of MS in CWS.

  18. The CRISPR-Cas system – from bacterial immunity to genome engineering

    Directory of Open Access Journals (Sweden)

    Maria Czarnek

    2016-09-01

    Full Text Available Precise and efficient genome modifications present a great value in attempts to comprehend the roles of particular genes and other genetic elements in biological processes as well as in various pathologies. In recent years novel methods of genome modification known as genome editing, which utilize so called “programmable” nucleases, came into use. A true revolution in genome editing has been brought about by the introduction of the CRISP-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated system, in which one of such nucleases, i.e. Cas9, plays a major role. This system is based on the elements of the bacterial and archaeal mechanism responsible for acquired immunity against phage infections and transfer of foreign genetic material. Microorganisms incorporate fragments of foreign DNA into CRISPR loci present in their genomes, which enables fast recognition and elimination of future infections. There are several types of CRISPR-Cas systems among prokaryotes but only elements of CRISPR type II are employed in genome engineering. CRISPR-Cas type II utilizes small RNA molecules (crRNA and tracrRNA to precisely direct the effector nuclease – Cas9 – to a specific site in the genome, i.e. to the sequence complementary to crRNA. Cas9 may be used to: (i introduce stable changes into genomes e.g. in the process of generation of knock-out and knock-in animals and cell lines, (ii activate or silence the expression of a gene of interest, and (iii visualize specific sites in genomes of living cells. The CRISPR-Cas-based tools have been successfully employed for generation of animal and cell models of a number of diseases, e.g. specific types of cancer. In the future, the genome editing by programmable nucleases may find wide application in medicine e.g. in the therapies of certain diseases of genetic origin and in the therapy of HIV-infected patients.

  19. Elevated carbon monoxide in the exhaled breath of mice during a systemic bacterial infection.

    Directory of Open Access Journals (Sweden)

    Alan G Barbour

    Full Text Available Blood is the specimen of choice for most laboratory tests for diagnosis and disease monitoring. Sampling exhaled breath is a noninvasive alternative to phlebotomy and has the potential for real-time monitoring at the bedside. Improved instrumentation has advanced breath analysis for several gaseous compounds from humans. However, application to small animal models of diseases and physiology has been limited. To extend breath analysis to mice, we crafted a means for collecting nose-only breath samples from groups and individual animals who were awake. Samples were subjected to gas chromatography and mass spectrometry procedures developed for highly sensitive analysis of trace volatile organic compounds (VOCs in the atmosphere. We evaluated the system with experimental systemic infections of severe combined immunodeficiency Mus musculus with the bacterium Borrelia hermsii. Infected mice developed bacterial densities of ∼10(7 per ml of blood by day 4 or 5 and in comparison to uninfected controls had hepatosplenomegaly and elevations of both inflammatory and anti-inflammatory cytokines. While 12 samples from individual infected mice on days 4 and 5 and 6 samples from uninfected mice did not significantly differ for 72 different VOCs, carbon monoxide (CO was elevated in samples from infected mice, with a mean (95% confidence limits effect size of 4.2 (2.8-5.6, when differences in CO2 in the breath were taken into account. Normalized CO values declined to the uninfected range after one day of treatment with the antibiotic ceftriaxone. Strongly correlated with CO in the breath were levels of heme oxygenase-1 protein in serum and HMOX1 transcripts in whole blood. These results (i provide further evidence of the informativeness of CO concentration in the exhaled breath during systemic infection and inflammation, and (ii encourage evaluation of this noninvasive analytic approach in other various other rodent models of infection and for utility in

  20. Suggested guidelines for using systemic antimicrobials in bacterial skin infections: part?2? antimicrobial choice, treatment regimens and compliance

    OpenAIRE

    Beco, L.; Guagu?re, E.; M?ndez, C. Lorente; Noli, C.; Nuttall, T.; Vroom, M.

    2013-01-01

    Systemic antimicrobials are critically important in veterinary healthcare, and resistance is a major concern. Antimicrobial stewardship will be important in maintaining clinical efficacy by reducing the development and spread of antimicrobial resistance. Bacterial skin infections are one of the most common reasons for using systemic antimicrobials in dogs and cats. Appropriate management of these infections is, therefore, crucial in any policy for responsible antimicrobial use. The goals of t...

  1. The Bacterial Type III Secretion System as a Target for Developing New Antibiotics

    Science.gov (United States)

    McShan, Andrew C.; De Guzman, Roberto N.

    2017-01-01

    Antibiotic resistance in pathogens requires new targets for developing novel antibacterials. The bacterial type III secretion system (T3SS) is an attractive target for developing antibacterials as it is essential in the pathogenesis of many Gram-negative bacteria. The T3SS consists of structural proteins, effectors and chaperones. Over 20 different structural proteins assemble into a complex nanoinjector that punctures a hole on the eukaryotic cell membrane to allow the delivery of effectors directly into the host cell cytoplasm. Defects in the assembly and function of the T3SS render bacteria non-infective. Two major classes of small molecules, salicylidene acylhydrazides and thiazolidinones, have been shown to inhibit multiple genera of bacteria through the T3SS. Many additional chemically and structurally diverse classes of small molecule inhibitors of the T3SS have been identified as well. While specific targets within the T3SS of a few inhibitors have been suggested, the vast majority of specific protein targets within the T3SS remain to be identified or characterized. Other T3SS inhibitors include polymers, proteins and polypeptides mimics. In addition, T3SS activity is regulated by its interaction with biologically relevant molecules, such as bile salts and sterols, which could serve as scaffolds for drug design. PMID:25521643

  2. The type VI secretion system impacts bacterial invasion and population dynamics in a model intestinal microbiota

    Science.gov (United States)

    Logan, Savannah L.; Shields, Drew S.; Hammer, Brian K.; Xavier, Joao B.; Parthasarathy, Raghuveer

    Animal gastrointestinal tracts are home to a diverse community of microbes. The mechanisms by which microbial species interact and compete in this dense, physically dynamic space are poorly understood, limiting our understanding of how natural communities are assembled and how different communities could be engineered. Here, we focus on a physical mechanism for competition: the type VI secretion system (T6SS). The T6SS is a syringe-like organelle used by certain bacteria to translocate effector proteins across the cell membranes of target bacterial cells, killing them. Here, we use T6SS+ and T6SS- strains of V. cholerae, the pathogen that causes cholera in humans, and light sheet fluorescence microscopy for in vivo imaging to show that the T6SS provides an advantage to strains colonizing the larval zebrafish gut. Furthermore, we show that T6SS+ bacteria can invade and alter an existing population of a different species in the zebrafish gut, reducing its abundance and changing the form of its population dynamics. This work both demonstrates a mechanism for altering the gut microbiota with an invasive species and explores the processes controlling the stability and dynamics of the gut ecosystem. Research Corporation, Gordon and Betty Moore Foundation, and the Simons Foundation.

  3. Long range excitonic transport in a biomimetic system inspired by the bacterial light-harvesting apparatus

    International Nuclear Information System (INIS)

    Harel, Elad

    2012-01-01

    Photosynthesis, the process by which energy from sunlight drives cellular metabolism, relies on a unique organization of light-harvesting and reaction center complexes. Recently, the organization of light-harvesting LH2 complexes and dimeric reaction center-light-harvesting I-PufX core complexes in membranes of purple non-sulfur bacteria was revealed by atomic force microscopy [S. Bahatyrova et al., Nature (London) 430, 1058 (2004)]. Here, we discuss optimal exciton transfer in a biomimetic system closely modeled on the structure of LH2 and its organization within the membrane using a Markovian quantum model with dissipation and trapping added phenomenologically. In a deliberate manner, we neglect the high level detail of the bacterial light-harvesting complex and its interaction with the phonon bath in order to elucidate a set of design principles that may be incorporated in artificial pigment-scaffold constructs in a supramolecular assembly. We show that our scheme reproduces many of the most salient features found in their natural counterpart and may be largely explained by simple electrostatic considerations. Most importantly, we show that quantum effects act primarily to enforce robustness with respect to spatial and spectral disorder between and within complexes. The implications of such an arrangement are discussed in the context of biomimetic photosynthetic analogs capable of transferring energy efficiently across tens to hundreds of nanometers.

  4. Platelets and the innate immune system: mechanisms of bacterial-induced platelet activation.

    Science.gov (United States)

    Cox, D; Kerrigan, S W; Watson, S P

    2011-06-01

    It has become clear that platelets are not simply cell fragments that plug the leak in a damaged blood vessel; they are, in fact, also key components in the innate immune system, which is supported by the presence of Toll-like receptors (TLRs) on platelets. As the cells that respond first to a site of injury, they are well placed to direct the immune response to deal with any resulting exposure to pathogens. The response is triggered by bacteria binding to platelets, which usually triggers platelet activation and the secretion of antimicrobial peptides. The main platelet receptors that mediate these interactions are glycoprotein (GP)IIb-IIIa, GPIbα, FcγRIIa, complement receptors, and TLRs. This process may involve direct interactions between bacterial proteins and the receptors, or can be mediated by plasma proteins such as fibrinogen, von Willebrand factor, complement, and IgG. Here, we review the variety of interactions between platelets and bacteria, and look at the potential for inhibiting these interactions in diseases such as infective endocarditis and sepsis. © 2011 International Society on Thrombosis and Haemostasis.

  5. [Bacterial biofilms on PVC tubing's inner surface of hemodialysis water treatment system].

    Science.gov (United States)

    Yang, Sha; Jia, Ke; Peng, Youming; Liu, Hong; Liu, Yinghong; Chen, Xing; Liu, Fuyou

    2009-10-01

    To determine the morphology, bacteria and endotoxin content of biofilms on the inner surface of PVC tubes in hemodialysis water treatment system. We dissolved biofilms of segments before and after reverse osmosis machine for bacterial count and identification. We studied biofilm structure of segments before and after reverse osmosis machine with eyes and scanning electron microscope. Biofilms of all 7 segments were dissolved for qualitative and quantitative assay of endotoxin. The inner surface of segment before reverse osmosis machine was homogeneously distributed with activated carbon powder deposition. The segment after reverse osmosis machine was normal. With scanning electron microscope, biofilm with successive surface and sandwich was found on the inner surface of segment before reverse osmosis machine, formed by clustering bacillus, activated carbon powder and some coccus. Bacteria of the same shape and length were found on segment after reverse osmosis machine, but fewer and looser. Bacterial culture and identification showed the former was mostly gram-negative bacillus, the latter was only a few micrococcus. Endotoxin of biofilm was between 2.0 EU/mL and 4.0 EU/mL. Quantitative assay showed: segment after softener (2.821+/-0.807) EU/mL; segment after active charcoal canister(3.635+/-0.427) EU/mL; segment before reverse osmosis machine (3.687+/-0.271) EU/mL; segment after reverse osmosis machine (2.041+/-0.295) EU/mL; exit of power pump (1.983+/-0.390)EU/mL;the 1st dead space (2.373+/-0.535) EU/mL; and the 2nd dead space (2.858+/-0.690)EU/mL. Biofilms are found on the inner surface of segment before and after reverse osmosis machine. Endotoxin level from high to low is as follows: segment before reverse osmosis machine, segment after active charcoal canister, the 2nd dead space, segment after softener, the 1st dead space, segment after reverse osmosis machine, exit of power pump. The character of the bacteria and endotoxin of the biofilm can help us find

  6. Molecular Mechanisms of the Whole DNA Repair System: A Comparison of Bacterial and Eukaryotic Systems

    Directory of Open Access Journals (Sweden)

    Rihito Morita

    2010-01-01

    Full Text Available DNA is subjected to many endogenous and exogenous damages. All organisms have developed a complex network of DNA repair mechanisms. A variety of different DNA repair pathways have been reported: direct reversal, base excision repair, nucleotide excision repair, mismatch repair, and recombination repair pathways. Recent studies of the fundamental mechanisms for DNA repair processes have revealed a complexity beyond that initially expected, with inter- and intrapathway complementation as well as functional interactions between proteins involved in repair pathways. In this paper we give a broad overview of the whole DNA repair system and focus on the molecular basis of the repair machineries, particularly in Thermus thermophilus HB8.

  7. Predictive value of the APACHE II, SAPS II, SOFA and GCS scoring systems in patients with severe purulent bacterial meningitis.

    Science.gov (United States)

    Pietraszek-Grzywaczewska, Iwona; Bernas, Szymon; Łojko, Piotr; Piechota, Anna; Piechota, Mariusz

    2016-01-01

    Scoring systems in critical care patients are essential for predicting of the patient outcome and evaluating the therapy. In this study, we determined the value of the Acute Physiology and Chronic Health Evaluation II (APACHE II), Simplified Acute Physiology Score II (SAPS II), Sequential Organ Failure Assessment (SOFA) and Glasgow Coma Scale (GCS) scoring systems in the prediction of mortality in adult patients admitted to the intensive care unit (ICU) with severe purulent bacterial meningitis. We retrospectively analysed data from 98 adult patients with severe purulent bacterial meningitis who were admitted to the single ICU between March 2006 and September 2015. Univariate logistic regression identified the following risk factors of death in patients with severe purulent bacterial meningitis: APACHE II, SAPS II, SOFA, and GCS scores, and the lengths of ICU stay and hospital stay. The independent risk factors of patient death in multivariate analysis were the SAPS II score, the length of ICU stay and the length of hospital stay. In the prediction of mortality according to the area under the curve, the SAPS II score had the highest accuracy followed by the APACHE II, GCS and SOFA scores. For the prediction of mortality in a patient with severe purulent bacterial meningitis, SAPS II had the highest accuracy.

  8. Bacterial diversity of bacteriomes and organs of reproductive, digestive and excretory systems in two cicada species (Hemiptera: Cicadidae.

    Directory of Open Access Journals (Sweden)

    Zhou Zheng

    Full Text Available Cicadas form intimate symbioses with bacteria to obtain nutrients that are scarce in the xylem fluid they feed on. The obligate symbionts in cicadas are purportedly confined to specialized bacteriomes, but knowledge of bacterial communities associated with cicadas is limited. Bacterial communities in the bacteriomes and organs of reproductive, digestive and excretory systems of two cicada species (Platypleura kaempferi and Meimuna mongolica were investigated using different methods, and the bacterial diversity and distribution patterns of dominant bacteria in different tissues were compared. Within each species, the bacterial communities of testes are significantly different from those of bacteriomes and ovaries. The dominant endosymbiont Candidatus Sulcia muelleri is found not only in the bacteriomes and reproductive organs, but also in the "filter chamber + conical segment" of both species. The transmission mode of this endosymbiont in the alimentary canal and its effect on physiological processes merits further study. A novel bacterium of Rhizobiales, showing ~80% similarity to Candidatus Hodgkinia cicadicola, is dominant in the bacteriomes and ovaries of P. kaempferi. Given that the genome of H. cicadicola exhibits rapid sequence evolution, it is possible that this novel bacterium is a related endosymbiont with beneficial trophic functions similar to that of H. cicadicola in some other cicadas. Failure to detect H. cicadicola in M. mongolica suggests that it has been subsequently replaced by another bacterium, a yeast or gut microbiota which compensates for the loss of H. cicadicola. The distribution of this novel Rhizobiales species in other cicadas and its identification require further investigation to help establish the definition of the bacterial genus Candidatus Hodgkinia and to provide more information on sequence divergence of related endosymbionts of cicadas. Our results highlight the complex bacterial communities of cicadas, and

  9. Symbiotic nitrogen-fixing bacterial populations trapped from soils under agroforestry systems in the Western Amazon

    Directory of Open Access Journals (Sweden)

    Paula Marcela Duque Jaramillo

    2013-12-01

    Full Text Available Cowpea (Vigna unguiculata is an important grain-producing legume that can forego nitrogen fertilization by establishing an efficient symbiosis with nitrogen-fixing bacteria. Although inoculating strains have already been selected for this species, little is known about the genotypic and symbiotic diversity of native rhizobia. Recently, Bradyrhizobium has been shown to be the genus most frequently trapped by cowpea in agricultural soils of the Amazon region. We investigated the genetic and symbiotic diversity of 148 bacterial strains with different phenotypic and cultural properties isolated from the nodules of the trap species cowpea, which was inoculated with samples from soils under agroforestry systems from the western Amazon. Sixty non-nodulating strains indicated a high frequency of endophytic strains in the nodules. The 88 authenticated strains had varying symbiotic efficiency. The SPAD (Soil Plant Analysis Development index (indirect measurement of chlorophyll content was more efficient at evaluating the contribution of symbiotic N2-fixation than shoot dry matter under axenic conditions. Cowpea-nodulating bacteria exhibited a high level of genetic diversity, with 68 genotypes identified by BOX-PCR. Sequencing of the 16S rRNA gene showed a predominance of the genus Bradyrhizobium, which accounted for 70 % of all strains sequenced. Other genera identified were Rhizobium, Ochrobactrum, Paenibacillus, Bosea, Bacillus, Enterobacter, and Stenotrophomonas. These results support the promiscuity of cowpea and demonstrate the high genetic and symbiotic diversity of rhizobia in soils under agroforestry systems, with some strains exhibiting potential for use as inoculants. The predominance of Bradyrhizobium in land uses with different plant communities and soil characteristics reflects the adaptation of this genus to the Amazon region.

  10. Bacterial mitosis

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Borch, Jonas; Dam, Mette

    2003-01-01

    Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system...... movement is powered by insertional polymerization of ParM. Consistently, we find that segregating plasmids are positioned at the ends of extending ParM filaments. Thus, the process of R1 plasmid segregation in E. coli appears to be mechanistically analogous to the actin-based motility operating...

  11. Structure of the phosphotransferase domain of the bifunctional aminoglycoside-resistance enzyme AAC(6')-Ie-APH(2'')-Ia.

    Science.gov (United States)

    Smith, Clyde A; Toth, Marta; Bhattacharya, Monolekha; Frase, Hilary; Vakulenko, Sergei B

    2014-06-01

    The bifunctional acetyltransferase(6')-Ie-phosphotransferase(2'')-Ia [AAC(6')-Ie-APH(2'')-Ia] is the most important aminoglycoside-resistance enzyme in Gram-positive bacteria, conferring resistance to almost all known aminoglycoside antibiotics in clinical use. Owing to its importance, this enzyme has been the focus of intensive research since its isolation in the mid-1980s but, despite much effort, structural details of AAC(6')-Ie-APH(2'')-Ia have remained elusive. The structure of the Mg2GDP complex of the APH(2'')-Ia domain of the bifunctional enzyme has now been determined at 2.3 Å resolution. The structure of APH(2'')-Ia is reminiscent of the structures of other aminoglycoside phosphotransferases, having a two-domain architecture with the nucleotide-binding site located at the junction of the two domains. Unlike the previously characterized APH(2'')-IIa and APH(2'')-IVa enzymes, which are capable of utilizing both ATP and GTP as the phosphate donors, APH(2'')-Ia uses GTP exclusively in the phosphorylation of the aminoglycoside antibiotics, and in this regard closely resembles the GTP-dependent APH(2'')-IIIa enzyme. In APH(2'')-Ia this GTP selectivity is governed by the presence of a `gatekeeper' residue, Tyr100, the side chain of which projects into the active site and effectively blocks access to the adenine-binding template. Mutation of this tyrosine residue to a less bulky phenylalanine provides better access for ATP to the NTP-binding template and converts APH(2'')-Ia into a dual-specificity enzyme.

  12. Effect of two different polishing systems on fluoride release, surface roughness and bacterial adhesion of newly developed restorative materials.

    Science.gov (United States)

    Bayrak, Gokcen Deniz; Sandalli, Nuket; Selvi-Kuvvetli, Senem; Topcuoglu, Nursen; Kulekci, Guven

    2017-11-12

    To evaluate the effects of two different polishing systems on fluoride release, surface roughness and bacterial adhesion of five restorative materials MATERIALS AND METHODS: The study groups were comprised of five different restorative materials, Beautifil II (B); GCP Glass Fill (G); Amalgomer CR (A); Dyract XP (D); Fuji IX GP (F) and 21 specimens were prepared from each material. Each group was divided into three subgroups according to the polishing system: Mylar (control) (C), Sof-lex (S), and Enhance-Pogo (EP). The amount of fluoride release was measured using a fluoride ion-selective electrode and surface roughness was investigated with a profilometer. Bacterial adhesion on the materials was evaluated by optical density readouts for S.mutans on a spectrophotometer. The highest amount of fluoride was released from specimens in the S subgroup of group G during all measurement days. Surface roughness values were significantly lower in subgroup C than the other polishing systems in all study groups except group G (P restorative materials especially in glass ionomer-based materials. This article stated that polishing promoted a significant increase of fluoride release on restorative materials especially in glass ionomer-based materials. Further, proper polishing systems must be chosen according to the structure and composition of materials to provide the best clinical benefits in terms of fluoride release, surface roughness and bacterial adhesion. © 2017 Wiley Periodicals, Inc.

  13. Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance

    Directory of Open Access Journals (Sweden)

    Cashman Kathleen A

    2008-06-01

    Full Text Available Abstract Background There is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP, glycoprotein 1 (GP1, and glycoprotein 2 (GP2. Results Full-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein (MBP fusions in the Rosetta strains of Escherichia coli (E. coli using pMAL-c2x vectors. Average fusion protein yields per liter of culture for MBP-NP, MBP-GP1, and MBP-GP2 were 10 mg, 9 mg, and 9 mg, respectively. Each protein was captured from cell lysates using amylose resin, cleaved with Factor Xa, and purified using size-exclusion chromatography (SEC. Fermentation cultures resulted in average yields per liter of 1.6 mg, 1.5 mg, and 0.7 mg of purified NP, GP1 and GP2, respectively. LASV-specific antibodies in human convalescent sera specifically detected each of the purified recombinant LASV proteins, highlighting their utility in diagnostic applications. In addition, mouse hyperimmune ascitic fluids (MHAF against a panel of Old and New World arenaviruses demonstrated selective cross reactivity with LASV proteins in Western blot and enzyme-linked immunosorbent assay (ELISA. Conclusion These results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination.

  14. Acanthamoeba keratitis: synergy between amebic and bacterial cocontaminants in contact lens care systems as a prelude to infection.

    Science.gov (United States)

    Bottone, E J; Madayag, R M; Qureshi, M N

    1992-01-01

    We encountered a patient with Acanthamoeba keratitis whose contact lens care solution contained numerous trophozoites and cysts admixed with Xanthomonas maltophilia organisms, many of which were adherent to the trophozoite surface and internalized within endocytic vacuoles. Because of this finding, we investigated the role of bacterial cocontaminants in contact lens care systems as substrates for the growth of Acanthamoeba spp. Individual cocultivation of Acanthamoeba castellanii and A. polyphaga with X. maltophilia, Flavobacterium breve, and Pseudomonas paucimobilis showed better enhancement (1.5x) of ameba growth after 96 h than that obtained in the presence of Staphylococcus aureus, S. epidermidis, and Escherichia coli, the standard cocultivation species used for isolation of amebae from clinical specimens. Our data suggest that contamination of contact lens care systems with Acanthamoeba spp. and a bacterial species capable of supporting amebic growth may be the first step in the pathogenesis of ameba-induced keratitis by the provision of large inocula of amebae. Images PMID:1401013

  15. A bacterial two-hybrid system that utilizes Gateway cloning for rapid screening of protein-protein interactions.

    Science.gov (United States)

    Karna, S L Rajasekhar; Zogaj, Xhavit; Barker, Jeffrey R; Seshu, Janakiram; Dove, Simon L; Klose, Karl E

    2010-11-01

    Comprehensive clone sets representing the entire genome now exist for a large number of organisms. The Gateway entry clone sets are a particularly useful means to study gene function, given the ease of introduction into any Gateway-suitable destination vector. We have adapted a bacterial two-hybrid system for use with Gateway entry clone sets, such that potential interactions between proteins encoded within these clone sets can be determined by new destination vectors. We show that utilizing the Gateway clone sets for Francisella tularensis and Vibrio cholerae, known interactions between F. tularensis IglA and IglB and V. cholerae VipA and VipB could be confirmed with these destination vectors. Moreover, the introduction of unique tags into each vector allowed for visualization of the expressed hybrid proteins via Western immunoblot. This Gateway-suitable bacterial two-hybrid system provides a new tool for rapid screening of protein-protein interactions.

  16. Development of a Low Input and sustainable Switchgrass Feedstock Production System Utilizing Beneficial Bacterial Endophytes

    Energy Technology Data Exchange (ETDEWEB)

    Mei, Chuansheng [IALR; Nowak, Jerzy [VPISU; Seiler, John [VPISU

    2014-10-24

    Switchgrass represents a promising feedstock crop for US energy sustainability. However, its broad utilization for bioenergy requires improvements of biomass yields and stress tolerance. In this DOE funded project, we have been working on harnessing beneficial bacterial endophytes to enhance switchgrass performance and to develop a low input feedstock production system for marginal lands that do not compete with the production of food crops. We have demonstrated that one of most promising plant growth-promoting bacterial endophytes, Burkholderia phytofirmans strain PsJN, is able to colonize roots and significantly promote growth of switchgrass cv. Alamo under in vitro, growth chamber, greenhouse, as well as field conditions. Furthermore, PsJN bacterization improved growth and development of switchgrass seedlings, significantly stimulated plant root and shoot growth, and tiller number in the field, and enhanced biomass accumulation on both poor (p<0.001) and rich (p<0.05) soils, with more effective stimulation of plant growth in low fertility soil. Plant physiology measurements showed that PsJN inoculated Alamo had consistently lower transpiration, lower stomatal conductance, and higher water use efficiency in greenhouse conditions. These physiological changes may significantly contribute to the recorded growth enhancement. PsJN inoculation rapidly results in an increase in photosynthetic rates which contributes to the advanced growth and development. Some evidence suggests that this initial growth advantage decreases with time when resources are not limited such as in greenhouse studies. Additionally, better drought resistance and drought hardening were observed in PsJN inoculated switchgrass. Using the DOE-funded switchgrass EST microarray, in a collaboration with the Genomics Core Facility at the Noble Foundation, we have determined gene expression profile changes in both responsive switchgrass cv. Alamo and non-responsive cv. Cave-in-Rock (CR) following Ps

  17. Impact of biofilm formation and detachment on the transmission of bacterial antibiotic resistance in drinking water distribution systems.

    Science.gov (United States)

    Zhang, Junpeng; Li, Weiying; Chen, Jiping; Qi, Wanqi; Wang, Feng; Zhou, Yanyan

    2018-03-22

    There is growing awareness of the antibiotic-resistance crisis and its implications for public health among clinicians, researchers, politicians, and the public. We studied bacterial antibiotic resistance transition and the role of biofilms in a drinking water distribution system (DWDS). We tracked several different antibiotic resistant bacteria (ARB) with resistance to tetracycline, sulfamethoxazole, clindamycin, and norfloxacin for one year in a DWDS. The results indicated that the amount of ARB increased in tap water, presumably due to biofilm detachment. The effect of biofilm detachment on the transmission of antibiotic resistance from biofilms to tap water was explored by using a bacterial annular reactor. The percentage of ARB of inlet water, outlet water, and biofilms ranged from 0.26% to 9.85%, 1.08%-16.29%, and 0.52%-29.97%, respectively in a chlorinated system, and from 0.23% to 9.89%, 0.84%-16.84%, and 0.35%-17.77%, respectively, in a chloraminated system. The relative abundances of antibiotic resistance Acinetobacter, Sphingomonas, and Bradyrhizobium were higher in outlet water than in inlet water, as determined by high throughout sequencing. The amount of ARB percentage varied with the concentration of viable but non-culturable (VBNC) cells (r = 0.21, n = 160, P resistance mutation rate in VBNC cells. Our results suggest that biofilm detachment was promoted by disinfectant and affected the overall bacterial antibiotic resistance of microbes in tap water. Copyright © 2018 Elsevier Ltd. All rights reserved.

  18. Influence of hydraulic regimes on bacterial community structure and composition in an experimental drinking water distribution system.

    Science.gov (United States)

    Douterelo, I; Sharpe, R L; Boxall, J B

    2013-02-01

    Microbial biofilms formed on the inner-pipe surfaces of drinking water distribution systems (DWDS) can alter drinking water quality, particularly if they are mechanically detached from the pipe wall to the bulk water, such as due to changes in hydraulic conditions. Results are presented here from applying 454 pyrosequencing of the 16S ribosomal RNA (rRNA) gene to investigate the influence of different hydrological regimes on bacterial community structure and to study the potential mobilisation of material from the pipe walls to the network using a full scale, temperature-controlled experimental pipeline facility accurately representative of live DWDS. Analysis of pyrosequencing and water physico-chemical data showed that habitat type (water vs. biofilm) and hydraulic conditions influenced bacterial community structure and composition in our experimental DWDS. Bacterial community composition clearly differed between biofilms and bulk water samples. Gammaproteobacteria and Betaproteobacteria were the most abundant phyla in biofilms while Alphaproteobacteria was predominant in bulk water samples. This suggests that bacteria inhabiting biofilms, predominantly species belonging to genera Pseudomonas, Zooglea and Janthinobacterium, have an enhanced ability to express extracellular polymeric substances to adhere to surfaces and to favour co-aggregation between cells than those found in the bulk water. Highest species richness and diversity were detected in 28 days old biofilms with this being accentuated at highly varied flow conditions. Flushing altered the pipe-wall bacterial community structure but did not completely remove bacteria from the pipe walls, particularly under highly varied flow conditions, suggesting that under these conditions more compact biofilms were generated. This research brings new knowledge regarding the influence of different hydraulic regimes on the composition and structure of bacterial communities within DWDS and the implication that this

  19. Defense Islands in Bacterial and Archaeal Genomes and Prediction of Novel Defense Systems ▿†‡

    Science.gov (United States)

    Makarova, Kira S.; Wolf, Yuri I.; Snir, Sagi; Koonin, Eugene V.

    2011-01-01

    The arms race between cellular life forms and viruses is a major driving force of evolution. A substantial fraction of bacterial and archaeal genomes is dedicated to antivirus defense. We analyzed the distribution of defense genes and typical mobilome components (such as viral and transposon genes) in bacterial and archaeal genomes and demonstrated statistically significant clustering of antivirus defense systems and mobile genes and elements in genomic islands. The defense islands are enriched in putative operons and contain numerous overrepresented gene families. A detailed sequence analysis of the proteins encoded by genes in these families shows that many of them are diverged variants of known defense system components, whereas others show features, such as characteristic operonic organization, that are suggestive of novel defense systems. Thus, genomic islands provide abundant material for the experimental study of bacterial and archaeal antivirus defense. Except for the CRISPR-Cas systems, different classes of defense systems, in particular toxin-antitoxin and restriction-modification systems, show nonrandom clustering in defense islands. It remains unclear to what extent these associations reflect functional cooperation between different defense systems and to what extent the islands are genomic “sinks” that accumulate diverse nonessential genes, particularly those acquired via horizontal gene transfer. The characteristics of defense islands resemble those of mobilome islands. Defense and mobilome genes are nonrandomly associated in islands, suggesting nonadaptive evolution of the islands via a preferential attachment-like mechanism underpinned by the addictive properties of defense systems such as toxins-antitoxins and an important role of horizontal mobility in the evolution of these islands. PMID:21908672

  20. A type VI secretion system is involved in Pseudomonas fluorescens bacterial competition.

    Directory of Open Access Journals (Sweden)

    Victorien Decoin

    Full Text Available Protein secretion systems are crucial mediators of bacterial interactions with other organisms. Among them, the type VI secretion system (T6SS is widespread in Gram-negative bacteria and appears to inject toxins into competitor bacteria and/or eukaryotic cells. Major human pathogens, such as Vibrio cholerae, Burkholderia and Pseudomonas aeruginosa, express T6SSs. Bacteria prevent self-intoxication by their own T6SS toxins by producing immunity proteins, which interact with the cognate toxins. We describe here an environmental P. fluorescens strain, MFE01, displaying an uncommon oversecretion of Hcp (hemolysin-coregulated protein and VgrG (valine-glycine repeat protein G into the culture medium. These proteins are characteristic components of a functional T6SS. The aim of this study was to attribute a role to this energy-consuming overexpression of the T6SS. The genome of MFE01 contains at least two hcp genes (hcp1 and hcp2, suggesting that there may be two putative T6SS clusters. Phenotypic studies have shown that MFE01 is avirulent against various eukaryotic cell models (amebas, plant or animal cell models, but has antibacterial activity against a wide range of competitor bacteria, including rhizobacteria and clinical bacteria. Depending on the prey cell, mutagenesis of the hcp2 gene in MFE01 abolishes or reduces this antibacterial killing activity. Moreover, the introduction of T6SS immunity proteins from S. marcescens, which is not killed by MFE01, protects E. coli against MFE01 killing. These findings suggest that the protein encoded by hcp2 is involved in the killing activity of MFE01 mediated by effectors of the T6SS targeting the peptidoglycan of Gram-negative bacteria. Our results indicate that MFE01 can protect potato tubers against Pectobacterium atrosepticum, which causes tuber soft rot. Pseudomonas fluorescens is often described as a major PGPR (plant growth-promoting rhizobacterium, and our results suggest that there may be a

  1. Rhizosphere-associated Pseudomonas induce systemic resistance to herbivores at the cost of susceptibility to bacterial pathogens.

    Science.gov (United States)

    Haney, Cara H; Wiesmann, Christina L; Shapiro, Lori R; Melnyk, Ryan A; O'Sullivan, Lucy R; Khorasani, Sophie; Xiao, Li; Han, Jiatong; Bush, Jenifer; Carrillo, Juli; Pierce, Naomi E; Ausubel, Frederick M

    2017-10-31

    Plant-associated soil microbes are important mediators of plant defence responses to diverse above-ground pathogen and insect challengers. For example, closely related strains of beneficial rhizosphere Pseudomonas spp. can induce systemic resistance (ISR), systemic susceptibility (ISS) or neither against the bacterial foliar pathogen Pseudomonas syringae pv. tomato DC3000 (Pto DC3000). Using a model system composed of root-associated Pseudomonas spp. strains, the foliar pathogen Pto DC3000 and the herbivore Trichoplusia ni (cabbage looper), we found that rhizosphere-associated Pseudomonas spp. that induce either ISS and ISR against Pto DC3000 all increased resistance to herbivory by T. ni. We found that resistance to T. ni and resistance to Pto DC3000 are quantitative metrics of the jasmonic acid (JA)/salicylic acid (SA) trade-off and distinct strains of rhizosphere-associated Pseudomonas spp. have distinct effects on the JA/SA trade-off. Using genetic analysis and transcriptional profiling, we provide evidence that treatment of Arabidopsis with Pseudomonas sp. CH267, which induces ISS against bacterial pathogens, tips the JA/SA trade-off towards JA-dependent defences against herbivores at the cost of a subset of SA-mediated defences against bacterial pathogens. In contrast, treatment of Arabidopsis with the ISR strain Pseudomonas sp. WCS417 disrupts JA/SA antagonism and simultaneously primes plants for both JA- and SA-mediated defences. Our findings show that ISS against the bacterial foliar pathogens triggered by Pseudomonas sp. CH267, which is a seemingly deleterious phenotype, may in fact be an adaptive consequence of increased resistance to herbivory. Our work shows that pleiotropic effects of microbiome modulation of plant defences are important to consider when using microbes to modify plant traits in agriculture. © 2017 John Wiley & Sons Ltd.

  2. A Bacterial Pathogen uses Distinct Type III Secretion Systems to Alternate between Host Kingdom

    Science.gov (United States)

    Gram-negative bacterial pathogens of eukaryotes often secrete proteins directly into host cells via a needle-like protein channel called a ‘type III secretion system’ (T3SS). Bacteria that are adapted to either animal or plant hosts use phylogenetically distinct T3SSs for secreting proteins. Here, ...

  3. Long-Term Bacterial Dynamics in a Full-Scale Drinking Water Distribution System

    NARCIS (Netherlands)

    Prest, E.I.E.D.; Weissbrodt, D.G.; Hammes, F; van Loosdrecht, Mark C.M.; Vrouwenvelder, J.S.

    2016-01-01

    Large seasonal variations in microbial drinking water quality can occur in distribution networks, but are often not taken into account when evaluating results from short-term water sampling campaigns. Temporal dynamics in bacterial community characteristics were investigated during a two-year

  4. An iron detection system determines bacterial swarming initiation and biofilm formation

    NARCIS (Netherlands)

    Lin, Chuan-Sheng; Tsai, Yu-Huan; Chang, Chih-Jung; Tseng, Shun-Fu; Wu, Tsung-Ru; Lu, Chia-Chen; Wu, Ting-Shu; Lu, Jang-Jih; Horng, Jim-Tong; Martel, Jan; Ojcius, David M.; Lai, Hsin-Chih; Young, John D.; Andrews, S. C.; Robinson, A. K.; Rodriguez-Quinones, F.; Touati, D.; Yeom, J.; Imlay, J. A.; Park, W.; Marx, J. J.; Braun, V.; Hantke, K.; Cornelis, P.; Wei, Q.; Vinckx, T.; Troxell, B.; Hassan, H. M.; Verstraeten, N.; Lewis, K.; Hall-Stoodley, L.; Costerton, J. W.; Stoodley, P.; Kearns, D. B.; Losick, R.; Butler, M. T.; Wang, Q.; Harshey, R. M.; Lai, S.; Tremblay, J.; Deziel, E.; Overhage, J.; Bains, M.; Brazas, M. D.; Hancock, R. E.; Partridge, J. D.; Kim, W.; Surette, M. G.; Givskov, M.; Rather, P. N.; Houdt, R. Van; Michiels, C. W.; Mukherjee, S.; Inoue, T.; Frye, J. G.; McClelland, M.; McCarter, L.; Silverman, M.; Matilla, M. A.; Wu, Y.; Outten, F. W.; Singh, P. K.; Parsek, M. R.; Greenberg, E. P.; Welsh, M. J.; Banin, E.; Vasil, M. L.; Wosten, M. M.; Kox, L. F.; Chamnongpol, S.; Soncini, F. C.; Groisman, E. A.; Laub, M. T.; Goulian, M.; Krell, T.; Lai, H. C.; Lin, C. S.; Soo, P. C.; Tsai, Y. H.; Wei, J. R.; Wyckoff, E. E.; Mey, A. R.; Leimbach, A.; Fisher, C. F.; Payne, S. M.; Livak, K. J.; Schmittgen, T. D.; Clarke, M. B.; Hughes, D. T.; Zhu, C.; Boedeker, E. C.; Sperandio, V.; Stintzi, A.; Clarke-Pearson, M. F.; Brady, S. F.; Drake, E. J.; Gulick, A. M.; Qaisar, U.; Rowland, M. A.; Deeds, E. J.; Garcia, C. A.; Alcaraz, E. S.; Franco, M. A.; Rossi, B. N. Passerini de; Mehi, O.; Skaar, E. P.; Visaggio, D.; Nishino, K.; Dietz, P.; Gerlach, G.; Beier, D.; Bustin, S. A.; Schwyn, B.; Neilands, J. B.

    2016-01-01

    Iron availability affects swarming and biofilm formation in various bacterial species. However, how bacteria sense iron and coordinate swarming and biofilm formation remains unclear. Using Serratia marcescens as a model organism, we identify here a stage-specific iron-regulatory machinery comprising

  5. Bacterial Community Analysis, New Exoelectrogen Isolation and Enhanced Performance of Microbial Electrochemical Systems Using Nano-Decorated Anodes

    Science.gov (United States)

    Xu, Shoutao

    Microbial electrochemical systems (MESs) have attracted much research attention in recent years due to their promising applications in renewable energy generation, bioremediation, and wastewater treatment. In a MES, microorganisms interact with electrodes via electrons, catalyzing oxidation and reduction reactions at the anode and the cathode. The bacterial community of a high power mixed consortium MESs (maximum power density is 6.5W/m2) was analyzed by using denature gradient gel electrophoresis (DGGE) and 16S DNA clone library methods. The bacterial DGGE profiles were relatively complex (more than 10 bands) but only three brightly dominant bands in DGGE results. These results indicated there are three dominant bacterial species in mixed consortium MFCs. The 16S DNA clone library method results revealed that the predominant bacterial species in mixed culture is Geobacter sp (66%), Arcobacter sp and Citrobacter sp. These three bacterial species reached to 88% of total bacterial species. This result is consistent with the DGGE result which showed that three bright bands represented three dominant bacterial species. Exoelectrogenic bacterial strain SX-1 was isolated from a mediator-less microbial fuel cell by conventional plating techniques with ferric citrate as electron acceptor under anaerobic conditions. Phylogenetic analysis of the 16S rDNA sequence revealed that it was related to the members of Citrobacter genus with Citrobacter sp. sdy-48 being the most closely related species. The bacterial strain SX-1 produced electricity from citrate, acetate, glucose, sucrose, glycerol, and lactose in MFCs with the highest current density of 205 mA/m2 generated from citrate. Cyclic voltammetry analysis indicated that membrane associated proteins may play an important role in facilitating electron transfer from the bacteria to the electrode. This is the first study that demonstrates that Citrobacter species can transfer electrons to extracellular electron acceptors

  6. An in vivo comparison of internal bacterial colonization in two dental implant systems: identification of a pathogenic reservoir.

    Science.gov (United States)

    Mawhinney, Joanne; Connolly, Eimear; Claffey, Noel; Moran, Gary; Polyzois, Ioannis

    2015-04-01

    The aim of this study was to compare internal bacterial colonization in two implant systems, one screw root form (SRF) with an external hexagon connection and one plateau root form (PRF) with a Morse taper internal connection. Thirty-two implants; 12 SRF and 20 PRF, were sampled in 15 patients. All implants had been in function for at least 6 months prior to sampling. The implant restoration was removed and 10 µl of sterile saline was introduced into the implant well via a sterile glass syringe. The saline was drawn back up and transferred to the laboratory for microbiological analysis. The number of aerobic and anaerobic colony forming units per millilitre was determined and the dominant micro-organism in each sample was identified by 16s rRNA gene amplicon sequencing. There was a significant difference between bleeding on probing around the SRF implants (3%) and the PRF implants (28%) (p = 0.0496). Bacterial colonization was identified at 11 SRF and 19 PRF implants. The numbers of anaerobic bacteria recovered from PRF implants was significantly higher than that from SRF implants (p = 0.0002). Streptococcus species and Enterococcus faecalis were found to dominate. This in vivo study demonstrated bacterial colonization in both types of implant systems, irrespective of the type of connection. Significantly greater anaerobic counts were found in the Morse taper internal connection implants.

  7. Evidence of bacterial biofilm in tubing from hydraulic pathway of hemodialysis system.

    Science.gov (United States)

    Man, N K; Degremont, A; Darbord, J C; Collet, M; Vaillant, P

    1998-07-01

    Biofilms consist of microorganisms immobilized at a substratum surface embedded in an organic polymer matrix of bacterial origin. Tubing drawn from the fluid pathways within dialysis machines of various models were investigated for biofilm. Scanning electron microscopy (SEM), performed on approximately 2 cm2 samples of the tubing inner surfaces revealed that the inner surfaces of the tubing were covered with biofilms consisting of numerous deposits and glycocalix at different stages of formation with components containing bacteria and algae. Evaluations of biomass were performed from tubing sections of various lengths and inner diameters put in tubes containing water for injection and immersed in an ultrasound washtub for 1 h to ensure sloughing of the biofilm. Living bacteria were identified by plating on nutrient agar media and incubation for 48 h at 37 degrees C. Epifluorescent stains were used for the total bacteria count. Lipopolysaccharide levels were determined by the endotoxin activity measurements. Polyoside contents were determined by the colometric method, and the chemical oxygen demand was measured to evaluate the amount of organic substance. Biofilms detached from tubing samples drawn from the water path, bicarbonate path, and fresh dialysate path within dialysis machines contained approximately 1.10(3)-1.10(6) total bacteria/cm2, yet only some living bacteria were found. Endotoxin levels ranged from 1 to 12 EU/cm2. In contrast in the dialysate fluid, no bacteria were found, and the endotoxin content was under the detection level of the method. The polyoside content and chemical oxygen demand of the biomass ranged from 11 to 83 microg/cm2 and from 53 to 234 mg/cm2, respectively. It is concluded that a germ- and endotoxin-free dialysate does not exclude the risks and hazards of bacteria and endotoxin discharge from biofilm developed on the fluid pathway tubing, acting as a reservoir for continuous contamination, and efforts in the optimization of

  8. Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands.

    Science.gov (United States)

    Vercoe, Reuben B; Chang, James T; Dy, Ron L; Taylor, Corinda; Gristwood, Tamzin; Clulow, James S; Richter, Corinna; Przybilski, Rita; Pitman, Andrew R; Fineran, Peter C

    2013-04-01

    In prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (Cas) proteins constitute a defence system against bacteriophages and plasmids. CRISPR/Cas systems acquire short spacer sequences from foreign genetic elements and incorporate these into their CRISPR arrays, generating a memory of past invaders. Defence is provided by short non-coding RNAs that guide Cas proteins to cleave complementary nucleic acids. While most spacers are acquired from phages and plasmids, there are examples of spacers that match genes elsewhere in the host bacterial chromosome. In Pectobacterium atrosepticum the type I-F CRISPR/Cas system has acquired a self-complementary spacer that perfectly matches a protospacer target in a horizontally acquired island (HAI2) involved in plant pathogenicity. Given the paucity of experimental data about CRISPR/Cas-mediated chromosomal targeting, we examined this process by developing a tightly controlled system. Chromosomal targeting was highly toxic via targeting of DNA and resulted in growth inhibition and cellular filamentation. The toxic phenotype was avoided by mutations in the cas operon, the CRISPR repeats, the protospacer target, and protospacer-adjacent motif (PAM) beside the target. Indeed, the natural self-targeting spacer was non-toxic due to a single nucleotide mutation adjacent to the target in the PAM sequence. Furthermore, we show that chromosomal targeting can result in large-scale genomic alterations, including the remodelling or deletion of entire pre-existing pathogenicity islands. These features can be engineered for the targeted deletion of large regions of bacterial chromosomes. In conclusion, in DNA-targeting CRISPR/Cas systems, chromosomal interference is deleterious by causing DNA damage and providing a strong selective pressure for genome alterations, which may have consequences for bacterial evolution and pathogenicity.

  9. Cytotoxic Chromosomal Targeting by CRISPR/Cas Systems Can Reshape Bacterial Genomes and Expel or Remodel Pathogenicity Islands

    Science.gov (United States)

    Vercoe, Reuben B.; Chang, James T.; Dy, Ron L.; Taylor, Corinda; Gristwood, Tamzin; Clulow, James S.; Richter, Corinna; Przybilski, Rita; Pitman, Andrew R.; Fineran, Peter C.

    2013-01-01

    In prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (Cas) proteins constitute a defence system against bacteriophages and plasmids. CRISPR/Cas systems acquire short spacer sequences from foreign genetic elements and incorporate these into their CRISPR arrays, generating a memory of past invaders. Defence is provided by short non-coding RNAs that guide Cas proteins to cleave complementary nucleic acids. While most spacers are acquired from phages and plasmids, there are examples of spacers that match genes elsewhere in the host bacterial chromosome. In Pectobacterium atrosepticum the type I-F CRISPR/Cas system has acquired a self-complementary spacer that perfectly matches a protospacer target in a horizontally acquired island (HAI2) involved in plant pathogenicity. Given the paucity of experimental data about CRISPR/Cas–mediated chromosomal targeting, we examined this process by developing a tightly controlled system. Chromosomal targeting was highly toxic via targeting of DNA and resulted in growth inhibition and cellular filamentation. The toxic phenotype was avoided by mutations in the cas operon, the CRISPR repeats, the protospacer target, and protospacer-adjacent motif (PAM) beside the target. Indeed, the natural self-targeting spacer was non-toxic due to a single nucleotide mutation adjacent to the target in the PAM sequence. Furthermore, we show that chromosomal targeting can result in large-scale genomic alterations, including the remodelling or deletion of entire pre-existing pathogenicity islands. These features can be engineered for the targeted deletion of large regions of bacterial chromosomes. In conclusion, in DNA–targeting CRISPR/Cas systems, chromosomal interference is deleterious by causing DNA damage and providing a strong selective pressure for genome alterations, which may have consequences for bacterial evolution and pathogenicity. PMID:23637624

  10. Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands.

    Directory of Open Access Journals (Sweden)

    Reuben B Vercoe

    2013-04-01

    Full Text Available In prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPRs and their associated (Cas proteins constitute a defence system against bacteriophages and plasmids. CRISPR/Cas systems acquire short spacer sequences from foreign genetic elements and incorporate these into their CRISPR arrays, generating a memory of past invaders. Defence is provided by short non-coding RNAs that guide Cas proteins to cleave complementary nucleic acids. While most spacers are acquired from phages and plasmids, there are examples of spacers that match genes elsewhere in the host bacterial chromosome. In Pectobacterium atrosepticum the type I-F CRISPR/Cas system has acquired a self-complementary spacer that perfectly matches a protospacer target in a horizontally acquired island (HAI2 involved in plant pathogenicity. Given the paucity of experimental data about CRISPR/Cas-mediated chromosomal targeting, we examined this process by developing a tightly controlled system. Chromosomal targeting was highly toxic via targeting of DNA and resulted in growth inhibition and cellular filamentation. The toxic phenotype was avoided by mutations in the cas operon, the CRISPR repeats, the protospacer target, and protospacer-adjacent motif (PAM beside the target. Indeed, the natural self-targeting spacer was non-toxic due to a single nucleotide mutation adjacent to the target in the PAM sequence. Furthermore, we show that chromosomal targeting can result in large-scale genomic alterations, including the remodelling or deletion of entire pre-existing pathogenicity islands. These features can be engineered for the targeted deletion of large regions of bacterial chromosomes. In conclusion, in DNA-targeting CRISPR/Cas systems, chromosomal interference is deleterious by causing DNA damage and providing a strong selective pressure for genome alterations, which may have consequences for bacterial evolution and pathogenicity.

  11. Bacterial meningitis

    NARCIS (Netherlands)

    Roos, Karen L.; van de Beek, Diederik

    2010-01-01

    Bacterial meningitis is a neurological emergency. Empiric antimicrobial and adjunctive therapy should be initiated as soon as a single set of blood cultures has been obtained. Clinical signs suggestive of bacterial meningitis include fever, headache, meningismus, vomiting, photophobia, and an

  12. Heparin-Related Thrombocytopenia Triggered by Severe Status of Systemic Lupus Erythematosus and Bacterial Infection

    Directory of Open Access Journals (Sweden)

    Satoshi Suzuki

    2016-01-01

    Full Text Available A patient with severe lupus nephritis developed thrombocytopenia during treatment with high-dose steroids. In addition to viral- or disease-induced cytopenia, the pathology was believed to arise from diverse contributing factors, such as thrombotic microangiopathy and heparin-related thrombocytopenia (HIT. By combining plasma exchange therapy and intravenous cyclophosphamide, we successfully controlled the SLE activity and improved the thrombocytopenia. An antecedent bacterial infection or SLE activity is believed to have contributed to the concurrent HIT.

  13. A short-time scale colloidal system reveals early bacterial adhesion dynamics.

    Directory of Open Access Journals (Sweden)

    Christophe Beloin

    2008-07-01

    Full Text Available The development of bacteria on abiotic surfaces has important public health and sanitary consequences. However, despite several decades of study of bacterial adhesion to inert surfaces, the biophysical mechanisms governing this process remain poorly understood, due, in particular, to the lack of methodologies covering the appropriate time scale. Using micrometric colloidal surface particles and flow cytometry analysis, we developed a rapid multiparametric approach to studying early events in adhesion of the bacterium Escherichia coli. This approach simultaneously describes the kinetics and amplitude of early steps in adhesion, changes in physicochemical surface properties within the first few seconds of adhesion, and the self-association state of attached and free-floating cells. Examination of the role of three well-characterized E. coli surface adhesion factors upon attachment to colloidal surfaces--curli fimbriae, F-conjugative pilus, and Ag43 adhesin--showed clear-cut differences in the very initial phases of surface colonization for cell-bearing surface structures, all known to promote biofilm development. Our multiparametric analysis revealed a correlation in the adhesion phase with cell-to-cell aggregation properties and demonstrated that this phenomenon amplified surface colonization once initial cell-surface attachment was achieved. Monitoring of real-time physico-chemical particle surface properties showed that surface-active molecules of bacterial origin quickly modified surface properties, providing new insight into the intricate relations connecting abiotic surface physicochemical properties and bacterial adhesion. Hence, the biophysical analytical method described here provides a new and relevant approach to quantitatively and kinetically investigating bacterial adhesion and biofilm development.

  14. Inflammatory Monocyte Recruitment Is Regulated by Interleukin-23 during Systemic Bacterial Infection

    Science.gov (United States)

    Indramohan, Mohanalaxmi; Sieve, Amy N.; Break, Timothy J.

    2012-01-01

    Listeria monocytogenes is a Gram-positive intracellular pathogen that causes meningitis and septicemia in immunocompromised individuals and spontaneous abortion in pregnant women. The innate immune response against L. monocytogenes is primarily mediated by neutrophils and monocytes. Interleukin-23 (IL-23) is an important proinflammatory cytokine well known for its role in neutrophil recruitment in various infectious and autoimmune diseases. We have previously shown that IL-23 is required for host resistance against L. monocytogenes and for neutrophil recruitment to the liver, but not the spleen, during infection. Despite efficient neutrophil recruitment to the spleen, IL-23p19 knockout (KO) mice have an increased bacterial burden in this organ, suggesting that IL-23 may regulate the recruitment/function of another cell type to the spleen. In this study, we show that specific depletion of neutrophils abrogated the differences in bacterial burdens in the livers but not the spleens of C57BL/6 (B6) and IL-23p19 KO mice. Interestingly, L. monocytogenes-infected IL-23p19 KO mice had fewer monocytes in the spleen than B6 mice, as well as a reduction in the monocyte-recruiting chemokines CCL2 and CCL7. Additionally, the overall concentrations of tumor necrosis factor alpha (TNF-α) and nitric oxide (NO•), as well as the percentages and total numbers of monocytes producing TNF-α and NO•, were reduced in IL-23p19 KO mice compared to levels in B6 mice, leading to increased bacterial burdens in the spleens of L. monocytogenes-infected IL-23p19 KO mice. Collectively, our data establish that IL-23 is required for the optimal recruitment of TNF-α- and NO•-producing inflammatory monocytes, thus revealing a novel mechanism by which this proinflammatory cytokine provides protection against bacterial infection. PMID:22966045

  15. Assessing Interactions of Multiple Agrichemicals by Using Bacterial Assemblages in a Wetland Mesocosm System

    Directory of Open Access Journals (Sweden)

    Bruce Libman

    2005-08-01

    Full Text Available Agrichemicals may enter wetlands located adjacent to or downstream from agricultural fields. We investigated the individual and interactive effects of three agrichemicals [atrazine, chlorpyrifos, and monosodium acid methanearsonate (MSMA] and methyl mercury on abundance and heterotrophic potential of wetland heterotrophic bacteria assemblages. We used a factorial experimental design, in which chemicals were introduced in all possible combinations to 66 500-liter mesocosms at the Biological Field Station of the University of Mississippi. Methyl mercury was added to bring the total mercury (HG concentration to 0.4 mg/Kg wet weight at the sediment surface. Atrazine, chlorpyrifos, and MSMA were added at concentrations of 192, 51, and 219μg/L, respectively. Over 32 days of exposure, microbial heterotrophic activity was sensitive to only the interactive effect of HG*ATR*CPF in the sediments and only CPF in the water. Total bacterial numbers did not exhibit any significant treatment effects. Therefore, the effects of agrichemicals were reflected on cell-specific bacterial heterotrophic activity rather than bacterial population size.

  16. The two-component signal transduction system YvcPQ regulates the bacterial resistance to bacitracin in Bacillus thuringiensis.

    Science.gov (United States)

    Zhang, Shumeng; Li, Xinfeng; Wang, Xun; Li, Zhou; He, Jin

    2016-10-01

    YvcPQ is one of the two-component signal transduction systems that respond to specific stimuli and enable cells to adjust multiple cellular functions. It consists of a histidine kinase YvcQ and a response regulator YvcP. In this study, through searching the consensus sequence recognized by YvcP, we found four YvcP-binding motifs in the promoter regions of genes yvcR (BMB171_C4100), BMB171_C4385, kapD (BMB171_C4525) and BMB171_C4835 in Bacillus thuringiensis BMB171 which is a representative of Bacillus cereus group, and confirmed that these genes are regulated by YvcP. We compared the sequence of yvcPQ and its downstream genes in genus Bacillus, and found two different kinds of yvc locus, one was the yvcPQ-RS in B. subtilis species and the other was the yvcPQ-R-S1S2 in B. cereus group. Furthermore, we found that YvcP activates the transcription of yvcS1S2 (downstream of yvcR) to promote bacterial resistance to bacitracin and deletion of either yvcPQ operon or yvcS1S2 operon renders the bacterial cells more sensitive to bacitracin. This study enriched our understanding of both the YvcPQ's function and the mechanism of bacterial resistance to bacitracin.

  17. Zinc metalloproteinase ZmpC suppresses experimental pneumococcal meningitis by inhibiting bacterial invasion of central nervous systems.

    Science.gov (United States)

    Yamaguchi, Masaya; Nakata, Masanobu; Sumioka, Ryuichi; Hirose, Yujiro; Wada, Satoshi; Akeda, Yukihiro; Sumitomo, Tomoko; Kawabata, Shigetada

    2017-11-17

    Streptococcus pneumoniae is a leading cause of bacterial meningitis. Here, we investigated whether pneumococcal paralogous zinc metalloproteases contribute to meningitis onset. Findings of codon-based phylogenetic analyses indicated 3 major clusters in the Zmp family; ZmpA, ZmpC, and ZmpB, with ZmpD as a subgroup. In vitro invasion assays of human brain microvascular endothelial cells (hBMECs) showed that deletion of the zmpC gene in S. pneumoniae strain TIGR4 significantly increased bacterial invasion into hBMECs, whereas deletion of either zmpA or zmpB had no effect. In a mouse meningitis model, the zmpC deletion mutant exhibited increased invasion of the brain and was associated with increased matrix metalloproteinase-9 in plasma and mortality as compared with the wild type. We concluded that ZmpC suppresses pneumococcal virulence by inhibiting bacterial invasion of the central nervous system. Furthermore, ZmpC illustrates the evolutional theory stating that gene duplication leads to acquisition of novel function to suppress excessive mortality.

  18. Differential effects of interleukin-17 receptor signaling on innate and adaptive immunity during central nervous system bacterial infection

    Directory of Open Access Journals (Sweden)

    Vidlak Debbie

    2012-06-01

    Full Text Available Abstract Although IL-17A (commonly referred to as IL-17 has been implicated in the pathogenesis of central nervous system (CNS autoimmune disease, its role during CNS bacterial infections remains unclear. To evaluate the broader impact of IL-17 family members in the context of CNS infection, we utilized IL-17 receptor (IL-17R knockout (KO mice that lack the ability to respond to IL-17, IL-17F and IL-17E (IL-25. In this article, we demonstrate that IL-17R signaling regulates bacterial clearance as well as natural killer T (NKT cell and gamma-delta (γδ T cell infiltrates during Staphylococcus aureus-induced brain abscess formation. Specifically, when compared with wild-type (WT animals, IL-17R KO mice exhibited elevated bacterial burdens at days 7 and 14 following S. aureus infection. Additionally, IL-17R KO animals displayed elevated neutrophil chemokine production, revealing the ability to compensate for the lack of IL-17R activity. Despite these differences, innate immune cell recruitment into brain abscesses was similar in IL-17R KO and WT mice, whereas IL-17R signaling exerted a greater influence on adaptive immune cell recruitment. In particular, γδ T cell influx was increased in IL-17R KO mice at day 7 post-infection. In addition, NK1.1high infiltrates were absent in brain abscesses of IL-17R KO animals and, surprisingly, were rarely detected in the livers of uninfected IL-17R KO mice. Although IL-17 is a key regulator of neutrophils in other infection models, our data implicate an important role for IL-17R signaling in regulating adaptive immunity during CNS bacterial infection.

  19. Bacterial dispersal promotes biodegradation in heterogeneous systems exposed to osmotic stress

    Directory of Open Access Journals (Sweden)

    Anja Worrich

    2016-08-01

    Full Text Available Contaminant biodegradation in soils is hampered by the heterogeneous distribution of degrading communities colonizing isolated microenvironments as a result of the soil architecture. Over the last years, soil salinization was recognized as an additional problem especially in arid and semiarid ecosystems as it drastically reduces the activity and motility of bacteria. Here, we studied the importance of different spatial processes for benzoate biodegradation at an environmentally relevant range of osmotic potentials (ΔΨo using model ecosystems exhibiting a heterogeneous distribution of the soil borne bacterium Pseudomonas putida KT2440. Three systematically manipulated scenarios allowed us to cover the effects of i substrate diffusion ii substrate diffusion and autonomous bacterial dispersal, and iii substrate diffusion and autonomous as well as mediated bacterial dispersal along glass fiber networks mimicking fungal hyphae. To quantify the relative importance of the different spatial processes, we compared these heterogeneous scenarios to a reference value obtained for each ΔΨo by means of a quasi optimal scenario in which degraders were ab initio homogenously distributed. Substrate diffusion as the sole spatial process was insufficient to counteract the disadvantage due to spatial degrader heterogeneity at ΔΨo ranging from 0 to 1 MPa. In this scenario, only 13.8 to 21.3 % of the quasi-optimal biodegradation performance could be achieved. In the same range of ΔΨo values, substrate diffusion in combination with bacterial dispersal allowed between 68.6 % and 36.2 % of the performance showing a clear downwards trend with decreasing ΔΨo. At ¬ 1.5 MPa, however, this scenario performed worse than the diffusion scenario, possibly as a result of energetic disadvantages associated with flagellum synthesis and emerging requirements to exceed a critical population density to resist osmotic stress. Network mediated bacterial dispersal kept

  20. Responses of bacterial communities in arable soils in a rice-wheat cropping system to different fertilizer regimes and sampling times.

    Directory of Open Access Journals (Sweden)

    Jun Zhao

    Full Text Available Soil physicochemical properties, soil microbial biomass and bacterial community structures in a rice-wheat cropping system subjected to different fertilizer regimes were investigated in two seasons (June and October. All fertilizer regimes increased the soil microbial biomass carbon and nitrogen. Both fertilizer regime and time had a significant effect on soil physicochemical properties and bacterial community structure. The combined application of inorganic fertilizer and manure organic-inorganic fertilizer significantly enhanced the bacterial diversity in both seasons. The bacterial communities across all samples were dominated by Proteobacteria, Acidobacteria and Chloroflexi at the phylum level. Permutational multivariate analysis confirmed that both fertilizer treatment and season were significant factors in the variation of the composition of the bacterial community. Hierarchical cluster analysis based on Bray-Curtis distances further revealed that bacterial communities were separated primarily by season. The effect of fertilizer treatment is significant (P = 0.005 and accounts for 7.43% of the total variation in bacterial community. Soil nutrients (e.g., available K, total N, total P and organic matter rather than pH showed significant correlation with the majority of abundant taxa. In conclusion, both fertilizer treatment and seasonal changes affect soil properties, microbial biomass and bacterial community structure. The application of NPK plus manure organic-inorganic fertilizer may be a sound fertilizer practice for sustainable food production.

  1. Procalcitonin, C-reactive protein and serum lactate dehydrogenase in the diagnosis of bacterial sepsis, SIRS and systemic candidiasis.

    Science.gov (United States)

    Miglietta, Fabio; Faneschi, Maria Letizia; Lobreglio, Giambattista; Palumbo, Claudio; Rizzo, Adriana; Cucurachi, Marco; Portaccio, Gerolamo; Guerra, Francesco; Pizzolante, Maria

    2015-09-01

    The aim of this study was to evaluate procalcitonin (PCT), C-reactive protein (CRP), platelet count (PLT) and serum lactate dehydrogenase (LDH) as early markers for diagnosis of SIRS, bacterial sepsis and systemic candidiasis in intensive care unit (ICU) patients. Based on blood culture results, the patients were divided into a sepsis group (70 patients), a SIRS group (42 patients) and a systemic candidiasis group (33 patients). PCT, CRP, LDH and PLT levels were measured on day 0 and on day 2 from the sepsis symptom onset. PCT levels were higher in Gram negative sepsis than those in Gram positive sepsis, although the P value between the two subgroups is not significant (P=0.095). Bacterial sepsis group had higher PCT and CRP levels compared with the systemic candidiasis group, whereas PLT and LDH levels showed similar levels in these two subgroups. The AUC for PCT (AUC: 0.892, P candidiasis groups (P=0.093 N.S.). In conclusion, PCT can be used as a preliminary marker in the event of clinical suspicion of systemic candidiasis; however, low PCT levels (candidiasis and SIRS groups.

  2. Bacterial Enzymes and Antibiotic Resistance- Oral Presentation

    Energy Technology Data Exchange (ETDEWEB)

    Maltz, Lauren [SLAC National Accelerator Lab., Menlo Park, CA (United States)

    2015-08-25

    By using protein crystallography and X-ray diffraction, structures of bacterial enzymes were solved to gain a better understanding of how enzymatic modification acts as an antibacterial resistance mechanism. Aminoglycoside phosphotransferases (APHs) are one of three aminoglycoside modifying enzymes that confer resistance to the aminoglycoside antibiotics via enzymatic modification, rendering many drugs obsolete. Specifically, the APH(2”) family vary in their substrate specificities and also in their preference for the phosphate donor (ADP versus GDP). By solving the structures of members of the APH(2”) family of enzymes, we can see how domain movements are important to their substrate specificity. Our structure of the ternary complex of APH(2”)-IIIa with GDP and kanamycin, when compared to the known structures of APH(2”)-IVa, reveals that there are real physical differences between these two enzymes, a structural finding that explains why the two enzymes differ in their preferences for certain aminoglycosides. Another important group of bacterial resistance enzymes are the Class D β-lactamases. Oxacillinase carbapenemases (OXAs) are part of this enzyme class and have begun to confer resistance to ‘last resort’ drugs, most notably carbapenems. Our structure of OXA-143 shows that the conformational flexibility of a conserved hydrophobic residue in the active site (Val130) serves to control the entry of a transient water molecule responsible for a key step in the enzyme’s mechanism. Our results provide insight into the structural mechanisms of these two different enzymes.

  3. Parallel evolution of a type IV secretion system in radiating lineages of the host-restricted bacterial pathogen Bartonella.

    Science.gov (United States)

    Engel, Philipp; Salzburger, Walter; Liesch, Marius; Chang, Chao-Chin; Maruyama, Soichi; Lanz, Christa; Calteau, Alexandra; Lajus, Aurélie; Médigue, Claudine; Schuster, Stephan C; Dehio, Christoph

    2011-02-10

    Adaptive radiation is the rapid origination of multiple species from a single ancestor as the result of concurrent adaptation to disparate environments. This fundamental evolutionary process is considered to be responsible for the genesis of a great portion of the diversity of life. Bacteria have evolved enormous biological diversity by exploiting an exceptional range of environments, yet diversification of bacteria via adaptive radiation has been documented in a few cases only and the underlying molecular mechanisms are largely unknown. Here we show a compelling example of adaptive radiation in pathogenic bacteria and reveal their genetic basis. Our evolutionary genomic analyses of the α-proteobacterial genus Bartonella uncover two parallel adaptive radiations within these host-restricted mammalian pathogens. We identify a horizontally-acquired protein secretion system, which has evolved to target specific bacterial effector proteins into host cells as the evolutionary key innovation triggering these parallel adaptive radiations. We show that the functional versatility and adaptive potential of the VirB type IV secretion system (T4SS), and thereby translocated Bartonella effector proteins (Beps), evolved in parallel in the two lineages prior to their radiations. Independent chromosomal fixation of the virB operon and consecutive rounds of lineage-specific bep gene duplications followed by their functional diversification characterize these parallel evolutionary trajectories. Whereas most Beps maintained their ancestral domain constitution, strikingly, a novel type of effector protein emerged convergently in both lineages. This resulted in similar arrays of host cell-targeted effector proteins in the two lineages of Bartonella as the basis of their independent radiation. The parallel molecular evolution of the VirB/Bep system displays a striking example of a key innovation involved in independent adaptive processes and the emergence of bacterial pathogens

  4. Parallel evolution of a type IV secretion system in radiating lineages of the host-restricted bacterial pathogen Bartonella.

    Directory of Open Access Journals (Sweden)

    Philipp Engel

    2011-02-01

    Full Text Available Adaptive radiation is the rapid origination of multiple species from a single ancestor as the result of concurrent adaptation to disparate environments. This fundamental evolutionary process is considered to be responsible for the genesis of a great portion of the diversity of life. Bacteria have evolved enormous biological diversity by exploiting an exceptional range of environments, yet diversification of bacteria via adaptive radiation has been documented in a few cases only and the underlying molecular mechanisms are largely unknown. Here we show a compelling example of adaptive radiation in pathogenic bacteria and reveal their genetic basis. Our evolutionary genomic analyses of the α-proteobacterial genus Bartonella uncover two parallel adaptive radiations within these host-restricted mammalian pathogens. We identify a horizontally-acquired protein secretion system, which has evolved to target specific bacterial effector proteins into host cells as the evolutionary key innovation triggering these parallel adaptive radiations. We show that the functional versatility and adaptive potential of the VirB type IV secretion system (T4SS, and thereby translocated Bartonella effector proteins (Beps, evolved in parallel in the two lineages prior to their radiations. Independent chromosomal fixation of the virB operon and consecutive rounds of lineage-specific bep gene duplications followed by their functional diversification characterize these parallel evolutionary trajectories. Whereas most Beps maintained their ancestral domain constitution, strikingly, a novel type of effector protein emerged convergently in both lineages. This resulted in similar arrays of host cell-targeted effector proteins in the two lineages of Bartonella as the basis of their independent radiation. The parallel molecular evolution of the VirB/Bep system displays a striking example of a key innovation involved in independent adaptive processes and the emergence of bacterial

  5. A systematic approach for the assessment of bacterial growth-controlling factors linked to biological stability of drinking water in distribution systems

    KAUST Repository

    Prest, E. I.

    2016-01-06

    A systematic approach is presented for the assessment of (i) bacterial growth-controlling factors in drinking water and (ii) the impact of distribution conditions on the extent of bacterial growth in full-scale distribution systems. The approach combines (i) quantification of changes in autochthonous bacterial cell concentrations in full-scale distribution systems with (ii) laboratoryscale batch bacterial growth potential tests of drinking water samples under defined conditions. The growth potential tests were done by direct incubation of water samples, without modification of the original bacterial flora, and with flow cytometric quantification of bacterial growth. This method was shown to be reproducible (ca. 4% relative standard deviation) and sensitive (detection of bacterial growth down to 5 μg L-1 of added assimilable organic carbon). The principle of step-wise assessment of bacterial growth-controlling factors was demonstrated on bottled water, shown to be primarily carbon limited at 133 (±18) × 103 cells mL-1 and secondarily limited by inorganic nutrients at 5,500 (±1,700) × 103 cells mL-1. Analysis of the effluent of a Dutch full-scale drinking water treatment plant showed (1) bacterial growth inhibition as a result of end-point chlorination, (2) organic carbon limitation at 192 (±72) × 103 cells mL-1 and (3) inorganic nutrient limitation at 375 (±31) × 103 cells mL-1. Significantly lower net bacterial growth was measured in the corresponding full-scale distribution system (176 (±25) × 103 cells mL-1) than in the laboratory-scale growth potential test of the same water (294 (±35) × 103 cells mL-1), highlighting the influence of distribution on bacterial growth. The systematic approach described herein provides quantitative information on the effect of drinking water properties and distribution system conditions on biological stability, which can assist water utilities in decision-making on treatment or distribution system improvements to

  6. Insight into the mechanism of chemical modification of antibacterial agents by antibiotic resistance enzyme O-phosphotransferase-IIIA.

    Science.gov (United States)

    Power, Blake Hollett; Smith, Nathan; Downer, Brandon; Alisaraie, Laleh

    2017-01-01

    In the present work, the mechanism of resistance to aminoglycoside antibiotics was investigated. We examined the conformational changes of the O-phosphotransferase-IIIa enzyme, complexed with the antibiotics using MD simulations. The inhibitory effects of a group of antibacterial peptides against the enzyme were also examined, among which CP10A showed the highest affinity and the results correlated with the measured IC50 values. The regioselectivity of the phosphorylation reaction was shown to be in favor of the OH at the 5″ position versus the 3' of the antibiotic. The binding mode of CP10A was evaluated by means of MD simulation that resulted in recognizing its Trp8 and Arg13 residues binding near to where residues at the 3' and 5″ positions of the antibiotic would bind; thus, they are essential for the peptide inhibitory effect. The major open, semi-open, and closed conformations of the binding sites were identified throughout the MD trajectory, which enable the enzyme to regulate the influx of molecules into these sites. Based on the enzyme crystal structure, it was assumed that the 'antibiotic loop' of the enzyme is stable in its liganded mode; however, MD results revealed that the loop is highly flexible in both liganded and ligand-free modes. © 2016 John Wiley & Sons A/S.

  7. Systems biology of bacterial nitrogen fixation: High-throughput technology and its integrative description with constraint-based modeling

    Directory of Open Access Journals (Sweden)

    Resendis-Antonio Osbaldo

    2011-07-01

    Full Text Available Abstract Background Bacterial nitrogen fixation is the biological process by which atmospheric nitrogen is uptaken by bacteroids located in plant root nodules and converted into ammonium through the enzymatic activity of nitrogenase. In practice, this biological process serves as a natural form of fertilization and its optimization has significant implications in sustainable agricultural programs. Currently, the advent of high-throughput technology supplies with valuable data that contribute to understanding the metabolic activity during bacterial nitrogen fixation. This undertaking is not trivial, and the development of computational methods useful in accomplishing an integrative, descriptive and predictive framework is a crucial issue to decoding the principles that regulated the metabolic activity of this biological process. Results In this work we present a systems biology description of the metabolic activity in bacterial nitrogen fixation. This was accomplished by an integrative analysis involving high-throughput data and constraint-based modeling to characterize the metabolic activity in Rhizobium etli bacteroids located at the root nodules of Phaseolus vulgaris (bean plant. Proteome and transcriptome technologies led us to identify 415 proteins and 689 up-regulated genes that orchestrate this biological process. Taking into account these data, we: 1 extended the metabolic reconstruction reported for R. etli; 2 simulated the metabolic activity during symbiotic nitrogen fixation; and 3 evaluated the in silico results in terms of bacteria phenotype. Notably, constraint-based modeling simulated nitrogen fixation activity in such a way that 76.83% of the enzymes and 69.48% of the genes were experimentally justified. Finally, to further assess the predictive scope of the computational model, gene deletion analysis was carried out on nine metabolic enzymes. Our model concluded that an altered metabolic activity on these enzymes induced

  8. Nanotechnology to rescue bacterial bidirectional extracellular electron transfer in bioelectrochemical systems

    KAUST Repository

    Kalathil, Shafeer

    2016-03-17

    An electrically active bacterium transports its metabolically generated electrons to insoluble substrates such as electrodes via a process known as extracellular electron transport (EET). Bacterial EET is a crucial process in the geochemical cycling of metals, bioremediation and bioenergy devices such as microbial fuel cells (MFCs). Recently, it has been found that electroactive bacteria can reverse their respiratory pathways by accepting electrons from a negatively poised electrode to produce high-value chemicals such as ethanol in a process termed as microbial electrosynthesis (MES). A poor electrical connection between bacteria and the electrode hinders the EET and MES processes significantly. Also, the bidirectional EET process is sluggish and needs to be improved drastically to extend its practical applications. Several attempts have been undertaken to improve the bidirectional EET by employing various advanced nanostructured materials such as carbon nanotubes and graphene. This review covers the recent progress in the bacterial bidirectional EET processes using advanced nanostructures in the light of current understandings of bacteria–nanomaterial interactions.

  9. IMPACT OF BACTERIAL QUORUM SENSING SYSTEM ON CHANGES OF ORGANOLEPTIC MARKERS OF STORAGE CABBAGE

    Directory of Open Access Journals (Sweden)

    Kamila Myszka

    2015-04-01

    Full Text Available The regulation of phenotypes of vegetable-associated bacteria can be mediated through the production of acylated homoserine lactones (AHLs. This knowledge is essential for successful control of bacterial diseases of vegetable. The purpose of these investigations was to define the AHL patterns of gram-negative bacteria presented in storage cabbage by LC/MS technique. The phenomenon of regulating the pectinolytic activity and the exopolysaccharide (EPS production by AHLs that are associated with microbial spoilage of cabbage was also examined. Among 100 strains isolated from storage cabbage, 47 isolates produced AHLs. The results of the 16S rDNA sequence analysis indicated that selected microflora was highly closely related to Pantoea agglomerans, Rahnella aquatilis, Pseudomonas fluorescens, Pseudomonas syringae, and Pseudomonas cedrina (approximately 98%-99% confidence. The chemical nature of AHLs produced by selected microflora differ from species to species. The pattern of AHLs of Ps. cedrina consisting of C8-HSL, 3-oxo-C10-HSL, and 3-hydroxy-C14-HSL, has not been previously reported. The present study demonstrates that bacterial spoilage of storage cabbage is influenced by quorum sensing. Application of furanone C-30 that acts as quorum sensing inhibitor, caused the significant reduction in the production of EPS and pectinolytic enzymes by examined bacteria.

  10. Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMAP system.

    Science.gov (United States)

    Dunbar, Sherry A; Vander Zee, Coe A; Oliver, Kerry G; Karem, Kevin L; Jacobson, James W

    2003-05-01

    Escherichia coli, Salmonella, Listeria monocytogenes and Campylobacter jejuni are bacterial pathogens commonly implicated in foodborne illnesses. Generally used detection methods (i.e., culture, biochemical testing, ELISA and nucleic acid amplification) can be laborious, time-consuming and require multiple tests to detect all of the pathogens. Our objective was to develop rapid assays to simultaneously detect these four organisms through the presence of antigen or DNA using the Luminex LabMAP system. For nucleic acid detection, organism-specific capture probes corresponding to the 23S ribosomal RNA gene (rrl) were coupled covalently to LabMAP microspheres. Target molecules included synthetic complementary oligonucleotides and genomic DNA isolated from ATCC type strains or other well-characterized strains of each organism. Universal PCR primers were designed to amplify variable regions of bacterial 23S ribosomal DNA, yielding biotinylated amplicons of 86 to 109 bp in length. Varying quantities of targets were hybridized to the combined microsphere sets, labeled with streptavidin-R-phycoerythrin and analyzed on the Luminex(100) system. Results of nucleic acid detection assays, obtained in 30 to 40 min following amplification, correctly and specifically identified each bacterial species with a detection sensitivity of 10(3) to 10(5) genome copies. Capture-sandwich immunoassays were developed with organism-specific antibodies coupled to different microsphere sets. Microspheres were incubated with organism-specific standards and reactivity was assessed with biotinylated detection antibodies and streptavidin-R-phycoerythrin. In the immunoassays, microsphere-associated fluorescence was organism concentration dependent with detectable response at detection of pathogens. The practical significance of this multiplexing approach would be to provide more timely, economical and comprehensive information than is available with conventional isolation and identification

  11. The bacterial interlocked process ONtology (BiPON): a systemic multi-scale unified representation of biological processes in prokaryotes.

    Science.gov (United States)

    Henry, Vincent J; Goelzer, Anne; Ferré, Arnaud; Fischer, Stephan; Dinh, Marc; Loux, Valentin; Froidevaux, Christine; Fromion, Vincent

    2017-11-23

    High-throughput technologies produce huge amounts of heterogeneous biological data at all cellular levels. Structuring these data together with biological knowledge is a critical issue in biology and requires integrative tools and methods such as bio-ontologies to extract and share valuable information. In parallel, the development of recent whole-cell models using a systemic cell description opened alternatives for data integration. Integrating a systemic cell description within a bio-ontology would help to progress in whole-cell data integration and modeling synergistically. We present BiPON, an ontology integrating a multi-scale systemic representation of bacterial cellular processes. BiPON consists in of two sub-ontologies, bioBiPON and modelBiPON. bioBiPON organizes the systemic description of biological information while modelBiPON describes the mathematical models (including parameters) associated with biological processes. bioBiPON and modelBiPON are related using bridge rules on classes during automatic reasoning. Biological processes are thus automatically related to mathematical models. 37% of BiPON classes stem from different well-established bio-ontologies, while the others have been manually defined and curated. Currently, BiPON integrates the main processes involved in bacterial gene expression processes. BiPON is a proof of concept of the way to combine formally systems biology and bio-ontology. The knowledge formalization is highly flexible and generic. Most of the known cellular processes, new participants or new mathematical models could be inserted in BiPON. Altogether, BiPON opens up promising perspectives for knowledge integration and sharing and can be used by biologists, systems and computational biologists, and the emerging community of whole-cell modeling.

  12. The effect of E coli virulence on bacterial translocation and systemic sepsis in the neonatal rabbit model.

    Science.gov (United States)

    Jackson, R J; Smith, S D; Wadowsky, R M; DePudyt, L; Rowe, M I

    1991-04-01

    In the surgical neonate, three factors that promote bacterial translocation and systemic infection are: (1) intestinal bacterial colonization and overgrowth; (2) compromised host defenses; and (3) disruption of the mucosal epithelial barrier. The newborn rabbit provides an excellent model to study these factors. Like the human, there is early closure of the gut mucosa to macromolecules, and nutrition can be maintained by breast or formula feeding. This study examines translocation and systemic sepsis after colonization with virulent K1 and avirulent K100 strains of Escherichia coli. New Zealand white rabbit pups (2 to 5 days old) were studied. The gastrointestinal tracts of 12 were colonized with K1 E coli; 14 were colonized with K100 E coli; 12 control animals were not inoculated. Mesenteric lymph node (MLN), liver, spleen, and colon homogenate were cultured 72 hours postinoculation. No bacteria were isolated from the colons of all but one control animal. Translocation or systemic sepsis did not occur. Translocation to the MLN was significantly increased (P less than .03) in K1 (50%) and K100 (36%) groups compared with controls (0%). Translocation to liver and spleen (systemic sepsis) was significantly increased (P less than .03) in K1 animals (67%) compared with K100 (0%) or controls (0%). Colonization by both strains of E coli led to translocation to the MLN, but only K1 E coli caused systemic sepsis. This suggests that although colonization by E coli in the newborn leads to translocation to the MLN, progression to systemic sepsis is the result of characteristics of the bacteria and/or neonatal host responses.

  13. A microtiter plate-based system for the semiautomated growth and assay of bacterial cells for beta-galactosidase activity.

    Science.gov (United States)

    Menzel, R

    1989-08-15

    The introduction of automated pipetting devices, microtiter readers, and microcomputers makes it possible to significantly increase the number of enzyme assays which can be performed as part of the analysis of a biological process. A number of difficulties must be overcome in any such integrated approach based on the microtiter plate. Among these are cell lysis, temperature control, the conversion of microtiter reader optical density values to standard 1-cm path length values, and data management. The utility of such a scheme can be extended to gene regulation and bacterial genetics studies, if bacterial cell culture techniques can be incorporated into the scheme. This paper addresses these issues in the application of a semiautomated system to the study of the induction of the gyrA promoter by treatment (of a gyrA-lac operon fusion-containing strain) with a gyrase inhibitor. This system is specific to the requirements of our studies into the modulation of gene expression by DNA relaxation. The general approach, however, can be readily adapted to other studies.

  14. Radiometric detection of bacterial metabolism

    International Nuclear Information System (INIS)

    Camargo, E.E.; Wagner Junior, H.N.

    1979-01-01

    The measurement of 14 CO 2 produced by the bacterial oxidation of labelled compounds is discussed as a means of evaluating the bacterial metabolism. The following items are discussed:automated radiometric detection, types of graphs, clinical applications of the radiometric system and influential factors. Complementary studies on bacterial assimilation of substances are presented. (M.A.) [pt

  15. Bacterial community dynamics during the early stages of biofilm formation in a chlorinated experimental drinking water distribution system: implications for drinking water discolouration

    Science.gov (United States)

    Douterelo, I; Sharpe, R; Boxall, J

    2014-01-01

    Aims To characterize bacterial communities during the early stages of biofilm formation and their role in water discolouration in a fully representative, chlorinated, experimental drinking water distribution systems (DWDS). Methods and Results Biofilm development was monitored in an experimental DWDS over 28 days; subsequently the system was disturbed by raising hydraulic conditions to simulate pipe burst, cleaning or other system conditions. Biofilm cell cover was monitored by fluorescent microscopy and a fingerprinting technique used to assess changes in bacterial community. Selected samples were analysed by cloning and sequencing of the 16S rRNA gene. Fingerprinting analysis revealed significant changes in the bacterial community structure over time (P < 0·05). Cell coverage increased over time accompanied by an increase in bacterial richness and diversity. Conclusions Shifts in the bacterial community structure were observed along with an increase in cell coverage, bacterial richness and diversity. Species related to Pseudomonas spp. and Janthinobacterium spp. dominated the process of initial attachment. Based on fingerprinting results, the hydraulic regimes did not affect the bacteriological composition of biofilms, but they did influence their mechanical stability. Significance and Importance of the Study This study gives a better insight into the early stages of biofilm formation in DWDS and will contribute to the improvement of management strategies to control the formation of biofilms and the risk of discolouration. PMID:24712449

  16. Bacterial Proteasomes.

    Science.gov (United States)

    Jastrab, Jordan B; Darwin, K Heran

    2015-01-01

    Interest in bacterial proteasomes was sparked by the discovery that proteasomal degradation is required for the pathogenesis of Mycobacterium tuberculosis, one of the world's deadliest pathogens. Although bacterial proteasomes are structurally similar to their eukaryotic and archaeal homologs, there are key differences in their mechanisms of assembly, activation, and substrate targeting for degradation. In this article, we compare and contrast bacterial proteasomes with their archaeal and eukaryotic counterparts, and we discuss recent advances in our understanding of how bacterial proteasomes function to influence microbial physiology.

  17. Dynamic instability in the hook-flagellum system that triggers bacterial flicks

    Science.gov (United States)

    Jabbarzadeh, Mehdi; Fu, Henry Chien

    2018-01-01

    Dynamical bending, buckling, and polymorphic transformations of the flagellum are known to affect bacterial motility, but run-reverse-flick motility of monotrichous bacteria also involves the even more flexible hook connecting the flagellum to its rotary motor. Although flick initiation has been hypothesized to involve either static Euler buckling or dynamic bending of the hook, the precise mechanism of flick initiation remains unknown. Here, we find that flicks initiate via a dynamic instability requiring flexibility in both the hook and flagellum. We obtain accurate estimates of forces and torques on the hook that suggest that flicks occur for stresses below the (static) Euler buckling criterion, then provide a mechanistic model for flick initiation that requires combined bending of the hook and flagellum. We calculate the triggering torque-stiffness ratio and find that our predicted onset of dynamic instability corresponds well with experimental observations.

  18. Metabolic Regulation of a Bacterial Cell System with Emphasis on Escherichia coli Metabolism

    Science.gov (United States)

    Shimizu, Kazuyuki

    2013-01-01

    It is quite important to understand the overall metabolic regulation mechanism of bacterial cells such as Escherichia coli from both science (such as biochemistry) and engineering (such as metabolic engineering) points of view. Here, an attempt was made to clarify the overall metabolic regulation mechanism by focusing on the roles of global regulators which detect the culture or growth condition and manipulate a set of metabolic pathways by modulating the related gene expressions. For this, it was considered how the cell responds to a variety of culture environments such as carbon (catabolite regulation), nitrogen, and phosphate limitations, as well as the effects of oxygen level, pH (acid shock), temperature (heat shock), and nutrient starvation. PMID:25937963

  19. The dynamic instability in the hook/flagellum system that triggers bacterial flicks

    Science.gov (United States)

    Jabbarzadeh, Mehdi; Fu, Henry

    2017-11-01

    Dynamical bending, buckling, and polymorphic transformations of the flagellum are known to affect bacterial motility, but run-reverse-flick motility of monotrichous bacteria also involves the even more flexible hook, which connects the flagellum to the cell body. Here, we identify the dynamic buckling mechanism that produces flicks in Vibrio alginolyticus. Estimates of forces and torques on the hook from experimental observations suggest that flicks are triggered at stresses below the hook's static Euler buckling criterion. Using an accurate linearization of the Kirchoff rod model for the hook in a model of a swimming bacterium with rigid flagellum, we show that as hook stiffness decreases there is a transition from on-axis flagellar rotation with small hook deflections to flagellar precession with large deflections. When flagellum flexibility is incorporated, the precession is disrupted by significant flagellar bending - i.e., incipient flicks. The predicted onset of dynamic instabilities corresponds well with experimentally observed flick events.

  20. Baseplate assembly of phage Mu: Defining the conserved core components of contractile-tailed phages and related bacterial systems.

    Science.gov (United States)

    Büttner, Carina R; Wu, Yingzhou; Maxwell, Karen L; Davidson, Alan R

    2016-09-06

    Contractile phage tails are powerful cell puncturing nanomachines that have been co-opted by bacteria for self-defense against both bacteria and eukaryotic cells. The tail of phage T4 has long served as the paradigm for understanding contractile tail-like systems despite its greater complexity compared with other contractile-tailed phages. Here, we present a detailed investigation of the assembly of a "simple" contractile-tailed phage baseplate, that of Escherichia coli phage Mu. By coexpressing various combinations of putative Mu baseplate proteins, we defined the required components of this baseplate and delineated its assembly pathway. We show that the Mu baseplate is constructed through the independent assembly of wedges that are organized around a central hub complex. The Mu wedges are comprised of only three protein subunits rather than the seven found in the equivalent structure in T4. Through extensive bioinformatic analyses, we found that homologs of the essential components of the Mu baseplate can be identified in the majority of contractile-tailed phages and prophages. No T4-like prophages were identified. The conserved simple baseplate components were also found in contractile tail-derived bacterial apparatuses, such as type VI secretion systems, Photorhabdus virulence cassettes, and R-type tailocins. Our work highlights the evolutionary connections and similarities in the biochemical behavior of phage Mu wedge components and the TssF and TssG proteins of the type VI secretion system. In addition, we demonstrate the importance of the Mu baseplate as a model system for understanding bacterial phage tail-derived systems.

  1. Mineralization in newborn calves contributes to health, improve the antioxidant system and reduces bacterial infections.

    Science.gov (United States)

    Glombowsky, Patrícia; da Silva, Aleksandro S; Soldá, Natan M; Galli, Gabriela M; Biazus, Angelisa H; Campigotto, Gabriela; Bottari, Nathieli B; Sousa, Rejane S; Brisola, Maiara C; Stefani, Lenita M; Baldissera, Matheus D; Leal, Marta L R; Morsch, Vera M; Schetinger, Maria Rosa C; Machado, Gustavo

    2018-01-01

    The first phase of life of dairy calves has elevated mortality indices linked with low immunity and sanitary challenges, mainly bacterial infections are involved in the pathogenesis of diarrhea, the leading cause of death. Also, other important problem is the nutritional deficiencies, such as the mineral deficiency. Thus, the aim of this study was to evaluate whether an intramuscular mineral supplementation based on selenium, copper, potassium, magnesium and phosphorus possess beneficial effects on health of dairy calves. For this, ten calves were divided in two groups: the group A was supplemented with injectable mineral, while the group B was used as control group (without mineral supplementation). The mineral complex was administrated via intramuscularly at dose of 3 mL/animal on days 2 and 14 post-birth. The total blood was collected on days 2, 10, 20 and 30 of life of animals in order to analyze the antioxidant enzymes (catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx)), blood count and seric biochemistry linked with proteic, lipid and carbohydrate metabolism. Feces samples were also collected on days 10, 20 and 30 of life of animals to perform the total bacterial count, parasitological exam and fecal consistency score. Moreover, the weight and corporal temperature were also evaluated. The mineral supplementation presented beneficial properties to calves from birth to the 30th of life through the increase on activity of antioxidant enzymes, improvement of immunity, and avoiding problems linked with diarrhea and anemia, can be considered an interesting approach to prevent these alterations linked with high mortality in the period of life. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Enteric dysbiosis promotes antibiotic-resistant bacterial infection: systemic dissemination of resistant and commensal bacteria through epithelial transcytosis.

    Science.gov (United States)

    Yu, Linda Chia-Hui; Shih, Yi-An; Wu, Li-Ling; Lin, Yang-Ding; Kuo, Wei-Ting; Peng, Wei-Hao; Lu, Kuo-Shyan; Wei, Shu-Chen; Turner, Jerrold R; Ni, Yen-Hsuan

    2014-10-15

    Antibiotic usage promotes intestinal colonization of antibiotic-resistant bacteria. However, whether resistant bacteria gain dominance in enteric microflora or disseminate to extraintestinal viscera remains unclear. Our aim was to investigate temporal diversity changes in microbiota and transepithelial routes of bacterial translocation after antibiotic-resistant enterobacterial colonization. Mice drinking water with or without antibiotics were intragastrically gavaged with ampicillin-resistant (Amp-r) nonpathogenic Escherichia coli (E. coli) and given normal water afterward. The composition and spatial distribution of intestinal bacteria were evaluated using 16S rDNA sequencing and fluorescence in situ hybridization. Bacterial endocytosis in epithelial cells was examined using gentamicin resistance assay and transmission electromicroscopy. Paracellular permeability was assessed by tight junctional immunostaining and measured by tissue conductance and luminal-to-serosal dextran fluxes. Our results showed that antibiotic treatment enabled intestinal colonization and transient dominance of orally acquired Amp-r E. coli in mice. The colonized Amp-r E. coli peaked on day 3 postinoculation and was competed out after 1 wk, as evidenced by the recovery of commensals, such as Escherichia, Bacteroides, Lachnospiraceae, Clostridium, and Lactobacillus. Mucosal penetration and extraintestinal dissemination of exogenous and endogenous enterobacteria were correlated with abnormal epithelial transcytosis but uncoupled with paracellular tight junctional damage. In conclusion, antibiotic-induced enteric dysbiosis predisposes to exogenous infection and causes systemic dissemination of both antibiotic-resistant and commensal enterobacteria through transcytotic routes across epithelial layers. These results may help explain the susceptibility to sepsis in antibiotic-resistant enteric bacterial infection. Copyright © 2014 the American Physiological Society.

  3. Effect of disinfectant residual on the interaction between bacterial growth and assimilable organic carbon in a drinking water distribution system.

    Science.gov (United States)

    Li, Weiying; Zhang, Junpeng; Wang, Feng; Qian, Lin; Zhou, Yanyan; Qi, Wanqi; Chen, Jiping

    2018-03-09

    Public health is threatened by deteriorated water quality due to bacterial regrowth and uncontrolled growth-related problems in drinking water distribution systems (DWDSs). To investigate the scope of this problem, a two-year field study was conducted in south China. The amount of assimilable organic carbon (AOC), total cell concentrations (TCC), and intact cell concentrations (ICC) of water samples were determined by flow cytometry. The results indicated that ICC was significantly correlated to AOC concentration when the chlorine concentration was less than 0.15 mg/L, and ICC was lower at chlorine concentrations greater than 0.15 mg/L, suggesting that free chlorine level had effect on AOC and ICC. To further analyze the effect of disinfectant on AOC and bacterial growth, we designed an orthogonal experiment with different dosages of two commonly used disinfectants, chlorine and chloramine. The results demonstrated that high concentrations of free chlorine (>0.15 mg/L) and chloramine (>0.4 mg/L) were associated with relatively low proportions of intact cells and cultivable bacteria. Compared with chlorine, chloramine tended to cause lower AOC level and intact cells, likely because the chlorinated disinfection byproducts (DBPs) were more easily absorbed by bacteria than the chloraminated DBPs. Based on the statistical analysis of 240 water samples, ICC was limited when AOC concentration was less than 135 μg/L, while temperature and the number of small-size particles showed positive effects on ICC (P<0.05). We conclude that the use of chloramine and controlling particle numbers should be suitable strategies to limit bacterial regrowth. Copyright © 2018 Elsevier Ltd. All rights reserved.

  4. Chimeric FimH adhesin of type 1 fimbriae: a bacterial surface display system for heterologous sequences

    DEFF Research Database (Denmark)

    Pallesen, L; Poulsen, LK; Christiansen, Gunna

    1995-01-01

    of heterologous DNA segments encoding two reporter sequences. In the selected positions such insertions did not significantly alter the function of the FimH protein with regard to surface location and adhesive ability. The system seemed to be quite flexible, since chimeric versions of the FimH adhesin containing......The FimH adhesin of type 1 fimbriae has been tested as a display system for heterologous protein segments on the surface of Escherichia coli. This was carried out by introduction of restriction site handles (BglII sites) in two different positions in the fimH gene, followed by in-frame insertion...... as many as 56 foreign amino acids were transported to the bacterial surface as components of the fimbrial organelles. Furthermore, the foreign protein segments were recognized by insert-specific antibodies when expressed within chimeric proteins on the surface of the bacteria. The results from...

  5. An integrated flow cytometry-based system for real-time, high sensitivity bacterial detection and identification.

    Directory of Open Access Journals (Sweden)

    Dan A Buzatu

    Full Text Available Foodborne illnesses occur in both industrialized and developing countries, and may be increasing due to rapidly evolving food production practices. Yet some primary tools used to assess food safety are decades, if not centuries, old. To improve the time to result for food safety assessment a sensitive flow cytometer based system to detect microbial contamination was developed. By eliminating background fluorescence and improving signal to noise the assays accurately measure bacterial load or specifically identify pathogens. These assays provide results in minutes or, if sensitivity to one cell in a complex matrix is required, after several hours enrichment. Conventional assessments of food safety require 48 to 56 hours. The assays described within are linear over 5 orders of magnitude with results identical to culture plates, and report live and dead microorganisms. This system offers a powerful approach to real-time assessment of food safety, useful for industry self-monitoring and regulatory inspection.

  6. Assessing the impact of water treatment on bacterial biofilms in drinking water distribution systems using high-throughput DNA sequencing.

    Science.gov (United States)

    Shaw, Jennifer L A; Monis, Paul; Fabris, Rolando; Ho, Lionel; Braun, Kalan; Drikas, Mary; Cooper, Alan

    2014-12-01

    Biofilm control in drinking water distribution systems (DWDSs) is crucial, as biofilms are known to reduce flow efficiency, impair taste and quality of drinking water and have been implicated in the transmission of harmful pathogens. Microorganisms within biofilm communities are more resistant to disinfection compared to planktonic microorganisms, making them difficult to manage in DWDSs. This study evaluates the impact of four unique drinking water treatments on biofilm community structure using metagenomic DNA sequencing. Four experimental DWDSs were subjected to the following treatments: (1) conventional coagulation, (2) magnetic ion exchange contact (MIEX) plus conventional coagulation, (3) MIEX plus conventional coagulation plus granular activated carbon, and (4) membrane filtration (MF). Bacterial biofilms located inside the pipes of each system were sampled under sterile conditions both (a) immediately after treatment application ('inlet') and (b) at a 1 km distance from the treatment application ('outlet'). Bacterial 16S rRNA gene sequencing revealed that the outlet biofilms were more diverse than those sampled at the inlet for all treatments. The lowest number of unique operational taxonomic units (OTUs) and lowest diversity was observed in the MF inlet. However, the MF system revealed the greatest increase in diversity and OTU count from inlet to outlet. Further, the biofilm communities at the outlet of each system were more similar to one another than to their respective inlet, suggesting that biofilm communities converge towards a common established equilibrium as distance from treatment application increases. Based on the results, MF treatment is most effective at inhibiting biofilm growth, but a highly efficient post-treatment disinfection regime is also critical in order to prevent the high rates of post-treatment regrowth. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Bacterial community radial-spatial distribution in biofilms along pipe wall in chlorinated drinking water distribution system of East China.

    Science.gov (United States)

    Liu, Jingqing; Ren, Hongxing; Ye, Xianbei; Wang, Wei; Liu, Yan; Lou, Liping; Cheng, Dongqing; He, Xiaofang; Zhou, Xiaoyan; Qiu, Shangde; Fu, Liusong; Hu, Baolan

    2017-01-01

    Biofilms in the pipe wall may lead to water quality deterioration and biological instability in drinking water distribution systems (DWDSs). In this study, bacterial community radial-spatial distribution in biofilms along the pipe wall in a chlorinated DWDS of East China was investigated. Three pipes of large diameter (300, 600, and 600 mm) were sampled in this DWDS, including a ductile cast iron pipe (DCIP) with pipe age of 11 years and two gray cast iron pipes (GCIP) with pipe ages of 17 and 19 years, and biofilms in the upper, middle, and lower parts of each pipe wall were collected. Real-time quantitative polymerase chain reaction (qPCR) and culture-based method were used to quantify bacteria. 454 pyrosequencing was used for bacterial community analysis. The results showed that the biofilm density and total solid (TS) and volatile solid (VS) contents increased gradually from the top to the bottom along the pipe wall. Microorganisms were concentrated in the upper and lower parts of the pipe wall, together accounting for more than 80 % of the total biomass in the biofilms. The bacterial communities in biofilms were significantly different in different areas of the pipe wall and had no strong interaction. Compared with the upper and lower parts of the pipe wall, the bacterial community in the middle of the pipe wall was distributed evenly and had the highest diversity. The 16S rRNA genes of various possible pathogens, including Escherichia coli, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Salmonella enterica, were detected in the biofilms, and the abundances of these possible pathogens were highest in the middle of the pipe wall among three areas. The detachment of the biofilms is the main reason for the deterioration of the water quality in DWDSs. The results of this study suggest that the biofilms in the middle of the pipe wall have highly potential risk for drinking water safety, which provides new ideas for the study of the microbial ecology in

  8. Bacterial adhesion

    NARCIS (Netherlands)

    Loosdrecht, van M.C.M.

    1988-01-01

    As mentioned in the introduction of this thesis bacterial adhesion has been studied from a variety of (mostly practice oriented) starting points. This has resulted in a range of widely divergent approaches. In order to elucidate general principles in bacterial adhesion phenomena, we felt it

  9. A Generalist Protist Predator Enables Coexistence in Multitrophic Predator-Prey Systems Containing a Phage and the Bacterial Predator Bdellovibrio

    Directory of Open Access Journals (Sweden)

    Julia Johnke

    2017-10-01

    Full Text Available Complex ecosystems harbor multiple predators and prey species whose direct and indirect interactions are under study. In particular, the combined effects of predator diversity and resource preference on prey removal are not known. To understand the effect of interspecies interactions, combinations of micro-predators—i.e., protists (generalists, predatory bacteria (semi-specialists, and phages (specialists—and bacterial prey were tracked over a 72-h period in miniature membrane bioreactors. While specialist predators alone drove their preferred prey to extinction, the inclusion of a generalist resulted in uniform losses among prey species. Most importantly, presence of a generalist predator enabled coexistence of all predators and prey. As the generalist predator also negatively affected the other predators, we suggest that resource partitioning between predators and the constant availability of resources for bacterial growth due to protist predation stabilizes the system and keeps its diversity high. The appearance of resistant prey strains and subsequent evolution of specialist predators unable to infect the ancestral prey implies that multitrophic communities are able to persist and stabilize themselves. Interestingly, the appearance of BALOs and phages unable to infect their prey was only observed for the BALO or phage in the absence of additional predators or prey species indicating that competition between predators might influence coevolutionary dynamics.

  10. Genome-wide analysis of bacterial determinants of plant growth promotion and induced systemic resistance by Pseudomonas fluorescens.

    Science.gov (United States)

    Cheng, Xu; Etalo, Desalegn W; van de Mortel, Judith E; Dekkers, Ester; Nguyen, Linh; Medema, Marnix H; Raaijmakers, Jos M

    2017-11-01

    Pseudomonas fluorescens strain SS101 (Pf.SS101) promotes growth of Arabidopsis thaliana, enhances greening and lateral root formation, and induces systemic resistance (ISR) against the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). Here, targeted and untargeted approaches were adopted to identify bacterial determinants and underlying mechanisms involved in plant growth promotion and ISR by Pf.SS101. Based on targeted analyses, no evidence was found for volatiles, lipopeptides and siderophores in plant growth promotion by Pf.SS101. Untargeted, genome-wide analyses of 7488 random transposon mutants of Pf.SS101 led to the identification of 21 mutants defective in both plant growth promotion and ISR. Many of these mutants, however, were auxotrophic and impaired in root colonization. Genetic analysis of three mutants followed by site-directed mutagenesis, genetic complementation and plant bioassays revealed the involvement of the phosphogluconate dehydratase gene edd, the response regulator gene colR and the adenylsulfate reductase gene cysH in both plant growth promotion and ISR. Subsequent comparative plant transcriptomics analyses strongly suggest that modulation of sulfur assimilation, auxin biosynthesis and transport, steroid biosynthesis and carbohydrate metabolism in Arabidopsis are key mechanisms linked to growth promotion and ISR by Pf.SS101. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  11. Pathophysiology of bacterial infection of the central nervous system and its putative role in the pathogenesis of behavioral changes

    Directory of Open Access Journals (Sweden)

    Tatiana Barichello

    2013-03-01

    Full Text Available Invasion of the central nervous system (CNS by microorganisms is a severe and frequently fatal event during the course of many infectious diseases. It may lead to deafness, blindness, cerebral palsy, hydrocephalus, cognitive impairment or permanent neurological dysfunction in survivors. Pathogens can cross the blood-brain barrier by transcellular migration, paracellular migration and in infected macrophages. Pathogens may breach the blood-brain barrier and be recognized by antigen-presenting cells through the binding of Toll-like receptors. This induces the activation of nuclear factor kappa B or mitogen-activated protein kinase pathways and subsequently induces leukocyte infiltration and proliferation and the expression of numerous proteins involved in inflammation and the immune response. Many brain cells can produce cytokines, chemokines and other pro-inflammatory molecules in response to bacteria stimuli; as a consequence, polymorphonuclear cells are attracted and activated, and release large amounts of superoxide anion and nitric oxide, leading to peroxynitrite formation and oxidative stress. This cascade leads to lipid peroxidation, mitochondrial damage and blood-brain barrier breakdown, contributing to cellular injury during neuronal infection. Current evidence suggests that bacterial CNS infections can play a role in the etiopathogenesis of behavioral disorders by increasing pro-inflammatory cytokines and bacterial virulence factors. The aim of this review is to summarize the current knowledge of the relevant pathophysiologic steps in CNS infections.

  12. Influence of the Biliary System on Biliary Bacteria Revealed by Bacterial Communities of the Human Biliary and Upper Digestive Tracts.

    Directory of Open Access Journals (Sweden)

    Fuqiang Ye

    Full Text Available Biliary bacteria have been implicated in gallstone pathogenesis, though a clear understanding of their composition and source is lacking. Moreover, the effects of the biliary environment, which is known to be generally hostile to most bacteria, on biliary bacteria are unclear. Here, we investigated the bacterial communities of the biliary tract, duodenum, stomach, and oral cavity from six gallstone patients by using 16S rRNA amplicon sequencing. We found that all observed biliary bacteria were detectable in the upper digestive tract. The biliary microbiota had a comparatively higher similarity with the duodenal microbiota, versus those of the other regions, but with a reduced diversity. Although the majority of identified bacteria were greatly diminished in bile samples, three Enterobacteriaceae genera (Escherichia, Klebsiella, and an unclassified genus and Pyramidobacter were abundant in bile. Predictive functional analysis indicated enhanced abilities of environmental information processing and cell motility of biliary bacteria. Our study provides evidence for the potential source of biliary bacteria, and illustrates the influence of the biliary system on biliary bacterial communities.

  13. Nitrogen removal capacity and bacterial community dynamics of a Canon biofilter system at different organic matter concentrations.

    Science.gov (United States)

    García-Ruiz, María J; Maza-Márquez, Paula; González-López, Jesús; Osorio, Francisco

    2018-02-01

    Three Canon bench-scale bioreactors with a volume of 2 L operating in parallel were configured as submerged biofilters. In the present study we investigated the effects of a high ammonium concentration (320 mgNH 4 + · L -1 ) and different concentrations of organic matter (0, 100 and 400 mgCOD·L -1 ) on the nitrogen removal capacity and the bacterial community structure. After 60 days, the Canon biofilters operated properly under concentrations of 0 and 100 mgCOD·L -1 of organic matter, with nitrogen removal efficiencies up to 85%. However, a higher concentration of organic matter (400 mgCOD·L -1 ) produced a partial inhibition of nitrogen removal (68.1% efficiency). The addition of higher concentrations of organic matter a modified the bacterial community structure in the Canon biofilter, increasing the proliferation of heterotrophic bacteria related to the genera of Thauera, Longilinea, Ornatilinea, Thermomarinilinea, unclassified Chlorobiales and Denitratisoma. However, heterotrophic bacteria co-exist with Nitrosomonas and Candidatus Scalindua. Thus, our study confirms the co-existence of different microbial activities (AOB, Anammox and denitrification) and the adaptation of a fixed-biofilm system to different concentrations of organic matter. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Bacterial biomes and potential human pathogens in irrigation water and leafy greens from different production systems described using pyrosequencing.

    Science.gov (United States)

    Jongman, M; Chidamba, L; Korsten, L

    2017-10-01

    To investigate the influence of irrigation water microbial quality on leafy green vegetables produced in commercial and small-scale farms as well as homestead gardens using pyrosequencing. Next generation sequencing analysis of the V1-V3 hypervariable region of bacterial 16S rDNA was used to compare bacterial diversity in irrigation water sources and on leafy vegetables. In all samples (12) analysed, the phylum Proteobacteria (64·5%), class Gammaproteobacteria (56·6%) and genus Aeromonas (14·4%) were found to be dominant. Of the total Escherichia sequences detected in tested samples, lettuce (16·3%) from the one commercial farm harboured more sequences than cabbage from the small-scale farm (1·3%) or homestead gardens (1·9%). Escherichia sequences were detected in both irrigation water (4·6%) and on cabbage (1·3%) samples from the small-scale farm. The genus Salmonella was absent in borehole water but was detected in the holding dam water (biomes in irrigation water and on leafy greens were described with pyrosequencing and revealed insights into prevalence of potential and opportunistic pathogens across different production systems. © 2017 The Society for Applied Microbiology.

  15. Biofilm structures (EPS and bacterial communities) in drinking water distribution systems are conditioned by hydraulics and influence discolouration.

    Science.gov (United States)

    Fish, K; Osborn, A M; Boxall, J B

    2017-09-01

    High-quality drinking water from treatment works is degraded during transport to customer taps through the Drinking Water Distribution System (DWDS). Interactions occurring at the pipe wall-water interface are central to this degradation and are often dominated by complex microbial biofilms that are not well understood. This study uses novel application of confocal microscopy techniques to quantify the composition of extracellular polymeric substances (EPS) and cells of DWDS biofilms together with concurrent evaluation of the bacterial community. An internationally unique, full-scale, experimental DWDS facility was used to investigate the impact of three different hydraulic patterns upon biofilms and subsequently assess their response to increases in shear stress, linking biofilms to water quality impacts such as discolouration. Greater flow variation during growth was associated with increased cell quantity but was inversely related to EPS-to-cell volume ratios and bacterial diversity. Discolouration was caused and EPS was mobilised during flushing of all conditions. Ultimately, biofilms developed under low-varied flow conditions had lowest amounts of biomass, the greatest EPS volumes per cell and the lowest discolouration response. This research shows that the interactions between hydraulics and biofilm physical and community structures are complex but critical to managing biofilms within ageing DWDS infrastructure to limit water quality degradation and protect public health. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  16. Fat storage-inducing transmembrane (FIT or FITM proteins are related to lipid phosphatase/phosphotransferase enzymes

    Directory of Open Access Journals (Sweden)

    Matthew J Hayes

    2017-12-01

    Full Text Available Fat storage-inducing transmembrane (FIT or FITM proteins have been implicated in the partitioning of triacylglycerol to lipid droplets and the budding of lipid droplets from the ER. At the molecular level, the sole relevant interaction is that FITMs directly bind to triacyglycerol and diacylglycerol, but how they function at the molecular level is not known. Saccharomyces cerevisiae has two FITM homologues: Scs3p and Yft2p. Scs3p was initially identified because deletion leads to inositol auxotrophy, with an unusual sensitivity to addition of choline. This strongly suggests a role for Scs3p in phospholipid biosynthesis. Looking at the FITM family as widely as possible, we found that FITMs are widespread throughout eukaryotes, indicating presence in the last eukaryotic common ancestor. Protein alignments also showed that FITM sequences contain the active site of lipid phosphatase/phosphotransferase (LPT enzymes. This large family transfers phosphate-containing headgroups either between lipids or in exchange for water. We confirmed the prediction that FITMs are related to LPTs by showing that single amino-acid substitutions in the presumptive catalytic site prevented their ability to rescue growth of the mutants on low inositol/high choline media when over-expressed. The substitutions also prevented rescue of other phenotypes associated with loss of FITM in yeast, including mistargeting of Opi1p, defective ER morphology, and aberrant lipid droplet budding. These results suggest that Scs3p, Yft2p and FITMs in general are LPT enzymes involved in an as yet unknown critical step in phospholipid metabolism.

  17. Bacterial antagonists of fungal pathogens also control root-knot nematodes by induced systemic resistance of tomato plants.

    Science.gov (United States)

    Adam, Mohamed; Heuer, Holger; Hallmann, Johannes

    2014-01-01

    The potential of bacterial antagonists of fungal pathogens to control the root-knot nematode Meloidogyne incognita was investigated under greenhouse conditions. Treatment of tomato seeds with several strains significantly reduced the numbers of galls and egg masses compared with the untreated control. Best performed Bacillus subtilis isolates Sb4-23, Mc5-Re2, and Mc2-Re2, which were further studied for their mode of action with regard to direct effects by bacterial metabolites or repellents, and plant mediated effects. Drenching of soil with culture supernatants significantly reduced the number of egg masses produced by M. incognita on tomato by up to 62% compared to the control without culture supernatant. Repellence of juveniles by the antagonists was shown in a linked twin-pot set-up, where a majority of juveniles penetrated roots on the side without inoculated antagonists. All tested biocontrol strains induced systemic resistance against M. incognita in tomato, as revealed in a split-root system where the bacteria and the nematodes were inoculated at spatially separated roots of the same plant. This reduced the production of egg masses by up to 51%, while inoculation of bacteria and nematodes in the same pot had only a minor additive effect on suppression of M. incognita compared to induced systemic resistance alone. Therefore, the plant mediated effect was the major reason for antagonism rather than direct mechanisms. In conclusion, the bacteria known for their antagonistic potential against fungal pathogens also suppressed M. incognita. Such "multi-purpose" bacteria might provide new options for control strategies, especially with respect to nematode-fungus disease complexes that cause synergistic yield losses.

  18. Chronic bacterial-viral vasculitis as manifestation of systemic inflammatory response syndrome in females.

    Science.gov (United States)

    Kvantaliani, T; Tsiklauri, P; Pargalava, N

    2005-09-01

    Study involved 52 women (mean age 43+/-1,3) without any evidence of CHD, suffering from dull or acute prolonged non-anginal chest pain, with undergoing chronic bacterial-viral infection--Chlamydia pneumonia in combination with HSV-I, CMV and/or EBV. 30--patients serum-positive to plasma markers (IgG antibodies) of previous infection were enclosed in group I, 22--with plasma consumption of IgA+IgG antibody complex demonstrating re-infection/reactivation phase of disease--in group II and 20 healthy serum-negative females (median age 47+/-2,8 years)--controls (group III). The intergroup analysis revealed the complex of disturbances in some plasma parameters of II group patients, namely significant elevation of CRP, F and LPO activity vs. I and III group data, along with reduced parameters of immune status in both groups of infected persons. The patients with mixed infections showed the high frequency of specific re-polarization phase abnormalities, cardiac rhythm and conduction disturbances. These changes together with intimate-medial wall injuries of inflammatory origin disclosed by DS technique enables us to suggest that in women with previous chronic untreated or insufficiently treated infection, in cases of their overload activity, recurrent or super-infection may provide deep immunosuppressive conditions leading to farther cardio-vascular abnormalities.

  19. Assessment of toxicity of a glyphosate-based formulation using bacterial systems in lake water.

    Science.gov (United States)

    Amorós, I; Alonso, J L; Romaguera, S; Carrasco, J M

    2007-05-01

    A new Aeromonas bioassay is described to assess the potential harmful effects of the glyphosate-based herbicide, Roundup, in the Albufera lake, a protected area near Valencia. Viability markers as membrane integrity, culturability and beta-galactosidase production of Aeromonas caviae were studied to determine the influence of the herbicide in the bacterial cells. Data from the multifactor analysis of variance test showed no significant differences (P>0.05) between A. caviae counts of viability markers at the studied concentrations (0, 50 and 100 mg l-1 of glyphosate). The effects of Roundup on microbial biota present in the lake were assessed by measuring the number of indigenous mesophilic Aeromonas in presence of different amounts of the herbicide at 0, 50 and 100 mg l-1 of glyphosate. In samples containing 50 and 100 mg l-1 of glyphosate a significant (PAlbufera lake water to Microtox luminescent bacterium (Vibrio fischeri) also was determined. The EC50 values obtained were 36.4 mg l-1 and 64.0 mgl-1 of glyphosate respectively. The acidity (pH 4.5) of the herbicide formulation was the responsible of the observed toxicity.

  20. Microfluidic system for the identification of bacterial pathogens causing urinary tract infections

    Science.gov (United States)

    Becker, Holger; Hlawatsch, Nadine; Haraldsson, Tommy; van der Wijngaart, Wouter; Lind, Anders; Malhotra-Kumar, Surbi; Turlej-Rogacka, Agata; Goossens, Herman

    2015-03-01

    Urinary tract infections (UTIs) are among the most common bacterial infections and pose a significant healthcare burden. The growing trend in antibiotic resistance makes it mandatory to develop diagnostic kits which allow not only the determination of a pathogen but also the antibiotic resistances. We have developed a microfluidic cartridge which takes a direct urine sample, extracts the DNA, performs an amplification using batch-PCR and flows the sample over a microarray which is printed into a microchannel for fluorescence detection. The cartridge is injection-molded out of COP and contains a set of two-component injection-molded rotary valves to switch between input and to isolate the PCR chamber during thermocycling. The hybridization probes were spotted directly onto a functionalized section of the outlet microchannel. We have been able to successfully perform PCR of E.coli in urine in this chip and perform a fluorescence detection of PCR products. An upgraded design of the cartridge contains the buffers and reagents in blisters stored on the chip.

  1. Construction of a high efficiency copper adsorption bacterial system via peptide display and its application on copper dye polluted wastewater.

    Science.gov (United States)

    Maruthamuthu, Murali Kannan; Nadarajan, Saravanan Prabhu; Ganesh, Irisappan; Ravikumar, Sambandam; Yun, Hyungdon; Yoo, Ik-Keun; Hong, Soon Ho

    2015-11-01

    For the construction of an efficient copper waste treatment system, a cell surface display strategy was employed. The copper adsorption ability of recombinant bacterial strains displaying three different copper binding peptides were evaluated in LB Luria-Bertani medium (LB), artificial wastewater, and copper phthalocyanine containing textile dye industry wastewater samples. Structural characteristics of the three peptides were also analyzed by similarity-based structure modeling. The best binding peptide was chosen for the construction of a dimeric peptide display and the adsorption ability of the monomeric and dimeric peptide displayed strains were compared. The dimeric peptide displayed strain showed superior copper adsorption in all three tested conditions (LB, artificial wastewater, and textile dye industry wastewater). When the strains were exposed to copper phthalocyanine dye polluted wastewater, the dimeric peptide display [543.27 µmol/g DCW dry cell weight (DCW)] showed higher adsorption of copper when compared with the monomeric strains (243.53 µmol/g DCW).

  2. A disposable bacterial lysis cartridge (BLC) suitable for an in situ water-borne pathogen detection system.

    Science.gov (United States)

    Lee, Eun-Hee; Lim, Hyun Jeong; Son, Ahjeong; Chua, Beelee

    2015-11-21

    We constructed a disposable bacterial lysis cartridge (BLC) suitable for an in situ pathogen detection system. It had an in-built micro corona discharge based ozone generator that provided ozone for cell lysis. Using a custom sample handling platform, its performance was evaluated with a Gram-positive bacterium of Bacillus subtilis. It was capable of achieving a similar degree of lysis as a commercial ultrasonic dismembrator with a P-1 microprobe in 10 min at an air pump flow rate of 29.4 ml min(-1) and an ozone generator operating voltage of 1600 V. The lysing duration could be significantly reduced to 5 min by increasing the air pump flow rate and the ozone generator operating voltage as well as by the addition of sodium dodecyl sulfate (SDS).

  3. Intra-Species Bacterial Quorum Sensing Studied at Single Cell Level in a Double Droplet Trapping System

    Directory of Open Access Journals (Sweden)

    Wilhelm T. S. Huck

    2013-05-01

    Full Text Available In this paper, we investigated the intra-species bacterial quorum sensing at the single cell level using a double droplet trapping system. Escherichia coli transformed to express the quorum sensing receptor protein, LasR, were encapsulated in microdroplets that were positioned adjacent to microdroplets containing the autoinducer, N-(3-oxododecanoyl-L-homoserine lactone (OdDHL. Functional activation of the LasR protein by diffusion of the OdDHL across the droplet interface was measured by monitoring the expression of green fluorescent protein (GFP from a LasR-dependent promoter. A threshold concentration of OdDHL was found to induce production of quorum-sensing associated GFP by E. coli. Additionally, we demonstrated that LasR-dependent activation of GFP expression was also initiated when the adjacent droplets contained single E. coli transformed with the OdDHL synthase gene, LasI, representing a simple quorum sensing circuit between two droplets.

  4. Changes in soil physicochemical properties and soil bacterial community in mulberry (Morus alba L.)/alfalfa (Medicago sativa L.) intercropping system.

    Science.gov (United States)

    Zhang, Meng-Meng; Wang, Ning; Hu, Yan-Bo; Sun, Guang-Yu

    2018-03-13

    A better understanding of tree-based intercropping effects on soil physicochemical properties and bacterial community has a potential contribution to improvement of agroforestry productivity and sustainability. In this study, we investigated the effects of mulberry/alfalfa intercropping on soil physicochemical properties and soil bacterial community by MiSeq sequencing of bacterial 16S rRNA gene. The results showed a significant increase in the contents of available nitrogen, available phosphate, available potassium, and total carbon in the rhizosphere soil of the intercropped alfalfa. Sequencing results showed that intercropping improved bacterial richness and diversity of mulberry and alfalfa based on richness estimates and diversity indices. The relative abundances of Proteobacteria, Actinobacteria, and Firmicutes were significantly higher in intercropping mulberry than in monoculture mulberry; and the abundances of Proteobacteria, Bacteroidetes, and Gemmatimonadetes in the intercropping alfalfa were markedly higher than that in monoculture alfalfa. Bacterial taxa with soil nutrients cycling were enriched in the intercropping system. There were higher relative abundances of Bacillus (0.32%), Pseudomonas (0.14%), and Microbacterium (0.07%) in intercropping mulberry soil, and Bradyrhizobium (1.0%), Sphingomonas (0.56%), Pseudomonas (0.18%), Microbacterium (0.15%), Rhizobium (0.09%), Neorhizobium (0.08%), Rhodococcus (0.06%), and Burkholderia (0.04%) in intercropping alfalfa soil. Variance partition analysis showed that planting pattern contributed 26.7% of the total variation of bacterial community, and soil environmental factors explained approximately 56.5% of the total variation. This result indicated that the soil environmental factors were more important than the planting pattern in shaping the bacterial community in the field soil. Overall, mulberry/alfalfa intercropping changed soil bacterial community, which was related to changes in soil total carbon

  5. Construction of a Novel Magnetic Targeting Anti-Tumor Drug Delivery System: Cytosine Arabinoside-Loaded Bacterial Magnetosome

    Directory of Open Access Journals (Sweden)

    Wenguo Wu

    2013-09-01

    Full Text Available To ease the side effects triggered by cytosine arabinoside (Ara-C for acute leukemia treatment, a novel magnetic targeting anti-tumor drug delivery system was constructed through bacterial magnetosomes (BMs from Magnetospirillum magneticum AMB-1 combined with Ara-C by crosslinking of genipin (GP. The results showed that Ara-C could be bonded onto the membrane surface of BMs effectively through chemical crosslinking induced by dual hand reagents GP. The average diameters of BMs and Ara-C-coupled BMs (ABMs were 42.0 ± 8.6 and 72.7 ± 6.0 nm respectively, and the zeta potentials (−38.1 ± 9.1 revealed that these systems were stable, confirming the stability of the system. The optimal encapsulation efficiency and drug loading were 89.05% ± 2.33% and 47.05% ± 0.64% respectively when crosslinking reaction lasted for 72 h. The system also presented long-term stability and release behaviors without initial burst release (Ara-C could be released 80% within three months. Our results indicate that BMs have great potential in biomedical and clinical fields as a novel anti-tumor drug carrier.

  6. Bacterial Vaginosis

    Science.gov (United States)

    ... Archive STDs Home Page Bacterial Vaginosis (BV) Chlamydia Gonorrhea Genital Herpes Hepatitis HIV/AIDS & STDs Human Papillomavirus ( ... of getting other STDs, such as chlamydia and gonorrhea . These bacteria can sometimes cause pelvic inflammatory disease ( ...

  7. Cooperative working of bacterial chromosome replication proteins generated by a reconstituted protein expression system

    Science.gov (United States)

    Fujiwara, Kei; Katayama, Tsutomu; Nomura, Shin-ichiro M.

    2013-01-01

    Replication of all living cells relies on the multirounds flow of the central dogma. Especially, expression of DNA replication proteins is a key step to circulate the processes of the central dogma. Here we achieved the entire sequential transcription–translation–replication process by autonomous expression of chromosomal DNA replication machineries from a reconstituted transcription–translation system (PURE system). We found that low temperature is essential to express a complex protein, DNA polymerase III, in a single tube using the PURE system. Addition of the 13 genes, encoding initiator, DNA helicase, helicase loader, RNA primase and DNA polymerase III to the PURE system gave rise to a DNA replication system by a coupling manner. An artificial genetic circuit demonstrated that the DNA produced as a result of the replication is able to provide genetic information for proteins, indicating the in vitro central dogma can sequentially undergo two rounds. PMID:23737447

  8. Sonic Hedgehog Signaling Regulates Hematopoietic Stem/Progenitor Cell Activation during the Granulopoietic Response to Systemic Bacterial Infection.

    Science.gov (United States)

    Shi, Xin; Wei, Shengcai; Simms, Kevin J; Cumpston, Devan N; Ewing, Thomas J; Zhang, Ping

    2018-01-01

    Activation and reprogramming of hematopoietic stem/progenitor cells play a critical role in the granulopoietic response to bacterial infection. Our current study determined the significance of Sonic hedgehog (SHH) signaling in the regulation of hematopoietic precursor cell activity during the host defense response to systemic bacterial infection. Bacteremia was induced in male Balb/c mice via intravenous injection (i.v.) of Escherichia coli (5 × 10 7 CFUs/mouse). Control mice received i.v. saline. SHH protein level in bone marrow cell (BMC) lysates was markedly increased at both 24 and 48 h of bacteremia. By contrast, the amount of soluble SHH ligand in marrow elutes was significantly reduced. These contrasting alterations suggested that SHH ligand release from BMCs was reduced and/or binding of soluble SHH ligand to BMCs was enhanced. At both 12 and 24 h of bacteremia, SHH mRNA expression by BMCs was significantly upregulated. This upregulation of SHH mRNA expression was followed by a marked increase in SHH protein expression in BMCs. Activation of the ERK1/2-SP1 pathway was involved in mediating the upregulation of SHH gene expression. The major cell type showing the enhancement of SHH expression in the bone marrow was lineage positive cells. Gli1 positioned downstream of the SHH receptor activation serves as a key component of the hedgehog (HH) pathway. Primitive hematopoietic precursor cells exhibited the highest level of baseline Gli1 expression, suggesting that they were active cells responding to SHH ligand stimulation. Along with the increased expression of SHH in the bone marrow, expression of Gli1 by marrow cells was significantly upregulated at both mRNA and protein levels following bacteremia. This enhancement of Gli1 expression was correlated with activation of hematopoietic stem/progenitor cell proliferation. Mice with Gli1 gene deletion showed attenuation in activation of marrow hematopoietic stem/progenitor cell proliferation and inhibition

  9. Behaviour of marine oil-degrading bacterial populations in a continuous culture system

    Digital Repository Service at National Institute of Oceanography (India)

    Mohandass, C.; David, J.J.; Nair, S.; LokaBharathi, P.A.; Chandramohan, D.

    In pursuit of developing an oil-degrading microbial consortium, we used the principle of "plasmid assisted molecular breeding" (PAMB) in a continuous culture system. Three marine bacteria, Pseudomonas putida, Brevibacterium epidermidis...

  10. Changes of bacterial diversity and tetracycline resistance in sludge from AAO systems upon exposure to tetracycline pressure

    International Nuclear Information System (INIS)

    Huang, Manhong; Qi, Fangfang; Wang, Jue; Xu, Qi; Lin, Li

    2015-01-01

    Highlights: • High-throughput sequencing was used to compare sludge bacteria with and without TC. • Bacterial diversity increased with TC addition despite of various oxygen conditions. • Total TRGs proliferated with TC addition in three kinds of sludge. • The concentration of efflux pump genes was the highest in the three groups of TRGs. - Abstract: Two lab-scale anaerobic-anoxic-oxic (AAO) systems were used to investigate the changes in tetracycline (TC) resistance and bacterial diversity upon exposure to TC pressure. High-throughput sequencing was used to detect diversity changes in microorganisms at the level of class in sludge from different bioreactors with and without TC. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect the abundances of eight tetracycline resistance genes (TRGs), tetA, tetB, tetC, tetE, tetM, tetO, tetS and tetX. The results showed that the diversities of the microbial communities of anoxic, anaerobic and aerobic sludge all increased with the addition of TC. TC substantially changed the structure of the microbial community regardless of oxygen conditions. Bacteroidetes and Proteobacteria were the dominant species in the three kinds of sludge and were substantially enriched with TC pressure. In sludge with TC added, almost all target TRGs proliferated more than those in sludge without TC except tetX, which decreased in anaerobic sludge with TC addition. The concentration of efflux pump genes, tet(A–C, E), was the highest among the three groups of TRGs in the different kinds of sludge

  11. Changes of bacterial diversity and tetracycline resistance in sludge from AAO systems upon exposure to tetracycline pressure

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Manhong, E-mail: egghmh@163.com; Qi, Fangfang; Wang, Jue; Xu, Qi; Lin, Li

    2015-11-15

    Highlights: • High-throughput sequencing was used to compare sludge bacteria with and without TC. • Bacterial diversity increased with TC addition despite of various oxygen conditions. • Total TRGs proliferated with TC addition in three kinds of sludge. • The concentration of efflux pump genes was the highest in the three groups of TRGs. - Abstract: Two lab-scale anaerobic-anoxic-oxic (AAO) systems were used to investigate the changes in tetracycline (TC) resistance and bacterial diversity upon exposure to TC pressure. High-throughput sequencing was used to detect diversity changes in microorganisms at the level of class in sludge from different bioreactors with and without TC. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect the abundances of eight tetracycline resistance genes (TRGs), tetA, tetB, tetC, tetE, tetM, tetO, tetS and tetX. The results showed that the diversities of the microbial communities of anoxic, anaerobic and aerobic sludge all increased with the addition of TC. TC substantially changed the structure of the microbial community regardless of oxygen conditions. Bacteroidetes and Proteobacteria were the dominant species in the three kinds of sludge and were substantially enriched with TC pressure. In sludge with TC added, almost all target TRGs proliferated more than those in sludge without TC except tetX, which decreased in anaerobic sludge with TC addition. The concentration of efflux pump genes, tet(A–C, E), was the highest among the three groups of TRGs in the different kinds of sludge.

  12. Strategies for production of active eukaryotic proteins in bacterial expression system

    OpenAIRE

    Orawan Khow; Sunutcha Suntrarachun

    2012-01-01

    Bacteria have long been the favorite expression system for recombinant protein production. However, the flaw of the system is that insoluble and inactive proteins are co-produced due to codon bias, protein folding, phosphorylation, glycosylation, mRNA stability and promoter strength. Factors are cited and the methods to convert to soluble and active proteins are described, for example a tight control of Escherichia coli milieu, refolding from inclusion body and through fusion technology.

  13. Understanding the impact of water distribution system conditions on the biodegradation of haloacetic acids and expression of bacterial dehalogenase genes.

    Science.gov (United States)

    Behbahani, Mohsen; Lin, Boren; Phares, Tamara L; Seo, Youngwoo

    2018-06-05

    The objective of this study is to evaluate the influence of water distribution system conditions (pH, total organic carbon, residual chlorine, and phosphate) on haloacetic acids (HAAs) biodegradation. A series of batch microcosm tests were conducted to determine biodegradation kinetics and collected biomass was used for real time quantitative reverse transcription polymerase chain reaction analyses to monitor how these drinking water distribution system conditions affect the relative expression of bacterial dehalogenase genes. It was observed that tested water distribution system conditions affected HAA biodegradation with different removal efficiencies (0-100%). HAA biodegradation was improved in tested samples with TOC (3 mg/L) and pH 8.5 compared to those of TOC (0 mg/L) and pH 7, respectively. However, slight improvement was observed with the increased PO 4 concentration (3.5 mg/L), and the presence of residual chlorine even at low concentration prohibited biodegradation of HAAs. The observed trend in the relative expression of dehII genes was compatible with the HAA biodegradation trend. Overall relative expression ratio of dehII genes was lower at pH 7, phosphate (0.5 mg/L), and TOC (0 mg/L) in comparison with pH 8.5, phosphate (3.5 mg/L), and TOC (3 mg/L) in the same experimental conditions. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. Engineering bacterial surface displayed human norovirus capsid proteins: A novel system to explore interaction between norovirus and ligands

    Directory of Open Access Journals (Sweden)

    Mengya eNiu

    2015-12-01

    Full Text Available Human noroviruses (HuNoVs are major contributors to acute nonbacterial gastroenteritis outbreaks. Many aspects of HuNoVs are poorly understood due to both the current inability to culture HuNoVs, and the lack of efficient small animal models. Surrogates for HuNoVs, such as recombinant viral like particles (VLPs expressed in eukaryotic system or P particles expressed in prokaryotic system, have been used for studies in immunology and interaction between the virus and its receptors. However, it is difficult to use VLPs or P particles to collect or isolate potential ligands binding to these recombinant capsid proteins. In this study, a new strategy was used to collect HuNoVs binding ligands through the use of ice nucleation protein (INP to display recombinant capsid proteins of HuNoVs on bacterial surfaces. The viral protein-ligand complex could be easily separated by a low speed centrifugation step. This system was also used to explore interaction between recombinant capsid proteins of HuNoVs and their receptors. In this system, the VP1 capsid encoding gene (ORF2 and the protruding domain (P domain encoding gene (3’ terminal fragment of ORF2 of HuNoVs GI.1 and GII.4 were fused with 5’ terminal fragment of ice nucleation protein encoding gene (inaQn. The results demonstrated that the recombinant VP1 and P domains of HuNoVs were expressed and anchored on the surface of Escherichia coli BL21 cells after the bacteria were transformed with the corresponding plasmids. Both cell surface displayed VP1 and P domains could be recognized by HuNoVs specific antibodies and interact with the viral histo-blood group antigens receptors. In both cases, displayed P domains had better binding abilities than VP1. This new strategy of using displayed HuNoVs capsid proteins on the bacterial surface could be utilized to separate HuNoVs binding components from complex samples, to investigate interaction between the virus and its receptors, as well as to develop an

  15. Vimentin in Bacterial Infections

    DEFF Research Database (Denmark)

    Mak, Tim N; Brüggemann, Holger

    2016-01-01

    filaments (IFs). IFs have not only roles in maintaining the structural integrity of the cell, but they are also involved in many cellular processes including cell adhesion, immune signaling, and autophagy, processes that are important in the context of bacterial infections. Here, we summarize the knowledge...... about the role of IFs in bacterial infections, focusing on the type III IF protein vimentin. Recent studies have revealed the involvement of vimentin in host cell defenses, acting as ligand for several pattern recognition receptors of the innate immune system. Two main aspects of bacteria......-vimentin interactions are presented in this review: the role of vimentin in pathogen-binding on the cell surface and subsequent bacterial invasion and the interaction of cytosolic vimentin and intracellular pathogens with regards to innate immune signaling. Mechanistic insight is presented involving distinct bacterial...

  16. Exploitation of eukaryotic ubiquitin signaling pathways by effectors translocated by bacterial type III and type IV secretion systems.

    Directory of Open Access Journals (Sweden)

    Aurélie Angot

    2007-01-01

    Full Text Available The specific and covalent addition of ubiquitin to proteins, known as ubiquitination, is a eukaryotic-specific modification central to many cellular processes, such as cell cycle progression, transcriptional regulation, and hormone signaling. Polyubiquitination is a signal for the 26S proteasome to destroy earmarked proteins, but depending on the polyubiquitin chain topology, it can also result in new protein properties. Both ubiquitin-orchestrated protein degradation and modification have also been shown to be essential for the host's immune response to pathogens. Many animal and plant pathogenic bacteria utilize type III and/or type IV secretion systems to inject effector proteins into host cells, where they subvert host signaling cascades as part of their infection strategy. Recent progress in the determination of effector function has taught us that playing with the host's ubiquitination system seems a general tactic among bacteria. Here, we discuss how bacteria exploit this system to control the timing of their effectors' action by programming them for degradation, to block specific intermediates in mammalian or plant innate immunity, or to target host proteins for degradation by mimicking specific ubiquitin/proteasome system components. In addition to analyzing the effectors that have been described in the literature, we screened publicly available bacterial genomes for mimicry of ubiquitin proteasome system subunits and detected several new putative effectors. Our understanding of the intimate interplay between pathogens and their host's ubiquitin proteasome system is just beginning. This exciting research field will aid in better understanding this interplay, and may also provide new insights into eukaryotic ubiquitination processes.

  17. Modeling the induced mutation process in bacterial cells with defects in excision repair system

    Science.gov (United States)

    Bugay, A. N.; Vasilyeva, M. A.; Krasavin, E. A.; Parkhomenko, A. Yu.

    2015-12-01

    A mathematical model of the UV-induced mutation process in Escherichia coli cells with defects in the uvrA and polA genes has been developed. The model describes in detail the reaction kinetics for the excision repair system. The number of mismatches as a result of translesion synthesis is calculated for both wild-type and mutant cells. The effect of temporal modulation of the number of single-stranded DNA during postreplication repair has been predicted. A comparison of effectiveness of different repair systems has been conducted.

  18. Bacterial Histidine Kinases as Novel Antibacterial Drug Targets

    NARCIS (Netherlands)

    Bem, A.E.; Velikova, N.R.; Pellicer, M.T.; Baarlen, van P.; Marina, A.; Wells, J.M.

    2015-01-01

    Bacterial histidine kinases (HKs) are promising targets for novel antibacterials. Bacterial HKs are part of bacterial two-component systems (TCSs), the main signal transduction pathways in bacteria, regulating various processes including virulence, secretion systems and antibiotic resistance. In

  19. Bacterial Composition of Biofilms Collected From Two Service Areas in a Metropolitan Drinking Water Distribution System

    Science.gov (United States)

    The development and succession of bacteria were examined by 16S rRNA gene clone libraries generated from various biofilms within a metropolitan water distribution system. Biofilms were obtained from off-line devices using polycarbonate coupons from annular reactors incubated for ...

  20. Inhibition of bacterial foodborne pathogens by the lactoperoxidase system in combination with monolaurin.

    Science.gov (United States)

    McLay, J C; Kennedy, M J; Orourke, A L; Elliot, R M; Simmonds, R S

    2002-02-25

    The lactoperoxidase system (LPS) and monolaurin (ML) are potential natural antimicrobial agents for use in foods. The LPS is considered to have greatest activity against Gram-negative bacteria while ML is usually considered to have greatest activity against Gram-positive bacteria. An LPS-ML combination system (utilizing lactoperoxidase (LPX) in the range 5-200 mg kg(-1) and ML in the range 50-1,000 ppm) inhibited growth of Escherichia coli O157:H7 and Staphylococcus aureus. Growth of S. aureus was inhibited more strongly in broth than in milk, in milk than in ground beef A similar pattern was observed for E. coli O157:H7, though enhanced inhibition by LPS-ML systems over that obtained in comparable LPS only systems was not observed in ground beef The inhibitory action of the LPS in combination with other lipids was also examined, with progressively weaker inhibition observed in combinations including palmitoleic acid, monopalmitolein, lauric acid, caprylic acid, and sodium lauryl sulphate.

  1. Bacterial activity dynamics in the water phase during start-up of recirculating aquaculture systems

    DEFF Research Database (Denmark)

    Rojas-Tirado, Paula Andrea; Pedersen, Per Bovbjerg; Pedersen, Lars-Flemming

    2017-01-01

    tMicrobial water quality in recirculating aquaculture systems (RAS) is important for successful RAS opera-tion but difficult to assess and control. There is a need to identify factors affecting changes in the bacterialdynamics – in terms of abundance and activity – to get the information needed...

  2. Saponin, an inhibitory agent of carbon dioxide production by white cells : its use in the microbiologic examination of blood components in an automated bacterial culture system

    NARCIS (Netherlands)

    van Doorne, Hans; van der Tuuk Adriani, W.P A; van der Ven, L.I; Bosch, E.H; de Natris, T; Smit Sibinga, C.Th.

    1998-01-01

    BACKGROUND: Blood components with a white cell count >100 x 10(9) per L may cause false-positive results when the BacT/Alert system is used for the microbiologic examination. The effects of different concentrations of saponin on bacterial growth and on carbon dioxide production by blood fractions

  3. Flavobacterium branchiophilum and F. succinicans associated with bacterial gill disease in rainbow trout Oncorhynchus mykiss (Walbaum) in water recirculation aquaculture systems

    Science.gov (United States)

    Raised rainbow trout Oncorhynchus mykiss in six replicated water recirculation aquaculture systems (WRAS), and manipulated environmental conditions to promote bacterial gill disease (BGD). For each episode of BGD, gill tissue was sampling from affected fish, unaffected fish within the same WRAS, and...

  4. An in vitro biological and anti-bacterial study on a sol-gel derived silver-incorporated bioglass system.

    Science.gov (United States)

    Balamurugan, A; Balossier, G; Laurent-Maquin, D; Pina, S; Rebelo, A H S; Faure, J; Ferreira, J M F

    2008-10-01

    The purpose of this study was to evaluate the in vitro antibacterial and biological activity of silver-incorporated bioactive glass system SiO2-CaO-P2O5-Ag2O (AgBG). The bacteriostatic and bactericidal properties of this new quaternary glass system along with the ternary sol-gel glass system SiO2-CaO-P2O5 (BG) have been studied using Escherichia coli as a test micro-organism. The AGBG system thus appears to be a promising material for dental applications, since similar effects might be produced on a film of bacteria and mucous that grows on the teeth. The SiO2-CaO-P2O5-Ag2O and SiO2-CaO-P2O5 glass systems were synthesized by the sol-gel technique and characterized for their physicho-chemical properties. The antibacterial activity and biological properties were evaluated by determining the minimum inhibitory concentrations (MICs). Release of Ag+ into the culture medium was measured by inductively coupled plasma (ICP) analysis. The in vitro antibacterial action of the SiO2-CaO-P2O5-Ag2O was compared with that of its ternary counterpart glass system. The concentrations of Ag-bioglass, in the range of 0.02-0.20 mg of Ag-bioglass per millilitre of culture medium, were found to inhibit the growth of these bacteria. The Ag-bioglass not only acts bacteriostatically but it also elicited a rapid bactericidal action. A complete bactericidal effect was elicited in the early stages of the incubation at Ag-bioglass concentration of 20 mg/ml and the ternary glass system had no effect on bacterial growth or viability. The antibacterial action of Ag-bioglass was exclusively attributed to the leaching of Ag+ ions from the glass matrix. One of the major advantages of incorporating silver ions into a gel glass system is that the porous glass matrix can allow for controlled sustained delivery of the antibacterial agent to dental material, used even under anaerobic conditions such as deep in the periodontal pocket. This glass system also provides long-term action required for systems

  5. A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair

    Science.gov (United States)

    Babu, Mohan; Beloglazova, Natalia; Flick, Robert; Graham, Chris; Skarina, Tatiana; Nocek, Boguslaw; Gagarinova, Alla; Pogoutse, Oxana; Brown, Greg; Binkowski, Andrew; Phanse, Sadhna; Joachimiak, Andrzej; Koonin, Eugene V.; Savchenko, Alexei; Emili, Andrew; Greenblatt, Jack; Edwards, Aled M.; Yakunin, Alexander F.

    2011-01-01

    Summary Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks, and 5′-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair. PMID:21219465

  6. A Highly Conserved Bacterial D-Serine Uptake System Links Host Metabolism and Virulence.

    Directory of Open Access Journals (Sweden)

    James P R Connolly

    2016-01-01

    Full Text Available The ability of any organism to sense and respond to challenges presented in the environment is critically important for promoting or restricting colonization of specific sites. Recent work has demonstrated that the host metabolite D-serine has the ability to markedly influence the outcome of infection by repressing the type III secretion system of enterohaemorrhagic Escherichia coli (EHEC in a concentration-dependent manner. However, exactly how EHEC monitors environmental D-serine is not understood. In this work, we have identified two highly conserved members of the E. coli core genome, encoding an inner membrane transporter and a transcriptional regulator, which collectively help to "sense" levels of D-serine by regulating its uptake from the environment and in turn influencing global gene expression. Both proteins are required for full expression of the type III secretion system and diversely regulated prophage-encoded effector proteins demonstrating an important infection-relevant adaptation of the core genome. We propose that this system acts as a key safety net, sampling the environment for this metabolite, thereby promoting colonization of EHEC to favorable sites within the host.

  7. Portal versus systemic drainage of small bowel allografts: comparative assessment of survival, function, rejection, and bacterial translocation.

    Science.gov (United States)

    Berney, Thierry; Kato, Tomoaki; Nishida, Seigo; Tector, A Joseph; Mittal, Naveen K; Madariaga, Juan; Nery, Jose R; Cantwell, G Patricia; Ruiz, Philip; Tzakis, Andreas G

    2002-12-01

    Portal venous drainage of small bowel grafts is theoretically more physiologic than systemic drainage, but is technically more demanding. Comparisons in animal models have not demonstrated a clear advantage of one technique over the other, but clinical data are lacking. Clinical records of 36 patients who underwent 37 small bowel transplantation procedures from January 1995 to August 2001 were reviewed. Portal drainage was performed in 19 patients (PD group). Systemic drainage was performed in 18 patients (SD group). Median followup was 531 days. PD and SD patients had similar ICU stays (median 7 versus 9 days) and endotracheal intubation durations (median 3 versus 5 days). All current survivors, with the exception of one patient in each group, are independent from parenteral nutrition. Liver function tests were similar in both groups. There was a twofold increase in tacrolimus dosage in the PD group to achieve similar trough levels indicating a "first-pass" hepatic clearance effect. Cumulative incidence of acute rejection episodes and OKT3-requiring rejection episodes were similar in both groups. To the contrary, a lower incidence of gram-negative rods of Enterococcus sp. in blood or bronchoalveolar lavage suggested that the clearance of translocated intestinal bacteria was more efficient in the PD group. Graft and patient survival rates were similar in both groups. Systemic venous drainage of small bowel transplants is a dependable technique, associated with similar results as portal venous drainage, in terms of overall mortality, morbidity, rejection, function, and patient and graft survival. But attention should be paid to an impaired clearance of intestinal bacterial translocation after systemic drainage.

  8. T3DB: an integrated database for bacterial type III secretion system

    OpenAIRE

    Wang, Yejun; Huang, He; Sun, Ming’an; Zhang, Qing; Guo, Dianjing

    2012-01-01

    Abstract Background Type III Secretion System (T3SS), which plays important roles in pathogenesis or symbiosis, is widely expressed in a variety of gram negative bacteria. However, lack of unique nomenclature for T3SS genes has hindered T3SS related research. It is necessary to set up a knowledgebase integrating T3SS-related research data to facilitate the communication between different research groups interested in different bacteria. Description A T3SS-related Database (T3DB) was developed...

  9. Platelets and the innate immune system: Mechanisms of bacterial-induced platelet activation.

    OpenAIRE

    Cox, Dermot; Kerrigan, Steven W; Watson, Steve

    2011-01-01

    It has become clear that platelets are not simply cell fragments that can plug the leak in a damaged blood vessel, they are in fact key components in the innate immune system which is supported by the presence of Toll-like receptors (TLRs) on platelets. As the first responding cell to a site of injury they are well placed to direct the immune response to deal with any resulting exposure to pathogens. The response is triggered by bacteria binding to platelets which usually triggers platelet ac...

  10. BACTERIAL CONSORTIUM

    Directory of Open Access Journals (Sweden)

    Payel Sarkar

    2013-01-01

    Full Text Available Petroleum aromatic hydrocarbons like benzen e, toluene, ethyl benzene and xylene, together known as BTEX, has almost the same chemical structure. These aromatic hydrocarbons are released as pollutants in th e environment. This work was taken up to develop a solvent tolerant bacterial cons ortium that could degrade BTEX compounds as they all share a common chemical structure. We have isolated almost 60 different types of bacterial strains from different petroleum contaminated sites. Of these 60 bacterial strains almost 20 microorganisms were screene d on the basis of capability to tolerate high concentration of BTEX. Ten differe nt consortia were prepared and the compatibility of the bacterial strains within the consortia was checked by gram staining and BTEX tolerance level. Four successful mi crobial consortia were selected in which all the bacterial strains concomitantly grew in presence of high concentration of BTEX (10% of toluene, 10% of benzene 5% ethyl benzene and 1% xylene. Consortium #2 showed the highest growth rate in pr esence of BTEX. Degradation of BTEX by consortium #2 was monitored for 5 days by gradual decrease in the volume of the solvents. The maximum reduction observed wa s 85% in 5 days. Gas chromatography results also reveal that could completely degrade benzene and ethyl benzene within 48 hours. Almost 90% degradation of toluene and xylene in 48 hours was exhibited by consortium #2. It could also tolerate and degrade many industrial solvents such as chloroform, DMSO, acetonitrile having a wide range of log P values (0.03–3.1. Degradation of aromatic hydrocarbon like BTEX by a solvent tolerant bacterial consortium is greatly significant as it could degrade high concentration of pollutants compared to a bacterium and also reduces the time span of degradation.

  11. [Diagnosis of bacterial vaginosis].

    Science.gov (United States)

    Djukić, Slobodanka; Ćirković, Ivana; Arsić, Biljana; Garalejić, Eliana

    2013-01-01

    Bacterial vaginosis is a common, complex clinical syndrome characterized by alterations in the normal vaginal flora. When symptomatic, it is associated with a malodorous vaginal discharge and on occasion vaginal burning or itching. Under normal conditions, lactobacilli constitute 95% of the bacteria in the vagina. Bacterial vaginosis is associated with severe reduction or absence of the normal H2O2-producing lactobacilli and overgrowth of anaerobic bacteria and Gardnerella vaginalis, Atopobium vaginae, Mycoplasma hominis and Mobiluncus species. Most types of infectious disease are diagnosed by culture, by isolating an antigen or RNA/DNA from the microbe, or by serodiagnosis to determine the presence of antibodies to the microbe. Therefore, demonstration of the presence of an infectious agent is often a necessary criterion for the diagnosis of the disease. This is not the case for bacterial vaginosis, since the ultimate cause of the disease is not yet known. There are a variety of methods for the diagnosis of bacterial vaginosis but no method can at present be regarded as the best. Diagnosing bacterial vaginosis has long been based on the clinical criteria of Amsel, whereby three of four defined criteria must be satisfied. Nugent's scoring system has been further developed and includes validation of the categories of observable bacteria structures. Up-to-date molecular tests are introduced, and better understanding of vaginal microbiome, a clear definition for bacterial vaginosis, and short-term and long-term fluctuations in vaginal microflora will help to better define molecular tests within the broader clinical context.

  12. Performance and bacterial communities of successive alkalinity-producing systems (SAPSs) in passive treatment processes treating mine drainages differing in acidity and metal levels.

    Science.gov (United States)

    Jung, Sokhee Philemon; Cheong, Youngwook; Yim, Giljae; Ji, Sangwoo; Kang, Hojeong

    2014-03-01

    Successive alkalinity-producing systems (SAPSs) is a key unit process in the passive treatment of acidic mine drainage. Physico-chemistry and pyrosequencing-based bacterial communities of two passive treatment processes in Gapjung (GJ) and Seokbong (SB) were analyzed. The influent of SB harbored higher levels of acidity and metals than that of GJ. SAPS-SB demonstrated better performance of acidity neutralization and metal removal than SAPS-GJ, despite its shorter hydraulic retention time and higher acidity. System diagnosis revealed that the capacities of SAPSs were not well predicted in the design steps. Bacterial diversity indices and composition were compared at the same sequence read number for fair evaluation. Most of the bacterial sequences were affiliated with uncultured species. A notable difference was observed in the bacterial community compositions of the SAPSs in GJ and SB. Classes of putative sulfate-reducing bacteria, Clostridia (8.3 %) and Deltaproteobacteria (6.1 %), were detected in SAPS-GJ, and Clostridia (14.6 %) was detected in SAPS-SB. Bacilli, which is not a known sulfate-reducing bacterial group, was the second largest class (12.8 %) in SAPS-GJ and the largest class (51.1 %) in SAPS-SB, suggesting that Bacilli may have a prominent role in SAPS. One hundred ninety operational taxonomic units were shared, which occupied ~10 % of each number of total operational taxonomic units in SAPS-GJ and SAPS-SB, respectively. Bacilli and Clostridia were the major shared classes, and Bacillus, Lysinibacillus, and Ureibacillus were the major shared genera. Rarefaction analysis, richness estimates, diversity estimates, and abundance rank analysis show that the sediment bacterial community of SAPS-GJ was more diverse and more evenly distributed than that of SAPS-SB.

  13. A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants.

    Science.gov (United States)

    Yus, Eva; Yang, Jae-Seong; Sogues, Adrià; Serrano, Luis

    2017-08-28

    Quantitative analysis of the sequence determinants of transcription and translation regulation is relevant for systems and synthetic biology. To identify these determinants, researchers have developed different methods of screening random libraries using fluorescent reporters or antibiotic resistance genes. Here, we have implemented a generic approach called ELM-seq (expression level monitoring by DNA methylation) that overcomes the technical limitations of such classic reporters. ELM-seq uses DamID (Escherichia coli DNA adenine methylase as a reporter coupled with methylation-sensitive restriction enzyme digestion and high-throughput sequencing) to enable in vivo quantitative analyses of upstream regulatory sequences. Using the genome-reduced bacterium Mycoplasma pneumoniae, we show that ELM-seq has a large dynamic range and causes minimal toxicity. We use ELM-seq to determine key sequences (known and putatively novel) of promoter and untranslated regions that influence transcription and translation efficiency. Applying ELM-seq to other organisms will help us to further understand gene expression and guide synthetic biology.Quantitative analysis of how DNA sequence determines transcription and translation regulation is of interest to systems and synthetic biologists. Here the authors present ELM-seq, which uses Dam activity as reporter for high-throughput analysis of promoter and 5'-UTR regions.

  14. Architecture and inherent robustness of a bacterial cell-cycle control system.

    Science.gov (United States)

    Shen, Xiling; Collier, Justine; Dill, David; Shapiro, Lucy; Horowitz, Mark; McAdams, Harley H

    2008-08-12

    A closed-loop control system drives progression of the coupled stalked and swarmer cell cycles of the bacterium Caulobacter crescentus in a near-mechanical step-like fashion. The cell-cycle control has a cyclical genetic circuit composed of four regulatory proteins with tight coupling to processive chromosome replication and cell division subsystems. We report a hybrid simulation of the coupled cell-cycle control system, including asymmetric cell division and responses to external starvation signals, that replicates mRNA and protein concentration patterns and is consistent with observed mutant phenotypes. An asynchronous sequential digital circuit model equivalent to the validated simulation model was created. Formal model-checking analysis of the digital circuit showed that the cell-cycle control is robust to intrinsic stochastic variations in reaction rates and nutrient supply, and that it reliably stops and restarts to accommodate nutrient starvation. Model checking also showed that mechanisms involving methylation-state changes in regulatory promoter regions during DNA replication increase the robustness of the cell-cycle control. The hybrid cell-cycle simulation implementation is inherently extensible and provides a promising approach for development of whole-cell behavioral models that can replicate the observed functionality of the cell and its responses to changing environmental conditions.

  15. A bicistronic expression system for bacterial production of authentic human interleukin-18.

    Science.gov (United States)

    Kirkpatrick, Robert B; McDevitt, Patrick J; Matico, Rosalie E; Nwagwu, Silas; Trulli, Stephen H; Mao, Joyce; Moore, Dwight D; Yorke, Adam F; McLaughlin, Megan M; Knecht, Kristin A; Elefante, Louis C; Calamari, Amy S; Fornwald, Jim A; Trill, John J; Jonak, Zdenka L; Kane, James; Patel, Pramathesh S; Sathe, Ganesh M; Shatzman, Allan R; Tapley, Peter M; Johanson, Kyung O

    2003-02-01

    Interleukin-18 (IL-18) is activated and released from immune effector cells to stimulate acquired and innate immune responses involving T and natural killer (NK) cells. The release of IL-18 from mammalian cells is linked to its proteolytic activation by caspases including interleukin 1 converting enzyme (ICE). The absence of a signal peptide sequence and the requirement for coupled activation and cellular release have presented challenges for the large-scale recombinant production of IL-18. In this study, we have explored methods for the direct production of authentic human IL-18 toward the development of a large-scale production system. Expression of mature IL-18 directly in Escherichia coli with a methionine initiating codon leads to the production of MetIL-18 that is dramatically less potent in bioassays than IL-18 produced as a pro-peptide and activated in vitro. To produce an authentic IL-18, we have devised a bicistronic expression system for the coupled transcription and translation of ProIL-18 with caspase-1 (ICE) or caspase-4 (ICE-rel II, TX, ICH-2). Mature IL-18 with an authentic N-terminus was produced and has a biological activity and potency comparable to that of in vitro processed mature IL-18. Optimization of this system for the maximal production yields can be accomplished by modulating the temperature, to affect the rate of caspase activation and to favor the accumulation of ProIL-18, prior to its proteolytic processing by activated caspase. The effect of temperature is particularly profound for the caspase-4 co-expression process, enabling optimized production levels of over 150 mg/L in shake flasks at 25 degrees C. An alternative bicistronic expression design utilizing a precise ubiquitin IL-18 fusion, processed by co-expressed ubiquitinase, was also successfully used to generate fully active IL-18, thereby demonstrating that the pro-sequence of IL-18 is not required for recombinant IL-18 production. Copyright 2002 Elsevier Science (USA)

  16. A secretory system for bacterial production of high-profile protein targets

    DEFF Research Database (Denmark)

    Kotzsch, Alexander; Vernet, Erik; Hammarström, Martin

    2011-01-01

    Escherichia coli represents a robust, inexpensive expression host for the production of recombinant proteins. However, one major limitation is that certain protein classes do not express well in a biologically relevant form using standard expression approaches in the cytoplasm of E. coli. To impr......Escherichia coli represents a robust, inexpensive expression host for the production of recombinant proteins. However, one major limitation is that certain protein classes do not express well in a biologically relevant form using standard expression approaches in the cytoplasm of E. coli...... membrane protein F (OmpF) and osmotically inducible protein Y (OsmY). Based on the results of this initial study, we carried out an extended expression screen employing the OsmY fusion and multiple constructs of a more diverse set of human proteins. Using this high-throughput compatible system, we clearly...

  17. Antigen 43-mediated autotransporter display, a versatile bacterial cell surface presentation system

    DEFF Research Database (Denmark)

    Kjærgaard, Kristian; Hasman, Henrik; Schembri, Mark

    2002-01-01

    to the outer membrane and secretion through the cell envelope is contained within the protein itself. Ag43 consists of two subunits (alpha and beta), where the beta-subunit forms an integral outer membrane translocator to which the alpha-subunit is noncovalently attached. The simplicity of the Ag43 system...... makes it ideally suited as a surface display scaffold. Here we demonstrate that the Ag43 alpha-module can accommodate and display correctly folded inserts and has the ability to display entire functional protein domains, exemplified by the FimH lectin domain. The presence of heterologous cysteine...... bridges does not interfere with surface display, and Ag43 chimeras are correctly processed into alpha- and beta-modules, offering optional and easy release of the chimeric alpha-subunits. Furthermore, Ag43 can be displayed in many gram-negative bacteria. This feature is exploited for display of our...

  18. Adult bacterial meningitis

    DEFF Research Database (Denmark)

    Meyer, C N; Samuelsson, I S; Galle, M

    2004-01-01

    Episodes of adult bacterial meningitis (ABM) at a Danish hospital in 1991-2000 were identified from the databases of the Department of Clinical Microbiology, and compared with data from the Danish National Patient Register and the Danish National Notification System. Reduced penicillin susceptibi......Episodes of adult bacterial meningitis (ABM) at a Danish hospital in 1991-2000 were identified from the databases of the Department of Clinical Microbiology, and compared with data from the Danish National Patient Register and the Danish National Notification System. Reduced penicillin...

  19. The innate immune and systemic response in honey bees to a bacterial pathogen, Paenibacillus larvae

    Directory of Open Access Journals (Sweden)

    Foster Leonard J

    2009-08-01

    Full Text Available Abstract Background There is a major paradox in our understanding of honey bee immunity: the high population density in a bee colony implies a high rate of disease transmission among individuals, yet bees are predicted to express only two-thirds as many immunity genes as solitary insects, e.g., mosquito or fruit fly. This suggests that the immune response in bees is subdued in favor of social immunity, yet some specific immune factors are up-regulated in response to infection. To explore the response to infection more broadly, we employ mass spectrometry-based proteomics in a quantitative analysis of honey bee larvae infected with the bacterium Paenibacillus larvae. Newly-eclosed bee larvae, in the second stage of their life cycle, are susceptible to this infection, but become progressively more resistant with age. We used this host-pathogen system to probe not only the role of the immune system in responding to a highly evolved infection, but also what other mechanisms might be employed in response to infection. Results Using quantitative proteomics, we compared the hemolymph (insect blood of five-day old healthy and infected honey bee larvae and found a strong up-regulation of some metabolic enzymes and chaperones, while royal jelly (food and energy storage proteins were down-regulated. We also observed increased levels of the immune factors prophenoloxidase (proPO, lysozyme and the antimicrobial peptide hymenoptaecin. Furthermore, mass spectrometry evidence suggests that healthy larvae have significant levels of catalytically inactive proPO in the hemolymph that is proteolytically activated upon infection. Phenoloxidase (PO enzyme activity was undetectable in one or two-day-old larvae and increased dramatically thereafter, paralleling very closely the age-related ability of larvae to resist infection. Conclusion We propose a model for the host response to infection where energy stores and metabolic enzymes are regulated in concert with direct

  20. Bacterial respiratory tract infections are promoted by systemic hyperglycemia after severe burn injury in pediatric patients.

    Science.gov (United States)

    Kraft, Robert; Herndon, David N; Mlcak, Ronald P; Finnerty, Celeste C; Cox, Robert A; Williams, Felicia N; Jeschke, Marc G

    2014-05-01

    Burns are associated with hyperglycemia leading to increased incidence of infections with pneumonia being one of the most prominent and adverse complications. Recently, various studies in critically ill patients indicated that increased pulmonary glucose levels with airway/blood glucose threshold over 150 mg/dl lead to an overwhelming growth of bacteria in the broncho-pulmonary system, subsequently resulting in an increased risk of pulmonary infections. The aim of the present study was to determine whether a similar cutoff value exists for severely burned pediatric patients. One-hundred six severely burned pediatric patients were enrolled in the study. Patients were divided in two groups: high (H) defined as daily average glucose levels >75% of LOS >150 mg/dl), and low (L) with daily average glucose levels >75% of the LOS <150 mg/dl). Incidences of pneumonia, atelectasis, and acute respiratory distress syndrome (ARDS) were assessed. Incidence of infections, sepsis, and respiratory parameters were recorded. Blood was analyzed for glucose and insulin levels. Statistical analysis was performed using Student's t-test and chi-square test. Significance was set at p<0.05. Patient groups were similar in demographics and injury characteristics. Pneumonia in patients on the mechanical ventilation (L: 21%, H: 32%) and off mechanical ventilation (L: 5%, H: 15%), as well as ARDS were significantly higher in the high group (L: 3%, H: 19%), p<0.05, while atelectasis was not different. Patients in the high group required significantly longer ventilation compared to low patients (p<0.05). Furthermore, incidence of infection and sepsis were significantly higher in the high group, p<0.05. Our results indicate that systemic glucose levels over 150 mg/dl are associated with a higher incidence of pneumonia confirming the previous studies in critically ill patients. Copyright © 2013 Elsevier Ltd and ISBI. All rights reserved.

  1. From the microbiome to the central nervous system, an update on the epidemiology and pathogenesis of bacterial meningitis in childhood

    Science.gov (United States)

    Janowski, Andrew B; Newland, Jason G

    2017-01-01

    In the past century, advances in antibiotics and vaccination have dramatically altered the incidence and clinical outcomes of bacterial meningitis. We review the shifting epidemiology of meningitis in children, including after the implementation of vaccines that target common meningitic pathogens and the introduction of intrapartum antibiotic prophylaxis offered to mothers colonized with Streptococcus agalactiae. We also discuss what is currently known about the pathogenesis of meningitis. Recent studies of the human microbiome have illustrated dynamic relationships of bacterial and viral populations with the host, which may potentiate the risk of bacterial meningitis. PMID:28184287

  2. Gene and transcript abundances of bacterial type III secretion systems from the rumen microbiome are correlated with methane yield in sheep.

    Science.gov (United States)

    Kamke, Janine; Soni, Priya; Li, Yang; Ganesh, Siva; Kelly, William J; Leahy, Sinead C; Shi, Weibing; Froula, Jeff; Rubin, Edward M; Attwood, Graeme T

    2017-08-08

    Ruminants are important contributors to global methane emissions via microbial fermentation in their reticulo-rumens. This study is part of a larger program, characterising the rumen microbiomes of sheep which vary naturally in methane yield (g CH 4 /kg DM/day) and aims to define differences in microbial communities, and in gene and transcript abundances that can explain the animal methane phenotype. Rumen microbiome metagenomic and metatranscriptomic data were analysed by Gene Set Enrichment, sparse partial least squares regression and the Wilcoxon Rank Sum test to estimate correlations between specific KEGG bacterial pathways/genes and high methane yield in sheep. KEGG genes enriched in high methane yield sheep were reassembled from raw reads and existing contigs and analysed by MEGAN to predict their phylogenetic origin. Protein coding sequences from Succinivibrio dextrinosolvens strains were analysed using Effective DB to predict bacterial type III secreted proteins. The effect of S. dextrinosolvens strain H5 growth on methane formation by rumen methanogens was explored using co-cultures. Detailed analysis of the rumen microbiomes of high methane yield sheep shows that gene and transcript abundances of bacterial type III secretion system genes are positively correlated with methane yield in sheep. Most of the bacterial type III secretion system genes could not be assigned to a particular bacterial group, but several genes were affiliated with the genus Succinivibrio, and searches of bacterial genome sequences found that strains of S. dextrinosolvens were part of a small group of rumen bacteria that encode this type of secretion system. In co-culture experiments, S. dextrinosolvens strain H5 showed a growth-enhancing effect on a methanogen belonging to the order Methanomassiliicoccales, and inhibition of a representative of the Methanobrevibacter gottschalkii clade. This is the first report of bacterial type III secretion system genes being associated with high

  3. International multicenter evaluation of the DiversiLab bacterial typing system for Escherichia coli and Klebsiella spp.

    Science.gov (United States)

    Voets, Guido M; Leverstein-van Hall, Maurine A; Kolbe-Busch, Susanne; van der Zanden, Adri; Church, Deirdre; Kaase, Martin; Grisold, Andrea; Upton, Mathew; Cloutman-Green, Elaine; Cantón, Rafael; Friedrich, Alexander W; Fluit, Ad C

    2013-12-01

    Successful multidrug-resistant clones are increasing in prevalence globally, which makes the ability to identify these clones urgent. However, adequate, easy-to-perform, and reproducible typing methods are lacking. We investigated whether DiversiLab (DL), an automated repetitive-sequence-based PCR bacterial typing system (bioMérieux), is suitable for comparing isolates analyzed at different geographic centers. A total of 39 Escherichia coli and 39 Klebsiella species isolates previously typed by the coordinating center were analyzed. Pulsed-field gel electrophoresis (PFGE) confirmed the presence of one cluster of 6 isolates, three clusters of 3 isolates, and three clusters of 2 isolates for each set of isolates. DL analysis was performed in 11 centers in six different countries using the same protocol. The DL profiles of 425 E. coli and 422 Klebsiella spp. were obtained. The DL system showed a lower discriminatory power for E. coli than did PFGE. The local DL data showed a low concordance, as indicated by the adjusted Rand and Wallace coefficients (0.132 to 0.740 and 0.070 to 1.0 [E. coli] and 0.091 to 0.864 and 0.056 to 1.0 [Klebsiella spp.], respectively). The central analysis showed a significantly improved concordance (0.473 to 1.0 and 0.290 to 1.0 [E. coli] and 0.513 to 0.965 and 0.425 to 1.0 [Klebsiella spp.], respectively). The misclassifications of profiles for individual isolates were mainly due to inconsistent amplification, which was most likely due to variations in the quality and amounts of the isolated DNA used for amplification. Despite local variations, the DL system has the potential to indicate the occurrence of clonal outbreaks in an international setting, provided there is strict adherence to standardized, reproducible DNA isolation methods and analysis protocols, all supported by a central database for profile comparisons.

  4. Phosphorus depletion in forest soils shapes bacterial communities towards phosphorus recycling systems.

    Science.gov (United States)

    Bergkemper, Fabian; Schöler, Anne; Engel, Marion; Lang, Friederike; Krüger, Jaane; Schloter, Michael; Schulz, Stefanie

    2016-06-01

    Phosphorus (P) is an important macronutrient for all biota on earth but similarly a finite resource. Microorganisms play on both sides of the fence as they effectively mineralize organic and solubilize precipitated forms of soil phosphorus but conversely also take up and immobilize P. Therefore, we analysed the role of microbes in two beech forest soils with high and low P content by direct sequencing of metagenomic deoxyribonucleic acid. For inorganic P solubilization, a significantly higher microbial potential was detected in the P-rich soil. This trait especially referred to Candidatus Solibacter usiatus, likewise one of the dominating species in the data sets. A higher microbial potential for efficient phosphate uptake systems (pstSCAB) was detected in the P-depleted soil. Genes involved in P starvation response regulation (phoB, phoR) were prevalent in both soils. This underlines the importance of effective phosphate (Pho) regulon control for microorganisms to use alternative P sources during phosphate limitation. Predicted genes were primarily harboured by Rhizobiales, Actinomycetales and Acidobacteriales. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  5. Bacterial Ecology

    DEFF Research Database (Denmark)

    Fenchel, Tom

    2011-01-01

    Bacterial ecology is concerned with the interactions between bacteria and their biological and nonbiological environments and with the role of bacteria in biogeochemical element cycling. Many fundamental properties of bacteria are consequences of their small size. Thus, they can efficiently exploit...

  6. Bacterial meningitis

    NARCIS (Netherlands)

    Heckenberg, Sebastiaan G. B.; Brouwer, Matthijs C.; van de Beek, Diederik

    2014-01-01

    Bacterial meningitis is a neurologic emergency. Vaccination against common pathogens has decreased the burden of disease. Early diagnosis and rapid initiation of empiric antimicrobial and adjunctive therapy are vital. Therapy should be initiated as soon as blood cultures have been obtained,

  7. Bacterial lipases

    NARCIS (Netherlands)

    Jaeger, Karl-Erich; Ransac, Stéphane; Dijkstra, Bauke W.; Colson, Charles; Heuvel, Margreet van; Misset, Onno

    Many different bacterial species produce lipases which hydrolyze esters of glycerol with preferably long-chain fatty acids. They act at the interface generated by a hydrophobic lipid substrate in a hydrophilic aqueous medium. A characteristic property of lipases is called interfacial activation,

  8. Bacterial Ecology

    DEFF Research Database (Denmark)

    Fenchel, Tom

    2011-01-01

    , the production and oxidation of methane, nitrate reduction and fixation of atmospheric nitrogen are exclusively carried out by different groups of bacteria. Some bacterial species – ‘extremophiles’ – thrive in extreme environments in which no eukaryotic organisms can survive with respect to temperature, salinity...

  9. Bacterial Vaginosis

    Science.gov (United States)

    ... that coats the walls of the vagina Vaginal discharge with an unpleasant or fishlike odor Vaginal pain or itching Burning during urination Doctors are unsure of the incubation period for bacterial vaginosis. How Is the Diagnosis Made? Your child’s pediatrician can make the diagnosis ...

  10. Bacterial stress

    Indian Academy of Sciences (India)

    First page Back Continue Last page Graphics. Bacterial stress. Physicochemical and chemical parameters: temperature, pressure, pH, salt concentration, oxygen, irradiation. Nutritional depravation: nutrient starvation, water shortage. Toxic compounds: Antibiotics, heavy metals, toxins, mutagens. Interactions with other cells: ...

  11. Changes of bacterial diversity and tetracycline resistance in sludge from AAO systems upon exposure to tetracycline pressure.

    Science.gov (United States)

    Huang, Manhong; Qi, Fangfang; Wang, Jue; Xu, Qi; Lin, Li

    2015-11-15

    Two lab-scale anaerobic-anoxic-oxic (AAO) systems were used to investigate the changes in tetracycline (TC) resistance and bacterial diversity upon exposure to TC pressure. High-throughput sequencing was used to detect diversity changes in microorganisms at the level of class in sludge from different bioreactors with and without TC. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect the abundances of eight tetracycline resistance genes (TRGs), tetA, tetB, tetC, tetE, tetM, tetO, tetS and tetX. The results showed that the diversities of the microbial communities of anoxic, anaerobic and aerobic sludge all increased with the addition of TC. TC substantially changed the structure of the microbial community regardless of oxygen conditions. Bacteroidetes and Proteobacteria were the dominant species in the three kinds of sludge and were substantially enriched with TC pressure. In sludge with TC added, almost all target TRGs proliferated more than those in sludge without TC except tetX, which decreased in anaerobic sludge with TC addition. The concentration of efflux pump genes, tet(A-C, E), was the highest among the three groups of TRGs in the different kinds of sludge. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

    Directory of Open Access Journals (Sweden)

    Weir Jerry P

    2007-05-01

    Full Text Available Abstract Background Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2 BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome. Results Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction. Conclusion Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors.

  13. Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes.

    Science.gov (United States)

    Schmeisser, Falko; Weir, Jerry P

    2007-05-14

    Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC) technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2) BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome. Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction. Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors.

  14. A multi-omic systems-based approach reveals metabolic markers of bacterial vaginosis and insight into the disease.

    Science.gov (United States)

    Yeoman, Carl J; Thomas, Susan M; Miller, Margret E Berg; Ulanov, Alexander V; Torralba, Manolito; Lucas, Sarah; Gillis, Marcus; Cregger, Melissa; Gomez, Andres; Ho, Mengfei; Leigh, Steven R; Stumpf, Rebecca; Creedon, Douglas J; Smith, Michael A; Weisbaum, Jon S; Nelson, Karen E; Wilson, Brenda A; White, Bryan A

    2013-01-01

    Bacterial vaginosis (BV) is the most common vaginal disorder of reproductive-age women. Yet the cause of BV has not been established. To uncover key determinants of BV, we employed a multi-omic, systems-biology approach, including both deep 16S rRNA gene-based sequencing and metabolomics of lavage samples from 36 women. These women varied demographically, behaviorally, and in terms of health status and symptoms. 16S rRNA gene-based community composition profiles reflected Nugent scores, but not Amsel criteria. In contrast, metabolomic profiles were markedly more concordant with Amsel criteria. Metabolomic profiles revealed two distinct symptomatic BV types (SBVI and SBVII) with similar characteristics that indicated disruption of epithelial integrity, but each type was correlated to the presence of different microbial taxa and metabolites, as well as to different host behaviors. The characteristic odor associated with BV was linked to increases in putrescine and cadaverine, which were both linked to Dialister spp. Additional correlations were seen with the presence of discharge, 2-methyl-2-hydroxybutanoic acid, and Mobiluncus spp., and with pain, diethylene glycol and Gardnerella spp. The results not only provide useful diagnostic biomarkers, but also may ultimately provide much needed insight into the determinants of BV.

  15. The bacterial two-hybrid system uncovers the involvement of acetylation in regulating of Lrp activity in Salmonella Typhimurium

    Directory of Open Access Journals (Sweden)

    Ran Qin

    2016-11-01

    Full Text Available Nε-lysine acetylation is an abundant and important Post-translational modification in bacteria. We used the bacterial two-hybrid system to screen the genome library of the Salmonella Typhimurium to identify potential proteins involved in acetyltransferase Pat - or deacetylase CobB-mediated acetylation. Then, the in vitro (deacetylation assays were used to validate the potential targets, such as STM14_1074, NrdF, RhaR. Lrp, a leucine-responsive regulatory protein and global regulator, was shown to interact with Pat. We further demonstrate that Lrp could be acetylated by Pat and deacetylated by NAD+-dependent CobB in vitro. Specifically, the conserved lysine residue 36 (K36 in helix-turn-helix (HTH DNA-binding domain of Lrp was acetylated. Acetylation of K36 impaired the function of Lrp through altering the affinity with the target promoter. The mutation of K36 in chromosome mimicking acetylation enhanced the transcriptional level of itself and attenuated the mRNA levels of Lrp-regulated genes including fimA, which was confirmed by yeast agglutination assay. These findings demonstrate that the acetylation regulates the DNA-binding activity of Lrp, suggesting that acetylation modification of transcription factors is a conserved regulatory manner to modulate gene expression in bacteria and eukaryotes.

  16. Glycerol as a Cheaper Carbon Source in Bacterial Cellulose (BC) Production by Gluconacetobacter Xylinus DSM46604 in Batch Fermentation System

    International Nuclear Information System (INIS)

    Azila Adnan; Nair, G.R.; Roslan Umar; Roslan Umar

    2015-01-01

    Bacterial cellulose (BC) is a polymer of glucose monomers, which has unique properties including high crystallinity and high strength. It has potential to be used in biomedical applications such as making artificial blood vessel, wound dressings, and in the paper making industry. Extensive study on BC aimed to improve BC production such as by using glycerol as a cheaper carbon source. BC was produced in shake flask culture using five different concentrations of glycerol (10, 20, 30, 40 and 50 g/ L). Using concentration of glycerol above 20 g/ L inhibited culture growth and BC production. Further experiments were performed in batch culture (3-L bioreactor) using 20 g/ L glycerol. It produced yield and productivity of 0.15 g/ g and 0.29 g/ L/ day BC, respectively. This is compared with the control medium, 50 g/ L glucose, which only gave yield and productivity of 0.05 g/ g and 0.23 g/ L/ day, respectively. Twenty g/ L of glycerol enhanced BC production by Gluconacetobacter xylinus DSM46604 in batch fermentation system. (author)

  17. Optimizing a portable biosensor system for bacterial detection in milk based mix for ice cream

    Directory of Open Access Journals (Sweden)

    A. Biscotti

    2018-04-01

    Full Text Available One of the primary focuses of the food industry is providing products compliant with safety standards. The microbiological analysis helps in the identification of the presence of pathogen microorganisms in the food. The analysis with Agar Plate is the classic method. This approach guarantees a high accuracy, but it needs a long detection time (twenty-four to forty-eight hours, beyond high costs and skilled technician. In recent times have been proposed many different methods to have a faster response, and between them there is the impedance method. One of its features is that it is fast, in fact it requires between three to fourteen hours to obtain a reliable measurement. The system is accurate, and suitable to be executed automatically. To test this method has been used UHT Ice Cream Mix. A known volume of mix has been inoculated with increasing percentage of cultures of E. coli. The measurement of the impedance of the inoculated mix has been done by an electronic board designed for the application, and by applying a sinusoidal voltage to the test tube. The signal was digitally generated by the microprocessor, and supplied externally through a D.A. converter. The signal was then filtered to delete from its spectrum the high frequency components typical of the digitally generated signals. The data obtained from impedance instrument showed a reliable correspondence with those from the plate count. By working in less time compared to traditional methods, this tool is well suited for in-situ preliminary analysis in commercial and professional foodservice environment.

  18. Bacterial Adhesion & Blocking Bacterial Adhesion

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk

    2008-01-01

    reduce or delay bacterial biofilm formation of a range of urinary tract infectious E.coli and Klebsiella isolates. Several other proteinaceous coatings were also found to display anti-adhesive properties, possibly providing a measure for controlling the colonization of implant materials. Several other...... components. These substances may both mediate and stabilize the bacterial biofilm. Finally, several adhesive structures were examined, and a novel physiological biofilm phenotype in E.coli biofilms was characterized, namely cell chain formation. The autotransporter protein, antigen 43, was implicated...

  19. T346Hunter: a novel web-based tool for the prediction of type III, type IV and type VI secretion systems in bacterial genomes.

    Directory of Open Access Journals (Sweden)

    Pedro Manuel Martínez-García

    Full Text Available T346Hunter (Type Three, Four and Six secretion system Hunter is a web-based tool for the identification and localisation of type III, type IV and type VI secretion systems (T3SS, T4SS and T6SS, respectively clusters in bacterial genomes. Non-flagellar T3SS (NF-T3SS and T6SS are complex molecular machines that deliver effector proteins from bacterial cells into the environment or into other eukaryotic or prokaryotic cells, with significant implications for pathogenesis of the strains encoding them. Meanwhile, T4SS is a more functionally diverse system, which is involved in not only effector translocation but also conjugation and DNA uptake/release. Development of control strategies against bacterial-mediated diseases requires genomic identification of the virulence arsenal of pathogenic bacteria, with T3SS, T4SS and T6SS being major determinants in this regard. Therefore, computational methods for systematic identification of these specialised machines are of particular interest. With the aim of facilitating this task, T346Hunter provides a user-friendly web-based tool for the prediction of T3SS, T4SS and T6SS clusters in newly sequenced bacterial genomes. After inspection of the available scientific literature, we constructed a database of hidden Markov model (HMM protein profiles and sequences representing the various components of T3SS, T4SS and T6SS. T346Hunter performs searches of such a database against user-supplied bacterial sequences and localises enriched regions in any of these three types of secretion systems. Moreover, through the T346Hunter server, users can visualise the predicted clusters obtained for approximately 1700 bacterial chromosomes and plasmids. T346Hunter offers great help to researchers in advancing their understanding of the biological mechanisms in which these sophisticated molecular machines are involved. T346Hunter is freely available at http://bacterial-virulence-factors.cbgp.upm.es/T346Hunter.

  20. Influence of straw incorporation with and without straw decomposer on soil bacterial community structure and function in a rice-wheat cropping system.

    Science.gov (United States)

    Zhao, Jun; Ni, Tian; Xun, Weibing; Huang, Xiaolei; Huang, Qiwei; Ran, Wei; Shen, Biao; Zhang, Ruifu; Shen, Qirong

    2017-06-01

    To study the influence of straw incorporation with and without straw decomposer on bacterial community structure and biological traits, a 3-year field experiments, including four treatments: control without fertilizer (CK), chemical fertilizer (NPK), chemical fertilizer plus 7500 kg ha -1 straw incorporation (NPKS), and chemical fertilizer plus 7500 kg ha -1 straw incorporation and 300 kg ha -1 straw decomposer (NPKSD), were performed in a rice-wheat cropping system in Changshu (CS) and Jintan (JT) city, respectively. Soil samples were taken right after wheat (June) and rice (October) harvest in both sites, respectively. The NPKS and NPKSD treatments consistently increased crop yields, cellulase activity, and bacterial abundance in both sampling times and sites. Moreover, the NPKS and NPKSD treatments altered soil bacterial community structure, particularly in the wheat harvest soils in both sites, separating from the CK and NPK treatments. In the rice harvest soils, both NPKS and NPKSD treatments had no considerable impacts on bacterial communities in CS, whereas the NPKSD treatment significantly shaped bacterial communities compared to the other treatments in JT. These practices also significantly shifted the bacterial composition of unique operational taxonomic units (OTUs) rather than shared OTUs. The relative abundances of copiotrophic bacteria (Proteobacteria, Betaproteobacteria, and Actinobacteria) were positively correlated with soil total N, available N, and available P. Taken together, these results indicate that application of straw incorporation with and without straw decomposer could particularly stimulate the copiotrophic bacteria, enhance the soil biological activity, and thus, contribute to the soil productivity and sustainability in agro-ecosystems.

  1. The Escherichia coli Phosphoenolpyruvate-Dependent Phosphotransferase System : Observation of Heterogeneity in the Amino Acid Composition of HPr

    NARCIS (Netherlands)

    Roossien, F.F.; Dooijewaard, G.; Robillard, G.T.

    1979-01-01

    Resonances of the aromatic protons of tyrosine have been observed in the proton nuclear magnetic resonance (1H NMR) spectrum of purified HPr from Escherichia coli. Analysis of the NMR spectrum of native HPr suggests that the tyrosine is located in a single position in the secondary structure and

  2. The NMR determination of the IIAmtl binding site on HPr of the Escherichia coli phosphoenol pyruvate-dependent phosphotransferase system

    NARCIS (Netherlands)

    Nuland, Nico A.J. van; Kroon, Gerard J.A.; Dijkstra, Klaas; Wolters, Gea K.; Scheek, Ruud M.; Robillard, George T.

    1993-01-01

    The region of the surface of the histidine-containing protein (HPr) which interacts with the A domain of the mannitol-specific Enzyme II (IIAmtl) has been mapped by titrating the A-domain into a solution of 15N-labeled HPr and monitoring the effects on the amide proton and nitrogen chemical shifts

  3. Detrimental effects of commercial zinc oxide and silver nanomaterials on bacterial populations and performance of wastewater systems

    Science.gov (United States)

    Mboyi, Anza-vhudziki; Kamika, Ilunga; Momba, MaggyN. B.

    2017-08-01

    The widespread use of commercial nanomaterials (NMs) in consumer products has raised environmental concerns as they can enter and affect the efficiency of the wastewater treatment plants. In this study the effect of various concentrations of zinc oxide NMs (nZnO) and silver NMs (nAg) on the selected wastewater bacterial species (Bacillus licheniformis, Brevibacillus laterosporus and Pseudomonas putida) was ascertained at different pH levels (pH 2, 7 and 10). Lethal concentrations (LC) of NMs and parameters such as chemical oxygen demand (COD) and dissolved oxygen (DO) were taken into consideration to assess the performance of a wastewater batch reactor. Bacterial isolates were susceptible to varying concentrations of both nZnO and nAg at pH 2, 7 and 10. It was found that a change in pH did not significantly affect the toxicity of test NMs towards target bacterial isolates. All bacterial species were significantly inhibited (p 0.05) in COD removal in the presence of increasing concentrations of NMs, which resulted in increasing releases of COD. Noticeably, there was no significant difference (p > 0.05) in the decrease in DO uptake in the presence of increasing NM concentrations for all bacterial isolates. The toxic effects of the target NMs on bacterial populations in wastewater may negatively impact the performance of biological treatment processes and may thus affect the efficiency of wastewater treatment plants in producing effluent of high quality.

  4. Bacterial meningitis in immunocompromised patients

    NARCIS (Netherlands)

    van Veen, K.E.B.

    2018-01-01

    Bacterial meningitis is an acute infection of the meninges, in The Netherlands most commonly caused by Streptococcus pneumoniae and Neisseria meningitides. Risk factors for acquiring bacterial meningitis include a decreased function of the immune system. The aim of this thesis was to study

  5. Bacterial lipases

    OpenAIRE

    Jaeger, Karl-Erich; Ransac, Stéphane; Dijkstra, Bauke W.; Colson, Charles; Heuvel, Margreet van; Misset, Onno

    1994-01-01

    Many different bacterial species produce lipases which hydrolyze esters of glycerol with preferably long-chain fatty acids. They act at the interface generated by a hydrophobic lipid substrate in a hydrophilic aqueous medium. A characteristic property of lipases is called interfacial activation, meaning a sharp increase in lipase activity observed when the substrate starts to form an emulsion, thereby presenting to the enzyme an interfacial area. As a consequence, the kinetics of a lipase rea...

  6. Bacterial blight of cotton

    Directory of Open Access Journals (Sweden)

    Aïda JALLOUL

    2015-04-01

    Full Text Available Bacterial blight of cotton (Gossypium ssp., caused by Xanthomonas citri pathovar malvacearum, is a severe disease occurring in all cotton-growing areas. The interactions between host plants and the bacteria are based on the gene-for-gene concept, representing a complex resistance gene/avr gene system. In light of the recent data, this review focuses on the understanding of these interactions with emphasis on (1 the genetic basis for plant resistance and bacterial virulence, (2 physiological mechanisms involved in the hypersensitive response to the pathogen, including hormonal signaling, the oxylipin pathway, synthesis of antimicrobial molecules and alteration of host cell structures, and (3 control of the disease.

  7. Early Innate Immunity to Bacterial Infection in the Lung Is Regulated Systemically by the Commensal Microbiota via Nod-Like Receptor Ligands

    Science.gov (United States)

    2014-01-01

    The commensal microbiota is a major regulator of the immune system. The majority of commensal bacteria inhabit the gastrointestinal tract and are known to regulate local mucosal defenses against intestinal pathogens. There is growing appreciation that the commensal microbiota also regulates immune responses at extraintestinal sites. Currently, however, it is unclear how this influences host defenses against bacterial infection outside the intestine. Microbiota depletion caused significant defects in the early innate response to lung infection by the major human pathogen Klebsiella pneumoniae. After microbiota depletion, early clearance of K. pneumoniae was impaired, and this could be rescued by administration of bacterial Nod-like receptor (NLR) ligands (the NOD1 ligand MurNAcTriDAP and NOD2 ligand muramyl dipeptide [MDP]) but not bacterial Toll-like receptor (TLR) ligands. Importantly, NLR ligands from the gastrointestinal, but not upper respiratory, tract rescued host defenses in the lung. Defects in early innate immunity were found to be due to reduced reactive oxygen species-mediated killing of bacteria by alveolar macrophages. These data show that bacterial signals from the intestine have a profound influence on establishing the levels of antibacterial defenses in distal tissues. PMID:25135683

  8. Tillage practices and straw-returning methods affect topsoil bacterial community and organic C under a rice-wheat cropping system in central China

    Science.gov (United States)

    Guo, Lijin; Zheng, Shixue; Cao, Cougui; Li, Chengfang

    2016-09-01

    The objective of this study was to investigate how the relationships between bacterial communities and organic C (SOC) in topsoil (0-5 cm) are affected by tillage practices [conventional intensive tillage (CT) or no-tillage (NT)] and straw-returning methods [crop straw returning (S) or removal (NS)] under a rice-wheat rotation in central China. Soil bacterial communities were determined by high-throughput sequencing technology. After two cycles of annual rice-wheat rotation, compared with CT treatments, NT treatments generally had significantly more bacterial genera and monounsaturated fatty acids/saturated fatty acids (MUFA/STFA), but a decreased gram-positive bacteria/gram-negative bacteria ratio (G+/G-). S treatments had significantly more bacterial genera and MUFA/STFA, but had decreased G+/G- compared with NS treatments. Multivariate analysis revealed that Gemmatimonas, Rudaea, Spingomonas, Pseudomonas, Dyella, Burkholderia, Clostridium, Pseudolabrys, Arcicella and Bacillus were correlated with SOC, and cellulolytic bacteria (Burkholderia, Pseudomonas, Clostridium, Rudaea and Bacillus) and Gemmationas explained 55.3% and 12.4% of the variance in SOC, respectively. Structural equation modeling further indicated that tillage and residue managements affected SOC directly and indirectly through these cellulolytic bacteria and Gemmationas. Our results suggest that Burkholderia, Pseudomonas, Clostridium, Rudaea, Bacillus and Gemmationas help to regulate SOC sequestration in topsoil under tillage and residue systems.

  9. Interfering with bacterial gossip

    DEFF Research Database (Denmark)

    Bjarnsholt, Thomas; Tolker-Nielsen, Tim; Givskov, Michael

    2011-01-01

    Biofilm resilience poses major challenges to the development of novel antimicrobial agents. Biofilm bacteria can be considered small groups of “Special Forces” capable of infiltrating the host and destroying important components of the cellular defense system with the aim of crippling the host...... defense. Antibiotics exhibit a rather limited effect on biofilms. Furthermore, antibiotics have an ‘inherent obsolescence’ because they select for development of resistance. Bacterial infections with origin in bacterial biofilms have become a serious threat in developed countries. Pseudomonas aeruginosa...... that appropriately target bacteria in their relevant habitat with the aim of mitigating their destructive impact on patients. In this review we describe molecular mechanisms involved in “bacterial gossip” (more scientifically referred to as quorum sensing (QS) and c-di-GMP signaling), virulence, biofilm formation...

  10. A novel approach to recycle bacterial culture waste for fermentation reuse via a microbial fuel cell-membrane bioreactor system.

    Science.gov (United States)

    Li, Jian; Zhu, Yuan; Zhuang, Liangpeng; Otsuka, Yuichiro; Nakamura, Masaya; Goodell, Barry; Sonoki, Tomonori; He, Zhen

    2015-09-01

    Biochemical production processes require water and nutrient resources for culture media preparation, but aqueous waste is generated after the target products are extracted. In this study, culture waste (including cells) produced from a lab-scale fermenter was fed into a microbial fuel cell-membrane bioreactor (MFC-MBR) system. Electrical energy was generated via the interaction between the microbial consortia and the solid electrode in the MFC. The treated wastewater was reclaimed in this process which was reused as a solvent and a nutrient source in subsequent fermentation. Polarization testing showed that the MFC produced a maximum current density of 37.53 A m(-3) with a maximum power density of 5.49 W m(-3). The MFC was able to generate 0.04 kWh of energy per cubic meter of culture waste treated. The lab-scale fermenters containing pure cultures of an engineered Pseudomonas spp. were used to generate 2-pyrone-4,6-dicarboxylic acid (PDC), a high value platform chemical. When the MFC-MBR-treated wastewater was used for the fermenter culture medium, a specific bacterial growth rate of 1.00 ± 0.05 h(-1) was obtained with a PDC production rate of 708.11 ± 64.70 mg PDC L(-1) h(-1). Comparable values for controls using pure water were 0.95 ± 0.06 h(-1) and 621.01 ± 22.09 mg PDC L(-1) h(-1) (P > 0.05), respectively. The results provide insight on a new approach for more sustainable bio-material production while at the same time generating energy, and suggest that the treated wastewater can be used as a solvent and a nutrient source for the fermentation production of high value platform chemicals.

  11. The photochemical thiol-ene reaction as a versatile method for the synthesis of glutathioneS-conjugates targeting the bacterial potassium efflux system Kef.

    Science.gov (United States)

    Healy, Jess; Rasmussen, Tim; Miller, Samantha; Booth, Ian R; Conway, Stuart J

    2016-04-29

    The thiol-ene coupling reaction is emerging as an important conjugation reaction that is suitable for use in a biological setting. Here, we explore the utility of this reaction for the synthesis of glutathione- S -conjugates (GSX) and present a general, operationally simple, protocol with a wide substrate scope. The GSX afforded are an important class of compounds and provide invaluable molecular tools to study glutathione-binding proteins. In this study we apply the diverse library of GSX synthesised to further our understanding of the structural requirements for binding to the glutathione-binding protein, Kef, a bacterial K + efflux system, found in many bacterial pathogens. This system is vital to the survival of bacteria upon exposure to electrophiles, and plays an essential role in the maintenance of intracellular pH and K + homeostasis. Consequently, Kef is an appealing target for the development of novel antibacterial drugs.

  12. The Non-Flagellar Type III Secretion System Evolved from the Bacterial Flagellum and Diversified into Host-Cell Adapted Systems

    Science.gov (United States)

    Abby, Sophie S.; Rocha, Eduardo P. C.

    2012-01-01

    Type 3 secretion systems (T3SSs) are essential components of two complex bacterial machineries: the flagellum, which drives cell motility, and the non-flagellar T3SS (NF-T3SS), which delivers effectors into eukaryotic cells. Yet the origin, specialization, and diversification of these machineries remained unclear. We developed computational tools to identify homologous components of the two systems and to discriminate between them. Our analysis of >1,000 genomes identified 921 T3SSs, including 222 NF-T3SSs. Phylogenomic and comparative analyses of these systems argue that the NF-T3SS arose from an exaptation of the flagellum, i.e. the recruitment of part of the flagellum structure for the evolution of the new protein delivery function. This reconstructed chronology of the exaptation process proceeded in at least two steps. An intermediate ancestral form of NF-T3SS, whose descendants still exist in Myxococcales, lacked elements that are essential for motility and included a subset of NF-T3SS features. We argue that this ancestral version was involved in protein translocation. A second major step in the evolution of NF-T3SSs occurred via recruitment of secretins to the NF-T3SS, an event that occurred at least three times from different systems. In rhizobiales, a partial homologous gene replacement of the secretin resulted in two genes of complementary function. Acquisition of a secretin was followed by the rapid adaptation of the resulting NF-T3SSs to multiple, distinct eukaryotic cell envelopes where they became key in parasitic and mutualistic associations between prokaryotes and eukaryotes. Our work elucidates major steps of the evolutionary scenario leading to extant NF-T3SSs. It demonstrates how molecular evolution can convert one complex molecular machine into a second, equally complex machine by successive deletions, innovations, and recruitment from other molecular systems. PMID:23028376

  13. The non-flagellar type III secretion system evolved from the bacterial flagellum and diversified into host-cell adapted systems.

    Directory of Open Access Journals (Sweden)

    Sophie S Abby

    2012-09-01

    Full Text Available Type 3 secretion systems (T3SSs are essential components of two complex bacterial machineries: the flagellum, which drives cell motility, and the non-flagellar T3SS (NF-T3SS, which delivers effectors into eukaryotic cells. Yet the origin, specialization, and diversification of these machineries remained unclear. We developed computational tools to identify homologous components of the two systems and to discriminate between them. Our analysis of >1,000 genomes identified 921 T3SSs, including 222 NF-T3SSs. Phylogenomic and comparative analyses of these systems argue that the NF-T3SS arose from an exaptation of the flagellum, i.e. the recruitment of part of the flagellum structure for the evolution of the new protein delivery function. This reconstructed chronology of the exaptation process proceeded in at least two steps. An intermediate ancestral form of NF-T3SS, whose descendants still exist in Myxococcales, lacked elements that are essential for motility and included a subset of NF-T3SS features. We argue that this ancestral version was involved in protein translocation. A second major step in the evolution of NF-T3SSs occurred via recruitment of secretins to the NF-T3SS, an event that occurred at least three times from different systems. In rhizobiales, a partial homologous gene replacement of the secretin resulted in two genes of complementary function. Acquisition of a secretin was followed by the rapid adaptation of the resulting NF-T3SSs to multiple, distinct eukaryotic cell envelopes where they became key in parasitic and mutualistic associations between prokaryotes and eukaryotes. Our work elucidates major steps of the evolutionary scenario leading to extant NF-T3SSs. It demonstrates how molecular evolution can convert one complex molecular machine into a second, equally complex machine by successive deletions, innovations, and recruitment from other molecular systems.

  14. tmRNA-mediated trans-translation as the major ribosome rescue system in a bacterial cell

    Directory of Open Access Journals (Sweden)

    Hyouta eHimeno

    2014-04-01

    Full Text Available tmRNA (transfer messenger RNA; also known as 10Sa RNA or SsrA RNA is a small RNA molecule that is conserved among bacteria. It has structural and functional similarities to tRNA: it has an upper half of the tRNA-like structure, its 5’ end is processed by RNase P, it has typical tRNA-specific base modifications, it is aminoacylated with alanine, it binds to EF-Tu after aminoacylation and it enters the ribosome with EF-Tu and GTP. However, tmRNA lacks an anticodon, and instead it has a coding sequence for a short peptide called tag-peptide. An elaborate interplay of actions of tmRNA as both tRNA and mRNA with the help of a tmRNA-binding protein, SmpB, facilitates trans-translation, which produces a single polypeptide from two mRNA molecules. Initially alanyl-tmRNA in complex with EF-Tu and SmpB enters the vacant A-site of the stalled ribosome like aminoacyl-tRNA but without a codon-anticodon interaction, and subsequently truncated mRNA is replaced with the tag-encoding region of tmRNA. During these processes, not only tmRNA but also SmpB structurally and functionally mimics both tRNA and mRNA. Thus trans-translation rescues the stalled ribosome, thereby allowing recycling of the ribosome. Since the tag-peptide serves as a target of AAA+ proteases, the trans-translation products are preferentially degraded so that they do not accumulate in the cell. Although alternative rescue systems have recently been revealed, trans-translation is the only system that universally exists in bacteria. Furthermore, it is unique in that it employs a small RNA and that it prevents accumulation of nonfunctional proteins from truncated mRNA in the cell. It might play the major role in rescuing the stalled translation in the bacterial cell.

  15. Rationale and prospects of targeting bacterial two-component systems for antibacterial treatment of cystic fibrosis patients

    NARCIS (Netherlands)

    Velikova, Nadya; Wells, Jerry M.

    2016-01-01

    Bacterial respiratory infections are the main reason of morbidity and mortality among cystic fibrosis (CF) patients. In early childhood, the respiratory infections are due to Staphylococcus aureus and Haemophilus influenzae. In older CF patients, pathogenic Gram-negative bacteria like Achromobacter

  16. Effect of crop management and sample year on abundance of soil bacterial communities in organic and conventional cropping systems.

    Science.gov (United States)

    Orr, C H; Stewart, C J; Leifert, C; Cooper, J M; Cummings, S P

    2015-07-01

    To identify changes in the bacterial community, at the phylum level brought about by varied crop management. Next-generation sequencing methods were used to compare the taxonomic structure of the bacterial community within 24 agricultural soils managed with either organic or conventional methods, over a 3-year period. Relative abundance of the proportionately larger phyla (e.g. Acidobacteria and Actinobacteria) was primarily affected by sample year rather than crop management. Changes of abundance in these phyla were correlated with changes in pH, organic nitrogen and soil basal respiration. Crop management affected some of the less dominant phyla (Chloroflexi, Nitrospirae, Gemmatimonadetes) which also correlated with pH and organic N. Soil diversity can vary with changing environmental variables and soil chemistry. If these factors remain constant, soil diversity can also remain constant even under changing land use. The impact of crop management on environmental variables must be considered when interpreting bacterial diversity studies in agricultural soils. Impact of land use change should always be monitored across different sampling time points. Further studies at the functional group level are necessary to assess whether management-induced changes in bacterial community structure are of biological and agronomic relevance. © 2015 The Society for Applied Microbiology.

  17. Compositional analysis of bacterial communities in seawater, sediment, and sponges in the Misool coral reef system, Indonesia

    NARCIS (Netherlands)

    Cleary, Daniel Francis Richard; Polónia, Ana Rita Moura; Becking, Leontine E.; Voogd, de Nicole Joy; Purwanto,; Gomes, Helder; Gomes, Newton Carlos Marcial

    2017-01-01

    Sponge species have been deemed high microbial abundance (HMA) or low microbial abundance (LMA) based on the composition and abundance of their microbial symbionts. In the present study, we evaluated the richness and composition of bacterial communities associated with one HMA sponge (Xestospongia

  18. Diagnosis of bacterial vaginosis

    Directory of Open Access Journals (Sweden)

    Đukić Slobodanka

    2013-01-01

    Full Text Available Bacterial vaginosis is a common, complex clinical syndrome characterized by alterations in the normal vaginal flora. When symptomatic, it is associated with a malodorous vaginal discharge and on occasion vaginal burning or itching. Under normal conditions, lactobacilli constitute 95% of the bacteria in the vagina. Bacterial vaginosis is associated with severe reduction or absence of the normal H2O2­producing lactobacilli and overgrowth of anaerobic bacteria and Gardnerella vaginalis, Atopobium vaginae, Mycoplasma hominis and Mobiluncus species. Most types of infectious disease are diagnosed by culture, by isolating an antigen or RNA/DNA from the microbe, or by serodiagnosis to determine the presence of antibodies to the microbe. Therefore, demonstration of the presence of an infectious agent is often a necessary criterion for the diagnosis of the disease. This is not the case for bacterial vaginosis, since the ultimate cause of the disease is not yet known. There are a variety of methods for the diagnosis of bacterial vaginosis but no method can at present be regarded as the best. Diagnosing bacterial vaginosis has long been based on the clinical criteria of Amsel, whereby three of four defined criteria must be satisfied. Nugent’s scoring system has been further developed and includes validation of the categories of observable bacteria structures. Up­to­date molecular tests are introduced, and better understanding of vaginal microbiome, a clear definition for bacterial vaginosis, and short­term and long­term fluctuations in vaginal microflora will help to better define molecular tests within the broader clinical context.

  19. Mechanisms of action of systemic antibiotics used in periodontal treatment and mechanisms of bacterial resistance to these drugs.

    Science.gov (United States)

    Soares, Geisla Mary Silva; Figueiredo, Luciene Cristina; Faveri, Marcelo; Cortelli, Sheila Cavalca; Duarte, Poliana Mendes; Feres, Magda

    2012-01-01

    Antibiotics are important adjuncts in the treatment of infectious diseases, including periodontitis. The most severe criticisms to the indiscriminate use of these drugs are their side effects and, especially, the development of bacterial resistance. The knowledge of the biological mechanisms involved with the antibiotic usage would help the medical and dental communities to overcome these two problems. Therefore, the aim of this manuscript was to review the mechanisms of action of the antibiotics most commonly used in the periodontal treatment (i.e. penicillin, tetracycline, macrolide and metronidazole) and the main mechanisms of bacterial resistance to these drugs. Antimicrobial resistance can be classified into three groups: intrinsic, mutational and acquired. Penicillin, tetracycline and erythromycin are broad-spectrum drugs, effective against gram-positive and gram-negative microorganisms. Bacterial resistance to penicillin may occur due to diminished permeability of the bacterial cell to the antibiotic; alteration of the penicillin-binding proteins, or production of β-lactamases. However, a very small proportion of the subgingival microbiota is resistant to penicillins. Bacteria become resistant to tetracyclines or macrolides by limiting their access to the cell, by altering the ribosome in order to prevent effective binding of the drug, or by producing tetracycline/macrolide-inactivating enzymes. Periodontal pathogens may become resistant to these drugs. Finally, metronidazole can be considered a prodrug in the sense that it requires metabolic activation by strict anaerobe microorganisms. Acquired resistance to this drug has rarely been reported. Due to these low rates of resistance and to its high activity against the gram-negative anaerobic bacterial species, metronidazole is a promising drug for treating periodontal infections.

  20. Mechanisms of action of systemic antibiotics used in periodontal treatment and mechanisms of bacterial resistance to these drugs

    Directory of Open Access Journals (Sweden)

    Geisla Mary Silva Soares

    2012-06-01

    Full Text Available Antibiotics are important adjuncts in the treatment of infectious diseases, including periodontitis. The most severe criticisms to the indiscriminate use of these drugs are their side effects and, especially, the development of bacterial resistance. The knowledge of the biological mechanisms involved with the antibiotic usage would help the medical and dental communities to overcome these two problems. Therefore, the aim of this manuscript was to review the mechanisms of action of the antibiotics most commonly used in the periodontal treatment (i.e. penicillin, tetracycline, macrolide and metronidazole and the main mechanisms of bacterial resistance to these drugs. Antimicrobial resistance can be classified into three groups: intrinsic, mutational and acquired. Penicillin, tetracycline and erythromycin are broad-spectrum drugs, effective against gram-positive and gram-negative microorganisms. Bacterial resistance to penicillin may occur due to diminished permeability of the bacterial cell to the antibiotic; alteration of the penicillin-binding proteins, or production of β-lactamases. However, a very small proportion of the subgingival microbiota is resistant to penicillins. Bacteria become resistant to tetracyclines or macrolides by limiting their access to the cell, by altering the ribosome in order to prevent effective binding of the drug, or by producing tetracycline/macrolide-inactivating enzymes. Periodontal pathogens may become resistant to these drugs. Finally, metronidazole can be considered a prodrug in the sense that it requires metabolic activation by strict anaerobe microorganisms. Acquired resistance to this drug has rarely been reported. Due to these low rates of resistance and to its high activity against the gram-negative anaerobic bacterial species, metronidazole is a promising drug for treating periodontal infections.

  1. Immunohistochemical analysis of MMP-9, MMP-2 and TIMP-1, TIMP-2 expression in the central nervous system following infection with viral and bacterial meningitis.

    Directory of Open Access Journals (Sweden)

    Lech Chyczewski

    2009-01-01

    Full Text Available Matrix metalloproteinases (MMPs are capable of degrading components of the basal lamina of cerebral vessels, thereby disrupting the blood-brain barrier and inducing leukocyte recruitment. This study provides comprehensive information regarding the cell specificity of matrix metalloproteinases (MMP-2, MMP-9 and their binding tissue inhibitors (TIMP-1, TIMP-2 in the central nervous system during viral and bacterial meningitis. Specifically, we evaluated the immunoreactivity of MMPs and TIMPs in various cell types in brain parenchyma and meninges obtained from autopsy tissues. We found that a higher proportion of endothelial cells were positive for MMP-9 during meningitis when compared to controls. In addition, the immunoreactivity of MMP-9 decreased and the immunoreactivity of TIMP-1 increased in astrocytes upon infection. Furthermore, the results of this study revealed that mononuclear cells were highly immunoreactive for TIMP-1, TIMP-2 and MMP-9 during viral meningitis and that the expression of TIMPs in polymorphonuclear cells was even higher during bacterial meningitis. Taken together the results of this study indicated that the central nervous system resident cells and inflammatory infiltrates contribute to MMPs activity and that the expression patterns vary between cell types and in response to viral and bacterial meningitis.

  2. Suggested guidelines for using systemic antimicrobials in bacterial skin infections: part 1—diagnosis based on clinical presentation, cytology and culture

    Science.gov (United States)

    Beco, L.; Guaguère, E.; Méndez, C. Lorente; Noli, C.; Nuttall, T.; Vroom, M.

    2013-01-01

    Systemic antimicrobials are critically important in veterinary healthcare, and resistance is a major concern. Antimicrobial stewardship will be important in maintaining clinical efficacy by reducing the development and spread of antimicrobial resistance. Bacterial skin infections are one of the most common reasons for using systemic antimicrobials in dogs and cats. Appropriate management of these infections is, therefore, crucial in any policy for responsible antimicrobial use. The goals of therapy are to confirm that an infection is present, identify the causative bacteria, select the most appropriate antimicrobial, ensure that the infection is treated correctly, and to identify and manage any underlying conditions. This is the first of two articles that will provide evidence-led guidelines to help practitioners address these issues. This article covers diagnosis, including descriptions of the different clinical presentations of surface, superficial and deep bacterial skin infections, how to perform and interpret cytology, and how to best use bacterial culture and sensitivity testing. Part 2 will discuss therapy, including choice of drug and treatment regimens. PMID:23292951

  3. Pyrosequencing the Bemisia tabaci transcriptome reveals a highly diverse bacterial community and a robust system for insecticide resistance.

    Directory of Open Access Journals (Sweden)

    Wen Xie

    Full Text Available BACKGROUND: Bemisia tabaci (Gennadius is a phloem-feeding insect poised to become one of the major insect pests in open field and greenhouse production systems throughout the world. The high level of resistance to insecticides is a main factor that hinders continued use of insecticides for suppression of B. tabaci. Despite its prevalence, little is known about B. tabaci at the genome level. To fill this gap, an invasive B. tabaci B biotype was subjected to pyrosequencing-based transcriptome analysis to identify genes and gene networks putatively involved in various physiological and toxicological processes. METHODOLOGY AND PRINCIPAL FINDINGS: Using Roche 454 pyrosequencing, 857,205 reads containing approximately 340 megabases were obtained from the B. tabaci transcriptome. De novo assembly generated 178,669 unigenes including 30,980 from insects, 17,881 from bacteria, and 129,808 from the nohit. A total of 50,835 (28.45% unigenes showed similarity to the non-redundant database in GenBank with a cut-off E-value of 10-5. Among them, 40,611 unigenes were assigned to one or more GO terms and 6,917 unigenes were assigned to 288 known pathways. De novo metatranscriptome analysis revealed highly diverse bacterial symbionts in B. tabaci, and demonstrated the host-symbiont cooperation in amino acid production. In-depth transcriptome analysis indentified putative molecular markers, and genes potentially involved in insecticide resistance and nutrient digestion. The utility of this transcriptome was validated by a thiamethoxam resistance study, in which annotated cytochrome P450 genes were significantly overexpressed in the resistant B. tabaci in comparison to its susceptible counterparts. CONCLUSIONS: This transcriptome/metatranscriptome analysis sheds light on the molecular understanding of symbiosis and insecticide resistance in an agriculturally important phloem-feeding insect pest, and lays the foundation for future functional genomics research of the

  4. Efficiency of N2 Gas Flushing Compared to the Lactoperoxidase System at Controlling Bacterial Growth in Bovine Raw Milk Stored at Mild Temperatures

    Science.gov (United States)

    Munsch-Alatossava, Patricia; Quintyn, Romanie; De Man, Ingrid; Alatossava, Tapani; Gauchi, Jean-Pierrre

    2016-01-01

    To prevent excessive bacterial growth in raw milk, the FAO recommends two options: either cold storage or activation of the lactoperoxidase system (LPs/HT) in milk with the addition of two chemical preservatives, hydrogen peroxide (H) and thiocyanate (T). N2 gas flushing of raw milk has shown great potential to control bacterial growth in a temperature range of 6–12°C without promoting undesired side effects. Here, the effect of N2 gas (N) was tested as a single treatment and in combination with the lactoperoxidase system (NHT) on seven raw milk samples stored at 15 or 25°C. For the ratio defined as bacterial counts from a certain treatment/counts on the corresponding control, a classical Analyse of Variance (ANOVA) was performed, followed by mean comparison with the Ryan-Einot-Gabriel-Welsch multiple range test (REGWQ). Altogether, the growth inhibition was slightly but significantly higher at 25°C than at 15°C. Except for one sample, all ratios were lower for HT than for N alone; however, these differences were not judged to be significant for five samples by the REGWQ test; in the remaining two samples, N was more effective than HT in one case and less effective in the other case. This study shows that N2 gas flushing, which inhibited bacterial growth in raw milk at 15 and 25°C for 24 and 12 h, respectively, could constitute an alternative to LPs where no cold storage facilities exist, especially as a replacement for adulterating substances. PMID:27313575

  5. Regulation Systems of Bacteria such as Escherichia coli in Response to Nutrient Limitation and Environmental Stresses

    Directory of Open Access Journals (Sweden)

    Kazuyuki Shimizu

    2013-12-01

    Full Text Available An overview was made to understand the regulation system of a bacterial cell such as Escherichia coli in response to nutrient limitation such as carbon, nitrogen, phosphate, sulfur, ion sources, and environmental stresses such as oxidative stress, acid shock, heat shock, and solvent stresses. It is quite important to understand how the cell detects environmental signals, integrate such information, and how the cell system is regulated. As for catabolite regulation, F1,6B P (FDP, PEP, and PYR play important roles in enzyme level regulation together with transcriptional regulation by such transcription factors as Cra, Fis, CsrA, and cAMP-Crp. αKG plays an important role in the coordinated control between carbon (C- and nitrogen (N-limitations, where αKG inhibits enzyme I (EI of phosphotransferase system (PTS, thus regulating the glucose uptake rate in accordance with N level. As such, multiple regulation systems are co-ordinated for the cell synthesis and energy generation against nutrient limitations and environmental stresses. As for oxidative stress, the TCA cycle both generates and scavenges the reactive oxygen species (ROSs, where NADPH produced at ICDH and the oxidative pentose phosphate pathways play an important role in coping with oxidative stress. Solvent resistant mechanism was also considered for the stresses caused by biofuels and biochemicals production in the cell.

  6. Survival of bacterial indicators and the functional diversity of native microbial communities in the Floridan aquifer system, south Florida

    Science.gov (United States)

    Lisle, John T.

    2014-01-01

    The Upper Floridan aquifer in the southern region of Florida is a multi-use, regional scale aquifer that is used as a potable water source and as a repository for passively recharged untreated surface waters, and injected treated surface water and wastewater, industrial wastes, including those which contain greenhouse gases (for example, carbon dioxide). The presence of confined zones within the Floridan aquifer that range in salinity from fresh to brackish allow regulatory agencies to permit the injection of these different types of product waters into specific zones without detrimental effects to humans and terrestrial and aquatic ecosystems. The type of recharge that has received the most regulatory attention in south Florida is aquifer storage and recovery (ASR). The treated water, prior to injection and during recovery, must meet primary and secondary drinking water standards. The primary microbiology drinking water standard is total coliforms, which have been shown to be difficult to inactivate below the regulatory standard during the treatment process at some ASR facilities. The inefficient inactivation of this group of indicator bacteria permits their direct injection into the storage zones of the Floridan aquifer. Prior to this study, the inactivation rates for any member of the total coliform group during exposure to native geochemical conditions in groundwater from any zone of the Floridan aquifer had not been derived. Aboveground flow through mesocosms and diffusion chambers were used to quantify the inactivation rates of two bacterial indicators, Escherichia coli and Pseudomonas aeruginosa, during exposure to groundwater from six wells. These wells collect water from two ASR storage zones: the Upper Floridan aquifer (UFA) and Avon Park Permeable Zone (APPZ). Both bacterial strains followed a biphasic inactivation model. The E. coli populations had slower inactivation rates in the UFA (range: 0.217–0.628 per hour (h-1)) during the first phase of the

  7. Belowground Interactions Impact the Soil Bacterial Community, Soil Fertility, and Crop Yield in Maize/Peanut Intercropping Systems.

    Science.gov (United States)

    Li, Qisong; Chen, Jun; Wu, Linkun; Luo, Xiaomian; Li, Na; Arafat, Yasir; Lin, Sheng; Lin, Wenxiong

    2018-02-22

    Intercropping has been widely used to control disease and improve yield in agriculture. In this study, maize and peanut were used for non-separation intercropping (NS), semi-separation intercropping (SS) using a nylon net, and complete separation intercropping (CS) using a plastic sheet. In field experiments, two-year land equivalent ratios (LERs) showed yield advantages due to belowground interactions when using NS and SS patterns as compared to monoculture. In contrast, intercropping without belowground interactions (CS) showed a yield disadvantage. Meanwhile, in pot experiments, belowground interactions (found in NS and SS) improved levels of soil-available nutrients (nitrogen (N) and phosphorus (P)) and enzymes (urease and acid phosphomonoesterase) as compared to intercropping without belowground interactions (CS). Soil bacterial community assay showed that soil bacterial communities in the NS and SS crops clustered together and were considerably different from the CS crops. The diversity of bacterial communities was significantly improved in soils with NS and SS. The abundance of beneficial bacteria, which have the functions of P-solubilization, pathogen suppression, and N-cycling, was improved in maize and peanut soils due to belowground interactions through intercropping. Among these bacteria, numbers of Bacillus , Brevibacillus brevis , and Paenibacillus were mainly increased in the maize rhizosphere. Burkholderia , Pseudomonas , and Rhizobium were mainly increased in the peanut rhizosphere. In conclusion, using maize and peanut intercropping, belowground interactions increased the numbers of beneficial bacteria in the soil and improved the diversity of the bacterial community, which was conducive to improving soil nutrient (N and P) supply capacity and soil microecosystem stability.

  8. Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

    OpenAIRE

    Weir Jerry P; Schmeisser Falko

    2007-01-01

    Abstract Background Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC) technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2) BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. Whi...

  9. Assessment of anaerobic bacterial diversity and its effects on anaerobic system stability and the occurrence of antibiotic resistance genes.

    Science.gov (United States)

    Aydin, Sevcan; Ince, Bahar; Ince, Orhan

    2016-05-01

    This study evaluated the link between anaerobic bacterial diversity and, the biodegradation of antibiotic combinations and assessed how amending antibiotic combination and increasing concentration of antibiotics in a stepwise fashion influences the development of resistance genes in anaerobic reactors. The biodegradation, sorption and occurrence of the known antibiotic resistance genes (ARGs) of erythromycin and tetracycline were investigated using the processes of UV-HPLC and qPCR analysis respectively. Ion Torrent sequencing was used to detect microbial community changes in response to the addition of antibiotics. The overall results indicated that changes in the structure of a microbial community lead to changes in biodegradation capacity, sorption of antibiotics combinations and occurrence of ARGs. The enhanced biodegradation efficiency appeared to generate variations in the structure of the bacterial community. The results suggested that controlling the ultimate Gram-negative bacterial community, especially Acinetobacter-related populations, may promote the successful biodegradation of antibiotic combinations and reduce the occurrence of ARGs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Diversity of Clinical and Environmental Isolates of Vibrio cholerae in Natural Transformation and Contact-Dependent Bacterial Killing Indicative of Type VI Secretion System Activity.

    Science.gov (United States)

    Bernardy, Eryn E; Turnsek, Maryann A; Wilson, Sarah K; Tarr, Cheryl L; Hammer, Brian K

    2016-05-01

    The bacterial pathogen Vibrio cholerae can occupy both the human gut and aquatic reservoirs, where it may colonize chitinous surfaces that induce the expression of factors for three phenotypes: chitin utilization, DNA uptake by natural transformation, and contact-dependent bacterial killing via a type VI secretion system (T6SS). In this study, we surveyed a diverse set of 53 isolates from different geographic locales collected over the past century from human clinical and environmental specimens for each phenotype outlined above. The set included pandemic isolates of serogroup O1, as well as several serogroup O139 and non-O1/non-O139 strains. We found that while chitin utilization was common, only 22.6% of the isolates tested were proficient at chitin-induced natural transformation, suggesting that transformation is expendable. Constitutive contact-dependent killing of Escherichia coli prey, which is indicative of a functional T6SS, was rare among clinical isolates (only 4 of 29) but common among environmental isolates (22 of 24). These results bolster the pathoadaptive model in which tight regulation of T6SS-mediated bacterial killing is beneficial in a human host, whereas constitutive killing by environmental isolates may give a competitive advantage in natural settings. Future sequence analysis of this set of diverse isolates may identify previously unknown regulators and structural components for both natural transformation and T6SS. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  11. Diversity of Clinical and Environmental Isolates of Vibrio cholerae in Natural Transformation and Contact-Dependent Bacterial Killing Indicative of Type VI Secretion System Activity

    Science.gov (United States)

    Bernardy, Eryn E.; Turnsek, Maryann A.; Wilson, Sarah K.; Tarr, Cheryl L.

    2016-01-01

    The bacterial pathogen Vibrio cholerae can occupy both the human gut and aquatic reservoirs, where it may colonize chitinous surfaces that induce the expression of factors for three phenotypes: chitin utilization, DNA uptake by natural transformation, and contact-dependent bacterial killing via a type VI secretion system (T6SS). In this study, we surveyed a diverse set of 53 isolates from different geographic locales collected over the past century from human clinical and environmental specimens for each phenotype outlined above. The set included pandemic isolates of serogroup O1, as well as several serogroup O139 and non-O1/non-O139 strains. We found that while chitin utilization was common, only 22.6% of the isolates tested were proficient at chitin-induced natural transformation, suggesting that transformation is expendable. Constitutive contact-dependent killing of Escherichia coli prey, which is indicative of a functional T6SS, was rare among clinical isolates (only 4 of 29) but common among environmental isolates (22 of 24). These results bolster the pathoadaptive model in which tight regulation of T6SS-mediated bacterial killing is beneficial in a human host, whereas constitutive killing by environmental isolates may give a competitive advantage in natural settings. Future sequence analysis of this set of diverse isolates may identify previously unknown regulators and structural components for both natural transformation and T6SS. PMID:26944842

  12. Membrane and core periplasmic Agrobacterium tumefaciens virulence Type IV secretion system components localize to multiple sites around the bacterial perimeter during lateral attachment to plant cells.

    Science.gov (United States)

    Aguilar, Julieta; Cameron, Todd A; Zupan, John; Zambryski, Patricia

    2011-01-01

    Type IV secretion systems (T4SS) transfer DNA and/or proteins into recipient cells. Here we performed immunofluorescence deconvolution microscopy to localize the assembled T4SS by detection of its native components VirB1, VirB2, VirB4, VirB5, VirB7, VirB8, VirB9, VirB10, and VirB11 in the C58 nopaline strain of Agrobacterium tumefaciens, following induction of virulence (vir) gene expression. These different proteins represent T4SS components spanning the inner membrane, periplasm, or outer membrane. Native VirB2, VirB5, VirB7, and VirB8 were also localized in the A. tumefaciens octopine strain A348. Quantitative analyses of the localization of all the above Vir proteins in nopaline and octopine strains revealed multiple foci in single optical sections in over 80% and 70% of the bacterial cells, respectively. Green fluorescent protein (GFP)-VirB8 expression following vir induction was used to monitor bacterial binding to live host plant cells; bacteria bind predominantly along their lengths, with few bacteria binding via their poles or subpoles. vir-induced attachment-defective bacteria or bacteria without the Ti plasmid do not bind to plant cells. These data support a model where multiple vir-T4SS around the perimeter of the bacterium maximize effective contact with the host to facilitate efficient transfer of DNA and protein substrates. Transfer of DNA and/or proteins to host cells through multiprotein type IV secretion system (T4SS) complexes that span the bacterial cell envelope is critical to bacterial pathogenesis. Early reports suggested that T4SS components localized at the cell poles. Now, higher-resolution deconvolution fluorescence microscopy reveals that all structural components of the Agrobacterium tumefaciens vir-T4SS, as well as its transported protein substrates, localize to multiple foci around the cell perimeter. These results lead to a new model of A. tumefaciens attachment to a plant cell, where A. tumefaciens takes advantage of the multiple

  13. Diagnostic accuracies of procalcitonin and C-reactive protein for bacterial infection in patients with systemic rheumatic diseases: a meta-analysis.

    Science.gov (United States)

    Song, Gwan Gyu; Bae, Sang-Cheol; Lee, Young Ho

    2015-01-01

    The purpose of this study was to compare the diagnostic performance of procalcitonin and C-reactive protein (CRP) for bacterial infection in patients with systemic rheumatic diseases. We searched Medline, Embase, and the Cochran library, and performed two meta-analyses on the diagnostic accuracy of procalcitonin and CRP for bacterial infection in systemic rheumatic disease patients. A total of eight studies including 668 patients in whom the patients with bacterial infection were 208 were available for the meta-analysis. The pooled sensitivity and specificity of procalcitonin were 66.8% (95% confidence interval [CI] 60.0-73.2) and 89.8% (86.6-92.4), respectively, and those of CRP were 81.3% (75.3-86.3) and 63.0% (58.5-67.5). Procalcitonin PLR, NLR, and DOR were 5.930 (3.593-9.786), 0.352 (0.229-0.539), and 19.33 (10.25-36.45), respectively, and those for CRP were 2.228 (1.376-3.608), 0.367 (0.252-0.534), and 7.066 (3.559-14.03), respectively. The AUC of procalcitonin was 0.884 and the Q* index was 0.814, while the AUC of CRP was 0.789 and the Q* index was 0.726, which indicated that the diagnostic accuracy of procalcitonin in patients with systemic rheumatic diseases is higher than that of CRP (difference of AUC 0.095, 95% CI 0.004-0.185, p=0.039). When the data were limited to SLE, the specificity of procalcitonin was also significantly higher than that of CRP (difference 0.219, 95% CI 0.127-0.310, prheumatic diseases.

  14. BACTERIAL PLASMIDS

    Directory of Open Access Journals (Sweden)

    Marina Dinic

    2007-12-01

    Full Text Available Plasmids, extrachromosomal DNA, were identified in bacteria pertaining to family of Enterobacteriacae for the very first time. After that, they were discovered in almost every single observed strain. The structure of plasmids is made of circular double chain DNA molecules which are replicated autonomously in a host cell. Their length may vary from few up to several hundred kilobase (kb. Among the bacteria, plasmids are mostly transferred horizontally by conjugation process. Plasmid replication process can be divided into three stages: initiation, elongation, and termination. The process involves DNA helicase I, DNA gyrase, DNA polymerase III, endonuclease, and ligase.Plasmids contain genes essential for plasmid function and their preservation in a host cell (the beginning and the control of replication. Some of them possess genes whichcontrol plasmid stability. There is a common opinion that plasmids are unnecessary fora growth of bacterial population and their vital functions; thus, in many cases they can be taken up or kicked out with no lethal effects to a plasmid host cell. However,there are numerous biological functions of bacteria related to plasmids. Plasmids identification and classification are based upon their genetic features which are presented permanently in all of them, and these are: abilities to preserve themselves in a host cell and to control a replication process. In this way, plasmids classification among incompatibility groups is performed. The method of replicon typing, which is based on genotype and not on phenotype characteristics, has the same results as in compatibility grouping.

  15. Bacterial production and growth rate estimation from ( sup 3 H)thymidine incorporation for attached and free-living bacteria in aquatic systems

    Energy Technology Data Exchange (ETDEWEB)

    Iriberri, J.; Unanue, M.; Ayo, B.; Barcina, I., Egea, L. (Univ. del Pais Vasco, Bilbao (Spain))

    1990-02-01

    Production and specific growth rates of attached and free-living bacteria were estimated in an oligotrophic marine system, La Salvaje Beach, Vizcaya, Spain, and in a freshwater system having a higher nutrient concentration, Butron River, Vizcaya, Spain. Production was calculated from (methyl-{sup 3}H)thymidine incorporation by estimating specific conversion factors (cells or micrograms of C produced per mole of thymidine incorporated) for attached and free-living bacteria, respectively, in each system. Conversion factors were not statistically different between attached and free-living bacteria: 6.812 {times} 10{sup 11} and 8.678 {times} 10{sup 11} {mu}g of C mol{sup {minus}1} for free-living and attached bacteria in the freshwater system, and 1.276 {times} 10{sup 11} and 1.354 {times} 10{sup 11} {mu}g of C mol{sup {minus}1} for free-living and attached bacteria in the marine system. Therefore, use of a unique conversion factor for the mixed bacterial population is well founded. However, conversion factors were higher in the freshwater system than in the marine system. This could be due to the different tropic conditions of the two systems. Free-living bacteria contributed the most to production in the two systems (85% in the marine system and 67% in the freshwater system) because of their greater contribution to total biomass. Specific growth rates calculated from production data and biomass data were similar for attached and free-living bacteria.

  16. Bacterial production and growth rate estimation from [3H]thymidine incorporation for attached and free-living bacteria in aquatic systems

    International Nuclear Information System (INIS)

    Iriberri, J.; Unanue, M.; Ayo, B.; Barcina, I.; Egea, L.

    1990-01-01

    Production and specific growth rates of attached and free-living bacteria were estimated in an oligotrophic marine system, La Salvaje Beach, Vizcaya, Spain, and in a freshwater system having a higher nutrient concentration, Butron River, Vizcaya, Spain. Production was calculated from [methyl- 3 H]thymidine incorporation by estimating specific conversion factors (cells or micrograms of C produced per mole of thymidine incorporated) for attached and free-living bacteria, respectively, in each system. Conversion factors were not statistically different between attached and free-living bacteria: 6.812 x 10 11 and 8.678 x 10 11 μg of C mol -1 for free-living and attached bacteria in the freshwater system, and 1.276 x 10 11 and 1.354 x 10 11 μg of C mol -1 for free-living and attached bacteria in the marine system. Therefore, use of a unique conversion factor for the mixed bacterial population is well founded. However, conversion factors were higher in the freshwater system than in the marine system. This could be due to the different tropic conditions of the two systems. Free-living bacteria contributed the most to production in the two systems (85% in the marine system and 67% in the freshwater system) because of their greater contribution to total biomass. Specific growth rates calculated from production data and biomass data were similar for attached and free-living bacteria

  17. Functional genomics to discover antibiotic resistance genes: The paradigm of resistance to colistin mediated by ethanolamine phosphotransferase in Shewanella algae MARS 14.

    Science.gov (United States)

    Telke, Amar A; Rolain, Jean-Marc

    2015-12-01

    Shewanella algae MARS 14 is a colistin-resistant clinical isolate retrieved from bronchoalveolar lavage of a hospitalised patient. A functional genomics strategy was employed to discover the molecular support for colistin resistance in S. algae MARS 14. A pZE21 MCS-1 plasmid-based genomic expression library was constructed in Escherichia coli TOP10. The estimated library size was 1.30×10(8) bp. Functional screening of colistin-resistant clones was carried out on Luria-Bertani agar containing 8 mg/L colistin. Five colistin-resistant clones were obtained after complete screening of the genomic expression library. Analysis of DNA sequencing results found a unique gene in all selected clones. Amino acid sequence analysis of this unique gene using the Integrated Microbial Genomes (IMG) and KEGG databases revealed that this gene encodes ethanolamine phosphotransferase (EptA, or so-called PmrC). Reverse transcription PCR analysis indicated that resistance to colistin in S. algae MARS 14 was associated with overexpression of EptA (27-fold increase), which plays a crucial role in the arrangement of outer membrane lipopolysaccharide. Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

  18. Two-colour fluorescence fluorimetric analysis for direct quantification of bacteria and its application in monitoring bacterial growth in cellulose degradation systems.

    Science.gov (United States)

    Duedu, Kwabena O; French, Christopher E

    2017-04-01

    monitoring of growth of live bacterial cells in 96-well microplates and in assessing in vivo activity of cellulose degrading enzyme systems. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Novel near-infrared BiFC systems from a bacterial phytochrome for imaging protein interactions and drug evaluation under physiological conditions.

    Science.gov (United States)

    Chen, Minghai; Li, Wei; Zhang, Zhiping; Liu, Sanying; Zhang, Xiaowei; Zhang, Xian-En; Cui, Zongqiang

    2015-04-01

    Monitoring protein-protein interactions (PPIs) in live subjects is critical for understanding these fundamental biological processes. Bimolecular fluorescence complementation (BiFC) provides a good technique for imaging PPIs; however, a BiFC system with a long wavelength remains to be pursued for in vivo imaging. Here, we conducted systematic screening of split reporters from a bacterial phytochrome-based, near-infrared fluorescent protein (iRFP). Several new near-infrared phytochrome BiFC systems were built based on selected split sites including the amino acids residues 97/98, 99/100, 122/123, and 123/124. These new near-infrared BiFC systems from a bacterial phytochrome were verified as powerful tools for imaging PPIs under physiological conditions in live cells and in live mice. The interaction between HIV-1 integrase (IN) and cellular cofactor protein Lens epithelium-derived growth factor (LEDGF/p75) was visualized in live cells using the newly constructed iRFP BiFC system because of its important roles in HIV-1 integration and replication. Because the HIV IN-LEDGF/p75 interaction is an attractive anti-HIV target, drug evaluation assays to inhibit the HIV IN-LEDGF/p75 interaction were also performed using the newly constructed BiFC system. The results showed that compound 6 and carbidopa inhibit the HIV IN-LEDGF/p75 interaction in a dose-dependent manner under physiological conditions in the BiFC assays. This study provides novel near-infrared BiFC systems for imaging protein interactions under physiological conditions and provides guidance for splitting other bacterial phytochrome-like proteins to construct BiFC systems. The study also provides a new method for drug evaluation in live cells based on iRFP BiFC systems and supplies some new information regarding candidate drugs for anti-HIV therapies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Bacterial Cell Wall Components

    Science.gov (United States)

    Ginsberg, Cynthia; Brown, Stephanie; Walker, Suzanne

    Bacterial cell-surface polysaccharides cells are surrounded by a variety of cell-surface structures that allow them to thrive in extreme environments. Components of the cell envelope and extracellular matrix are responsible for providing the cells with structural support, mediating intercellular communication, allowing the cells to move or to adhere to surfaces, protecting the cells from attack by antibiotics or the immune system, and facilitating the uptake of nutrients. Some of the most important cell wall components are polysaccharide structures. This review discusses the occurrence, structure, function, and biosynthesis of the most prevalent bacterial cell surface polysaccharides: peptidoglycan, lipopolysaccharide, arabinogalactan, and lipoarabinomannan, and capsular and extracellular polysaccharides. The roles of these polysaccharides in medicine, both as drug targets and as therapeutic agents, are also described.

  1. Bacterial community variations in an alfalfa-rice rotation system revealed by 16S rRNA gene 454-pyrosequencing.

    Science.gov (United States)

    Lopes, Ana R; Manaia, Célia M; Nunes, Olga C

    2014-03-01

    Crop rotation is a practice harmonized with the sustainable rice production. Nevertheless, the implications of this empirical practice are not well characterized, mainly in relation to the bacterial community composition and structure. In this study, the bacterial communities of two adjacent paddy fields in the 3rd and 4th year of the crop rotation cycle and of a nonseeded subplot were characterized before rice seeding and after harvesting, using 454-pyrosequencing of the 16S rRNA gene. Although the phyla Acidobacteria, Proteobacteria, Chloroflexi, Actinobacteria and Bacteroidetes predominated in all the samples, there were variations in relative abundance of these groups. Samples from the 3rd and 4th years of the crop rotation differed on the higher abundance of groups of presumable aerobic bacteria and of presumable anaerobic and acidobacterial groups, respectively. Members of the phylum Nitrospira were more abundant after rice harvest than in the previously sampled period. Rice cropping was positively correlated with the abundance of members of the orders Acidobacteriales and 'Solibacterales' and negatively with lineages such as Chloroflexi 'Ellin6529'. Studies like this contribute to understand variations occurring in the microbial communities in soils under sustainable rice production, based on real-world data. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  2. Correlation between system performance and bacterial composition under varied mixing intensity in thermophilic anaerobic digestion of food waste

    KAUST Repository

    Ghanimeh, Sophia A.

    2017-12-07

    This study examines the stability and efficiency of thermophilic anaerobic digesters treating food waste under various mixing velocities (50–160 rpm). The results showed that high velocities (120 and 160 rpm) were harmful to the digestion process with 18–30% reduction in methane generation and 1.8 to 3.8 times increase in volatile fatty acids (VFA) concentrations, compared to mild mixing (50 and 80 rpm). Also, the removal rate of soluble COD dropped from 75 to 85% (at 50–80 rpm) to 20–59% (at 120–160 rpm). Similarly, interrupted mixing caused adverse impacts and led to near-failure conditions with excessive VFA accumulation (15.6 g l), negative removal rate of soluble COD and low methane generation (132 ml gVS). The best efficiency and stability were achieved under mild mixing (50 and 80 rpm). In particular, the 50 rpm stirring speed resulted in the highest methane generation (573 ml gVS). High-throughput sequencing of 16S rRNA genes revealed that the digesters were dominated by one bacterial genus (Petrotoga; phylym Thermotogae) at all mixing velocities except at 0 rpm, where the community was dominated by one bacterial genus (Anaerobaculum; phylum Synergistetes). The Petrotoga genus seems to have played a major role in the degradation of organic matter.

  3. Response of the bacterial community in an on-farm biopurification system, to which diverse pesticides are introduced over an agricultural season.

    Science.gov (United States)

    Holmsgaard, Peter N; Dealtry, Simone; Dunon, Vincent; Heuer, Holger; Hansen, Lars H; Springael, Dirk; Smalla, Kornelia; Riber, Leise; Sørensen, Søren J

    2017-10-01

    A biopurification system (BPS) is used on-farm to clean pesticide-contaminated wastewater. Due to high pesticide loads, a BPS represents a hot spot for the proliferation and selection as well as the genetic adaptation of discrete pesticide degrading microorganisms. However, while considerable knowledge exists on the biodegradation of specific pesticides in BPSs, the bacterial community composition of these systems has hardly been explored. In this work, the Shannon diversity, the richness and the composition of the bacterial community within an operational BPS receiving wastewater contaminated with various pesticides was, for the first time, elucidated over the course of an agricultural season, using DGGE profiling and pyrosequencing of 16S rRNA gene fragments amplified from total community DNA. During the agricultural season, an increase in the concentration of pesticides in the BPS was observed along with the detection of significant community changes including a decrease in microbial diversity. Additionally, a significant increase in the relative abundance of Proteobacteria, mainly the Gammaproteobacteria, was found, and OTUs (operational taxonomic units) affiliated to Pseudomonas responded positively during the course of the season. Furthermore, a banding-pattern analysis of 16S rRNA gene-based DGGE fingerprinting, targeting the Alpha- and Betaproteobacteria as well as the Actinobacteria, indicated that the Betaproteobacteria might play an important role. Interestingly, a decrease of Firmicutes and Bacteroidetes was observed, indicating their selective disadvantage in a BPS, to which pesticides have been introduced. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. An Amperometric Biosensor for the Determination of Bacterial Sepsis Biomarker, Secretory Phospholipase Group 2-IIA Using a Tri-Enzyme System

    Directory of Open Access Journals (Sweden)

    Nik Nurhanan Nik Mansor

    2018-02-01

    Full Text Available A tri-enzyme system consisting of choline kinase/choline oxidase/horseradish peroxidase was used in the rapid and specific determination of the biomarker for bacterial sepsis infection, secretory phospholipase Group 2-IIA (sPLA2-IIA. These enzymes were individually immobilized onto the acrylic microspheres via succinimide groups for the preparation of an electrochemical biosensor. The reaction of sPLA2-IIA with its substrate initiated a cascading enzymatic reaction in the tri-enzyme system that led to the final production of hydrogen peroxide, which presence was indicated by the redox characteristics of potassium ferricyanide, K3Fe(CN6. An amperometric biosensor based on enzyme conjugated acrylic microspheres and gold nanoparticles composite coated onto a carbon-paste screen printed electrode (SPE was fabricated and the current measurement was performed at a low potential of 0.20 V. This enzymatic biosensor gave a linear range 0.01–100 ng/mL (R2 = 0.98304 with a detection limit recorded at 5 × 10−3 ng/mL towards sPLA2-IIA. Moreover, the biosensor showed good reproducibility (relative standard deviation (RSD of 3.04% (n = 5. The biosensor response was reliable up to 25 days of storage at 4 °C. Analysis of human serum samples for sPLA2-IIA indicated that the biosensor has potential for rapid bacterial sepsis diagnosis in hospital emergency department.

  5. Bacterial communities hitching a hike - a guide to the river system of the Red river, Disko Island, West Greenland

    DEFF Research Database (Denmark)

    Hauptmann, Aviaja Zenia Edna Lyberth; Markussen, Thor N.; Stibal, Marek

    Glacier melting and altered precipitation patterns influence Arctic freshwater and coastal ecosystems. Arctic rivers are central to Arctic water ecosystems linking glacier meltwaters and precipitation with the ocean through transport of particulate matter and microorganisms. However, the impact...... of different water sources on the microbial communities in Arctic rivers and estuaries remains unknown. In this study we used 16S rRNA gene amplicon sequencing to assess a small river and its estuary on Disko Island, West Greenland (69°N). We describe the bacterial community through a river into the estuary......, including communities originating in a glacier and a proglacial lake. Our results show that water from the glacier and lake transports distinct communities into the river in terms of diversity and community composition. Bacteria of terrestrial origin were among the dominating OTUs in the main river, while...

  6. The Yersinia enterocolitica type 3 secretion system (T3SS) as toolbox for studying the cell biological effects of bacterial Rho GTPase modulating T3SS effector proteins.

    Science.gov (United States)

    Wölke, Stefan; Ackermann, Nikolaus; Heesemann, Jürgen

    2011-09-01

    The bacterial effector proteins IpgB(1) and IpgB(2) of Shigella and Map of Escherichia coli activate the Rho GTPases Rac1, RhoA and Cdc42, respectively, whereas YopE and YopT of Yersinia inhibit these Rho family GTPases. We established a Yersinia toolbox which allows to study the cellular effects of these effectors in different combinations in the context of Yersinia type 3 secretion system (Ysc)-T3SS-mediated injection into HeLa cells. For this purpose hybrid proteins were constructed by fusion of YopE with the effector protein of interest. As expected, injected hybrid proteins induced membrane ruffles and Yersinia uptake for IpgB(1) , stress fibres for IpgB(2) and microspikes for Map. By co-infection experiments we could demonstrate (i) IpgB(2) -mediated and ROCK-dependent inhibition of IpgB(1) -mediated Rac1 effects, (ii) YopT-mediated suppression of IpgB(1) -induced Yersinia invasion and (iii) failure of YopE-mediated suppression of IpgB(1) -induced Yersinia invasion, presumably due to preferential inhibition of RhoG by YopE GAP function. By infecting polarized MDCK cells we could demonstrate that Map or IpgB(1) but not IpgB(2) affects cell monolayer integrity. In summary, the Yersinia toolbox is suitable to study cellular effects of effector proteins of diverse bacterial species separately or in combination in the context of bacterial T3SS-mediated injection. © 2011 Blackwell Publishing Ltd.

  7. Composite membrane of bacterially-derived cellulose and molecularly imprinted polymer for use as a transdermal enantioselective controlled-release system of racemic propranolol.

    Science.gov (United States)

    Bodhibukkana, Chatchada; Srichana, Teerapol; Kaewnopparat, Sanae; Tangthong, Naruedom; Bouking, Pisit; Martin, Gary P; Suedee, Roongnapa

    2006-06-12

    A composite membrane for transdermal delivery of S-propranolol enantiomer was developed based on the controlled pore functionalization of bacterial cellulose membranes using a molecularly imprinted polymer (MIP) layer synthesis. The reactive pore-filling of an asymmetric porous cellulose membrane with a MIP thin-layer was effected using a silanized coupler as an additional anchor for the MIP. MIP thin-layers with specific binding sites for S-propranolol were synthesized by copolymerization of methacrylic acid with a cross-linker, ethylene glycol dimethacrylate in the presence of S-propranolol as the template molecule and the latter was subsequently extracted. Selective transport of S-propranolol through the MIP composite membrane was obtained, although this was determined mostly by the parent cellulose membrane with some ancillary contributory effect from the MIP layer. In addition, an enantioselectivity in the transport of propranolol prodrug enantiomers was found, suggesting that the shape and functional groups orientation, which are similar to that of the print molecule were essential for enantiomeric recognition of the MIP composite membrane. The enantioselectivity of S-MIP membranes was also shown when the release of propranolol enantiomers was studied in vitro using rat skin, with racemic propranolol contained in the donor compartment. The composite membrane of bacterially-derived cellulose and molecularly imprinted polymer may have great potential for use as a transdermal enantioselective controlled-release system for racemic propranolol.

  8. Structure of the phosphotransferase domain of the bifunctional aminoglycoside-resistance enzyme AAC(6′)-Ie-APH(2′′)-Ia

    Science.gov (United States)

    Smith, Clyde A.; Toth, Marta; Bhattacharya, Monolekha; Frase, Hilary; Vakulenko, Sergei B.

    2014-01-01

    The bifunctional acetyltransferase(6′)-Ie-phosphotransfer­ase(2′′)-Ia [AAC(6′)-Ie-APH(2′′)-Ia] is the most important aminoglycoside-resistance enzyme in Gram-positive bacteria, conferring resistance to almost all known aminoglycoside antibiotics in clinical use. Owing to its importance, this enzyme has been the focus of intensive research since its isolation in the mid-1980s but, despite much effort, structural details of AAC(6′)-Ie-APH(2′′)-Ia have remained elusive. The structure of the Mg2GDP complex of the APH(2′′)-Ia domain of the bifunctional enzyme has now been determined at 2.3 Å resolution. The structure of APH(2′′)-Ia is reminiscent of the structures of other aminoglycoside phosphotransferases, having a two-domain architecture with the nucleotide-binding site located at the junction of the two domains. Unlike the previously characterized APH(2′′)-IIa and APH(2′′)-IVa enzymes, which are capable of utilizing both ATP and GTP as the phosphate donors, APH(2′′)-Ia uses GTP exclusively in the phosphorylation of the aminoglycoside antibiotics, and in this regard closely resembles the GTP-dependent APH(2′′)-IIIa enzyme. In APH(2′′)-Ia this GTP selectivity is governed by the presence of a ‘gatekeeper’ residue, Tyr100, the side chain of which projects into the active site and effectively blocks access to the adenine-binding template. Mutation of this tyrosine residue to a less bulky phenylalanine provides better access for ATP to the NTP-binding template and converts APH(2′′)-Ia into a dual-specificity enzyme. PMID:24914967

  9. Variation in the ovine cortisol response to systemic bacterial endotoxin challenge is predominantly determined by signalling within the hypothalamic-pituitary-adrenal axis

    International Nuclear Information System (INIS)

    You Qiumei; Karrow, Niel A.; Cao Honghe; Rodriguez, Alexander; Mallard, Bonnie A.; Boermans, Herman J.

    2008-01-01

    Bi-directional communication between the neuroendocrine and immune systems is designed, in part, to maintain or restore homeostasis during physiological stress. Exposure to endotoxin during Gram-negative bacterial infection for example, elicits the release of pro-inflammatory cytokines that activate the hypothalamic-pituitary-adrenal axis (HPAA). The secretion of adrenal glucocorticoids subsequently down regulates the host inflammatory response, minimizing potential tissue damage. Sequence and epigenetic variants in genes involved in regulating the neuroendocrine and immune systems are likely to contribute to individual differences in the HPAA response, and this may influence the host anti-inflammatory response to toxin exposure and susceptibility to inflammatory disease. In this study, high (HCR) and low (LCR) cortisol responders were selected from a normal population of 110 female sheep challenged iv with Escherichia coli endotoxin (400 ng/kg) to identify potential determinants that contribute to variation in the cortisol response phenotype. This phenotype was stable over several years in the HCR and LCR animals, and did not appear to be attributed to differences in expression of hepatic immune-related genes or systemic pro-inflammatory cytokine concentrations. Mechanistic studies using corticotrophin-releasing factor (0.5 μg/kg body weight), arginine vasopressin (0.5 μg/kg), and adrenocorticotropic hormone (0.5 μg/kg) administered iv demonstrated that variation in this phenotype is largely determined by signalling within the HPAA. Future studies will use this ovine HCR/LCR model to investigate potential genetic and epigenetic variants that may contribute to variation in cortisol responsiveness to bacterial endotoxin

  10. Bacterial dynamics in model cheese systems, aiming at safety and quality of Portuguese-style traditional ewe's cheeses.

    Science.gov (United States)

    Pereira, Cláudia I; Graça, João A; Ogando, Natacha S; Gomes, Ana M P; Malcata, F Xavier

    2009-11-01

    An experiment using model ewe's milk cheeses was designed to characterize microbial interactions that arise in actual raw milk cheese environments. These model cheeses were manufactured according to Portuguese artisanal practices, except that the microbial load and biodiversity were fully controlled: single potential pathogens and spoilage bacteria, or a combination thereof, were combined at various initial inoculum levels in sterilized raw ewe's milk with several lactic acid bacteria (LAB) normally found in traditional cheeses. Viable microbial counts were monitored throughout a 60-day ripening period. Two alternative mathematical approaches were used to fit the experimental data generated in terms of population dynamics: percent of inhibition and D-values. These were able to explain the complex competitive interactions between the contaminant microorganisms and the LAB adventitious populations. In general, the tested LAB were less able to inhibit contaminants present in combination and in higher concentrations. Lactococcus lactis, with its strong acidifying potential, was the most effective factor in controlling the unwanted bacterial population, especially single Staphylococcus aureus. The two lactobacilli studied, especially Lactobacillus brevis, were shown to be less effective; Escherichia coli and Listeria innocua were the contaminants least inhibited by the LAB.

  11. The XylS/Pm regulator/promoter system and its use in fundamental studies of bacterial gene expression, recombinant protein production and metabolic engineering.

    Science.gov (United States)

    Gawin, Agnieszka; Valla, Svein; Brautaset, Trygve

    2017-07-01

    The XylS/Pm regulator/promoter system originating from the Pseudomonas putida TOL plasmid pWW0 is widely used for regulated low- and high-level recombinant expression of genes and gene clusters in Escherichia coli and other bacteria. Induction of this system can be graded by using different cheap benzoic acid derivatives, which enter cells by passive diffusion, operate in a dose-dependent manner and are typically not metabolized by the host cells. Combinatorial mutagenesis and selection using the bla gene encoding β-lactamase as a reporter have demonstrated that the Pm promoter, the DNA sequence corresponding to the 5' untranslated end of its cognate mRNA and the xylS coding region can be modified and improved relative to various types of applications. By combining such mutant genetic elements, altered and extended expression profiles were achieved. Due to their unique properties, obtained systems serve as a genetic toolbox valuable for heterologous protein production and metabolic engineering, as well as for basic studies aiming at understanding fundamental parameters affecting bacterial gene expression. The approaches used to modify XylS/Pm should be adaptable for similar improvements also of other microbial expression systems. In this review, we summarize constructions, characteristics, refinements and applications of expression tools using the XylS/Pm system. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  12. Novel insights in preventing Gram-negative bacterial infection in cirrhotic patients: review on the effects of GM-CSF in maintaining homeostasis of the immune system.

    Science.gov (United States)

    Xu, Dong; Zhao, Manzhi; Song, Yuhu; Song, Jianxin; Huang, Yuancheng; Wang, Junshuai

    2015-01-01

    Cirrhotic patients with dysfunctional and/or low numbers of leukocytes are often infected with bacteria, especially Gram-negative bacteria, which is characterized by producing lipopolysaccharide (LPS). Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that influences the production, maturation, function, and survival of various immune cells. In this paper, we reviewed not only Toll-like receptors 4 (TLR4) signaling pathway and its immunological effect, but also the specific stimulating function and autocrine performance of GM-CSF on hematopoietic cells, as well as the recent discovery of innate response activator-B cells in protection against microbial sepsis and the direct LPS-TLR4 signaling on hematopoiesis. Thus we concluded that GM-CSF might play important roles in preventing Gram-negative bacterial infections in cirrhotic patients through maintaining immune system functions and homeostasis.

  13. RND type efflux pump system MexAB-OprM of pseudomonas aeruginosa selects bacterial languages, 3-oxo-acyl-homoserine lactones, for cell-to-cell communication

    Directory of Open Access Journals (Sweden)

    Minagawa Shu

    2012-05-01

    Full Text Available Abstract Background Bacteria release a wide variety of small molecules including cell-to-cell signaling compounds. Gram-negative bacteria use a variety of self-produced autoinducers such as acylated homoserine lactones (acyl-HSLs as signal compounds for quorum sensing (QS within and between bacterial species. QS plays a significant role in the pathogenesis of infectious diseases and in beneficial symbiosis by responding to acyl-HSLs in Pseudomonas aeruginosa. It is considered that the selection of bacterial languages is necessary to regulate gene expression and thus it leads to the regulation of virulence and provides a growth advantage in several environments. In this study, we hypothesized that RND-type efflux pump system MexAB-OprM of P. aeruginosa might function in the selection of acyl-HSLs, and we provide evidence to support this hypothesis. Results Loss of MexAB-OprM due to deletion of mexB caused increases in QS responses, as shown by the expression of gfp located downstream of the lasB promoter and LasB elastase activity, which is regulated by a LasR-3-oxo-C12-HSL complex. Either complementation with a plasmid containing wild-type mexB or the addition of a LasR-specific inhibitor, patulin, repressed these high responses to 3-oxo-acyl-HSLs. Furthermore, it was shown that the acyl-HSLs-dependent response of P. aeruginosa was affected by the inhibition of MexB transport activity and the mexB mutant. The P. aeruginosa MexAB-OprM deletion mutant showed a strong QS response to 3-oxo-C10-HSL produced by Vibrio anguillarum in a bacterial cross-talk experiment. Conclusion This work demonstrated that MexAB-OprM does not control the binding of LasR to 3-oxo-Cn-HSLs but rather accessibility of non-cognate acyl-HSLs to LasR in P. aeruginosa. MexAB-OprM not only influences multidrug resistance, but also selects acyl-HSLs and regulates QS in P. aeruginosa. The results demonstrate a new QS regulation mechanism via the efflux system MexAB-OprM in P

  14. RND type efflux pump system MexAB-OprM of Pseudomonas aeruginosa selects bacterial languages, 3-oxo-acyl-homoserine lactones, for cell-to-cell communication.

    Science.gov (United States)

    Minagawa, Shu; Inami, Hiroyuki; Kato, Tomohisa; Sawada, Shinji; Yasuki, Tatsuya; Miyairi, Shinichi; Horikawa, Manabu; Okuda, Jun; Gotoh, Naomasa

    2012-05-10

    Bacteria release a wide variety of small molecules including cell-to-cell signaling compounds. Gram-negative bacteria use a variety of self-produced autoinducers such as acylated homoserine lactones (acyl-HSLs) as signal compounds for quorum sensing (QS) within and between bacterial species. QS plays a significant role in the pathogenesis of infectious diseases and in beneficial symbiosis by responding to acyl-HSLs in Pseudomonas aeruginosa. It is considered that the selection of bacterial languages is necessary to regulate gene expression and thus it leads to the regulation of virulence and provides a growth advantage in several environments. In this study, we hypothesized that RND-type efflux pump system MexAB-OprM of P. aeruginosa might function in the selection of acyl-HSLs, and we provide evidence to support this hypothesis. Loss of MexAB-OprM due to deletion of mexB caused increases in QS responses, as shown by the expression of gfp located downstream of the lasB promoter and LasB elastase activity, which is regulated by a LasR-3-oxo-C12-HSL complex. Either complementation with a plasmid containing wild-type mexB or the addition of a LasR-specific inhibitor, patulin, repressed these high responses to 3-oxo-acyl-HSLs. Furthermore, it was shown that the acyl-HSLs-dependent response of P. aeruginosa was affected by the inhibition of MexB transport activity and the mexB mutant. The P. aeruginosa MexAB-OprM deletion mutant showed a strong QS response to 3-oxo-C10-HSL produced by Vibrio anguillarum in a bacterial cross-talk experiment. This work demonstrated that MexAB-OprM does not control the binding of LasR to 3-oxo-Cn-HSLs but rather accessibility of non-cognate acyl-HSLs to LasR in P. aeruginosa. MexAB-OprM not only influences multidrug resistance, but also selects acyl-HSLs and regulates QS in P. aeruginosa. The results demonstrate a new QS regulation mechanism via the efflux system MexAB-OprM in P. aeruginosa.

  15. A common theme in interaction of bacterial immunoglobulin-binding proteins with immunoglobulins illustrated in the equine system.

    Science.gov (United States)

    Lewis, Melanie J; Meehan, Mary; Owen, Peter; Woof, Jenny M

    2008-06-20

    The M protein of Streptococcus equi subsp. equi known as fibrinogen-binding protein (FgBP) is a cell wall-associated protein with antiphagocytic activity that binds IgG. Recombinant versions of the seven equine IgG subclasses were used to investigate the subclass specificity of FgBP. FgBP bound predominantly to equine IgG4 and IgG7, with little or no binding to the other subclasses. Competitive binding experiments revealed that FgBP could inhibit the binding of staphylococcal protein A and streptococcal protein G to both IgG4 and IgG7, implicating the Fc interdomain region in binding to FgBP. To identify which of the two IgG Fc domains contributed to the interaction with FgBP, we tested two human IgG1/IgA1 domain swap mutants and found that both domains are required for full binding, with the CH3 domain playing a critical role. The binding site for FgBP was further localized using recombinant equine IgG7 antibodies with single or double point mutations to residues lying at the CH2-CH3 interface. We found that interaction of FgBP with equine IgG4 and IgG7 was able to disrupt C1q binding and antibody-mediated activation of the classical complement pathway, demonstrating an effective means by which S. equi may evade the immune response. The mode of interaction of FgBP with IgG fits a common theme for bacterial Ig-binding proteins. Remarkably, for those interactions studied in detail, it emerges that all the Ig-binding proteins target the CH2-CH3 domain interface, regardless of specificity for IgG or IgA, streptococcal or staphylococcal origin, or host species (equine or human).

  16. Nitrate source apportionment using a combined dual isotope, chemical and bacterial property, and Bayesian model approach in river systems

    Science.gov (United States)

    Xia, Yongqiu; Li, Yuefei; Zhang, Xinyu; Yan, Xiaoyuan

    2017-01-01

    Nitrate (NO3-) pollution is a serious problem worldwide, particularly in countries with intensive agricultural and population activities. Previous studies have used δ15N-NO3- and δ18O-NO3- to determine the NO3- sources in rivers. However, this approach is subject to substantial uncertainties and limitations because of the numerous NO3- sources, the wide isotopic ranges, and the existing isotopic fractionations. In this study, we outline a combined procedure for improving the determination of NO3- sources in a paddy agriculture-urban gradient watershed in eastern China. First, the main sources of NO3- in the Qinhuai River were examined by the dual-isotope biplot approach, in which we narrowed the isotope ranges using site-specific isotopic results. Next, the bacterial groups and chemical properties of the river water were analyzed to verify these sources. Finally, we introduced a Bayesian model to apportion the spatiotemporal variations of the NO3- sources. Denitrification was first incorporated into the Bayesian model because denitrification plays an important role in the nitrogen pathway. The results showed that fertilizer contributed large amounts of NO3- to the surface water in traditional agricultural regions, whereas manure effluents were the dominant NO3- source in intensified agricultural regions, especially during the wet seasons. Sewage effluents were important in all three land uses and exhibited great differences between the dry season and the wet season. This combined analysis quantitatively delineates the proportion of NO3- sources from paddy agriculture to urban river water for both dry and wet seasons and incorporates isotopic fractionation and uncertainties in the source compositions.

  17. Individuality, phenotypic differentiation, dormancy and ‘persistence’ in culturable bacterial systems: commonalities shared by environmental, laboratory, and clinical microbiology

    Science.gov (United States)

    Kell, Douglas; Potgieter, Marnie; Pretorius, Etheresia

    2015-01-01

    For bacteria, replication mainly involves growth by binary fission. However, in a very great many natural environments there are examples of phenotypically dormant, non-growing cells that do not replicate immediately and that are phenotypically ‘nonculturable’ on media that normally admit their growth. They thereby evade detection by conventional culture-based methods. Such dormant cells may also be observed in laboratory cultures and in clinical microbiology. They are usually more tolerant to stresses such as antibiotics, and in clinical microbiology they are typically referred to as ‘persisters’. Bacterial cultures necessarily share a great deal of relatedness, and inclusive fitness theory implies that there are conceptual evolutionary advantages in trading a variation in growth rate against its mean, equivalent to hedging one’s bets. There is much evidence that bacteria exploit this strategy widely. We here bring together data that show the commonality of these phenomena across environmental, laboratory and clinical microbiology. Considerable evidence, using methods similar to those common in environmental microbiology, now suggests that many supposedly non-communicable, chronic and inflammatory diseases are exacerbated (if not indeed largely caused) by the presence of dormant or persistent bacteria (the ability of whose components to cause inflammation is well known). This dormancy (and resuscitation therefrom) often reflects the extent of the availability of free iron. Together, these phenomena can provide a ready explanation for the continuing inflammation common to such chronic diseases and its correlation with iron dysregulation. This implies that measures designed to assess and to inhibit or remove such organisms (or their access to iron) might be of much therapeutic benefit. PMID:26629334

  18. Pyrosequencing Reveals Bacterial Communities in Unchlorinated Drinking Water Distribution System: An Integral Study of Bulk Water, Suspended Solids, Loose Deposits, and Pipe Wall Biofilm

    KAUST Repository

    Liu, G.

    2014-05-20

    The current understanding of drinking water distribution system (DWDS) microbiology is limited to pipe wall biofilm and bulk water; the contributions of particle-associated bacteria (from suspended solids and loose deposits) have long been neglected. Analyzing the composition and correlation of bacterial communities from different phases helped us to locate where most of the bacteria are and understand the interactions among these phases. In the present study, the bacteria from four critical phases of an unchlorinated DWDS, including bulk water, pipe wall biofilm, suspended solids, and loose deposits, were quantified and identified by adenosine triphosphate analysis and pyrosequencing, respectively. The results showed that the bulk water bacteria (including the contribution of suspended solids) contributed less than 2% of the total bacteria. The bacteria associated with loose deposits and pipe wall biofilm that accumulated in the DWDS accounted for over 98% of the total bacteria, and the contributions of bacteria in loose deposits and pipe wall biofilm were comparable. Depending on the amount of loose deposits, its contribution can be 7-fold higher than the pipe wall biofilm. Pyrosequencing revealed relatively stable bacterial communities in bulk water, pipe wall biofilm, and suspended solids throughout the distribution system; however, the communities present in loose deposits were dependent on the amount of loose deposits locally. Bacteria within the phases of suspended solids, loose deposits, and pipe wall biofilm were similar in phylogenetic composition. The bulk water bacteria (dominated by Polaromonas spp.) were clearly different from the bacteria from the other three phases (dominated by Sphingomonas spp.). This study highlighted that the integral DWDS ecology should include contributions from all of the four phases, especially the bacteria harbored by loose deposits. The accumulation of loose deposits and the aging process create variable microenvironments

  19. Pyrosequencing reveals bacterial communities in unchlorinated drinking water distribution system: an integral study of bulk water, suspended solids, loose deposits, and pipe wall biofilm.

    Science.gov (United States)

    Liu, G; Bakker, G L; Li, S; Vreeburg, J H G; Verberk, J Q J C; Medema, G J; Liu, W T; Van Dijk, J C

    2014-05-20

    The current understanding of drinking water distribution system (DWDS) microbiology is limited to pipe wall biofilm and bulk water; the contributions of particle-associated bacteria (from suspended solids and loose deposits) have long been neglected. Analyzing the composition and correlation of bacterial communities from different phases helped us to locate where most of the bacteria are and understand the interactions among these phases. In the present study, the bacteria from four critical phases of an unchlorinated DWDS, including bulk water, pipe wall biofilm, suspended solids, and loose deposits, were quantified and identified by adenosine triphosphate analysis and pyrosequencing, respectively. The results showed that the bulk water bacteria (including the contribution of suspended solids) contributed less than 2% of the total bacteria. The bacteria associated with loose deposits and pipe wall biofilm that accumulated in the DWDS accounted for over 98% of the total bacteria, and the contributions of bacteria in loose deposits and pipe wall biofilm were comparable. Depending on the amount of loose deposits, its contribution can be 7-fold higher than the pipe wall biofilm. Pyrosequencing revealed relatively stable bacterial communities in bulk water, pipe wall biofilm, and suspended solids throughout the distribution system; however, the communities present in loose deposits were dependent on the amount of loose deposits locally. Bacteria within the phases of suspended solids, loose deposits, and pipe wall biofilm were similar in phylogenetic composition. The bulk water bacteria (dominated by Polaromonas spp.) were clearly different from the bacteria from the other three phases (dominated by Sphingomonas spp.). This study highlighted that the integral DWDS ecology should include contributions from all of the four phases, especially the bacteria harbored by loose deposits. The accumulation of loose deposits and the aging process create variable microenvironments

  20. Physical and chemical quality, biodiversity, and thermodynamic prediction of adhesion of bacterial isolates from a water purification system: a case study

    Directory of Open Access Journals (Sweden)

    Roberta Barbosa Teodoro Alves

    2017-06-01

    Full Text Available ABSTRACT The objective of this study was to evaluate the quality of water purification system and identify the bacteria this system, predict bacterial adherence according to the hydrophobicity of these microorganisms and of the polypropylene distribution loop for purified water. The assessment of drinking water that supplies the purification system allowed good-quality physical, chemical, and microbiological specifications. The physicochemical specifications of the distributed purified water were approved, but the heterotrophic bacteria count was higher than allowed (>2 log CFU mL-1.The sanitation of the storage tank with chlorine decreased the number of bacteria adhered to the surface (4.34 cycles log. By sequencing of the 16SrDNA genes, six species of bacteria were identified. The contact angle was determined and polypropylene surface and all bacteria were considered to be hydrophilic, and adhesion was thermodynamically unfavorable. This case study showed the importance of monitoring the water quality in the purified water systems and the importance of sanitization with chemical agents. The count of heterotrophic bacteria on the polypropylene surface was consistent with the predicted thermodynamics results because the number of adhered cells reached approximate values of 5 log CFU cm-2.

  1. Comparison of the EntericBio multiplex PCR system with routine culture for detection of bacterial enteric pathogens.

    LENUS (Irish Health Repository)

    O'Leary, James

    2009-11-01

    The EntericBio system uses a multiplex PCR assay for the simultaneous detection of Campylobacter spp., Salmonella enterica, Shigella spp., and Escherichia coli O157 from feces. It combines overnight broth enrichment with PCR amplification and detection by hybridization. An evaluation of this system was conducted by comparing the results obtained with the system with those obtained by routine culture, supplemented with alternative PCR detection methods. In a study of 773 samples, routine culture and the EntericBio system yielded 94.6 and 92.4% negative results, respectively. Forty-two samples had positive results by culture, and all of these were positive with the EntericBio system. This system detected an additional 17 positive samples (Campylobacter spp., n = 12; Shigella spp., n = 1; E. coli O157, n = 4), but the results for 5 samples (Campylobacter spp., n = 2; Shigella spp., n = 1; E. coli O157, n = 2) could not be confirmed. The target for Shigella spp. detected by the EntericBio system is the ipaH gene, and the molecular indication of the presence of Shigella spp. was investigated by sequence analysis, which confirmed that the ipaH gene was present in a Klebsiella pneumoniae isolate from the patient. The sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 99.3%, 91.5%, and 100%, respectively. Turnaround times were significantly reduced with the EntericBio system, and a result was available between 24 and 32 h after receipt of the sample in the laboratory. In addition, the amount of laboratory waste was significantly reduced by use of this system. In summary, the EntericBio system proved convenient to use, more sensitive than the conventional culture used in this study, and highly specific; and it generated results significantly faster than routine culture for the pathogens tested.

  2. Two-Component Signaling System VgrRS Directly Senses Extracytoplasmic and Intracellular Iron to Control Bacterial Adaptation under Iron Depleted Stress.

    Directory of Open Access Journals (Sweden)

    Li Wang

    2016-12-01

    Full Text Available Both iron starvation and excess are detrimental to cellular life, especially for animal and plant pathogens since they always live in iron-limited environments produced by host immune responses. However, how organisms sense and respond to iron is incompletely understood. Herein, we reveal that in the phytopathogenic bacterium Xanthomonas campestris pv. campestris, VgrS (also named ColS is a membrane-bound receptor histidine kinase that senses extracytoplasmic iron limitation in the periplasm, while its cognate response regulator, VgrR (ColR, detects intracellular iron excess. Under iron-depleted conditions, dissociation of Fe3+ from the periplasmic sensor region of VgrS activates the VgrS autophosphorylation and subsequent phosphotransfer to VgrR, an OmpR-family transcription factor that regulates bacterial responses to take up iron. VgrR-VgrS regulon and the consensus DNA binding motif of the transcription factor VgrR were dissected by comparative proteomic and ChIP-seq analyses, which revealed that in reacting to iron-depleted environments, VgrR directly or indirectly controls the expressions of hundreds of genes that are involved in various physiological cascades, especially those associated with iron-uptake. Among them, we demonstrated that the phosphorylated VgrR tightly represses the transcription of a special TonB-dependent receptor gene, tdvA. This regulation is a critical prerequisite for efficient iron uptake and bacterial virulence since activation of tdvA transcription is detrimental to these processes. When the intracellular iron accumulates, the VgrR-Fe2+ interaction dissociates not only the binding between VgrR and the tdvA promoter, but also the interaction between VgrR and VgrS. This relieves the repression in tdvA transcription to impede continuous iron uptake and avoids possible toxic effects of excessive iron accumulation. Our results revealed a signaling system that directly senses both extracytoplasmic and intracellular

  3. Bacterial Prostatitis: Bacterial Virulence, Clinical Outcomes, and New Directions.

    Science.gov (United States)

    Krieger, John N; Thumbikat, Praveen

    2016-02-01

    Four prostatitis syndromes are recognized clinically: acute bacterial prostatitis, chronic bacterial prostatitis, chronic prostatitis/chronic pelvic pain syndrome, and asymptomatic prostatitis. Because Escherichia coli represents the most common cause of bacterial prostatitis, we investigated the importance of bacterial virulence factors and antimicrobial resistance in E. coli strains causing prostatitis and the potential association of these characteristics with clinical outcomes. A structured literature review revealed that we have limited understanding of the virulence-associated characteristics of E. coli causing acute prostatitis. Therefore, we completed a comprehensive microbiological and molecular investigation of a unique strain collection isolated from healthy young men. We also considered new data from an animal model system suggesting certain E. coli might prove important in the etiology of chronic prostatitis/chronic pelvic pain syndrome. Our human data suggest that E. coli needs multiple pathogenicity-associated traits to overcome anatomic and immune responses in healthy young men without urological risk factors. The phylogenetic background and accumulation of an exceptional repertoire of extraintestinal pathogenic virulence-associated genes indicate that these E. coli strains belong to a highly virulent subset of uropathogenic variants. In contrast, antibiotic resistance confers little added advantage to E. coli strains in these healthy outpatients. Our animal model data also suggest that certain pathogenic E. coli may be important in the etiology of chronic prostatitis/chronic pelvic pain syndrome through mechanisms that are dependent on the host genetic background and the virulence of the bacterial strain.

  4. Role of the Genes of Type VI Secretion System in Virulence of Rice Bacterial Brown Stripe Pathogen Acidovorax avenae subsp. avenae Strain RS-2

    Directory of Open Access Journals (Sweden)

    Md. Mahidul Islam Masum

    2017-09-01

    Full Text Available The Type VI secretion system (T6SS is a class of macromolecular machine that is required for the virulence of gram-negative bacteria. However, it is still not clear what the role of T6SS in the virulence of rice bacterial brown stripe pathogen Acidovorax avenae subsp. avenae (Aaa is. The aim of the current study was to investigate the contribution of T6SS in Aaa strain RS2 virulence using insertional deletion mutation and complementation approaches. This strain produced weak virulence but contains a complete T6SS gene cluster based on a genome-wide analysis. Here we compared the virulence-related phenotypes between the wild-type (RS-2 and 25 T6SS mutants, which were constructed using homologous recombination methods. The mutation of 15 T6SS genes significantly reduced bacterial virulence and the secretion of Hcp protein. Additionally, the complemented 7 mutations ΔpppA, ΔclpB, Δhcp, ΔdotU, ΔicmF, ΔimpJ, and ΔimpM caused similar virulence characteristics as RS-2. Moreover, the mutant ΔpppA, ΔclpB, ΔicmF, ΔimpJ and ΔimpM genes caused by a 38.3~56.4% reduction in biofilm formation while the mutants ΔpppA, ΔclpB, ΔicmF and Δhcp resulted in a 37.5~44.6% reduction in motility. All together, these results demonstrate that T6SS play vital roles in the virulence of strain RS-2, which may be partially attributed to the reductions in Hcp secretion, biofilm formation and motility. However, differences in virulence between strain RS-1 and RS-2 suggest that other factors may also be involved in the virulence of Aaa.

  5. Role of the Genes of Type VI Secretion System in Virulence of Rice Bacterial Brown Stripe Pathogen Acidovorax avenae subsp. avenae Strain RS-2.

    Science.gov (United States)

    Masum, Md Mahidul Islam; Yang, Yingzi; Li, Bin; Olaitan, Ogunyemi Solabomi; Chen, Jie; Zhang, Yang; Fang, Yushi; Qiu, Wen; Wang, Yanli; Sun, Guochang

    2017-09-21

    The Type VI secretion system (T6SS) is a class of macromolecular machine that is required for the virulence of gram-negative bacteria. However, it is still not clear what the role of T6SS in the virulence of rice bacterial brown stripe pathogen Acidovorax avenae subsp. avenae (Aaa) is. The aim of the current study was to investigate the contribution of T6SS in Aaa strain RS2 virulence using insertional deletion mutation and complementation approaches. This strain produced weak virulence but contains a complete T6SS gene cluster based on a genome-wide analysis. Here we compared the virulence-related phenotypes between the wild-type (RS-2) and 25 T6SS mutants, which were constructed using homologous recombination methods. The mutation of 15 T6SS genes significantly reduced bacterial virulence and the secretion of Hcp protein. Additionally, the complemented 7 mutations Δ pppA , Δ clpB , Δ hcp , Δ dotU , Δ icmF , Δ impJ , and Δ impM caused similar virulence characteristics as RS-2. Moreover, the mutant Δ pppA , Δ clpB , Δ icmF , Δ impJ and Δ impM genes caused by a 38.3~56.4% reduction in biofilm formation while the mutants Δ pppA , Δ clpB , Δ icmF and Δ hcp resulted in a 37.5~44.6% reduction in motility. All together, these results demonstrate that T6SS play vital roles in the virulence of strain RS-2, which may be partially attributed to the reductions in Hcp secretion, biofilm formation and motility. However, differences in virulence between strain RS-1 and RS-2 suggest that other factors may also be involved in the virulence of Aaa.

  6. Identification of two proteins that interact with the Erp virulence factor from Mycobacterium tuberculosis by using the bacterial two-hybrid system

    Directory of Open Access Journals (Sweden)

    Cataldi Angel A

    2009-01-01

    Full Text Available Abstract Background The exported repetitive protein (erp gene encodes a secreted 36-kDa protein with a central domain containing several proline-glycine-leucine-threonine-serine (PGLTS repeats. It has been demonstrated that erp is a virulence-associated factor since the disruption of this gene impairs the growth of Mycobacterium bovis and Mycobacterium tuberculosis in mice. Results In order to elucidate the function of Erp we searched for Erp-binding proteins from M. tuberculosis by using a bacterial two-hybrid system. Our results indicate that Erp interacts specifically with two putative membrane proteins, Rv1417 and Rv2617c. Further analysis revealed that the latter two interact with each other, indicating that Rv1417, Rv2617c and Erp are connected through multiple interactions. While Rv1417 is disseminated in several Actinomycetales genera, orthologues of Rv2617c are exclusively present in members of the M. tuberculosis complex (MTC. The central and amino-terminal regions of Erp were determined to be involved in the interaction with Rv1417 and Rv2627c. Erp forms from Mycobacterium smegmatis and Mycobacterium leprae were not able to interact with Rv2617c in two-hybrid assays. Immunolocalization experiments showed that Rv1417 and Rv2617c are found on the cell membrane and Erp on the bacterial cell wall. Finally, comparative genomics and expression studies revealed a possible role of Rv1417 in riboflavin metabolism. Conclusion We identified interactive partners of Erp, an M. tuberculosis protein involved in virulence, which will be the focus of future investigation to decipher the function of the Erp family protein.

  7. A systemic vaccine based on Escherichia coli O157:H7 bacterial ghosts (BGs) reduces the excretion of E. coli O157:H7 in calves.

    Science.gov (United States)

    Vilte, D A; Larzábal, M; Mayr, U B; Garbaccio, S; Gammella, M; Rabinovitz, B C; Delgado, F; Meikle, V; Cantet, R J C; Lubitz, P; Lubitz, W; Cataldi, A; Mercado, E C

    2012-04-15

    Cattle are the main reservoir of enterohemorrhagic Escherichia coli O157:H7, a bacterium that, in humans, causes hemorrhagic colitis and hemolytic uremic syndrome (HUS), a life-threatening disease, especially in children and older people. Therefore, the development of vaccines preventing colonization of cattle by E. coli O157:H7 could be a main tool for an HUS control program. In the present study, we evaluated bacterial ghosts (BGs) of E. coli O157:H7 as an experimental vaccine against this pathogen. BGs are empty envelopes of Gram-negative bacteria, which retain the morphological surface make-up of their living counterparts and are produced by controlled expression of the cloned protein E, which causes loss of all the cytoplasm content. In this work, E. coli O157:H7 BGs were used for subcutaneous immunization of calves. The vaccinated animals elicited significant levels of BG-specific IgG but not IgA antibodies in serum. Low levels of IgA and IgG antibodies against BGs were detected in saliva from vaccinated animals. Following oral challenge with E. coli O157:H7, a significant reduction in both the duration and total bacterial shedding was observed in vaccinated calves compared to the nonimmunized group. We demonstrated that systemic vaccination with E. coli O157 BGs provides protection in a bovine experimental model. Further research is needed to reach a higher mucosal immune response leading to an optimal vaccine. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. IL-1RI (Interleukin-1 Receptor Type I Signalling is Essential for Host Defence and Hemichannel Activity During Acute Central Nervous System Bacterial Infection

    Directory of Open Access Journals (Sweden)

    Juan Xiong

    2012-03-01

    Full Text Available Staphylococcus aureus is a common aetiological agent of bacterial brain abscesses. We have previously established that a considerable IL-1 (interleukin-1 response is elicited immediately following S. aureus infection, where the cytokine can exert pleiotropic effects on glial activation and blood–brain barrier permeability. To assess the combined actions of IL-1α and IL-1β during CNS (central nervous system infection, host defence responses were evaluated in IL-1RI (IL-1 receptor type I KO (knockout animals. IL-1RI KO mice were exquisitely sensitive to intracerebral S. aureus infection, as demonstrated by enhanced mortality rates and bacterial burdens within the first 24 h following pathogen exposure compared with WT (wild-type animals. Loss of IL-1RI signalling also dampened the expression of select cytokines and chemokines, concomitant with significant reductions in neutrophil and macrophage infiltrates into the brain. In addition, the opening of astrocyte hemichannels during acute infection was shown to be dependent on IL-1RI activity. Collectively, these results demonstrate that IL-1RI signalling plays a pivotal role in the genesis of immune responses during the acute stage of brain abscess development through S. aureus containment, inflammatory mediator production, peripheral immune cell recruitment, and regulation of astrocyte hemichannel activity. Taken in the context of previous studies with MyD88 (myeloid differentiation primary response gene 88 and TLR2 (Toll-like receptor 2 KO animals, the current report advances our understanding of MyD88-dependent cascades and implicates IL-1RI signalling as a major antimicrobial effector pathway during acute brain-abscess formation.

  9. Microbact™ 24E system identifiation and antimicrobial sensitivity pattern of bacterial flra from raw milk of apparently healthy lactating cows in Gwagwalada, Nigeria

    Directory of Open Access Journals (Sweden)

    Samuel Mailafia

    2017-08-01

    Full Text Available Objective: To determine the prevalence of bacterial organisms from raw milk of cows in Gwagwalada and determine their susceptibility to commonly used antimicrobial agents. Methods: A total of 120 samples of milk were obtained from lactating cows that were at different stages of postpartum from six different locations in Gwagwalada metropolis. Samples were subjected to Microbact™ 24E system identification, isolation and characterization of isolates, and antibiotics susceptibility test. Results: The most prevalent organisms were Staphylococcus aureus (34.1%, Escherichia coli (27.3% and Bacillus species (18.2% while the least isolated were Salmonella species (11.4% and Pseudomonas aeruginosa (9.0%. The highest resistance patterns were shown by Staphylococcus aureus which displayed resistance to five drugs: amoxicillin, ampiclox, levofloxacin, norfloxacin, chloramphenicol and streptomycin. The least resistance was displayed by Bacillus species which were resistant to only two drugs, norfloxacin and chloramphenicol. Pseudomonas aeroginosa dissipated the highest pattern susceptibility to ciprofloxacin, norfloxacin, gentamicin and streptomycin while Salmonella species showed the lowest pattern of susceptibility to ciprofloxacin only. Other microorganisms dissipated susceptibility patterns ranging from 16.6%–100.0%. Conclusions: This study documented the occurrence of bacterial flora in raw milk of apparently healthy lactating cows in the Gwagwalada area. The variation in patterns of multidrug resistance and susceptibilities in our studies may lead to possibility of transfer of antibiotic resistance from raw milk consumers. More studies are required using higher molecular techniques to expose different species of microorganisms causing milk borne illness and their antibiotic resistant genes.

  10. One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system.

    Science.gov (United States)

    Wang, Wei-Ming; Lee, A-Young; Chiang, Cheng-Ming

    2008-05-01

    The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stress responses. Although the importance of AP-1 has long been recognized, the biochemical characterization of AP-1 remains limited in part due to the difficulty in purifying full-length, reconstituted dimers with active DNA-binding and transcriptional activity. Using a combination of bacterial coexpression and epitope-tagging methods, we successfully purified all 12 heterodimers (3 Junx4 Fos) of full-length human AP-1 complexes as well as c-Jun/c-Jun, JunD/JunD, and c-Jun/JunD dimers from bacterial inclusion bodies using one-step nickel-NTA affinity tag purification following denaturation and renaturation of coexpressed AP-1 subunits. Coexpression of two constitutive components in a dimeric AP-1 complex helps stabilize the proteins when compared with individual protein expression in bacteria. Purified dimeric AP-1 complexes are functional in sequence-specific DNA binding, as illustrated by electrophoretic mobility shift assays and DNase I footprinting, and are also active in transcription with in vitro-reconstituted human papillomavirus (HPV) chromatin containing AP-1-binding sites in the native configuration of HPV nucleosomes. The availability of these recombinant full-length human AP-1 complexes has greatly facilitated mechanistic studies of AP-1-regulated gene transcription in many biological systems.

  11. Do host genetic traits in the bacterial sensing system play a role in the development of Chlamydia trachomatis-associated tubal pathology in subfertile women?

    Directory of Open Access Journals (Sweden)

    Ito James I

    2006-07-01

    Full Text Available Abstract Background In women, Chlamydia (C. trachomatis upper genital tract infection can cause distal tubal damage and occlusion, increasing the risk of tubal factor subfertility and ectopic pregnancy. Variations, like single nucleotide polymorphisms (SNPs, in immunologically important host genes are assumed to play a role in the course and outcome of a C. trachomatis infection. We studied whether genetic traits (carrying multiple SNPs in different genes in the bacterial sensing system are associated with an aberrant immune response and subsequently with tubal pathology following a C. trachomatis infection. The genes studied all encode for pattern recognition receptors (PRRs involved in sensing bacterial components. Methods Of 227 subfertile women, serum was available for C. trachomatis IgG antibody testing and genotyping (common versus rare allele of the PRR genes TLR9, TLR4, CD14 and CARD15/NOD2. In all women, a laparoscopy was performed to assess the grade of tubal pathology. Tubal pathology was defined as extensive peri-adnexal adhesions and/or distal occlusion of at least one tube. Results Following a C. trachomatis infection (i.e. C. trachomatis IgG positive, subfertile women carrying two or more SNPs in C. trachomatis PRR genes were at increased risk of tubal pathology compared to women carrying less than two SNPs (73% vs 33% risk. The differences were not statistically significant (P = 0.15, but a trend was observed. Conclusion Carrying multiple SNPs in C. trachomatis PRR genes tends to result in an aberrant immune response and a higher risk of tubal pathology following a C. trachomatis infection. Larger studies are needed to confirm our preliminary findings.

  12. Detection of Ca2+-dependent acid phosphatase activity identifies neuronal integrity in damaged rat central nervous system after application of bacterial melanin

    Directory of Open Access Journals (Sweden)

    Tigran R Petrosyan

    2016-01-01

    Full Text Available The study aims to confirm the neuroregenerative effects of bacterial melanin (BM on central nervous system injury using a special staining method based on the detection of Ca2+-dependent acid phosphatase activity. Twenty-four rats were randomly assigned to undergo either unilateral destruction of sensorimotor cortex (group I; n = 12 or unilateral rubrospinal tract transection at the cervical level (C3–4 (group II; n = 12. In each group, six rats were randomly selected after surgery to undergo intramuscular injection of BM solution (BM subgroup and the remaining six rats were intramuscularly injected with saline (saline subgroup. Neurological testing confirmed that BM accelerated the recovery of motor function in rats from both BM and saline subgroups. Two months after surgery, Ca2+-dependent acid phosphatase activity detection in combination with Chilingarian's calcium adenoside triphosphate method revealed that BM stimulated the sprouting of fibers and dilated the capillaries in the brain and spinal cord. These results suggest that BM can promote the recovery of motor function of rats with central nervous system injury; and detection of Ca2+-dependent acid phosphatase activity is a fast and easy method used to study the regeneration-promoting effects of BM on the injured central nervous system.

  13. Bacterial computing with engineered populations.

    Science.gov (United States)

    Amos, Martyn; Axmann, Ilka Maria; Blüthgen, Nils; de la Cruz, Fernando; Jaramillo, Alfonso; Rodriguez-Paton, Alfonso; Simmel, Friedrich

    2015-07-28

    We describe strategies for the construction of bacterial computing platforms by describing a number of results from the recently completed bacterial computing with engineered populations project. In general, the implementation of such systems requires a framework containing various components such as intracellular circuits, single cell input/output and cell-cell interfacing, as well as extensive analysis. In this overview paper, we describe our approach to each of these, and suggest possible areas for future research. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

  14. Lactose Transport System of Streptococcus thermophilus : a Hybrid Protein with Homology to the Melibiose Carrier and Enzyme III of Phosphoenolpyruvate-Dependent Phosphotransferase Systems

    NARCIS (Netherlands)

    Poolman, Bert; Royer, Theresa J.; Mainzer, Stanley E.; Schmidt, Brian F.

    1989-01-01

    The gene responsible for the transport of lactose into Streptococcus thermophilus (lacS) was cloned in Escherichia coli as a 4.2-kilobase fragment from an EcoRI library of chromosomal DNA by using the vector pKK223-3. From deletion analysis, the gene for lactose transport mapped to two HindIII

  15. Engineering of self-sustaining systems: substituting the yeast glucose transporter plus hexokinase for the Lactococcus lactis phosphotransferase system in a Lactococcus lactis network in silico

    NARCIS (Netherlands)

    Adamczyk, M.; Westerhoff, H.V.

    2012-01-01

    The success rate of introducing new functions into a living species is still rather unsatisfactory. Much of this is due to the very essence of the living state, i.e. its robustness towards perturbations. Living cells are bound to notice that metabolic engineering is being effected, through changes

  16. Potential for Combined Biocontrol Activity against Fungal Fish and Plant Pathogens by Bacterial Isolates from a Model Aquaponic System

    Directory of Open Access Journals (Sweden)

    Ivaylo Sirakov

    2016-11-01

    Full Text Available One of the main challenges in aquaponics is disease control. One possible solution for this is biological control with organisms exerting inhibitory effects on fish and plant pathogens. The aim of this study was to examine the potential of isolating microorganisms that exert an inhibitory effect on both plant and fish pathogens from an established aquaponic system. We obtained 924 isolates on selective King’s B agar and 101 isolates on MRS agar from different compartments of a model aquaponic system and tested them for antagonism against the plant pathogen Pythium ultimum and fish pathogen Saprolegnia parasitica. Overall, 42 isolates were able to inhibit both fungi. Although not yet tested in vivo, these findings open new options for the implementation of biological control of diseases in aquaponics, where plants and fish are cultivated in the same water recirculating system.

  17. Structural Studies of Bacterial Enzymes and their Relation to Antibiotic Resistance Mechanisms - Final Paper

    Energy Technology Data Exchange (ETDEWEB)

    Maltz, Lauren [SLAC National Accelerator Lab., Menlo Park, CA (United States)

    2015-08-27

    By using protein crystallography and X-ray diffraction, structures of bacterial enzymes were solved to gain a better understanding of how enzymatic modification acts as an antibacterial resistance mechanism. Aminoglycoside phosphotransferases (APHs) are one of three aminoglycoside modifying enzymes that confer resistance to the aminoglycoside antibiotics via enzymatic modification, rendering many drugs obsolete. Specifically, the APH(2”) family vary in their substrate specificities and also in their preference for the phosphate donor (ADP versus GDP). By solving the structures of members of the APH(2”) family of enzymes, we can see how domain movements are important to their substrate specificity. Our structure of the ternary complex of APH(2”)-IIIa with GDP and kanamycin, when compared to the known structures of APH(2”)-IVa, reveals that there are real physical differences between these two enzymes, a structural finding that explains why the two enzymes differ in their preferences for certain aminoglycosides. Another important group of bacterial resistance enzymes are the Class D β- lactamases. Oxacillinase carbapenemases (OXAs) are part of this enzyme class and have begun to confer resistance to ‘last resort’ drugs, most notably carbapenems. Our structure of OXA-143 shows that the conformational flexibility of a conserved hydrophobic residue in the active site (Val130) serves to control the entry of a transient water molecule responsible for a key step in the enzyme’s mechanism. Our results provide insight into the structural mechanisms of these two different enzymes

  18. Prevention of bacterial adhesion

    DEFF Research Database (Denmark)

    Klemm, Per; Vejborg, Rebecca Munk; Hancock, Viktoria

    2010-01-01

    Management of bacterial infections is becoming increasingly difficult due to the emergence and increasing prevalence of bacterial pathogens that are resistant to available antibiotics. Conventional antibiotics generally kill bacteria by interfering with vital cellular functions, an approach...... that imposes selection pressure for resistant bacteria. New approaches are urgently needed. Targeting bacterial virulence functions directly is an attractive alternative. An obvious target is bacterial adhesion. Bacterial adhesion to surfaces is the first step in colonization, invasion, and biofilm formation....... As such, adhesion represents the Achilles heel of crucial pathogenic functions. It follows that interference with adhesion can reduce bacterial virulence. Here, we illustrate this important topic with examples of techniques being developed that can inhibit bacterial adhesion. Some of these will become...

  19. First multi-epitope subunit vaccine against extraintestinal pathogenic Escherichia coli delivered by a bacterial type-3 secretion system (T3SS).

    Science.gov (United States)

    Wieser, Andreas; Magistro, Giuseppe; Nörenberg, Dominik; Hoffmann, Christiane; Schubert, Sören

    2012-01-01

    Infections due to extraintestinal pathogenic E. coli (ExPEC) are very common in humans as well as in animals. In humans ExPEC infections include urinary tract infections (UTI), septicemia, and wound infections, which result in significant morbidity, mortality, and substantial healthcare costs. In view of the increasing number of ExPEC infections caused by more and more resistant strains, effective prevention would be desirable. Given the rising treatment costs, a vaccine may be cost-effective in selected patient groups, such as women with recurrent UTI, patients with neurologic disorders impairing bladder function and men with prostate hyperplasia. Previous vaccine studies used single target proteins or whole inactivated ExPEC cells. Here, we describe a vaccine system for oral application based on artificial multiple subunit vaccine proteins. Those multi-epitope proteins are composed of predicted epitopes derived from ExPEC virulence-associated proteins. As ExPEC are known to form intracellular biofilms in the urothelium and can also resist killing by non-activated macrophages, T-cell responses are supposed to be an important measure to counteract these stages of ExPEC during infection. Therefore, a live bacterial antigen delivery system based upon the Salmonella type-III secretion system (T3SS) was used in this study to directly deliver the vaccine proteins into the cytoplasm of the host cells. Epitope-rich domains of the proteins FyuA, IroN, ChuA, IreA, Iha, and Usp were expressed in an attenuated Salmonella enterica serovar Typhimurium strain and translocated into target cells for extended periods of time inducing a strong T-cell response. No significant antibody titre increase against the secreted vaccine proteins could be detected in vaginal wash or serum. Despite that, one of the vaccine proteins was able to significantly reduce bacterial load in the challenge model of intraperitoneal sepsis. This study shows that a vaccine encompassing distinct epitopes of

  20. Construction and Testing of a Bacterial Luciferase Reporter Gene System for in Vivo Measurement of Nonsense Suppression in Streptomyces

    Czech Academy of Sciences Publication Activity Database

    Weiser, Jaroslav; Buriánková, Karolína; Kalachová, Ladislava; Branny, Pavel; Pernodet, J.-L.

    2006-01-01

    Roč. 51, č. 1 (2006), s. 62-64 ISSN 0015-5632 R&D Projects: GA ČR GA310/03/0292 Institutional research plan: CEZ:AV0Z50200510 Keywords : streptomyces * reporter gene system Subject RIV: EE - Microbiology, Virology Impact factor: 0.963, year: 2006

  1. IDENTIFICATION OF ACTIVE BACTERIAL COMMUNITIES IN A MODEL DRINKING WATER BIOFILM SYSTEM USING 16S RRNA-BASED CLONE LIBRARIES

    Science.gov (United States)

    Recent phylogenetic studies have used DNA as the target molecule for the development of environmental 16S rDNA clone libraries. As DNA may persist in the environment, DNA-based libraries cannot be used to identify metabolically active bacteria in water systems. In this study, a...

  2. Extracellular secretion of Carocin S1 in Pectobacterium carotovorum subsp. carotovorum occurs via the type III secretion system integral to the bacterial flagellum.

    Science.gov (United States)

    Chan, Yung-Chieh; Wu, Huang-Pin; Chuang, Duen-Yau

    2009-08-27

    Pectobacterium carotovorum subsp. carotovorum is a phytopathogenic enterobacterium responsible for soft rot, a disease characterized by extensive maceration of the affected plant tissue. This species also produces two or more antibacterial substances called bacteriocins, which enhance its competitiveness against related rival species. However, the secretion mechanism for low-molecular-weight bacteriocin is still unknown. A mutant (flhC::Tn5) that did not secrete the low-molecular-weight bacteriocin (LMWB), Carocin S1, was generated by Tn5 insertional mutagenesis. Sequence analysis indicated that this insertion disrupted open reading frame 2 (ORF2) and ORF3 of this strain. Deletion and rescue experiments indicated that ORF2 and ORF3 were both required for extracellular LMWB secretion. The ORF2 and ORF3 sequences showed high homology with the flhD and flhC gene sequences of Pectobacterium carotovorum subsp. atroseptica, Serratia marcescens, Yersinia enterocolitica, and Escherichia coli, indicating that they likely encoded key regulatory components of the type III flagella secretion system. Thus, the extracellular export of Carocin S1 by Pectobacterium carotovorum subsp. carotovorum appears to utilize the type III secretion system integral to bacterial flagella.

  3. Extracellular secretion of Carocin S1 in Pectobacterium carotovorum subsp. carotovorum occurs via the type III secretion system integral to the bacterial flagellum

    Directory of Open Access Journals (Sweden)

    Chuang Duen-yau

    2009-08-01

    Full Text Available Abstract Background Pectobacterium carotovorum subsp. carotovorum is a phytopathogenic enterobacterium responsible for soft rot, a disease characterized by extensive maceration of the affected plant tissue. This species also produces two or more antibacterial substances called bacteriocins, which enhance its competitiveness against related rival species. However, the secretion mechanism for low-molecular-weight bacteriocin is still unknown. Results A mutant (flhC::Tn5 that did not secrete the low-molecular-weight bacteriocin (LMWB, Carocin S1, was generated by Tn5 insertional mutagenesis. Sequence analysis indicated that this insertion disrupted open reading frame 2 (ORF2 and ORF3 of this strain. Deletion and rescue experiments indicated that ORF2 and ORF3 were both required for extracellular LMWB secretion. The ORF2 and ORF3 sequences showed high homology with the flhD and flhC gene sequences of Pectobacterium carotovorum subsp. atroseptica, Serratia marcescens, Yersinia enterocolitica, and Escherichia coli, indicating that they likely encoded key regulatory components of the type III flagella secretion system. Conclusion Thus, the extracellular export of Carocin S1 by Pectobacterium carotovorum subsp. carotovorum appears to utilize the type III secretion system integral to bacterial flagella.

  4. [Effect of mixed edaphic bacterial inoculants in the early development of improved cocoa cultivars (Theobroma cacao L.) in a traditional agroforestry system of Oaxaca, Mexico].

    Science.gov (United States)

    Hipólito-Romero, E; Carcaño-Montiel, M G; Ramos-Prado, J M; Vázquez-Cabañas, E A; López-Reyes, L; Ricaño-Rodríguez, J

    Cocoa plant (Theobroma cacao L.) is native from South America and it represents one of the most significant "bio-cultural" resources of Mesoamerica, since it is a region where it was domesticated and had a relevance as ritual drink and as currency in many pre-hispanic cultures until the arrival of the Spaniards who spread its use worldwide, and became it one of the most consumed commodity goods. Through this research, an alternative is proposed to address the problem of cultivars through the introduction of a wide variety of cocoa plants in traditional agroforestry systems, in synergy with the inoculation of nitrogen-fixing and insoluble phosphor solubilizing edaphic bacterial consortia. Four cultivars of improved grafted cocoa plants were introduced in a traditional agroforestry plot and three fertilization treatments were applied: application of biofertilizer, application of chemical fertilizer and control. Measurements of height, stem diameter, number of leaves and branches were recorded at 2 and 12 months after planting and rhizosphere microbial populations were characterized. Growth results showed good potential for all studied cultivars and it was observed that biofertilization foresees significant effects in some of the growth indicators of cocoa plant. Thereby, plant associations in an agroforestry system could be favorable to promote fruit development and resistance to pests and diseases. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  5. Modification of CusSR bacterial two-component systems by the introduction of an inducible positive feedback loop.

    Science.gov (United States)

    Ravikumar, Sambandam; Pham, Van Dung; Lee, Seung Hwan; Yoo, Ik-Keun; Hong, Soon Ho

    2012-06-01

    The CusSR two-component system (TCS) is a copper-sensing apparatus of E. coli that is responsible for regulating the copper-related homeostatic system. The dynamic characteristics of the CusSR network were modified by the introduction of a positive feedback loop. To construct the feedback loop, the CusR, which is activated by the cusC promoter, was cloned downstream of the cusC promoter and reporter protein. The feedback loop system, once activated by environmental copper, triggers the activation of the cusC promoter, which results in the amplification of a reporter protein and CusR expression. The threshold copper concentration for the activation of the modified CusSR TCS network was lowered from 2,476.5 μg/l to 247.7 μg/l, which indicates a tenfold increase in sensitivity. The intensity of the output signal was increased twofold, and was maintained for 16 h. The strategy proposed in this study can also be applied to modify the dynamic characteristics of other TCSs.

  6. The bacterial biofilms in dialysis water systems and the effect of the sub inhibitory concentrations of chlorine on them.

    Science.gov (United States)

    Suman, Ethel; Varghese, Benji; Joseph, Neethu; Nisha, Kumari; Kotian, M Shashidhar

    2013-05-01

    The presence of bacteria in the form of biofilms poses a problem in the fluid pathways of haemodialysis plants and procedures which are aimed to detach and neutralize biofilms are necessary to improve the patient safety and the quality of the healthcare. The present study was therefore aimed at isolating the organisms which colonized dialysis water systems as biofilms, as well as to study the effect of the sub inhibitory concentrations of chlorine on the biofilms which were produced by these isolates. Swabs were used to collect the biofilms which were produced on the internal surface of the dialysis tubing from the dialysis units. This study was conducted at the Department of Microbiology, Kasturba Medical College (KMC), Mangalore, India. The cultures were performed on MacConkey's agar and blood agar. The organisms which were isolated were identified and antibiotic sensitivity tests were performed. The biofilm production was done by the microtitre plate method of O'Toole and Kolter. The biofilm production was also studied in the presence of sub inhibitory concentrations of chlorine. Acinetobacter spp and Pseudomonas aeruginosa were the two predominant organisms which colonized the dialysis water systems as biofilms. The sub inhibitory concentrations of chlorine did not bring about any decrease in the biofilm production by the isolates. On the contrary, there was an increase in the biofilm production. Our study highlighted the importance of using appropriate methods to improve the quality of the water in dialysis units. This in turn, may help in reducing the biofilm formation in the water systems of dialysis units and thus, contribute to the prevention of hospital acquired infections in the patients who need haemodialysis.

  7. Role of β1 integrins and bacterial adhesins for Yop injection into leukocytes in Yersinia enterocolitica systemic mouse infection.

    Science.gov (United States)

    Deuschle, Eva; Keller, Birgit; Siegfried, Alexandra; Manncke, Birgit; Spaeth, Tanja; Köberle, Martin; Drechsler-Hake, Doreen; Reber, Julia; Böttcher, Ralph T; Autenrieth, Stella E; Autenrieth, Ingo B; Bohn, Erwin; Schütz, Monika

    2016-02-01

    Injection of Yersinia outer proteins (Yops) into host cells by a type III secretion system is an important immune evasion mechanism of Yersinia enterocolitica (Ye). In this process Ye invasin (Inv) binds directly while Yersinia adhesin A (YadA) binds indirectly via extracellular matrix (ECM) proteins to β1 integrins on host cells. Although leukocytes turned out to be an important target of Yop injection by Ye, it was unclear which Ye adhesins and which leukocyte receptors are required for Yop injection. To explain this, we investigated the role of YadA, Inv and β1 integrins for Yop injection into leukocytes and their impact on the course of systemic Ye infection in mice. Ex vivo infection experiments revealed that adhesion of Ye via Inv or YadA is sufficient to promote Yop injection into leukocytes as revealed by a β-lactamase reporter assay. Serum factors inhibit YadA- but not Inv-mediated Yop injection into B and T cells, shifting YadA-mediated Yop injection in the direction of neutrophils and other myeloid cells. Systemic Ye mouse infection experiments demonstrated that YadA is essential for Ye virulence and Yop injection into leukocytes, while Inv is dispensable for virulence and plays only a transient and minor role for Yop injection in the early phase of infection. Ye infection of mice with β1 integrin-depleted leukocytes demonstrated that β1 integrins are dispensable for YadA-mediated Yop injection into leukocytes, but contribute to Inv-mediated Yop injection. Despite reduced Yop injection into leukocytes, β1 integrin-deficient mice exhibited an increased susceptibility for Ye infection, suggesting an important role of β1 integrins in immune defense against Ye. This study demonstrates that Yop injection into leukocytes by Ye is largely mediated by YadA exploiting, as yet unknown, leukocyte receptors. Copyright © 2015. Published by Elsevier GmbH.

  8. Aerotaxis in Bacterial Turbulence

    Science.gov (United States)

    Fernandez, Vicente; Bisson, Antoine; Bitton, Cindy; Waisbord, Nicolas; Smriga, Steven; Rusconi, Roberto; Stocker, Roman

    2012-11-01

    Concentrated suspensions of motile bacteria exhibit correlated dynamics on spatial scales much larger than an individual bacterium. The resulting flows, visually similar to turbulence, can increase mixing and decrease viscosity. However, it remains unclear to what degree the collective dynamics depend on the motile behavior of bacteria at the individual level. Using a new microfluidic device to create controlled horizontal oxygen gradients, we studied the two dimensional behavior of dense suspensions of Bacillus subtilis. This system makes it possible to assess the interplay between the coherent large-scale motions of the suspension, oxygen transport, and the directional response of cells to oxygen gradients (aerotaxis). At the same time, this device has enabled us to examine the onset of bacterial turbulence and its influence on the propagation of the diffusing oxygen front, as the bacteria begin in a dormant state and transition to swimming when exposed to oxygen.

  9. Escherichia coli Nissle 1917 bacterial ghosts retain crucial surface properties and express chlamydial antigen: an imaging study of a delivery system for the ocular surface.

    Science.gov (United States)

    Montanaro, Jacqueline; Inic-Kanada, Aleksandra; Ladurner, Angela; Stein, Elisabeth; Belij, Sandra; Bintner, Nora; Schlacher, Simone; Schuerer, Nadine; Mayr, Ulrike Beate; Lubitz, Werner; Leisch, Nikolaus; Barisani-Asenbauer, Talin

    2015-01-01

    To target chronic inflammatory ocular surface diseases, a drug delivery platform is needed that is safe, possesses immunomodulatory properties, and can be used either for drug delivery, or as a foreign antigen carrier. A new therapeutic approach that we have previously proposed uses nonliving bacterial ghosts (BGs) as a carrier-delivery system which can be engineered to carry foreign antigens and/or be loaded with therapeutic drugs. The parent strain chosen for development of our BG delivery system is the probiotic Escherichia coli strain Nissle 1917 (EcN), whose intrinsic properties trigger the innate immune system with the flagella and fimbriae used to attach and stimulate epithelial cells. In previous studies, we have shown that EcN BGs are safe for the ocular surface route, but evidence that EcN BGs retain flagella and fimbriae after transformation, has never been visually confirmed. In this study, we used different visualization techniques to determine whether flagella and fimbriae are retained on EcN BGs engineered either for drug delivery or as a foreign antigen carrier. We have also shown by immunoelectron microscopy that EcN retains two foreign antigens after processing to become EcN BGs. Furthermore, we demonstrated that BGs derived from EcN and expressing a foreign antigen attachment to conjunctival epithelial cells in vitro without causing reduced cell viability. These results are an important step in constructing a delivery system based on a nonliving probiotic that is suitable for use in ocular surface diseases pairing immunomodulation and targeted delivery.

  10. Construction and testing of a bacterial luciferase reporter gene system for in vivo measurement of nonsense suppression in Streptomyces.

    Science.gov (United States)

    Weiser, J; Buriánková, K; Kalachová, L; Branny, P; Pernodet, J L

    2006-01-01

    A reporter gene system, based on luciferase genes from Vibrio harvei, was constructed for measurement of translation nonsense suppression in Streptomyces. Using the site-directed mutagenesis the TCA codon in position 13 of the luxB gene was replaced by all of the three stop codons individually. By cloning of luxA and luxB genes under the control of strong constitutive Streptomyces promoter ermE* in plasmid pUWL201 we created Wluxl with the wild-type sequence and pWlux2, pWlux3 and pWlux4 plasmids containing TGA-, TAG- and TAA-stop codons, respectively. Streptomyces lividans TK 24 was transformed with the plasmids and the reporter system was tested by growth of the strain in the presence of streptomycin as a translation accuracy modulator. Streptomycin increased nonsense suppression on UAA nearly 10-fold and more than 20-fold on UAG. On the other hand, UGA, the most frequent stop signal in Streptomyces, the effect was negligible.

  11. Functional components of the bacterial CzcCBA efflux system reduce cadmium uptake and accumulation in transgenic tobacco plants.

    Science.gov (United States)

    Nesler, Andrea; DalCorso, Giovanni; Fasani, Elisa; Manara, Anna; Di Sansebastiano, Gian Pietro; Argese, Emanuele; Furini, Antonella

    2017-03-25

    Cadmium (Cd) is a toxic trace element released into the environment by industrial and agricultural practices, threatening the health of plants and contaminating the food/feed chain. Biotechnology can be used to develop plant varieties with a higher capacity for Cd accumulation (for use in phytoremediation programs) or a lower capacity for Cd accumulation (to reduce Cd levels in food and feed). Here we generated transgenic tobacco plants expressing components of the Pseudomonas putida CzcCBA efflux system. Plants were transformed with combinations of the CzcC, CzcB and CzcA genes, and the impact on Cd mobilization was analysed. Plants expressing PpCzcC showed no differences in Cd accumulation, whereas those expressing PpCzcB or PpCzcA accumulated less Cd in the shoots, but more Cd in the roots. Plants expressing both PpCzcB and PpCzcA accumulated less Cd in the shoots and roots compared to controls, whereas plants expressing all three genes showed a significant reduction in Cd levels only in shoots. These results show that components of the CzcCBA system can be expressed in plants and may be useful for developing plants with a reduced capacity to accumulate Cd in the shoots, potentially reducing the toxicity of food/feed crops cultivated in Cd-contaminated soils. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Role of antibiosis on suppression of bacterial common blight ...

    African Journals Online (AJOL)

    Magesh

    2012-07-26

    Jul 26, 2012 ... Paenibacillus polymyxa strain HKA-15, a soybean bacterial endophyte showed strong antagonism against bacterial common ... with positive chemical control in suppression of bacterial common blight disease in French bean plants. ... known to induce systemic resistance in host plant and competing out the ...

  13. A two pulse drug delivery system for amoxicillin: an attempt to counter the scourge of bacterial resistance against antibiotics.

    Science.gov (United States)

    Akhter, Habban; Saigal, Nitin; Baboota, Sanjula; Faisal, Shah; Ali, Javed

    2011-09-01

    Bearing in mind the present scenario of the increasing biological tolerance of bacteria against antibiotics, a time controlled two pulse dosage form of amoxicillin was developed. The compression coating inlay tablet approach was used to deliver the drug in two pulses to different parts of the GIT after a well defined lag time between the two releases. This was made possible by formulating a core containing one of the two drug fractions (intended to be delivered as the second pulse), which was spray coated with a suspension of ethyl cellulose and a hydrophilic but water insoluble agent as a pore former (microcrystalline cellulose). Coating of up to 5% (m/m) was applied over the core tablet, giving a corresponding lag of 3, 5, 7 and 12 h. Increasing the level of coating led to retardation of the water uptake capacity of the core, leading to prolongation of the lag time. Microcrystalline cellulose was used as a hydrophilic but water insoluble porosity modifier in the barrier layer, varying the concentration of which had a significant effect on shortening or prolongation of the lag time. This coated system was further partially compression coated with the remaining drug fraction (to be released as the first immediate release pulse) with a disintegrant, giving a final tablet. The core tablet and the final two pulse inlay tablet were further investigated for their in vitro performance.

  14. Functional exploration of the bacterial type VI secretion system in mutualism: Azorhizobium caulinodans ORS571-Sesbania rostrata as a research model.

    Science.gov (United States)

    Lin, Hsiao-Han; Huang, Hsin-Mei; Yu, Manda; Lai, Erh-Min; Chien, Hsiao-Lin; Liu, Chi-Te

    2018-03-08

    The bacterial type VI secretion system (T6SS) has been considered the armed force of bacteria because it can deliver toxin effectors to prokaryotic or eukaryotic cells for survival and fitness. Although many legume symbiotic rhizobacteria encode T6SS in their genome, the biological function of T6SS in these bacteria is still unclear. To elucidate this issue, we used Azorhizobium caulinodans ORS571 and its symbiotic host Sesbania rostrata as our research model. By using T6SS gene deletion mutants, we found that T6SS provides A. caulinodans with better symbiotic competitiveness when co-infected with a T6SS-lacking strain, as demonstrated by two independent T6SS-deficient mutants. Meanwhile, the symbiotic effectiveness was not affected by T6SS because the nodule phenotype, nodule size, and nodule nitrogen fixation ability did not differ between the T6SS mutants and the wild-type when infected alone. Our data also suggest that under several lab culture conditions tested, A. caulinodans showed no T6SS-dependent interbacterial competition activity. Therefore, instead of being an antihost or antibacterial weapon of the bacterium, the T6SS in A. caulinodans ORS571 seems to participate specifically in symbiosis by increasing its symbiotic competitiveness.

  15. Persistence of antibiotic resistance genes and bacterial community changes in drinking water treatment system: From drinking water source to tap water.

    Science.gov (United States)

    Su, Hao-Chang; Liu, You-Sheng; Pan, Chang-Gui; Chen, Jun; He, Liang-Ying; Ying, Guang-Guo

    2018-03-01

    As emerging contaminants, antibiotic resistance genes (ARGs) have become a public concern. This study aimed to investigate the occurrence and diversity of ARGs, and variation in the composition of bacterial communities in source water, drinking water treatment plants, and tap water in the Pearl River Delta region, South China. Various ARGs were present in the different types of water. Among the 27 target ARGs, floR and sul1 dominated in source water from three large rivers in the region. Pearson correlation analysis suggested that sul1, sul2, floR, and cmlA could be potential indicators for ARGs in water samples. The total abundance of the detected ARGs in tap water was much lower than that in source water. Sand filtration and sedimentation in drinking water treatment plants could effectively remove ARGs; in contrast, granular activated carbon filtration increased the abundance of ARGs. It was found that Pseudomonas may be involved in the proliferation and dissemination of ARGs in the studied drinking water treatment system. Bacteria and ARGs were still present in tap water after treatment, though they were significantly reduced. More research is needed to optimize the water treatment process for ARG removal. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Mechanism of Asp24 Upregulation in Brucella abortus Rough Mutant with a Disrupted O-Antigen Export System and Effect of Asp24 in Bacterial Intracellular Survival

    Science.gov (United States)

    Tian, Mingxing; Qu, Jing; Han, Xiangan; Ding, Chan; Wang, Shaohui; Peng, Daxin

    2014-01-01

    We previously showed that Brucella abortus rough mutant strain 2308 ΔATP (called the ΔrfbE mutant in this study) exhibits reduced intracellular survival in RAW264.7 cells and attenuated persistence in BALB/c mice. In this study, we performed microarray analysis to detect genes with differential expression between the ΔrfbE mutant and wild-type strain S2308. Interestingly, acid shock protein 24 gene (asp24) expression was significantly upregulated in the ΔrfbE mutant compared to S2308, as confirmed by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting. Further studies using additional strains indicated that the upregulation of asp24 occurred only in rough mutants with disrupted O-antigen export system components, including the ATP-binding protein gene rfbE (bab1_0542) and the permease gene rfbD (bab1_0543), while the ΔwboA rough mutant (which lacks an O-antigen synthesis-related glycosyltransferase) and the RB51 strain (a vaccine strain with the rough phenotype) showed no significant changes in asp24 expression compared to S2308. In addition, abolishing the intracellular O-antigen synthesis of the ΔrfbE mutant by deleting the wboA gene (thereby creating the ΔrfbE ΔwboA double-knockout strain) recovered asp24 expression. These results indicated that asp24 upregulation is associated with intracellular O-antigen synthesis and accumulation but not with the bacterial rough phenotype. Further studies indicated that asp24 upregulation in the ΔrfbE mutant was associated neither with bacterial adherence and invasion nor with cellular necrosis on RAW264.7 macrophages. However, proper expression of the asp24 gene favors intracellular survival of Brucella in RAW264.7 cells and HeLa cells during an infection. This study reveals a novel mechanism for asp24 upregulation in B. abortus mutants. PMID:24752516

  17. Studying bacterial multispecies biofilms

    DEFF Research Database (Denmark)

    Røder, Henriette Lyng; Sørensen, Søren Johannes; Burmølle, Mette

    2016-01-01

    , but the identity and significance of interspecies bacterial interactions is neglected in these analyses. There is therefore an urgent need for bridging the gap between metagenomic analysis and in vitro models suitable for studies of bacterial interactions.Bacterial interactions and coadaptation are important......The high prevalence and significance of multispecies biofilms have now been demonstrated in various bacterial habitats with medical, industrial, and ecological relevance. It is highly evident that several species of bacteria coexist and interact in biofilms, which highlights the need for evaluating...

  18. Functional replacement of the Saccharomyces cerevisiae fatty acid synthase with a bacterial type II system allows flexible product profiles.

    Science.gov (United States)

    Fernandez-Moya, Ruben; Leber, Christopher; Cardenas, Javier; Da Silva, Nancy A

    2015-12-01

    The native yeast type I fatty acid synthase (FAS) is a complex, rigid enzyme, and challenging to engineer for the production of medium- or short-chain fatty acids. Introduction of a type II FAS is a promising alternative as it allows expression control for each discrete enzyme and the addition of heterologous thioesterases. In this study, the native Saccharomyces cerevisiae FAS was functionally replaced by the Escherichia coli type II FAS (eFAS) system. The E. coli acpS + acpP (together), fabB, fabD, fabG, fabH, fabI, fabZ, and tesA were expressed in individual S. cerevisiae strains, and enzyme activity was confirmed by in vitro activity assays. Eight genes were then integrated into the yeast genome, while tesA or an alternate thioesterase gene, fatB from Ricinus communis or TEII from Rattus novergicus, was expressed from a multi-copy plasmid. Native FAS activity was eliminated by knocking out the yeast FAS2 gene. The strains expressing only the eFAS as de novo fatty acid source grew without fatty acid supplementation demonstrating that this type II FAS is able to functionally replace the native yeast FAS. The engineered strain expressing the R. communis fatB thioesterase increased total fatty acid titer 1.7-fold and shifted the fatty acid profile towards C14 production, increasing it from <1% in the native strain to more than 30% of total fatty acids, and reducing C18 production from 39% to 8%. © 2015 Wiley Periodicals, Inc.

  19. Bacterial melanin promotes recovery after sciatic nerve injury in rats

    OpenAIRE

    Olga V Gevorkyan; Irina B Meliksetyan; Tigran R Petrosyan; Anichka S Hovsepyan

    2015-01-01

    Bacterial melanin, obtained from the mutant strain of Bacillus Thuringiensis, has been shown to promote recovery after central nervous system injury. It is hypothesized, in this study, that bacterial melanin can promote structural and functional recovery after peripheral nerve injury. Rats subjected to sciatic nerve transection were intramuscularly administered bacterial melanin. The sciatic nerve transected rats that did not receive intramuscular administration of bacterial melanin served as...

  20. Flip-Flop HSV-BAC: bacterial artificial chromosome based system for rapid generation of recombinant herpes simplex virus vectors using two independent site-specific recombinases

    Directory of Open Access Journals (Sweden)

    Todo Tomoki

    2006-09-01

    Full Text Available Abstract Background Oncolytic herpes simplex virus (HSV vectors that specifically replicate in and kill tumor cells sparing normal cells are a promising cancer therapy. Traditionally, recombinant HSV vectors have been generated through homologous recombination between the HSV genome and a recombination plasmid, which usually requires laborious screening or selection and can take several months. Recent advances in bacterial artificial chromosome (BAC technology have enabled cloning of the whole HSV genome as a BAC plasmid and subsequent manipulation in E. coli. Thus, we sought a method to generate recombinant oncolytic HSV vectors more easily and quickly using BAC technology. Results We have developed an HSV-BAC system, termed the Flip-Flop HSV-BAC system, for the rapid generation of oncolytic HSV vectors. This system has the following features: (i two site-specific recombinases, Cre and FLPe, are used sequentially to integrate desired sequences and to excise the BAC sequences, respectively; and (ii the size of the HSV-BAC-insert genome exceeds the packaging limit of HSV so only correctly recombined virus grows efficiently. We applied this to the construction of an HSV-BAC plasmid that can be used for the generation of transcriptionally-targeted HSV vectors. BAC sequences were recombined into the UL39 gene of HSV ICP4-deletion mutant d120 to generate M24-BAC virus, from which HSV-BAC plasmid pM24-BAC was isolated. An ICP4 expression cassette driven by an exogenous promoter was re-introduced to pM24-BAC by Cre-mediated recombination and nearly pure preparations of recombinant virus were obtained typically in two weeks. Insertion of the ICP4 coding sequence alone did not restore viral replication and was only minimally better than an ICP4-null construct, whereas insertion of a CMVIE promoter-ICP4 transgene (bM24-CMV efficiently drove viral replication. The levels of bM24-CMV replication in tumor cells varied considerably compared to hrR3 (UL39

  1. Escherichia coli Nissle 1917 bacterial ghosts retain crucial surface properties and express chlamydial antigen: an imaging study of a delivery system for the ocular surface

    Directory of Open Access Journals (Sweden)

    Montanaro J

    2015-07-01

    Full Text Available Jacqueline Montanaro,1 Aleksandra Inic-Kanada,1 Angela Ladurner,1 Elisabeth Stein,1 Sandra Belij,1 Nora Bintner,1 Simone Schlacher,1 Nadine Schuerer,1 Ulrike Beate Mayr,2 Werner Lubitz,2 Nikolaus Leisch,3 Talin Barisani-Asenbauer11Laura Bassi Centres of Expertise, OCUVAC – Centre of Ocular Inflammation and Infection, Centre for Pathophysiology, Infectiology, and Immunology, Medical University of Vienna, Vienna, Austria; 2BIRD-C GmbH & Co KG, Kritzendorf, Austria; 3Department of Ecogenomics and Systems Biology, University of Vienna, Vienna, AustriaAbstract: To target chronic inflammatory ocular surface diseases, a drug delivery platform is needed that is safe, possesses immunomodulatory properties, and can be used either for drug delivery, or as a foreign antigen carrier. A new therapeutic approach that we have previously proposed uses nonliving bacterial ghosts (BGs as a carrier-delivery system which can be engineered to carry foreign antigens and/or be loaded with therapeutic drugs. The parent strain chosen for development of our BG delivery system is the probiotic Escherichia coli strain Nissle 1917 (EcN, whose intrinsic properties trigger the innate immune system with the flagella and fimbriae used to attach and stimulate epithelial cells. In previous studies, we have shown that EcN BGs are safe for the ocular surface route, but evidence that EcN BGs retain flagella and fimbriae after transformation, has never been visually confirmed. In this study, we used different visualization techniques to determine whether flagella and fimbriae are retained on EcN BGs engineered either for drug delivery or as a foreign antigen carrier. We have also shown by immunoelectron microscopy that EcN retains two foreign antigens after processing to become EcN BGs. Furthermore, we demonstrated that BGs derived from EcN and expressing a foreign antigen attachment to conjunctival epithelial cells in vitro without causing reduced cell viability. These results

  2. Multilevel analysis of bacterial counts from chronic periodontitis after root planing/scaling, surgery, and systemic and local antibiotics: 2-year results

    Directory of Open Access Journals (Sweden)

    Ibrahimu Mdala

    2013-07-01

    Full Text Available Aim: To follow changes (over 2 years in subgingival bacterial counts of five microbial complexes including health-related Actinomyces spp. in deeper pockets (≥5 mm after periodontal treatments. Methods: Eight different treatments were studied: (1 scaling+root planing (SRP; (2 periodontal surgery (SURG+systemic amoxicillin (AMOX+systemic metronidazole (MET; (3 SURG+locally delivered tetracycline (TET; (4 SURG; (5 AMOX+MET+TET; (6 AMOX+MET; (7 TET; and (8 SURG+AMOX+MET+TET. Antibiotics were given immediately following SRP. Subgingival plaque was collected mesiobuccally from each tooth, except third molars, from 176 subjects, completing the study, at baseline, 3, 6, 12, 18, and 24 months post-treatment and analysed for 40 different bacteria using checkerboard hybridization. A negative binomial (NB generalized estimating equation (NB GEE model was used to analyze count data and a logistic GEE was used for proportions. Results: We observed short-term beneficial changes in the composition of the red complex of up to 3 months by treating subjects with AMOX+MET+TET. Similar short-term improvements with the same treatment were observed for Tannerella forsythia and Treponema denticola of the red complex. SURG had also short-term beneficial effect on Porphyromonas gingivalis. No periodontal treatments applied to severely affected sites promoted the growth of Actinomyces. Smoking elevated counts of both the red and orange complex while bleeding on probing (BOP and gingival redness were also predictors of more red complex counts. Comparatively similar findings were obtained by analyzing counts and by analyzing proportions. Conclusions: Although short-term reductions in the counts of the red complex were observed in sites that were treated with AMOX+MET+TET, long-term significant effects were not observed with any of the eight treatments. Poor oral hygiene in patients with severe chronic periodontitis diminished the beneficial effects of treatment.

  3. In vitro and in vivo uptake study of Escherichia coli Nissle 1917 bacterial ghosts: cell-based delivery system to target ocular surface diseases.

    Science.gov (United States)

    Stein, Elisabeth; Inic-Kanada, Aleksandra; Belij, Sandra; Montanaro, Jacqueline; Bintner, Nora; Schlacher, Simone; Mayr, Ulrike Beate; Lubitz, Werner; Stojanovic, Marijana; Najdenski, Hristo; Barisani-Asenbauer, Talin

    2013-09-24

    For the successful topical administration of drugs or vaccines to treat ocular surface diseases, efficient and well-tolerated delivery systems/carriers for conjunctival delivery are crucial in the development of new treatment strategies. The present study investigated the efficiency of internalization of bacterial ghosts (BGs) produced from probiotic Escherichia coli Nissle 1917 (EcN) by human conjunctival epithelial (HCjE) cell line, the EcN BGs cytotoxicity for HCjE cells, and in vivo uptake of EcN BGs by conjunctival guinea pig epithelial cells. The uptake of EcN BGs by HCjE cells was analyzed by laser scanning microscopy and flow cytometry. Immunohistochemistry was used to localize the EcN BGs in the guinea pig conjunctival tissue. Cytotoxicity of EcN BGs on HCjE cells was evaluated by measurement of LDH. Laser scanning microscopy and flow cytometry revealed that EcN BGs internalization by HCjE cells was time- and dose dependent. No cytotoxic effect on HCjE cells was observed after EcN BGs inoculation for 30 and 120 minutes, as well as 24 hours. In addition, the uptake of EcN BGs was detected in the conjunctival cells after in vivo administration of EcN BGs into the eye of the guinea pig. The findings that EcN BGs are nontoxic and effectively internalized in vitro by human and in vivo by guinea pig conjunctival cells comprise an important contribution to the future use of BGs as a system for conjunctival delivery of drugs and vaccines, either to treat or prevent ocular surface diseases.

  4. Reconsidering 'appropriate technology': the effects of operating conditions on the bacterial removal performance of two household drinking-water filter systems

    Energy Technology Data Exchange (ETDEWEB)

    Baumgartner, Jill [Harvard School of Public Health, Boston, MA (United States); Murcott, Susan [Massachusetts Institute of Technology, Cambridge, MA (United States); Ezzati, Majid [Harvard School of Public Health, Boston, MA (United States)

    2007-04-01

    We examined the performance of two household water treatment and safe storage (HWTS) systems, the Danvor plastic biosand filter and the Potters for Peace Filtron ceramic filter, under ideal as well as modified operating conditions using systematic and comparable measurements. The operating variables for the biosand filter were (i) pause times between filtration runs (ii) water-dosing volumes and (iii) the effluent volume at which a filtered water sample was collected. For the ceramic filter we examined overflow filtration versus standard filtration. We used the bacterial indicators of total coliforms and Escherichia coli to quantify microbiological removal. With the biosand filter, a 12 h pause time had significantly higher total coliform removal than a 36 h pause time at the 20 l collection point (79.1% versus 73.7%; p < 0.01) and borderline significance at the 10 l collection point (81.0% versus 78.3%; p = 0.07). High-volume filtration (20 l) had significantly lower total coliform removal efficacy than low-volume (10 l) filtration at the 10 l collection point (81.0% versus 84.2%; p = 0.03). We observed a decreasing trend in total coliform removal by sample collection volume with the highest removal efficacy at the 5 l sample collection point (versus at the 10 and 20 l collection points). Using the ceramic filter, mean total coliform and E. coli removal were significantly lower (p < 0.01) in overflow filtration than in standard filtration. The findings indicate that operating conditions can reduce the effectiveness of the systems in a field-based setting and increase environmental risk exposure.

  5. Dialkylresorcinols as bacterial signaling molecules.

    Science.gov (United States)

    Brameyer, Sophie; Kresovic, Darko; Bode, Helge B; Heermann, Ralf

    2015-01-13

    It is well recognized that bacteria communicate via small diffusible molecules, a process termed quorum sensing. The best understood quorum sensing systems are those that use acylated homoserine lactones (AHLs) for communication. The prototype of those systems consists of a LuxI-like AHL synthase and a cognate LuxR receptor that detects the signal. However, many proteobacteria possess LuxR receptors, yet lack any LuxI-type synthase, and thus these receptors are referred to as LuxR orphans or solos. In addition to the well-known AHLs, little is known about the signaling molecules that are sensed by LuxR solos. Here, we describe a novel cell-cell communication system in the insect and human pathogen Photorhabdus asymbiotica. We identified the LuxR homolog PauR to sense dialkylresorcinols (DARs) and cyclohexanediones (CHDs) instead of AHLs as signals. The DarABC synthesis pathway produces the molecules, and the entire system emerged as important for virulence. Moreover, we have analyzed more than 90 different Photorhabdus strains by HPLC/MS and showed that these DARs and CHDs are specific to the human pathogen P. asymbiotica. On the basis of genomic evidence, 116 other bacterial species are putative DAR producers, among them many human pathogens. Therefore, we discuss the possibility of DARs as novel and widespread bacterial signaling molecules and show that bacterial cell-cell communication goes far beyond AHL signaling in nature.

  6. Electromagnetism of Bacterial Growth

    Science.gov (United States)

    Ainiwaer, Ailiyasi

    2011-10-01

    There has been increasing concern from the public about personal health due to the significant rise in the daily use of electrical devices such as cell phones, radios, computers, GPS, video games and television. All of these devices create electromagnetic (EM) fields, which are simply magnetic and electric fields surrounding the appliances that simultaneously affect the human bio-system. Although these can affect the human system, obstacles can easily shield or weaken the electrical fields; however, magnetic fields cannot be weakened and can pass through walls, human bodies and most other objects. The present study was conducted to examine the possible effects of bacteria when exposed to magnetic fields. The results indicate that a strong causal relationship is not clear, since different magnetic fields affect the bacteria differently, with some causing an increase in bacterial cells, and others causing a decrease in the same cells. This phenomenon has yet to be explained, but the current study attempts to offer a mathematical explanation for this occurrence. The researchers added cultures to the magnetic fields to examine any effects to ion transportation. Researchers discovered ions such as potassium and sodium are affected by the magnetic field. A formula is presented in the analysis section to explain this effect.

  7. Environmental T4-Family Bacteriophages Evolve to Escape Abortive Infection via Multiple Routes in a Ba