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Sample records for bacterial protein produced

  1. Decreased Bacterial Attachment and Protein Adsorption to Coatings Produced by Low Enegy Plasma Polymerization

    DEFF Research Database (Denmark)

    Andersen, T.E.; Kingshott, Peter; Benter, M.;

    Introduction Silicone rubber is among the most biocompatible materials available, exhibiting low levels of extractables, absence of plasticizers and additives and fairly low activation of blood thrombogenesis components. However untreated silicone rubber does not efficiently resist protein...... with a surface less prone to the adsorption of biological matter. In the current study two different hydrophilic nanoscale coatings were produced by low energy plasma polymerization [3] and investigated· f()rl()w ... pr()tein adsorption and bacterial attachment properties. Methods were setup to enable...... the measurement of both initial adhesion of clinically isolated bacteria on silicone and subsequent biofilm formation during prolonged growth under liquid flow. The extend of adsorption of relevant proteins to the surfaces was also investigated using quartz crystal microbalance with dissipation (QCM...

  2. Secreted and immunogenic proteins produced by the honeybee bacterial pathogen, Paenibacillus larvae.

    Science.gov (United States)

    Antúnez, Karina; Anido, Matilde; Evans, Jay D; Zunino, Pablo

    2010-03-24

    American Foulbrood is a severe disease affecting larvae of honeybee Apis mellifera, causing significant decrease in the honeybee population, beekeeping industries and agricultural production. In spite of its importance, little is known about the virulence factors secreted by Paenibacillus larvae during larval infection. The aim of the present work was to perform a first approach to the identification and characterization of P. larvae secretome. P. larvae secreted proteins were analyzed by SDS-PAGE and identified by MALDI-TOF. Protein toxicity was evaluated using an experimental model based on feeding of A. mellifera larvae and immunogenicity was evaluated by Western blot, using an antiserum raised against cells and spores of P. larvae. Ten different proteins were identified among P. larvae secreted proteins, including proteins involved in transcription, metabolism, translation, cell envelope, transport, protein folding, degradation of polysaccharides and motility. Although most of these proteins are cytosolic, many of them have been previously detected in the extracellular medium of different Bacillus spp. cultures and have been related to virulence. The secreted proteins resulted highly toxic and immunogenic when larvae were exposed using an experimental model. This is the first description of proteins secreted by the honeybee pathogen P. larvae. This information may be relevant for the elucidation of bacterial pathogenesis mechanisms. PMID:19781868

  3. Nitrogen and energy balance in growing mink (Mustela vison) fed different levels of bacterial protein meal produced with natural gas

    DEFF Research Database (Denmark)

    Hellwing, Anne Louise Frydendahl; Tauson, Anne-Helene; Ahlstrøm, Øystein;

    2005-01-01

    The objective of this study was to estimate the effect of increasing the dietary content of bacterial protein meal (BPM) on energy and protein metabolism in growing mink kits. Sixteen male mink kits of the standard brown genotype were randomly fed one of four diets: A control (Diet III) and 60......% (Diet IV) of the digested nitrogen (DN) was replaced with BPM. Nitrogen balance and respiration experiments (indirect calorimetry) were carried out when the animals were approximately 9.5, 14.5, 17.5, 23.5 and 28.5 weeks of age. The apparent digestibility of crude protein and energy decreased...... significantly with increasing dietary BPM. The retained nitrogen was 0.45, 0.54, 0.52 and 0.40 g/kg0,75 on Diets I, II, III and IV, respectively, the observed differences between diets being non-significant (p=0.06). Heat production (HE) was between 645 and 665 kJ/kg0.75 on all diets (p=0.78). retained energy...

  4. Growh performance, nitrogen balance and urinary purine derivatives in growing-furring mink (Mustela vison) fed bacterial protein produced from natural gas

    DEFF Research Database (Denmark)

    Ahlstrøm, Ø.; Tauson, Anne-Helene; Hellwing, Anne Louise Frydendahl;

    2006-01-01

    A bacterial protein meal (BPM), containing 70% crude protein and produced on natural gas, was evaluated versus fish meal as protein source for mink in the growing-furring period (June 29-November 26). BPM, rich in nucleic acids, accounted for 0 (control), 20 and 40% of dietary crude protein...... corresponding to 0,4 and 8% of the wet diets, respectively. Each diet was given to 48 animals, 24 males and 24 females. The inclusion of BPM tended to reduce feed intake and body weight gain during the first half of the experimental period, but this was compensated for during the last part of the experiment......, except for males on the 8% BPM diet. Balance experiments carried out with 18 and 28 weeks old males, revealed similar digestibility of main nutrients except for fat that were reduced with BPM inclusion. N-retentions were similar for the dietary groups. Daily excretion of urine was lower with the 8% BPM...

  5. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten;

    2010-01-01

    Bacteria and Eukarya share essentially the same family of protein-serine/threonine kinases, also known as the Hanks-type kinases. However, when it comes to protein-tyrosine phosphorylation, bacteria seem to have gone their own way. Bacterial protein-tyrosine kinases (BY-kinases) are bacterial...... enzymes that are unique in exploiting the ATP/GTP-binding Walker motif to catalyze phosphorylation of protein tyrosine residues. Characterized for the first time only a decade ago, BY-kinases have now come to the fore. Important regulatory roles have been linked with these enzymes, via their involvement...... in exopolysaccharide production, virulence, DNA metabolism, stress response and other key functions of the bacterial cell. BY-kinases act through autophosphorylation (mainly in exopolysaccharide production) and phosphorylation of other proteins, which have in most cases been shown to be activated by...

  6. Engineered fluorescent proteins illuminate the bacterial periplasm

    Directory of Open Access Journals (Sweden)

    Thorben Dammeyer

    2012-10-01

    Full Text Available The bacterial periplasm is of special interest whenever cell factories are designed and engineered. Recombinantely produced proteins are targeted to the periplasmic space of Gram negative bacteria to take advantage of the authentic N-termini, disulfide bridge formation and easy accessibility for purification with less contaminating cellular proteins. The oxidizing environment of the periplasm promotes disulfide bridge formation - a prerequisite for proper folding of many proteins into their active conformation. In contrast, the most popular reporter protein in all of cell biology, Green Fluorescent Protein (GFP, remains inactive if translocated to the periplasmic space prior to folding. Here, the self-catalyzed chromophore maturation is blocked by formation of covalent oligomers via interchain disulfide bonds in the oxidizing environment. However, different protein engineering approaches addressing folding and stability of GFP resulted in improved proteins with enhanced folding properties. Recent studies describe GFP variants that are not only active if translocated in their folded form via the twin-arginine translocation (Tat pathway, but actively fold in the periplasm following general secretory pathway (Sec and signal recognition particle (SRP mediated secretion. This mini-review highlights the progress that enables new insights into bacterial export and periplasmic protein organization, as well as new biotechnological applications combining the advantages of the periplasmic production and the Aequorea-based fluorescent reporter proteins.

  7. Bacterial cell division proteins as antibiotic targets

    NARCIS (Netherlands)

    T. den Blaauwen; J.M. Andreu; O. Monasterio

    2014-01-01

    Proteins involved in bacterial cell division often do not have a counterpart in eukaryotic cells and they are essential for the survival of the bacteria. The genetic accessibility of many bacterial species in combination with the Green Fluorescence Protein revolution to study localization of protein

  8. Recent advances in bacterial heme protein biochemistry

    OpenAIRE

    Mayfield, Jeffery A.; Dehner, Carolyn A.; Dubois, Jennifer L.

    2011-01-01

    Recent progress in genetics, fed by the burst in genome sequence data, has led to the identification of a host of novel bacterial heme proteins that are now being characterized in structural and mechanistic terms. The following short review highlights very recent work with bacterial heme proteins involved in the uptake, biosynthesis, degradation, and use of heme in respiration and sensing.

  9. Produced water exposure alters bacterial response to biocides.

    Science.gov (United States)

    Vikram, Amit; Lipus, Daniel; Bibby, Kyle

    2014-11-01

    Microbial activity during the holding and reuse of wastewater from hydraulic fracturing operations, termed produced water, may lead to issues with corrosion, sulfide release, and fouling. Biocides are applied to control biological activity, often with limited efficacy, which is typically attributed to chemical interactions with the produced water. However, it is unknown whether there is a biologically driven mechanism to biocide tolerance in produced water. Here, we demonstrate that produced water exposure results in an enhanced tolerance against the typically used biocide glutaraldehyde and increased susceptibility to the oxidative biocide hypochlorite in a native and a model bacteria and that this altered resistance is due to the salinity of the produced water. In addition, we elucidate the genetic response of the model organism Pseudomonas fluorescens to produced water exposure to provide a mechanistic interpretation of the altered biocide resistance. The RNA-seq data demonstrated the induction of genes involved in osmotic stress, energy production and conversion, membrane integrity, and protein transport following produced water exposure, which facilitates bacterial survival and alters biocide tolerance. Efforts to fundamentally understand biocide resistance mechanisms, which enable the optimization of biocide application, hold significant implications for greening of the fracturing process through encouraging produced water recycling. Specifically, these results suggest the necessity of optimizing biocide application at the level of individual shale plays, rather than historical experience, based upon produced water characteristics and salinity. PMID:25279933

  10. BLANNOTATOR: enhanced homology-based function prediction of bacterial proteins

    Directory of Open Access Journals (Sweden)

    Kankainen Matti

    2012-02-01

    Full Text Available Abstract Background Automated function prediction has played a central role in determining the biological functions of bacterial proteins. Typically, protein function annotation relies on homology, and function is inferred from other proteins with similar sequences. This approach has become popular in bacterial genomics because it is one of the few methods that is practical for large datasets and because it does not require additional functional genomics experiments. However, the existing solutions produce erroneous predictions in many cases, especially when query sequences have low levels of identity with the annotated source protein. This problem has created a pressing need for improvements in homology-based annotation. Results We present an automated method for the functional annotation of bacterial protein sequences. Based on sequence similarity searches, BLANNOTATOR accurately annotates query sequences with one-line summary descriptions of protein function. It groups sequences identified by BLAST into subsets according to their annotation and bases its prediction on a set of sequences with consistent functional information. We show the results of BLANNOTATOR's performance in sets of bacterial proteins with known functions. We simulated the annotation process for 3090 SWISS-PROT proteins using a database in its state preceding the functional characterisation of the query protein. For this dataset, our method outperformed the five others that we tested, and the improved performance was maintained even in the absence of highly related sequence hits. We further demonstrate the value of our tool by analysing the putative proteome of Lactobacillus crispatus strain ST1. Conclusions BLANNOTATOR is an accurate method for bacterial protein function prediction. It is practical for genome-scale data and does not require pre-existing sequence clustering; thus, this method suits the needs of bacterial genome and metagenome researchers. The method and a

  11. Demodex-associated bacterial proteins induce neutrophil activation.

    LENUS (Irish Health Repository)

    2012-02-01

    Background: Patients with rosacea demonstrate a higher density of Demodex mites in their skin than controls. A bacterium isolated from a Demodex mite from a patient with papulopustular rosacea (PPR) was previously shown to provoke an immune response in patients with PPR or ocular rosacea thus suggesting a possible role for bacterial proteins in the etiology of this condition. Objectives: To examine the response of neutrophils to proteins derived from a bacterium isolated from a Demodex mite. Methods: Bacterial cells were lysed and proteins were partially purified by AKTA-FPLC. Isolated neutrophils were exposed to bacterial proteins and monitored for alterations in migration, degranulation and cytokine production. Results: Neutrophils exposed to proteins from Bacillus cells demonstrated increased levels of migration and elevated release of MMP-9, an enzyme known to degrade collagen and cathelicidin, an antimicrobial peptide. In addition neutrophils exposed to the bacterial proteins demonstrated elevated rates of Il-8 and TNF-alpha production. Conclusions: Proteins produced by a bacterium isolated from a Demodex mite have the ability to increase the migration, degranulation and cytokine production abilities of neutrophils. These results suggest that bacteria may play a role in the inflammatory erythema associated with rosacea.

  12. C-reactive protein and bacterial meningitis

    DEFF Research Database (Denmark)

    Gerdes, Lars Ulrik; Jørgensen, P E; Nexø, E;

    1998-01-01

    The aim of the study was to review published articles on the diagnostic accuracy of C-reactive protein (CRP) tests with cerebrospinal fluid and serum in diagnosing bacterial meningitis. The literature from 1980 and onwards was searched using the electronic databases of MEDLINE, and we used summary...

  13. Ice nucleation protein as a bacterial surface display protein

    OpenAIRE

    Sarhan Mohammed A.A.

    2011-01-01

    Surface display technology can be defined as that phenotype (protein or peptide) which is linked to a genotype (DNA or RNA) through an appropriate anchoring motif. A bacterial surface display system is based on expressing recombinant proteins fused to sorting signals (anchoring motifs) that direct their incorporation on the cell surface.

  14. Fluorescent sensors based on bacterial fusion proteins

    International Nuclear Information System (INIS)

    Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker. The pH dependence of the fluorescence intensity of the S-layer fusion proteins, monitored by fluorescence spectroscopy, showed that the ECFP tag was more stable than EGFP. Furthermore, the fluorescent fusion proteins were reassembled on silica particles modified with cationic and anionic polyelectrolytes. Zeta potential measurements confirmed the particle coatings and indicated their colloidal stability. Flow cytometry and fluorescence microscopy showed that the fluorescence of the fusion proteins was pH dependent and sensitive to the underlying polyelectrolyte coating. This might suggest that the fluorescent tag is not completely exposed to the bulk media as an independent moiety. Finally, it was found out that viscosity enhanced the fluorescence intensity of the three fluorescent S-layer proteins. (paper)

  15. Fluorescent sensors based on bacterial fusion proteins

    Science.gov (United States)

    Prats Mateu, Batirtze; Kainz, Birgit; Pum, Dietmar; Sleytr, Uwe B.; Toca-Herrera, José L.

    2014-06-01

    Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker. The pH dependence of the fluorescence intensity of the S-layer fusion proteins, monitored by fluorescence spectroscopy, showed that the ECFP tag was more stable than EGFP. Furthermore, the fluorescent fusion proteins were reassembled on silica particles modified with cationic and anionic polyelectrolytes. Zeta potential measurements confirmed the particle coatings and indicated their colloidal stability. Flow cytometry and fluorescence microscopy showed that the fluorescence of the fusion proteins was pH dependent and sensitive to the underlying polyelectrolyte coating. This might suggest that the fluorescent tag is not completely exposed to the bulk media as an independent moiety. Finally, it was found out that viscosity enhanced the fluorescence intensity of the three fluorescent S-layer proteins.

  16. Membrane fatty acids as markers of bacterial antibiotic-producers

    Czech Academy of Sciences Publication Activity Database

    Elhottová, Dana; Petrásek, Jiří; Krištůfek, Václav; Jágr, Michal; Chroňáková, Alica; Petříček, Miroslav

    Cairns : International Society for Microbial Ecology, 2008. [International Symposium on Microbial Ecology ISME 12. Microbial Diversity - Sustaining the Blue Planet /12./. 17.08.2008-22.08.2008, Cairns] R&D Projects: GA AV ČR IAA600660607; GA MŠk 2B06154 Institutional research plan: CEZ:AV0Z60660521; CEZ:AV0Z50200510 Keywords : membrane fatty acids * bacterial antibiotic -producers * microorganisms Subject RIV: EH - Ecology, Behaviour

  17. Frequency of Bacterial Frequency of Bacterial Contamination in Traditional Ice Cream Produced in Arak, Iran (2011)

    OpenAIRE

    M. Rezaei; Ghasemi khah , R. (PhD); M. Parviz; Zarei, D. (MSc

    2014-01-01

    Background and Objective: Ice cream is a suitable environment for microbial growth due to its chemical structure, ingredients, and its increased supply and demand. In the absence of hygienic considerations, it can cause poisoning. This study aimed to determine bacterial contamination in traditional ice cream produced in Arak city in 2011. Material and Methods: The samples (n= 30) were randomly obtained from different parts of Arak in, 2011. The Samples were shipped in cold conditions and tota...

  18. Frequency of Bacterial Frequency of Bacterial Contamination in Traditional Ice Cream Produced in Arak, Iran (2011

    Directory of Open Access Journals (Sweden)

    Rezaei, M. (MSc

    2014-05-01

    Full Text Available Background and Objective: Ice cream is a suitable environment for microbial growth due to its chemical structure, ingredients, and its increased supply and demand. In the absence of hygienic considerations, it can cause poisoning. This study aimed to determine bacterial contamination in traditional ice cream produced in Arak city in 2011. Material and Methods: The samples (n= 30 were randomly obtained from different parts of Arak in, 2011. The Samples were shipped in cold conditions and total count of microorganisms test was performed according to Iranian national standards. Results: In 16.66%, the microbial contamination was below the limit of microbial load (5×104, and in 83.3% the contamination was more than allowed level. Conclusion: This study highlights the dire situation for bacterial contamination of traditional ice cream in Arak city. Keywords: Arak, Ice Cream, Microbial Contamination

  19. Bacterial Protein N-Glycosylation: New Perspectives and Applications*

    OpenAIRE

    Nothaft, Harald; Szymanski, Christine M.

    2013-01-01

    Protein glycosylation is widespread throughout all three domains of life. Bacterial protein N-glycosylation and its application to engineering recombinant glycoproteins continue to be actively studied. Here, we focus on advances made in the last 2 years, including the characterization of novel bacterial N-glycosylation pathways, examination of pathway enzymes and evolution, biological roles of protein modification in the native host, and exploitation of the N-glycosylation pathways to create ...

  20. Convergent evolution among immunoglobulin G-binding bacterial proteins.

    OpenAIRE

    Frick, I M; Wikström, M.; Forsén, S.; Drakenberg, T; Gomi, H.; Sjöbring, U; Björck, L

    1992-01-01

    Protein G, a bacterial cell-wall protein with high affinity for the constant region of IgG (IgGFc) antibodies, contains homologous repeats responsible for the interaction with IgGFc. A synthetic peptide corresponding to an 11-amino acid-long sequence in the COOH-terminal region of the repeats was found to bind to IgGFc and block the interaction with protein G. Moreover, two other IgGFc-binding bacterial proteins (proteins A and H), which do not contain any sequences homologous to the peptide,...

  1. Bacterially produced recombinant influenza vaccines based on virus-like particles.

    Directory of Open Access Journals (Sweden)

    Andrea Jegerlehner

    Full Text Available Although current influenza vaccines are effective in general, there is an urgent need for the development of new technologies to improve vaccine production timelines, capacities and immunogenicity. Herein, we describe the development of an influenza vaccine technology which enables recombinant production of highly efficient influenza vaccines in bacterial expression systems. The globular head domain of influenza hemagglutinin, comprising most of the protein's neutralizing epitopes, was expressed in E. coli and covalently conjugated to bacteriophage-derived virus-like particles produced independently in E.coli. Conjugate influenza vaccines produced this way were used to immunize mice and found to elicit immune sera with high antibody titers specific for the native influenza hemagglutinin protein and high hemagglutination-inhibition titers. Moreover vaccination with these vaccines induced full protection against lethal challenges with homologous and highly drifted influenza strains.

  2. Integration of bacterial expansin-like proteins into cellulosome promotes the cellulose degradation.

    Science.gov (United States)

    Chen, Chao; Cui, Zhenling; Song, Xiangfei; Liu, Ya-Jun; Cui, Qiu; Feng, Yingang

    2016-03-01

    Cellulosomes are multi-enzyme complexes assembled by cellulases and hemicellulases through dockerin-cohesin interactions, which are the most efficient system for the degradation of lignocellulosic resources in nature. Recent genomic analysis of a cellulosome-producing anaerobe Clostridium clariflavum DSM 19732 revealed that two expansin-like proteins, Clocl_1298 and Clocl_1862, contain a dockerin module, which suggests that they are components of the cellulosome. Bacterial expansin-like proteins do not have hydrolytic activities, but can facilitate the degradation of cellulosic biomass via synergistic effects with cellulases. In this study, the synergistic effect of the expansin-like proteins with both native and designer cellulosomes was investigated. The free expansin-like proteins, including expansin-like domains of Clocl_1298 and Clocl_1862, as well as a well-studied bacterial expansin-like protein BsEXLX1 from Bacillus subtilis, promoted the cellulose degradation by native cellulosomes, indicating the cellulosomal expansin-like proteins have the synergistic function. When they were integrated into a trivalent designer cellulosome, the synergistic effect was further amplified. The sequence and structure analyses indicated that these cellulosomal expansin-like proteins share the conserved functional mechanism with other bacterial expansin-like proteins. These results indicated that non-catalytic expansin-like proteins in the cellulosome can enhance the activity of the cellulosome in lignocellulose degradation. The involvement of functional expansin-like proteins in the cellulosome also implies new physiological functions of bacterial expansin-like proteins and cellulosomes. PMID:26521249

  3. Data presenting a modified bacterial expression vector for expressing and purifying Nus solubility-tagged proteins.

    Science.gov (United States)

    Gupta, Nidhi; Wu, Heng; Terman, Jonathan R

    2016-09-01

    Bacteria are the predominant source for producing recombinant proteins but while many exogenous proteins are expressed, only a fraction of those are soluble. We have found that a new actin regulatory enzyme Mical is poorly soluble when expressed in bacteria but the use of a Nus fusion protein tag greatly increases its solubility. However, available vectors containing a Nus tag have been engineered in a way that hinders the separation of target proteins from the Nus tag during protein purification. We have now used recombinant DNA approaches to overcome these issues and reengineer a Nus solubility tag-containing bacterial expression vector. The data herein present a modified bacterial expression vector useful for expressing proteins fused to the Nus solubility tag and separating such target proteins from the Nus tag during protein purification. PMID:27547802

  4. No role for bacterially produced salicylic Acid in rhizobacterial induction of systemic resistance in Arabidopsis.

    Science.gov (United States)

    Ran, L X; van Loon, L C; Bakker, P A H M

    2005-11-01

    ABSTRACT The role of bacterially produced salicylic acid (SA) in the induction of systemic resistance in plants by rhizobacteria is far from clear. The strong SA producer Pseudomonas fluorescens WCS374r induces resistance in radish but not in Arabidopsis thaliana, whereas application of SA leads to induction of resistance in both plant species. In this study, we compared P. fluorescens WCS374r with three other SA-producing fluorescent Pseudomonas strains, P. fluorescens WCS417r and CHA0r, and P. aeruginosa 7NSK2 for their abilities to produce SA under different growth conditions and to induce systemic resistance in A. thaliana against bacterial speck, caused by P. syringae pv. tomato. All strains produced SA in vitro, varying from 5 fg cell(-1) for WCS417r to >25 fg cell(-1) for WCS374r. Addition of 200 muM FeCl(3) to standard succinate medium abolished SA production in all strains. Whereas the incubation temperature did not affect SA production by WCS417r and 7NSK2, strains WCS374r and CHA0r produced more SA when grown at 33 instead of 28 degrees C. WCS417r, CHA0r, and 7NSK2 induced systemic resistance apparently associated with their ability to produce SA, but WCS374r did not. Conversely, a mutant of 7NSK2 unable to produce SA still triggered induced systemic resistance (ISR). The possible involvement of SA in the induction of resistance was evaluated using SA-nonaccumulating transgenic NahG plants. Strains WCS417r, CHA0r, and 7NSK2 induced resistance in NahG Arabidopsis. Also, WCS374r, when grown at 33 or 36 degrees C, triggered ISR in these plants, but not in ethylene-insensitive ein2 or in non-plant pathogenesis- related protein-expressing npr1 mutant plants, irrespective of the growth temperature of the bacteria. These results demonstrate that, whereas WCS374r can be manipulated to trigger ISR in Arabidopsis, SA is not the primary determinant for the induction of systemic resistance against bacterial speck disease by this bacterium. Also, for the other

  5. Exploring the diversity of protein modifications: special bacterial phosphorylation systems

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Grangeasse, Christophe; Turgay, Kürşad

    2016-01-01

    physiology, and regulatory networks. Investigating these unusual bacterial kinase and phosphatases is not only important to understand their role in bacterial physiology but will help to generally understand the full potential and evolution of protein phosphorylation for signal transduction, protein...... has been most thoroughly investigated. Unlike in eukarya, a large diversity of enzyme families has been shown to phosphorylate and dephosphorylate proteins on various amino acids with different chemical properties in bacteria. In this review, after a brief overview of the known bacterial...... phosphorylation systems, we focus on more recently discovered and less widely known kinases and phosphatases. Namely, we describe in detail tyrosine- and arginine-phosphorylation together with some examples of unusual serine-phosphorylation systems and discuss their potential role and function in bacterial...

  6. Bacterial S-layer protein coupling to lipids

    DEFF Research Database (Denmark)

    Weygand, M.; Wetzer, B.; Pum, D.; Sleytr, U.B.; Cuvillier, N.; Kjær, K.; Howes, P.B.; Lösche, M.

    1999-01-01

    The coupling of bacterial surface (S)-layer proteins to lipid membranes is studied in molecular detail for proteins from Bacillus sphaericus CCM2177 and B. coagulans E38-66 recrystallized at dipalmitoylphosphatidylethanolamine (DPPE) monolayers on aqueous buffer. A comparison of the monolayer str...

  7. Partial Characteristics of Hydrogen Production by Fermentative Hydrogen-producing Bacterial Strain B49

    Institute of Scientific and Technical Information of China (English)

    Wang Xiangjing(王相晶); Ren Nanqi; Xiang Wensheng; Lin Ming; Guo Wanqian

    2003-01-01

    To investigate the characteristics of hydrogen production by a novel fermentative hydrogen-producing bacterial strain B49 (AF481148 in EMBL), batch experiments are conducted under different conditions. Hydrogen production has a correlation with cell growth and the consumption of glucose and soluble protein. The optimum pH for cell growth is 4.5±0.15. At acidic pH 4.0±0.15, the bacteria has the maximum accumulated hydrogen volume of 2382 ml/L culture and the maximum hydrogen evolution rate of 339.9 ml/L culture*h with 1% glucose. The optimum temperature for cell growth and hydrogen production is 35℃. In addition, fermentative hydrogen-producing bacterial strain B49 can generate hydrogen from the decomposition of other organic substrates such as wheat, soybean, corn, and potato. Moreover, it can also produce hydrogen from molasses wastewater and brewage wastewater, and hydrogen yields are 137.9 ml H2/g COD and 49.9 ml H2/g COD, respectively.

  8. Protein quality control in the bacterial periplasm.

    Science.gov (United States)

    Merdanovic, Melisa; Clausen, Tim; Kaiser, Markus; Huber, Robert; Ehrmann, Michael

    2011-01-01

    Protein quality control involves sensing and treatment of defective or incomplete protein structures. Misfolded or mislocalized proteins trigger dedicated signal transduction cascades that upregulate the production of protein quality-control factors. Corresponding proteases and chaperones either degrade or repair damaged proteins, thereby reducing the level of aggregation-prone molecules. Because the periplasm of gram-negative bacteria is particularly exposed to environmental changes and respective protein-folding stresses connected with the presence of detergents, low or high osmolarity of the medium, elevated temperatures, and the host's immune response, fine-tuned protein quality control systems are essential for survival under these unfavorable conditions. This review discusses recent advances in the identification and characterization of the key cellular factors and the emerging general principles of the underlying molecular mechanisms. PMID:21639788

  9. Prolonged inhibition of bacterial protein synthesis abolishes Salmonella invasion.

    OpenAIRE

    MacBeth, K J; Lee, C. A.

    1993-01-01

    We have found that prolonged inhibition of bacterial protein synthesis abolishes the ability of Salmonella typhimurium to enter HEp-2 cells. Our results suggest that an essential invasion factor has a functional half-life that is seen as a gradual loss of invasiveness in the absence of protein synthesis. Therefore, Salmonella invasiveness appears to be a transient phenotype that is lost unless protein synthesis is maintained. This finding may explain why salmonellae grown to stationary phase ...

  10. Influence of anerobic fungi of biogas producing bacterial community

    Czech Academy of Sciences Publication Activity Database

    Mrázek, Jakub; Štrosová, Lenka; Fliegerová, Kateřina

    Aberdeen : Rowett Institute -INRA, 2010. s. 1-1. [7th joint symposium of Rowett - INRA. 23.06. - 25.06.2010, Aberdeen] R&D Projects: GA ČR GPP503/10/P394; GA ČR GD525/08/H060 Institutional research plan: CEZ:AV0Z50450515 Keywords : anaerobic fungi * biogas * bacterial community Subject RIV: EE - Microbiology, Virology

  11. Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA

    OpenAIRE

    Belcourt, Michael F.; Penketh, Philip G.; Hodnick, William F.; Johnson, David A.; David H Sherman; Rockwell, Sara; Sartorelli, Alan C.

    1999-01-01

    The mitomycin C-resistance gene, mcrA, of Streptomyces lavendulae produces MCRA, a protein that protects this microorganism from its own antibiotic, the antitumor drug mitomycin C. Expression of the bacterial mcrA gene in mammalian Chinese hamster ovary cells causes profound resistance to mitomycin C and to its structurally related analog porfiromycin under aerobic conditions but produces little change in drug sensitivity under hypoxia. The mitomycins are prodrugs that are enzymatically reduc...

  12. The Chaotic Structure of Bacterial Virulence Protein Sequences

    Directory of Open Access Journals (Sweden)

    Sevdanur Genc

    2015-01-01

    Full Text Available Bacterial virulence proteins, which have been class ified on structure of virulence, causes several diseases. For instance, Adhesins play an important role in th e host cells. They are inserted DNA sequences for a variety of virulence properties. Several important methods conducted for the prediction of bacterial virulence proteins for finding new drugs or vaccines. In this study, we propose a method for feature sele ction about classification of bacterial virulence protein. The features are constituted dir ectly from the amino acid sequence of a given protein. Amino acids form proteins, which are criti cal to life, and have many important functions in living cells. They occurring with diff erent physicochemical properties by a vector of 20 numerical values, and collected in AAIndex datab ases of known 544 indices. For all that, this approach have two steps. Firstly , the amino acid sequence of a given protein analysed with Lyapunov Exponents that they have a chaotic structure in accordance wi th the chaos theory. After that, if the results show chara cterization over the complete distribution in the phase space from the point of deterministic sys tem, it means related protein will show a chaotic structure. Empirical results revealed that generated feature v ectors give the best performance with chaotic structure of physicochemical features of amino acid s with Adhesins and non-Adhesins data sets.

  13. Identification of ligands for bacterial sensor proteins.

    Science.gov (United States)

    Fernández, Matilde; Morel, Bertrand; Corral-Lugo, Andrés; Rico-Jiménez, Miriam; Martín-Mora, David; López-Farfán, Diana; Reyes-Darias, José Antonio; Matilla, Miguel A; Ortega, Álvaro; Krell, Tino

    2016-02-01

    Bacteria have evolved a variety of different signal transduction mechanisms. However, the cognate signal molecule for the very large amount of corresponding sensor proteins is unknown and their functional annotation represents a major bottleneck in the field of signal transduction. The knowledge of the signal molecule is an essential prerequisite to understand the signalling mechanisms. Recently, the identification of signal molecules by the high-throughput protein screening of commercially available ligand collections using differential scanning fluorimetry has shown promise to resolve this bottleneck. Based on the analysis of a significant number of different ligand binding domains (LBDs) in our laboratory, we identified two issues that need to be taken into account in the experimental design. Since a number of LBDs require the dimeric state for ligand recognition, it has to be assured that the protein analysed is indeed in the dimeric form. A number of other examples demonstrate that purified LBDs can contain bound ligand which prevents further binding. In such cases, the apo-form can be generated by denaturation and subsequent refolding. We are convinced that this approach will accelerate the functional annotation of sensor proteins which will help to understand regulatory circuits in bacteria. PMID:26511375

  14. Bacterial Microcompartment Organelles: Protein Shell Structure and Evolution

    OpenAIRE

    Yeates, Todd O; Crowley, Christopher S.; Tanaka, Shiho

    2010-01-01

    Some bacteria contain organelles or microcompartments consisting of a large virion-like protein shell encapsulating sequentially acting enzymes. These organized microcompartments serve to enhance or protect key metabolic pathways inside the cell. The variety of bacterial microcompartments provide diverse metabolic functions, ranging from CO2 fixation to the degradation of small organic molecules. Yet they share an evolutionarily related shell, which is defined by a conserved protein domain th...

  15. Surface display of proteins by Gram-negative bacterial autotransporters

    Directory of Open Access Journals (Sweden)

    Mourez Michael

    2006-06-01

    Full Text Available Abstract Expressing proteins of interest as fusions to proteins of the bacterial envelope is a powerful technique with many biotechnological and medical applications. Autotransporters have recently emerged as a good tool for bacterial surface display. These proteins are composed of an N-terminal signal peptide, followed by a passenger domain and a translocator domain that mediates the outer membrane translocation of the passenger. The natural passenger domain of autotransporters can be replaced by heterologous proteins that become displayed at the bacterial surface by the translocator domain. The simplicity and versatility of this system has made it very attractive and it has been used to display functional enzymes, vaccine antigens as well as polypeptides libraries. The recent advances in the study of the translocation mechanism of autotransporters have raised several controversial issues with implications for their use as display systems. These issues include the requirement for the displayed polypeptides to remain in a translocation-competent state in the periplasm, the requirement for specific signal sequences and "autochaperone" domains, and the influence of the genetic background of the expression host strain. It is therefore important to better understand the mechanism of translocation of autotransporters in order to employ them to their full potential. This review will focus on the recent advances in the study of the translocation mechanism of autotransporters and describe practical considerations regarding their use for bacterial surface display.

  16. Bacterial characterization using protein profiling in a microchip separations platform.

    Science.gov (United States)

    Pizarro, Shelly A; Lane, Pamela; Lane, Todd W; Cruz, Evelyn; Haroldsen, Brent; VanderNoot, Victoria A

    2007-12-01

    A rapid microanalytical protein-based approach to bacterial characterization is presented. Chip gel electrophoresis (CGE) coupled with LIF detection was used to analyze lysates from different bacterial cell lines to obtain signature profiles of the soluble protein composition. The study includes Escherichia coli, Bacillus subtilis, and Bacillus anthracis (Delta Sterne strain) vegetative cells as well as endospores formed from the latter two species as model organisms to demonstrate the method. A unified protein preparation protocol was developed for both cell types to streamline the benchtop process and aid future automation. Cells and spores were lysed and proteins solubilized using a combination of thermal and chemical lysis methods. Reducing agents, necessary to solubilize spore proteins, were eliminated using a small-scale rapid size-exclusion chromatography step to eliminate interference with down-stream protein labeling. This approach was found to be compatible with nonspore cells (i.e., vegetative cells) as well, not adversely impacting the protein signatures. Data are presented demonstrating distinct CGE protein signatures for our model organisms, suggesting the potential for discrimination of organisms on the basis of empirical protein patterns. The goal of this work is to develop a fast and field-portable method for characterizing bacteria via their proteomes. PMID:18008300

  17. Prolonged inhibition of bacterial protein synthesis abolishes Salmonella invasion.

    Science.gov (United States)

    MacBeth, K J; Lee, C A

    1993-01-01

    We have found that prolonged inhibition of bacterial protein synthesis abolishes the ability of Salmonella typhimurium to enter HEp-2 cells. Our results suggest that an essential invasion factor has a functional half-life that is seen as a gradual loss of invasiveness in the absence of protein synthesis. Therefore, Salmonella invasiveness appears to be a transient phenotype that is lost unless protein synthesis is maintained. This finding may explain why salmonellae grown to stationary phase lose their ability to enter cultured cells. In addition, a short-lived capacity to enter cells may be important during infection so that bacterial invasiveness is limited to certain times and host sites during pathogenesis. PMID:8454361

  18. Structural Aspects of Bacterial Outer Membrane Protein Assembly.

    Science.gov (United States)

    Calmettes, Charles; Judd, Andrew; Moraes, Trevor F

    2015-01-01

    The outer membrane of Gram-negative bacteria is predominantly populated by β-Barrel proteins and lipid anchored proteins that serve a variety of biological functions. The proper folding and assembly of these proteins is essential for bacterial viability and often plays a critical role in virulence and pathogenesis. The β-barrel assembly machinery (Bam) complex is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, whereas the localization of lipoproteins (Lol) system is required for proper targeting of lipoproteins to the outer membrane. PMID:26621472

  19. Recombinant Expression Screening of P. aeruginosa Bacterial Inner Membrane Proteins

    Directory of Open Access Journals (Sweden)

    Jeffery Constance J

    2010-11-01

    Full Text Available Abstract Background Transmembrane proteins (TM proteins make up 25% of all proteins and play key roles in many diseases and normal physiological processes. However, much less is known about their structures and molecular mechanisms than for soluble proteins. Problems in expression, solubilization, purification, and crystallization cause bottlenecks in the characterization of TM proteins. This project addressed the need for improved methods for obtaining sufficient amounts of TM proteins for determining their structures and molecular mechanisms. Results Plasmid clones were obtained that encode eighty-seven transmembrane proteins with varying physical characteristics, for example, the number of predicted transmembrane helices, molecular weight, and grand average hydrophobicity (GRAVY. All the target proteins were from P. aeruginosa, a gram negative bacterial opportunistic pathogen that causes serious lung infections in people with cystic fibrosis. The relative expression levels of the transmembrane proteins were measured under several culture growth conditions. The use of E. coli strains, a T7 promoter, and a 6-histidine C-terminal affinity tag resulted in the expression of 61 out of 87 test proteins (70%. In this study, proteins with a higher grand average hydrophobicity and more transmembrane helices were expressed less well than less hydrophobic proteins with fewer transmembrane helices. Conclusions In this study, factors related to overall hydrophobicity and the number of predicted transmembrane helices correlated with the relative expression levels of the target proteins. Identifying physical characteristics that correlate with protein expression might aid in selecting the "low hanging fruit", or proteins that can be expressed to sufficient levels using an E. coli expression system. The use of other expression strategies or host species might be needed for sufficient levels of expression of transmembrane proteins with other physical

  20. Inactivation of indispensable bacterial proteins by early proteins of bacteriophages: implication in antibacterial drug discovery.

    Science.gov (United States)

    Sau, S; Chattoraj, P; Ganguly, T; Chanda, P K; Mandal, N C

    2008-06-01

    Bacteriophages utilize host bacterial cellular machineries for their own reproduction and completion of life cycles. The early proteins that phage synthesize immediately after the entry of their genomes into bacterial cells participate in inhibiting host macromolecular biosynthesis, initiating phage-specific replication and synthesizing late proteins. Inhibition of synthesis of host macromolecules that eventually leads to cell death is generally performed by the physical and/or chemical modification of indispensable host proteins by early proteins. Interestingly, most modified bacterial proteins were shown to take part actively in phage-specific transcription and replication. Research on phages in last nine decades has demonstrated such lethal early proteins that interact with or chemically modify indispensable host proteins. Among the host proteins inhibited by lethal phage proteins, several are not inhibited by any chemical inhibitor available today. Under the context of widespread dissemination of antibiotic-resistant strains of pathogenic bacteria in recent years, the information of lethal phage proteins and cognate host proteins could be extremely invaluable as they may lead to the identification of novel antibacterial compounds. In this review, we summarize the current knowledge about some early phage proteins, their cognate host proteins and their mechanism of action and also describe how the above interacting proteins had been exploited in antibacterial drug discovery. PMID:18537683

  1. Bacterial protein meal in diets for pigs and minks

    DEFF Research Database (Denmark)

    Hellwing, Anne Louise Frydendahl; Tauson, Anne-Helene; Skrede, Anders;

    2007-01-01

    The effect of increasing the dietary content of bacterial protein meal (BPM) on protein turnover rate, and on nucleic acid and creatinine metabolism in growing minks and pigs was investigated in two experiments. In each experiment, 16 animals were allocated to four experimental diets. The diets...... containing no BPM served as controls, i.e. for minks diet M1, for pigs P1; the experimental diets contained increasing levels of BPM to replace fish meal (minks) or soybean meal (pigs), so that up to 17% (P2), 20% (M2), 35% (P3), 40% (M3), 52% (P4), and 60% (M4) of digestible N was BPM derived. Protein...... turnover rate was measured by means of the end-product method using [15N]glycine as tracer and urinary nitrogen as end-product. In minks, protein flux, synthesis, and breakdown increased significantly with increasing dietary BPM. In pigs, diet had no observed effect on protein turnover rate. The intake...

  2. Function, structure, and mechanism in bacterial photosensory LOV proteins

    OpenAIRE

    Herrou, Julien; Crosson, Sean

    2011-01-01

    LOV domains are protein photosensors conserved in bacteria, archaea, plants and fungi that detect blue light via a flavin cofactor. In the bacterial kingdom, LOV domains are present in both chemotrophic and phototrophic species, where they are found N-terminally of signaling and regulatory domains such as sensor histidine kinases, diguanylate cyclases/phosphodiesterases, DNA-binding domains, and σ factor regulators. In this review, we describe the current state of knowledge on the function of...

  3. Methods of producing protoporphyrin IX and bacterial mutants therefor

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Jizhong; Qiu, Dongru; He, Zhili; Xie, Ming

    2016-03-01

    The presently disclosed inventive concepts are directed in certain embodiments to a method of producing protoporphyrin IX by (1) cultivating a strain of Shewanella bacteria in a culture medium under conditions suitable for growth thereof, and (2) recovering the protoporphyrin IX from the culture medium. The strain of Shewanella bacteria comprises at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX. In certain embodiments of the method, the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or of shew_1140. In other embodiments, the presently disclosed inventive concepts are directed to mutant strains of Shewanella bacteria having at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX during cultivation of the bacteria. In certain embodiments the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or shew_1140.

  4. Biodegradable films produced from the bacterial polysaccharide FucoPol.

    Science.gov (United States)

    Ferreira, Ana R V; Torres, Cristiana A V; Freitas, Filomena; Reis, Maria A M; Alves, Vítor D; Coelhoso, Isabel M

    2014-11-01

    FucoPol, an exopolysaccharide produced by Enterobacter A47, grown in bioreactor with glycerol as carbon source, was used with citric acid to obtain biodegradable films by casting. The films were characterized in terms of optical, hygroscopic, mechanical and barrier properties. These films have shown to be transparent, but with a brown tone, imparting small colour changes when applied over coloured surfaces. They were hydrophilic, with high permeability to water vapour (1.01×10(-11)mol/msPa), but presented good barrier properties to oxygen and carbon dioxide (0.7×10(-16)molm/m(2)sPa and 42.7×10(-16)molm/m(2)sPa, respectively). Furthermore, films have shown mechanical properties under tensile tests characteristic of ductile films with high elongation at break, low tension at break and low elastic modulus. Although the obtained results are promising, films properties can be improved, namely by testing alternative plasticizers, crosslinking agents and blends with other biopolymers. Taking into account the observed ductile mechanical properties, good barrier properties to gases when low water content is used and their hydrophilic character, it is foreseen a good potential for FucoPol films to be incorporated as inner layer of a multilayer packaging material. PMID:24769364

  5. Slow Onset Inhibition of Bacterial β-Ketoacyl-acyl Carrier Protein Synthases by Thiolactomycin*

    OpenAIRE

    Machutta, Carl A.; Bommineni, Gopal R.; Luckner, Sylvia R.; Kapilashrami, Kanishk; Ruzsicska, Bela; Simmerling, Carlos; Kisker, Caroline; Tonge, Peter J.

    2009-01-01

    Thiolactomycin (TLM), a natural product thiolactone antibiotic produced by species of Nocardia and Streptomyces, is an inhibitor of the β-ketoacyl-acyl carrier protein synthase (KAS) enzymes in the bacterial fatty acid synthase pathway. Using enzyme kinetics and direct binding studies, TLM has been shown to bind preferentially to the acyl-enzyme intermediates of the KASI and KASII enzymes from Mycobacterium tuberculosis and Escherichia coli. These studies, which utilized acyl-enzyme mimics in...

  6. Chemiluminescence enzyme immunoassay using ProteinA-bacterial magnetite complex

    Science.gov (United States)

    Matsunaga, Tadashi; Sato, Rika; Kamiya, Shinji; Tanaka, Tsuyosi; Takeyama, Haruko

    1999-04-01

    Bacterial magnetic particles (BMPs) which have ProteinA expressed on their surface were constructed using magA which is a key gene in BMP biosynthesis in the magnetic bacterium Magnetospirillum sp. AMB-1. Homogenous chemiluminescence enzyme immunoassay using antibody bound ProteinA-BMP complexes was developed for detection of human IgG. A good correlation between the luminescence yield and the concentration of human IgG was obtained in the range of 1-10 3 ng/ml.

  7. Near-infrared fluorescent proteins engineered from bacterial phytochromes.

    Science.gov (United States)

    Shcherbakova, Daria M; Baloban, Mikhail; Verkhusha, Vladislav V

    2015-08-01

    Near-infrared fluorescent proteins (NIR FPs), photoactivatable NIR FPs and NIR reporters of protein-protein interactions developed from bacterial phytochrome photoreceptors (BphPs) have advanced non-invasive deep-tissue imaging. Here we provide a brief guide to the BphP-derived NIR probes with an emphasis on their in vivo applications. We describe phenotypes of NIR FPs and their photochemical and intracellular properties. We discuss NIR FP applications for imaging of various cell types, tissues and animal models in basic and translational research. In this discussion, we focus on NIR FPs that efficiently incorporate endogenous biliverdin chromophore and therefore can be used as straightforward as GFP-like proteins. We also overview a usage of NIR FPs in different imaging platforms, from planar epifluorescence to tomographic and photoacoustic technologies. PMID:26115447

  8. Bacterial Growth on Photochemically Transformed Leachates from Aquatic and Terrestrial Primary Producers

    DEFF Research Database (Denmark)

    Anesio, A.M.; Nielsen, Jon Theil; Granéli, W.

    2000-01-01

    We measured bacterial growth on phototransformed dissolved organic matter (DOM) leached from eight different primary producers. Leachates (10 mg C liter-1) were exposed to artificial UVA + UVB radiation, or kept in darkness, for 20 h. DOM solutions were subsequently inoculated with lake water...... leachate and type of bacterial growth criterion. Bacterial carbon utilization (biomass production plus respiration) over the entire incubation period (120 h) was enhanced by UV radiation of leachate from the terrestrial leaves, relative to carbon utilization in non-irradiated leachates. Conversely, carbon...... utilization was reduced by radiation of the leachates from aquatic macrophytes. In a separate experiment, the stable C and N isotope composition of bacteria grown on irradiated and non-irradiated DOM was estimated. Bacterial growth on UV-irradiated DOM was enriched in 13C relative to the bacteria in the non...

  9. Bacterial Protein Synthesis as a Target for Antibiotic Inhibition.

    Science.gov (United States)

    Arenz, Stefan; Wilson, Daniel N

    2016-01-01

    Protein synthesis occurs on macromolecular machines, called ribosomes. Bacterial ribosomes and the translational machinery represent one of the major targets for antibiotics in the cell. Therefore, structural and biochemical investigations into ribosome-targeting antibiotics provide not only insight into the mechanism of action and resistance of antibiotics, but also insight into the fundamental process of protein synthesis. This review summarizes the recent advances in our understanding of protein synthesis, particularly with respect to X-ray and cryoelectron microscopy (cryo-EM) structures of ribosome complexes, and highlights the different steps of translation that are targeted by the diverse array of known antibiotics. Such findings will be important for the ongoing development of novel and improved antimicrobial agents to combat the rapid emergence of multidrug resistant pathogenic bacteria. PMID:27481773

  10. Bacterial Canker (Clavibacter michiganensis subsp. michiganensis) of tomato in commercial seed produced in Indonesia

    NARCIS (Netherlands)

    Anwar, A.; Zouwen, van der P.S.; Ilyas, S.; Wolf, van der J.M.

    2004-01-01

    In 2002, Clavibacter michiganensis subsp. michiganensis (Smith) Davis, the causal organism of bacterial canker of tomato (Lycopersicon esculentum), was isolated from two of six commercial asymptomatic tomato seed lots produced on Java in Indonesia. C. michiganensis subsp. michiganensis has not been

  11. Fusion proteins useful for producing pinene

    Energy Technology Data Exchange (ETDEWEB)

    Peralta-Yahya, Pamela P.; Keasling, Jay D

    2016-06-28

    The present invention provides for a modified host cell comprising a heterologous pinene synthase (PS), or enzymatically active fragment or variant thereof, and optionally a geranyl pyrophosphate synthase (GPPS), or enzymatically active fragment or variant thereof, or a fusion protein comprising: (a) a PS and (b) a GPPS linked by a linker.

  12. Bacterial protein meal in diets for growing pigs

    DEFF Research Database (Denmark)

    Hellwing, Anne Louise Frydendahl; Tauson, Anne-Helene; Kjos, N.P.;

    2007-01-01

    This experiment investigated the effects of increasing the dietary content of bacterial protein meal (BPM) on the protein and energy metabolism of pigs from weaning to a live weight of 80 kg. FOur litters with four castrated male pigs in each litter were used. The litters were divided into two...... blocks according to age. One pig from each litter was fed one of the four experimental diets. Soya-bean meal was replaced with BPM on the basis of digestible protein, and the BPM contents in the four diets were 0% (BP0), 5% (BP5), 10% (BP10) and 15% (BP15), corresponding to 0%, 17%, 35% and 52...... by inclusion level of BPM. Retention of energy was 620 (BP0), 696 (BP5), 613 (BP10) and 664 kJ/kg0.75 per day (BP15), the differences among diets being non-significant. The N-free respiratory quotient was similar on all diets. It was concluded that the overall protein and energy metabolism in growing pigs were...

  13. Holo- And Apo- Structures of Bacterial Periplasmic Heme Binding Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ho, W.W.; Li, H.; Eakanunkul, S.; Tong, Y.; Wilks, A.; Guo, M.; Poulos, T.L.

    2009-06-01

    An essential component of heme transport in Gram-negative bacterial pathogens is the periplasmic protein that shuttles heme between outer and inner membranes. We have solved the first crystal structures of two such proteins, ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa. Both share a common architecture typical of Class III periplasmic binding proteins. The heme binds in a narrow cleft between the N- and C-terminal binding domains and is coordinated by a Tyr residue. A comparison of the heme-free (apo) and -bound (holo) structures indicates little change in structure other than minor alterations in the heme pocket and movement of the Tyr heme ligand from an 'in' position where it can coordinate the heme iron to an 'out' orientation where it points away from the heme pocket. The detailed architecture of the heme pocket is quite different in ShuT and PhuT. Although Arg{sup 228} in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg{sup 228}, and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B{sub 12}-binding protein BtuF and the hydroxamic-type siderophore-binding protein FhuD, the only two other structurally characterized Class III periplasmic binding proteins, demonstrates that PhuT/ShuT more closely resembles BtuF, which reflects the closer similarity in ligands, heme and B{sub 12}, compared with ligands for FhuD, a peptide siderophore.

  14. Bacterial Hydrolysis of Protein and Methylated Protein and Its Implications for Studies of Protein Degradation in Aquatic Systems

    OpenAIRE

    Keil, Richard G.; Kirchman, David L.

    1992-01-01

    Ribulose 1,5-bisphosphate carboxylase was radiolabelled by in vitro translation, resulting in uniformly labelled ribulose 1,5-bisphosphate carboxylase, and also by reductive methylation. We investigated the degradation of the two forms of radiolabelled protein by natural bacterial populations. Although total hydrolysis of uniformly labelled protein and methylated protein was nearly equal, percent assimilation, respiration, and release as low-molecular-weight material were different. Radioacti...

  15. Antibacterial synergy of curcumin with antibiotics against biofilm producing clinical bacterial isolates

    Science.gov (United States)

    Kali, Arunava; Bhuvaneshwar, Devaraj; Charles, Pravin M. V.; Seetha, Kunigal Srinivasaiah

    2016-01-01

    Introduction: The role of natural bioactive substances in treating infections has been rediscovered as bacterial resistance become common to most of the antibiotics. Curcumin is a bioactive substance from turmeric. Owing to antimicrobial properties, its prospect as an antibacterial agent is currently under focus. Materials and Methods: We have evaluated the in vitro synergy of curcumin with antibiotics against sixty biofilm producing bacterial isolates. Congo red agar method was used to identify the biofilm producing isolates. Curcumin minimum inhibitory concentration (MIC) was determined by agar dilution method. Its antibiotic synergy was identified by the increase in disc diffusion zone size on Mueller-Hinton agar with 32 mg/L curcumin. Results: The mean MICs of curcumin against Gram-positive and Gram-negative isolates were 126.9 mg/L and 117.4 mg/L, respectively. Maximum synergy was observed with ciprofloxacin among Gram-positive and amikacin, gentamicin, and cefepime among Gram-negative isolates. Conclusions: Curcumin per se as well as in combination with other antibiotics has a demonstrable antibacterial action against biofilm producing bacterial isolates. It may have a beneficial role in supplementing antibiotic therapy. PMID:27330262

  16. Insect Larvae: A New Platform to Produce Commercial Recombinant Proteins.

    Science.gov (United States)

    Targovnik, Alexandra M; Arregui, Mariana B; Bracco, Lautaro F; Urtasun, Nicolas; Baieli, Maria F; Segura, Maria M; Simonella, Maria A; Fogar, Mariela; Wolman, Federico J; Cascone, Osvaldo; Miranda, Maria V

    2016-01-01

    In Biotechnology, the expression of recombinant proteins is a constantly growing field and different hosts are used for this purpose. Some valuable proteins cannot be produced using traditional systems. Insects from the order Lepidoptera infected with recombinant baculovirus have appeared as a good choice to express high levels of proteins, especially those with post-translational modifications. Lepidopteran insects, which are extensively distributed in the world, can be used as small protein factories, the new biofactories. Species like Bombyx mori (silkworm) have been analyzed in Asian countries to produce a great number of recombinant proteins for use in basic and applied science and industry. Many proteins expressed in this larva have been commercialized. Several recombinant proteins produced in silkworms have already been commercialized. On the other hand, species like Spodoptera frugiperda, Heliothis virescens, Rachiplusia nu, Helicoverpa zea and Trichoplusia ni are widely distributed in both the occidental world and Europe. The expression of recombinant proteins in larvae has the advantage of its low cost in comparison with insect cell cultures. A wide variety of recombinant proteins, including enzymes, hormones and vaccines, have been efficiently expressed with intact biological activity. The expression of pharmaceutically proteins, using insect larvae or cocoons, has become very attractive. This review describes the use of insect larvae as an alternative to produce commercial recombinant proteins. PMID:26956108

  17. Low protein diets produce divergent effects on energy balance

    Science.gov (United States)

    Pezeshki, Adel; Zapata, Rizaldy C.; Singh, Arashdeep; Yee, Nicholas J.; Chelikani, Prasanth K.

    2016-01-01

    Diets deficient in protein often increase food consumption, body weight and fat mass; however, the underlying mechanisms remain poorly understood. We compared the effects of diets varying in protein concentrations on energy balance in obesity-prone rats. We demonstrate that protein-free (0% protein calories) diets decreased energy intake and increased energy expenditure, very low protein (5% protein) diets increased energy intake and expenditure, whereas moderately low protein (10% protein) diets increased energy intake without altering expenditure, relative to control diet (15% protein). These diet-induced alterations in energy expenditure are in part mediated through enhanced serotonergic and β-adrenergic signaling coupled with upregulation of key thermogenic markers in brown fat and skeletal muscle. The protein-free and very low protein diets decreased plasma concentrations of multiple essential amino acids, anorexigenic and metabolic hormones, but these diets increased the tissue expression and plasma concentrations of fibroblast growth factor-21. Protein-free and very low protein diets induced fatty liver, reduced energy digestibility, and decreased lean mass and body weight that persisted beyond the restriction period. In contrast, moderately low protein diets promoted gain in body weight and adiposity following the period of protein restriction. Together, our findings demonstrate that low protein diets produce divergent effects on energy balance. PMID:27122299

  18. Low protein diets produce divergent effects on energy balance.

    Science.gov (United States)

    Pezeshki, Adel; Zapata, Rizaldy C; Singh, Arashdeep; Yee, Nicholas J; Chelikani, Prasanth K

    2016-01-01

    Diets deficient in protein often increase food consumption, body weight and fat mass; however, the underlying mechanisms remain poorly understood. We compared the effects of diets varying in protein concentrations on energy balance in obesity-prone rats. We demonstrate that protein-free (0% protein calories) diets decreased energy intake and increased energy expenditure, very low protein (5% protein) diets increased energy intake and expenditure, whereas moderately low protein (10% protein) diets increased energy intake without altering expenditure, relative to control diet (15% protein). These diet-induced alterations in energy expenditure are in part mediated through enhanced serotonergic and β-adrenergic signaling coupled with upregulation of key thermogenic markers in brown fat and skeletal muscle. The protein-free and very low protein diets decreased plasma concentrations of multiple essential amino acids, anorexigenic and metabolic hormones, but these diets increased the tissue expression and plasma concentrations of fibroblast growth factor-21. Protein-free and very low protein diets induced fatty liver, reduced energy digestibility, and decreased lean mass and body weight that persisted beyond the restriction period. In contrast, moderately low protein diets promoted gain in body weight and adiposity following the period of protein restriction. Together, our findings demonstrate that low protein diets produce divergent effects on energy balance. PMID:27122299

  19. Ribosome reinitiation at leader peptides increases translation of bacterial proteins.

    Science.gov (United States)

    Korolev, Semen A; Zverkov, Oleg A; Seliverstov, Alexandr V; Lyubetsky, Vassily A

    2016-01-01

    Short leader genes usually do not encode stable proteins, although their importance in expression control of bacterial genomes is widely accepted. Such genes are often involved in the control of attenuation regulation. However, the abundance of leader genes suggests that their role in bacteria is not limited to regulation. Specifically, we hypothesize that leader genes increase the expression of protein-coding (structural) genes via ribosome reinitiation at the leader peptide in the case of a short distance between the stop codon of the leader gene and the start codon of the structural gene. For instance, in Actinobacteria, the frequency of leader genes at a distance of 10-11 bp is about 70 % higher than the mean frequency within the 1 to 65 bp range; and it gradually decreases as the range grows longer. A pronounced peak of this frequency-distance relationship is also observed in Proteobacteria, Bacteroidetes, Spirochaetales, Acidobacteria, the Deinococcus-Thermus group, and Planctomycetes. In contrast, this peak falls to the distance of 15-16 bp and is not very pronounced in Firmicutes; and no such peak is observed in cyanobacteria and tenericutes. Generally, this peak is typical for many bacteria. Some leader genes located close to a structural gene probably play a regulatory role as well. PMID:27084079

  20. Primordial soup was edible: abiotically produced Miller-Urey mixture supports bacterial growth.

    Science.gov (United States)

    Xie, Xueshu; Backman, Daniel; Lebedev, Albert T; Artaev, Viatcheslav B; Jiang, Liying; Ilag, Leopold L; Zubarev, Roman A

    2015-01-01

    Sixty years after the seminal Miller-Urey experiment that abiotically produced a mixture of racemized amino acids, we provide a definite proof that this primordial soup, when properly cooked, was edible for primitive organisms. Direct admixture of even small amounts of Miller-Urey mixture strongly inhibits E. coli bacteria growth due to the toxicity of abundant components, such as cyanides. However, these toxic compounds are both volatile and extremely reactive, while bacteria are highly capable of adaptation. Consequently, after bacterial adaptation to a mixture of the two most abundant abiotic amino acids, glycine and racemized alanine, dried and reconstituted MU soup was found to support bacterial growth and even accelerate it compared to a simple mixture of the two amino acids. Therefore, primordial Miller-Urey soup was perfectly suitable as a growth media for early life forms. PMID:26412575

  1. Surface Proteins of Streptococcus agalactiae and Related Proteins in Other Bacterial Pathogens.

    OpenAIRE

    Lindahl, Gunnar; Stålhammar-Carlemalm, Margaretha; Areschoug, Thomas

    2005-01-01

    Streptococcus agalactiae (group B Streptococcus) is the major cause of invasive bacterial disease, including meningitis, in the neonatal period. Although prophylactic measures have contributed to a substantial reduction in the number of infections, development of a vaccine remains an important goal. While much work in this field has focused on the S. agalactiae polysaccharide capsule, which is an important virulence factor that elicits protective immunity, surface proteins have received incre...

  2. Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain

    OpenAIRE

    Florea, Michael; Hagemann, Henrik; Santosa, Gabriella; Abbott, James; Micklem, Chris N.; Spencer-Milnes, Xenia; de Arroyo Garcia, Laura; Paschou, Despoina; Lazenbatt, Christopher; Kong, Deze; Chughtai, Haroon; Jensen, Kirsten; Freemont, Paul S.; Kitney, Richard; Reeve, Benjamin

    2016-01-01

    Bacterial cellulose is a remarkable material that is malleable, biocompatible, and over 10-times stronger than plant-based cellulose. It is currently used to create materials for tissue engineering, medicine, defense, electronics, acoustics, and fabrics. We describe here a bacterial strain that is readily amenable to genetic engineering and produces high quantities of bacterial cellulose in low-cost media. To reprogram this organism for biotechnology applications, we created a set of genetic ...

  3. Genome sequence and plasmid transformation of the model high-yield bacterial cellulose producer Gluconacetobacter hansenii ATCC 53582

    OpenAIRE

    Michael Florea; Benjamin Reeve; James Abbott; Freemont, Paul S.; Tom Ellis

    2016-01-01

    Bacterial cellulose is a strong, highly pure form of cellulose that is used in a range of applications in industry, consumer goods and medicine. Gluconacetobacter hansenii ATCC 53582 is one of the highest reported bacterial cellulose producing strains and has been used as a model organism in numerous studies of bacterial cellulose production and studies aiming to increased cellulose productivity. Here we present a high-quality draft genome sequence for G. hansenii ATCC 53582 and find that in ...

  4. Effect of bacterial protein meal on protein and energy metabolism in growing chickens

    DEFF Research Database (Denmark)

    Hellwing, Anne Louise Frydendahl; Tauson, Anne-Helene; Skrede, Anders

    2006-01-01

    This experiment investigates the effect of increasing the dietary content of bacterial protein meal (BPM) on the protein and energy metabolism, and carcass chemical composition of growing chickens. Seventy-two Ross male chickens were allocated to four diets, each in three replicates with 0% (D0), 2......% (D2), 4% D4), and 6% BPM (D6), BPM providing up to 20% of total dietary N. Five balance experiments were conducted when the chickens were 3-7, 10-14, 17-21, 23-27, and 30-34 days old. During the same periods, 22-h respiration experiments (indirect calorimetry) were performed with troups of 6 chickens...... for protein and energy retention found in the balance and respiration experiments. It was concluded that the overall protein and energy metabolism as well as carcass composition were not influenced by a dietary content of up to 6% BPM corresponding to 20% of dietary N....

  5. Effect of Organic Acids on Bacterial Cellulose Produced by Acetobacter xylinum

    Directory of Open Access Journals (Sweden)

    Hongmei Lu

    2016-03-01

    Full Text Available Based on the difference of bacterial cellulose production from rice saccharificate medium and chemical medium under static cultivation, effect of organic acids in the process of bacterial cellulose produced by A. xylinum was studied. The results showed that the kinds and contents of organic acids were different in both culture medium, in which accumulated oxalic acid and tartaric acid inhibited A. xylinum producing BC in chemical medium, while pyruvic acid, malic acid, lactic acid, acetic acid, citric acid and succinic acid, as ethanol, promoted A. xylinum to produce BC. Compared to the blank BC production 1.48 g/L, the optimum addition concentrations of pyruvic acid, malic acid, lactic acid, acetic acid, citric acid, succinic acid, and ethanol in chemical medium were 0.15%, 0.1%, 0.3%, 0.4%, 0.1%, 0.2% , 4% and the BC productions were 2.49 g/L, 2.83 g/L, 2.12 g/L, 2.54 g/L, 2.27 g/L, 1.88 g/L , 2.63 g/L, respectively. The co-existence of above organic acids and ethanol increased BC production even further.

  6. Mechanism of Excretion of a Bacterial Proteinase: Demonstration of Two Proteolytic Enzymes Produced by a Sarcina Strain (Coccus P)

    Energy Technology Data Exchange (ETDEWEB)

    SARNER, NITZA Z; BISSELL, MINA J; GIROLAMO, MARIO Di; GORINI, LUIGI

    1970-06-29

    A Sarcina strain (Coccus P) produces two proteolytic enzymes. One is found only extracellularly, is far more prevalent, and is actively excreted during exponential growth. It is the enzyme responsible for the known strong proteolytic activity of the cultures of this strain. A second protease is, however, produced which remains associated with the intact cells but is released by the protoplasts. The two enzymes appear unrelated in their derivation. Calcium ions play an essential role in preventing autodigestion of the excreted enzyme. Bacterial proteins are found outside the cell boundary as a consequence either of passive processes such as leakage or lysis or of active excretion. Under conditions in which leakage and lysis do not occur, as during exponential growth, the cell boundary is a barrier causing a complete separation of the bulk of the intracellular proteins from the one or very few extracellular proteins, with no trace of either type being detectable on the wrong side of the boundary. Since in bacteria there is no evidence of protein being produced other than internally, the separation into intraand extracellular proteins should occur after peptide chain formation. The question arises as to whether the structure of the cell boundary or that of the excreted proteins themselves determines this separation. Coccus P, a Sarcina closely related to Micrococcus lysodeikticus (3), produces an extracellular proteinase during the exponential phase of growth so that the process appears to be active excretion. The organism grows exponentially in a defined synthetic medium (12) to relatively high cell density (10{sup 9} cells/ml); therefore the mechanism of excretion can be studied over an extended period of time without the difficulties of changing growth rates. Coagulation of reconstituted skim milk provides a simple and sensitive assay for enzyme activity (I 1). The extracellular proteinase has also been purified and partially characterized (6-8). It has been shown

  7. Nutritional and fermentation parameters of Xaraés grass silage produced with bacterial additive

    Directory of Open Access Journals (Sweden)

    Erickson Tiago Pinheiro da Silva

    2014-07-01

    Full Text Available The use of bacterial additives in forage silages with low content of dry matter prevents undesirable fermentation and reduces losses by gases (PG and effluents (PE during the ensiling process. This study aimed to evaluate the fermentation parameters, chemical composition and in vitro digestibility of silage of Urochloa brizantha cv. Xaraés produced with bacterial additive. The inoculant contained the following strains: Propionibacterium acidipropionici + Lactobacillus plantarum; Lactobacillus buchnari; Propionibacterium acipropionici + commercial enzymes and Lactobacillus plantarum and Pediococcus pentosaccus, at 0, 25, 50, 75, 100, and 125% of the recommended level for sugarcane (2 g ton-1. The experiment was a completely randomized design with four replications, and six levels of inoculant (0, 25, 50, 75, 100, and 125%. There was a quadratic relationship between the inoculant addition and the levels of pH, PE, DMIVD, NDF, ADF and LIG of the silage. PG and MM increased linearly with the addition of inoculant. The N-NH3, DM, CP, CEL, HEM and EE were not affected by the inoculant. Bacterial additive at 50% provided increased DMIVD. Appropriate values were found for pH and NH3.

  8. Effects of bacterial action on waste rock producing acid drainage in the Brazilian first uranium mine

    International Nuclear Information System (INIS)

    This work is an evolution of the methodology showed in the paper 'Study of waste of waste rock piles producing acid drainage in the Brazilian first uranium mine', also submitted for INAC2009. Therefore, the present work also related to the determination of chemical species leaching from waste rock pile 4 (WRP4) of the Uranium Mine and Milling Facility located in the Pocos de Caldas Plateau, as well as the generation of acid waters. With the previous experimental setup, it has been observed that not only water and available oxygen are significant to pyrite oxidation reaction, but bacterial activity as well. As a first approach, the present work addresses the same experiment, but now testing without the influence of bacterial action. Therefore, the new methodology and experimental setup is now capable of determining the acidity of water in contact with material from the WRP4 and the concentration of chemical species dissolved as function of time. Such would also show the extent of bacterial action interference on the pyrite oxidation reaction. Results are based on mass balances comparing concentrations of chemical species in the waste rock before the experiment and in the waste rock plus the remaining water after the experiment. In addition, the evolution of the pH and EMF (electromotive force) values along with chemical species quantified through the experiment are presented through graphics. That is followed by discussions on the significance of such results in terms of concentration of the involved chemical species. The present work has also shown the need of improving the injection of air into the system. A more sophisticated experimental setup should be assembled in the near future, which would allow the quantification of differences between experimental tests with and without bacterial action. (author)

  9. Specific interaction between adenoviral 55-kDa E1B protein and in vivo produced p53 fusion proteins.

    Science.gov (United States)

    Chumakov, A; Koeffler, H P

    1993-09-15

    Several protein fusion systems have been used in recent years to study protein-protein and DNA-protein interactions. Most of them use bacterially produced proteins which have several inherent disadvantages, notably, the absence of correct post-translational modifications and the frequent insolubility of recombinant proteins. We sought to develop a system to study proteins interacting with the nuclear phosphoprotein p53, which is believed to be a tumor suppressor. To prepare fusions of p53, we developed a convenient system that permits both in vivo and in vitro production and easy affinity purification of peptides and protein fragments as glutathione-transferase fusions. We placed the coding sequence of the Schistosoma japonica glutathione S-transferase (GST) under the control of the strong CMV/T7 promoter and SV40 splice and polyadenylation signals. An extensive polylinker (MCS) at the 3' end of the GST gene is preceded by the sequence encoding the cleavage site of the site-specific protease. We cloned the complete coding sequences of human wild-type p53, as well as p53 mutants representing all four mutational hotspots (codons 141, 175, 248, and 273), into our expression vector. In vitro transcription using the upstream T7 promoter and translation in reticulocyte lysates form an easy way to produce hybrid proteins; affinity purification on a glutathione-agarose column removes proteins that are present in reticulocyte lysates. We have also studied specific in vivo interactions of human p53 with the adenoviral 55-kDa E1B protein by transfecting expression constructs of GST-p53 fusions into human Ad5-transformed 293 cells. PMID:8406015

  10. Surface modification of polyester to produce a bacterial cellulose-based vascular prosthetic device

    Science.gov (United States)

    Charpentier, Paul A.; Maguire, Anne; Wan, Wan-kei

    2006-07-01

    The surface of medical grade polyesters was modified to impart hydrophilic character for attachment to bacterial synthesized cellulose to produce a vascular prosthetic device. The polyesters were treated with UV/ozone, air plasma, and nitrogen plasma for various lengths of time. The unmodified and modified surfaces were analyzed by X-ray photoelectron spectroscopy (XPS) and advancing contact angle measurements. The surfaces were then coated with bacterial produced cellulose to study adhesion properties through tensile testing (peel testing). UV/ozone and plasma treatment XPS results indicated an increase in the oxygen concentration in the form of C sbnd O(H) on the treated polyester surfaces. The treatment time to reach steady state in the case of air and nitrogen plasmas took the order of seconds, while 7 min and longer were required for UV/ozone treatment. Peel strength tests to measure adhesion of modified polyester to cellulose reached their maximum values when the C sbnd O(H) concentrations were at the highest level. It was also at this level that the contact angle measurements showed no further decrease.

  11. Identification and Characterization of Inhibitors of Bacterial Enoyl-Acyl Carrier Protein Reductase

    OpenAIRE

    Ling, Losee L.; Xian, Jun; Ali, Syed; Geng, Bolin; Fan, Jun; Mills, Debra M.; Arvanites, Anthony C.; Orgueira, Hernan; Ashwell, Mark A.; Carmel, Gilles; Xiang, Yibin; Moir, Donald T.

    2004-01-01

    Bacterial enoyl-acyl carrier protein reductase (ENR) catalyzes an essential step in fatty acid biosynthesis. ENR is an attractive target for narrow-spectrum antibacterial drug discovery because of its essential role in metabolism and its sequence conservation across many bacterial species. In addition, the bacterial ENR sequence and structural organization are distinctly different from those of mammalian fatty acid biosynthesis enzymes. High-throughput screening to identify inhibitors of Esch...

  12. Isolation and identification of bacterial endophytes from pharmaceutical agarwood-producing Aquilaria species

    Directory of Open Access Journals (Sweden)

    Subhash J Bhore

    2013-01-01

    Full Text Available Background: Resins and gums are used in traditional medicine and do have potential applications in pharmacy and medicine. Agarwood is the fragrant resinous wood, which is an important commodity from Aquilaria species and has been used as a sedative, analgesic, and digestive in traditional medicine. Endophytic bacteria are potentially important in producing pharmaceutical compounds found in the plants. Hence, it was important to understand which types of endophytic bacteria are associated with pharmaceutical agarwood-producing Aquilaria species. Objective: This study was undertaken to isolate and identify endophytic bacteria associated with agarwood-producing seven (7 Aquilaria species from Malaysia. Materials and Methods: Botanical samples of seven Aquilaria species were collected, and endophytic bacteria were isolated from surface-sterilized-tissue samples. The 16S rRNA gene fragments were amplified using PCR method, and endophytic bacterial isolates (EBIs were identified based on 16S rRNA gene sequence similarity based method. Results: Culturable, 77 EBIs were analyzed, and results of 16S rRNA gene sequences analysis suggest that 18 different types of endophytic bacteria are associated with (seven Aquilaria species. From 77 EBIs, majority (36.4% of the isolates were of Bacillus pumilus. Conclusion: These findings indicate that agarwood-producing Aquilaria species are harboring 18 different types of culturable endophytic bacteria.

  13. Ability of sea-water bacterial consortium to produce electricity and denitrify water

    Science.gov (United States)

    Maruvada, Nagasamrat V. V.; Tommasi, Tonia; Kaza, Kesava Rao; Ruggeri, Bernardo

    Sea is a store house for varied types of microbes with an ability to reduce and oxidize substances like iron, sulphur, carbon dioxide, etc. Most of these processes happen in the sea water environment, but can be applied for purification of wastewater. In the present paper, we discuss the use of a consortium of seawater bacteria in a fuel cell to produce electricity by oxidizing organic matter and reducing nitrates. We also discuss how the growth of the bacterial consortium can lead to an increased electricity production and decreased diffusional resistance in the cell. The analysis was done using electrochemical impedance spectroscopy (EIS), and linear sweep voltammetry (LSV). Here, we use bicarbonate buffered solution, which is the natural buffering agent found in sea. We show that the seawater bacterial consortium can be used in both the anode and cathode parts of the cell. The results confirm the adaptability of the seawater bacteria to different environments and can be used for various applications. Heritage, Erasmus Mundus Programme, European Commission.

  14. Dengue-2 Structural Proteins Associate with Human Proteins to Produce a Coagulation and Innate Immune Response Biased Interactome

    Directory of Open Access Journals (Sweden)

    Soares Luis RB

    2011-01-01

    Full Text Available Abstract Background Dengue virus infection is a public health threat to hundreds of millions of individuals in the tropical regions of the globe. Although Dengue infection usually manifests itself in its mildest, though often debilitating clinical form, dengue fever, life-threatening complications commonly arise in the form of hemorrhagic shock and encephalitis. The etiological basis for the virus-induced pathology in general, and the different clinical manifestations in particular, are not well understood. We reasoned that a detailed knowledge of the global biological processes affected by virus entry into a cell might help shed new light on this long-standing problem. Methods A bacterial two-hybrid screen using DENV2 structural proteins as bait was performed, and the results were used to feed a manually curated, global dengue-human protein interaction network. Gene ontology and pathway enrichment, along with network topology and microarray meta-analysis, were used to generate hypothesis regarding dengue disease biology. Results Combining bioinformatic tools with two-hybrid technology, we screened human cDNA libraries to catalogue proteins physically interacting with the DENV2 virus structural proteins, Env, cap and PrM. We identified 31 interacting human proteins representing distinct biological processes that are closely related to the major clinical diagnostic feature of dengue infection: haemostatic imbalance. In addition, we found dengue-binding human proteins involved with additional key aspects, previously described as fundamental for virus entry into cells and the innate immune response to infection. Construction of a DENV2-human global protein interaction network revealed interesting biological properties suggested by simple network topology analysis. Conclusions Our experimental strategy revealed that dengue structural proteins interact with human protein targets involved in the maintenance of blood coagulation and innate anti

  15. Transcriptional abundance is not the single force driving the evolution of bacterial proteins

    OpenAIRE

    Wei, Wen; Zhang, Tao; Lin, Dan; Yang, Zu-Jun; Guo, Feng-Biao

    2013-01-01

    Background Despite rapid progress in understanding the mechanisms that shape the evolution of proteins, the relative importance of various factors remain to be elucidated. In this study, we have assessed the effects of 16 different biological features on the evolutionary rates (ERs) of protein-coding sequences in bacterial genomes. Results Our analysis of 18 bacterial species revealed new correlations between ERs and constraining factors. Previous studies have suggested that transcriptional a...

  16. Screening of bacterial strains for pectinolytic activity: characterization of the polygalacturonase produced by Bacillus sp

    Directory of Open Access Journals (Sweden)

    Soares Márcia M.C.N.

    1999-01-01

    Full Text Available One hundred sixty eight bacterial strains, isolated from soil and samples of vegetable in decomposition, were screened for the use of citrus pectin as the sole carbon source. 102 were positive for pectinase depolymerization in assay plates as evidenced by clear hydrolization halos. Among them, 30% presented considerable pectinolytic activity. The cultivation of these strains by submerged and semi-solid fermentation for polygalacturonase production indicated that five strains of Bacillus sp produced high quantities of the enzyme. The physico-chemical characteristics, such as optimum pH of 6.0 - 7.0, optimum temperatures between 45oC and 55oC, stability at temperatures above 40oC and in neutral and alkaline pH, were determined.

  17. The Challenge of Producing Ubiquitinated Proteins for Structural Studies

    Directory of Open Access Journals (Sweden)

    Serena Faggiano

    2014-06-01

    Full Text Available Protein ubiquitination is an important post-translational modification involved in several essential signalling pathways. It has different effects on the target protein substrate, i.e., it can trigger the degradation of the protein in the proteasome, change the interactions of the modified protein with its partners, or affect its localization and activity. In order to understand the molecular mechanisms underlying the consequences of protein ubiquitination, scientists have to face the challenging task of producing ubiquitinated proteins for structural characterization with X-ray crystallography and/or nuclear magnetic resonance (NMR spectroscopy. These techniques require milligrams of homogeneous samples of high purity. The strategies proposed so far for the production of ubiquitinated proteins can be divided into two groups, i.e., chemical (or non-enzymatic and enzymatic methodologies. In this review, we summarize the still very sparse examples available in the literature that describe successful production of ubiquitinated proteins amenable for biochemical and structural studies, and discuss advantages and disadvantages of the techniques proposed. We also give a perspective of the direction in which the field might evolve.

  18. EEVD motif of heat shock cognate protein 70 contributes to bacterial uptake by trophoblast giant cells

    Directory of Open Access Journals (Sweden)

    Kim Suk

    2009-12-01

    Full Text Available Abstract Background The uptake of abortion-inducing pathogens by trophoblast giant (TG cells is a key event in infectious abortion. However, little is known about phagocytic functions of TG cells against the pathogens. Here we show that heat shock cognate protein 70 (Hsc70 contributes to bacterial uptake by TG cells and the EEVD motif of Hsc70 plays an important role in this. Methods Brucella abortus and Listeria monocytogenes were used as the bacterial antigen in this study. Recombinant proteins containing tetratricopeptide repeat (TPR domains were constructed and confirmation of the binding capacity to Hsc70 was assessed by ELISA. The recombinant TPR proteins were used for investigation of the effect of TPR proteins on bacterial uptake by TG cells and on pregnancy in mice. Results The monoclonal antibody that inhibits bacterial uptake by TG cells reacted with the EEVD motif of Hsc70. Bacterial TPR proteins bound to the C-terminal of Hsc70 through its EEVD motif and this binding inhibited bacterial uptake by TG cells. Infectious abortion was also prevented by blocking the EEVD motif of Hsc70. Conclusions Our results demonstrate that surface located Hsc70 on TG cells mediates the uptake of pathogenic bacteria and proteins containing the TPR domain inhibit the function of Hsc70 by binding to its EEVD motif. These molecules may be useful in the development of methods for preventing infectious abortion.

  19. Characterization of Bacterial Mannanase for Hydrolyzing Palm Kernel Cake to Produce Manno-oligosaccharides Prebiotics

    Directory of Open Access Journals (Sweden)

    W. Utami

    2013-12-01

    Full Text Available Palm kernel cake (PKC is a promising source of prebiotics, since it contains high amount of β-mannan which can be further hydrolyzed to manno-oligasaccharides (MOS, a prebiotic. Therefore, this research was carried out to analyze the capability of a bacterial isolate (A2 isolates previously isolated from soils sample from around IPB campus to hydrolyze PKC. Based on 16S-DNA analysis, isolate A2 was identified as Brevibacillus borstelensis. Mannanase of A2 isolate had an optimum condition at 90 oC and pH 7. Mannanase activity of crude extracts using Locust Bean Gum (LBG and PKC as substrates were 0.37U/mL and 0.032U/mL, respectively. However, the most favorable production of oligosaccharides based on the degree of polymerization was obtained after 72-h of incubation with the ratio of substrate:enzyme, 1.2:1, on 1.5% PKC as substrate. The manno-oligosaccharides prebio-tic obtained was found to interfere the growth of both lactic acid bacteria (Lactobacillus casei and pathogenic microflora (Escherichia coli. E. coli apparently could not use this prebiotic as the carbon sources, in contrast to L. casei. Substitution of carbon source in medium with prebiotics reduced the capability of L. casei to produce organic acids. It is concluded that local A2 isolate (B. borstelensis produces mannanase which can be used to produce prebiotics from PKC.

  20. Magnesium improves hydrogen production by a novel fermentative hydrogen-producing bacterial strain B49

    Institute of Scientific and Technical Information of China (English)

    WANG Xiang-jing; REN Nan-qi; XIANG Wen-sheng

    2005-01-01

    Batch experiments were conducted to investigate the effects of magnesium on glucose metabolism, including growth and hydrogen-producing capacity of fermentative hydrogen-producing bacterial strain B49. These abilities were enhanced with an increase in magnesium concentration. At the end of fermentation from 10 g/L ratio of ethanol amount (mg/L) to acetate amount (mg/L) was 1.1, and the accumulated hydrogen volume hydrogen volume was increased to 2 360. 5 mL H2/L culture, the ratio of ethanol amount (mg/L) to acetate amount (mg/L) was increased to 1.3 and polysaccharide was decreased to 2. 5 mg/L. Moreover, the magnesium solution addition to the medium at different fermentation times affected hydrogen-producing ability. However,the later the addition time was postponed, the less the effect was on hydrogen evolution. Further experiments confirmed the enhancement was dependent on magnesium ions and not on the other inorganic ions such as SO42- or Cl-, which constituted the magnesium salts.

  1. Hemolysin, Protease, and EPS Producing Pathogenic Aeromonas hydrophila Strain An4 Shows Antibacterial Activity against Marine Bacterial Fish Pathogens

    Directory of Open Access Journals (Sweden)

    Anju Pandey

    2010-01-01

    Full Text Available A pathogenic Aeromonas hydrophila strain An4 was isolated from marine catfish and characterized with reference to its proteolytic and hemolytic activity along with SDS-PAGE profile (sodium dodecyl sulphate-Polyacrylamide gel electrophoresis of ECPs (extracellular proteins showing hemolysin (approximately 50 kDa. Agar well diffusion assay using crude cell extract of the bacterial isolate clearly demonstrated antibacterial activity against indicator pathogenic bacteria, Staphylococcus arlettae strain An1, Acinetobacter sp. strain An2, Vibrio parahaemolyticus strain An3, and Alteromonas aurentia SE3 showing inhibitory zone >10 mm well comparable to common antibiotics. Further GC-MS analysis of crude cell extract revealed several metabolites, namely, phenolics, pyrrolo-pyrazines, pyrrolo-pyridine, and butylated hydroxytoluene (well-known antimicrobials. Characterization of EPS using FTIR indicated presence of several protein-related amine and amide groups along with peaks corresponding to carboxylic and phenyl rings which may be attributed to its virulent and antibacterial properties, respectively. Besides hemolysin, EPS, and protease, Aeromonas hydrophila strain An4 also produced several antibacterial metabolites.

  2. Blocking of bacterial biofilm formation by a fish protein coating

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk; Klemm, Per

    2008-01-01

    proteinaceous coating is characterized with regards to its biofilm-reducing properties by using a range of urinary tract infectious isolates with various pathogenic and adhesive properties. The antiadhesive coating significantly reduced or delayed biofilm formation by all these isolates under every condition......Bacterial biofilm formation on inert surfaces is a significant health and economic problem in a wide range of environmental, industrial, and medical areas. Bacterial adhesion is generally a prerequisite for this colonization process and, thus, represents an attractive target for the development of...... biofilm-preventive measures. We have previously found that the preconditioning of several different inert materials with an aqueous fish muscle extract, composed primarily of fish muscle alpha-tropomyosin, significantly discourages bacterial attachment and adhesion to these surfaces. Here, this...

  3. Improving bacterial cellulose for blood vessel replacement: functionalization with a chimeric protein containing a cellulose-binding module and an adhesion peptide

    OpenAIRE

    Andrade, Fábia K.; Costa, Raquel; Domingues, Lucília; Soares, Raquel; Gama, F. M.

    2010-01-01

    Chimeric proteins containing a cellulose-binding module (CBM) and an adhesion peptide (RGD or GRGDY) were produced and used to improve the adhesion of human microvascular endothelial cells (HMEC) to bacterial cellulose (BC). The effect of these proteins on the HMEC–BC interaction was studied. The results obtained demonstrated that recombinant proteins containing adhesion sequences were able to significantly increase the attachment of HMEC to BC surfaces, especially the RGD sequenc...

  4. Imaging bacterial protein expression using genetically encoded sensors composed of RNA

    OpenAIRE

    Song, Wenjiao; Strack, Rita L.; Jaffrey, Samie R.

    2013-01-01

    We show that the difficulties in imaging the dynamics of protein expression in live bacterial cells can be overcome using fluorescent sensors based on Spinach, an RNA that activates the fluorescence of a small-molecule fluorophore. These RNAs selectively bind target proteins, and exhibit fluorescence increases that enable protein expression to be imaged in living cells. These sensors provide a general strategy to image protein expression in single bacteria in real-time.

  5. Bacterial endophyte Sphingomonas sp. LK11 produces gibberellins and IAA and promotes tomato plant growth.

    Science.gov (United States)

    Khan, Abdul Latif; Waqas, Muhammad; Kang, Sang-Mo; Al-Harrasi, Ahmed; Hussain, Javid; Al-Rawahi, Ahmed; Al-Khiziri, Salima; Ullah, Ihsan; Ali, Liaqat; Jung, Hee-Young; Lee, In-Jung

    2014-08-01

    Plant growth promoting endophytic bacteria have been identified as potential growth regulators of crops. Endophytic bacterium, Sphingomonas sp. LK11, was isolated from the leaves of Tephrosia apollinea. The pure culture of Sphingomonas sp. LK11 was subjected to advance chromatographic and spectroscopic techniques to extract and isolate gibberellins (GAs). Deuterated standards of [17, 17-(2)H2]-GA4, [17, 17-(2)H2]-GA9 and [17, 17-(2)H2]-GA20 were used to quantify the bacterial GAs. The analysis of the culture broth of Sphingomonas sp. LK11 revealed the existence of physiologically active gibberellins (GA4: 2.97 ± 0.11 ng/ml) and inactive GA9 (0.98 ± 0.15 ng/ml) and GA20 (2.41 ± 0.23). The endophyte also produced indole acetic acid (11.23 ± 0.93 μM/ml). Tomato plants inoculated with endophytic Sphingomonas sp. LK11 showed significantly increased growth attributes (shoot length, chlorophyll contents, shoot, and root dry weights) compared to the control. This indicated that such phyto-hormones-producing strains could help in increasing crop growth. PMID:24994010

  6. Textile Dye Removal from Wastewater Effluents Using Bioflocculants Produced by Indigenous Bacterial Isolates

    Directory of Open Access Journals (Sweden)

    Balakrishna Pillay

    2012-11-01

    Full Text Available Bioflocculant-producing bacteria were isolated from activated sludge of a wastewater treatment plant located in Durban, South Africa, and identified using standard biochemical tests as well as the analysis of their 16S rRNA gene sequences. The bioflocculants produced by these organisms were ethanol precipitated, purified using 2% (w/v cetylpyridinium chloride solution and evaluated for removal of wastewater dyes under different pH, temperature and nutritional conditions. Bioflocculants from these indigenous bacteria were very effective for decolourizing the different dyes tested in this study, with a removal rate of up to 97.04%. The decolourization efficiency was largely influenced by the type of dye, pH, temperature, and flocculant concentration. A pH of 7 was found to be optimum for the removal of both whale and mediblue dyes, while the optimum pH for fawn and mixed dye removal was found to be between 9 and 10. Optimum temperature for whale and mediblue dye removal was 35 °C, and that for fawn and mixed dye varied between 40–45 °C and 35–40 °C, respectively. These bacterial bioflocculants may provide an economical and cleaner alternative to replace or supplement present treatment processes for the removal of dyes from wastewater effluents, since they are biodegradable and easily sustainable.

  7. Antimicrobial Activity of Monoramnholipids Produced by Bacterial Strains Isolated from the Ross Sea (Antarctica).

    Science.gov (United States)

    Tedesco, Pietro; Maida, Isabel; Palma Esposito, Fortunato; Tortorella, Emiliana; Subko, Karolina; Ezeofor, Chidinma Christiana; Zhang, Ying; Tabudravu, Jioji; Jaspars, Marcel; Fani, Renato; de Pascale, Donatella

    2016-05-01

    Microorganisms living in extreme environments represent a huge reservoir of novel antimicrobial compounds and possibly of novel chemical families. Antarctica is one of the most extraordinary places on Earth and exhibits many distinctive features. Antarctic microorganisms are well known producers of valuable secondary metabolites. Specifically, several Antarctic strains have been reported to inhibit opportunistic human pathogens strains belonging to Burkholderia cepacia complex (Bcc). Herein, we applied a biodiscovery pipeline for the identification of anti-Bcc compounds. Antarctic sub-sea sediments were collected from the Ross Sea, and used to isolate 25 microorganisms, which were phylogenetically affiliated to three bacterial genera (Psychrobacter, Arthrobacter, and Pseudomonas) via sequencing and analysis of 16S rRNA genes. They were then subjected to a primary cell-based screening to determine their bioactivity against Bcc strains. Positive isolates were used to produce crude extracts from microbial spent culture media, to perform the secondary screening. Strain Pseudomonas BNT1 was then selected for bioassay-guided purification employing SPE and HPLC. Finally, LC-MS and NMR structurally resolved the purified bioactive compounds. With this strategy, we achieved the isolation of three rhamnolipids, two of which were new, endowed with high (MIC < 1 μg/mL) and unreported antimicrobial activity against Bcc strains. PMID:27128927

  8. Antimicrobial Activity of Monoramnholipids Produced by Bacterial Strains Isolated from the Ross Sea (Antarctica) †

    Science.gov (United States)

    Tedesco, Pietro; Maida, Isabel; Palma Esposito, Fortunato; Tortorella, Emiliana; Subko, Karolina; Ezeofor, Chidinma Christiana; Zhang, Ying; Tabudravu, Jioji; Jaspars, Marcel; Fani, Renato; de Pascale, Donatella

    2016-01-01

    Microorganisms living in extreme environments represent a huge reservoir of novel antimicrobial compounds and possibly of novel chemical families. Antarctica is one of the most extraordinary places on Earth and exhibits many distinctive features. Antarctic microorganisms are well known producers of valuable secondary metabolites. Specifically, several Antarctic strains have been reported to inhibit opportunistic human pathogens strains belonging to Burkholderia cepacia complex (Bcc). Herein, we applied a biodiscovery pipeline for the identification of anti-Bcc compounds. Antarctic sub-sea sediments were collected from the Ross Sea, and used to isolate 25 microorganisms, which were phylogenetically affiliated to three bacterial genera (Psychrobacter, Arthrobacter, and Pseudomonas) via sequencing and analysis of 16S rRNA genes. They were then subjected to a primary cell-based screening to determine their bioactivity against Bcc strains. Positive isolates were used to produce crude extracts from microbial spent culture media, to perform the secondary screening. Strain Pseudomonas BNT1 was then selected for bioassay-guided purification employing SPE and HPLC. Finally, LC-MS and NMR structurally resolved the purified bioactive compounds. With this strategy, we achieved the isolation of three rhamnolipids, two of which were new, endowed with high (MIC < 1 μg/mL) and unreported antimicrobial activity against Bcc strains. PMID:27128927

  9. H2-producing bacterial communities from a heat-treated soil inoculum.

    Science.gov (United States)

    Iyer, Prabha; Bruns, Mary Ann; Zhang, Husen; Van Ginkel, Steve; Logan, Bruce E

    2004-12-01

    Hydrogen gas (approximately 60% H(2)) was produced in a continuous flow bioreactor inoculated with heat-treated soil, and fed synthetic wastewater containing glucose (9.5 g l(-1)). The pH in the bioreactor was maintained at 5.5 to inhibit consumption of H(2) by methanogens. The objective of this study was to characterize bacterial communities in the reactor operated under two different hydraulic retention times (HRTs of 30-h and 10-h) and temperatures (30 degrees C and 37 degrees C). At 30-h HRT, the H(2) production rate was 80 ml h(-1) and yield was 0.91 mol H(2)/mol glucose. At 10-h HRT, the H(2) production rate was more than 5 times higher at 436 ml h(-1), and yield was 1.61 mol H(2)/mol glucose. Samples were removed from the reactor under steady-state conditions for PCR-based detection of bacterial populations by ribosomal intergenic spacer analysis (RISA). Populations detected at 30-h HRT were more diverse than at 10-h HRT and included representatives of Bacillaceae, Clostridiaceae, and Enterobacteriaceae. At 10-h HRT, only Clostridiaceae were detected. When the temperature of the 10-h HRT reactor was increased from 30 degrees C to 37 degrees C, the steady-state H(2) production rate increased slightly to 463 ml h(-1) and yield was 1.8 mol H(2)/mol glucose. Compared to 30 degrees C, RISA fingerprints at 37 degrees C from the 10-h HRT bioreactor exhibited a clear shift from populations related to Clostridium acidisoli (subcluster Ic) to populations related to Clostridium acetobutylicum (subcluster Ib). PMID:15558274

  10. Producing Recombinant mTEX101; a Murine Testis Specific Protein

    Science.gov (United States)

    Barzegar Yarmohammadi, Leila; Modarresi, Mohammad Hossein; Talebi, Saeed; Hadavi, Reza; Ostad Karampour, Mahyar; Mahmoudi, Ahmad Reza; Akhondi, Mohammad Mehdi; Rabbani, Hodjattallah; Jeddi-Tehrani, Mahmood

    2009-01-01

    Introduction Production of antibodies against specific proteins of testis germ cells is of great significance for the investigation of processes involved in spermatogenesis, study of infertility problems and determination of the probable role of these proteins as cancer-testis antigens. Murine Testis Specific Recombinant Protein 101 (mTEX101) is a 38kDa, GPI-anchored protein which is expressed in testis germ cells of adult mice but it seems to be absent in other tissues. The structure and function of mTEX101 is not completely understood yet, but it is speculated that it may transduce biochemical signals into the cytoplasm since mTEX101 does not have an intracellular domain but the precise mechanisms are still ambiguous. Materials and Methods RNA was extracted from three adult mice testis. The RNA was used in RT-PCR, employing a pair of specific primers for mTEX101 ORF region. TA-cloning technique was performed by the insertion of mTEX101 into a pGEM-T Easy Vector, followed by its subcloning into a His-tagged expression vector, pET-28a (+). The recombinant mTEX101 was then produced by transfection of the expression vector into BL 21 (DE3) E. coli strain. Results A recombinant protein, weighing 27kDa, was produced upon IPTG-induction of the bacterial host. The presence of mTEX101 protein was detected through Western blot analysis by anti-mTEX101 peptide antibodies. Conclusion We produced mTEX101 recombinant protein that could be used for the production of mono and polyclonal antibodies. PMID:23926468

  11. The diagnostic value of c-reactive protein estimation in differentiating bacterial from viral meningitis

    International Nuclear Information System (INIS)

    Objective: To evaluate the efficacy of serum and CSF C-reactive protein (C-rp) in differentiating bacterial from viral meningitis. Design: An observational, respective hospital-based study. Place and duration of study: It was conducted at the Department of Medicine and Department of Pediatrics, Shaikh Zayed Postgraduate Medical Institute Lahore, Over a Period of one year between march, 1999 and March, 2000. Subject and Methods: A randomized group of thirty patients, who presented with clinical features, suggestive of meningitis, were included in the study. C-reactive protein determinations were performed by latex agglutination method on the serum and cerebrospinal fluid (CSF) of these patients. Results: In the present study, c-reactive protein was found to be a more sensitive test for differentiating bacterial from non-bacterial meningitis on initial examination than the usual conventional methods used to diagnose bacterial meningitis. CSF C-reactive protein had a greater sensitivity (92% as compared to serum C-reactive protein (71%). Conclusion: C-reactive protein determination in CSF was found to be a useful indicator of bacterial meningitis that can be used to distinguish it from viral meningitis. (author)

  12. Bacterial diversity and composition in major fresh produce growing soils affected by physiochemical properties and geographic locations.

    Science.gov (United States)

    Ma, Jincai; Ibekwe, A Mark; Yang, Ching-Hong; Crowley, David E

    2016-09-01

    Microbial diversity of agricultural soils has been well documented, but information on leafy green producing soils is limited. In this study, we investigated microbial diversity and community structures in 32 (16 organic, 16 conventionally managed soils) from California (CA) and Arizona (AZ) using pyrosequencing, and identified factors affecting bacterial composition. Results of detrended correspondence analysis (DCA) and dissimilarity analysis showed that bacterial community structures of conventionally managed soils were similar to that of organically managed soils; while the bacterial community structures in soils from Salinas, California were different (Psoils from Yuma, Arizona and Imperial Valley, California. Canonical correspondence analysis (CCA) and artificial neural network (ANN) analysis of bacterial community structures and soil variables showed that electrical conductivity (EC), clay content, water-holding capacity (WHC), pH, total nitrogen (TN), and organic carbon (OC) significantly (Psoil physical properties (clay, EC, and WHC), soil chemical variables (pH, TN, and OC) and sampling location explained 16.3%, 12.5%, and 50.9%, respectively, of total variations in bacterial community structure, leaving 13% of the total variation unexplained. Our current study showed that bacterial community composition and diversity in major fresh produce growing soils from California and Arizona is a function of soil physiochemical characteristics and geographic distances of sampling sites. PMID:27135583

  13. Interactions That Drive Sec-Dependent Bacterial Protein Transport†

    OpenAIRE

    Rusch, Sharyn L.; Kendall, Debra A.

    2007-01-01

    Understanding the transport of hydrophilic proteins across biological membranes continues to be an important undertaking. The general secretory (Sec) pathway in Escherichia coli transports the majority of E. coli proteins from their point of synthesis in the cytoplasm to their sites of final localization, associating sequentially with a number of protein components of the transport machinery. The targeting signals for these substrates must be discriminated from those of proteins transported v...

  14. Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA.

    Science.gov (United States)

    Belcourt, M F; Penketh, P G; Hodnick, W F; Johnson, D A; Sherman, D H; Rockwell, S; Sartorelli, A C

    1999-08-31

    The mitomycin C-resistance gene, mcrA, of Streptomyces lavendulae produces MCRA, a protein that protects this microorganism from its own antibiotic, the antitumor drug mitomycin C. Expression of the bacterial mcrA gene in mammalian Chinese hamster ovary cells causes profound resistance to mitomycin C and to its structurally related analog porfiromycin under aerobic conditions but produces little change in drug sensitivity under hypoxia. The mitomycins are prodrugs that are enzymatically reduced and activated intracellularly, producing cytotoxic semiquinone anion radical and hydroquinone reduction intermediates. In vitro, MCRA protects DNA from cross-linking by the hydroquinone reduction intermediate of these mitomycins by oxidizing the hydroquinone back to the parent molecule; thus, MCRA acts as a hydroquinone oxidase. These findings suggest potential therapeutic applications for MCRA in the treatment of cancer with the mitomycins and imply that intrinsic or selected mitomycin C resistance in mammalian cells may not be due solely to decreased bioactivation, as has been hypothesized previously, but instead could involve an MCRA-like mechanism. PMID:10468636

  15. Visualizing translocation and localization of bacterial type III effector proteins using a genetically encoded reporter system

    OpenAIRE

    Gawthorne, Jayde A.; Audry, Laurent; McQuitty, Claire; Dean, Paul; Christie, John M.; Enninga, Jost; Andrew J. Roe

    2016-01-01

    Bacterial Type Three Secretion System (T3SS) effector proteins are critical determinants of infection for many animal and plant pathogens. However, monitoring of the translocation and delivery of these important virulence determinants has proved to be technically challenging. Here, we used a genetically engineered LOV (light-oxygen-voltage) sensing domain derivative to monitor the expression, translocation and localization of bacterial T3SS effectors. We found the Escherichia coli O157:H7 bac...

  16. Proteolytic activation of human pancreatitis associated protein is required for peptidoglycan binding and bacterial aggregation

    OpenAIRE

    Medveczky, Péter; Szmola, Richárd; Sahin-Tóth, Miklós

    2009-01-01

    Pancreatitis associated protein (PAP) is a 16 kDa lectin-like protein, which becomes robustly upregulated in the pancreatic juice during acute pancreatitis. Trypsin cleaves the N terminus of PAP, which in turn forms insoluble fibrils. PAP and its paralog the pancreatic stone protein induce bacterial aggregation and, more recently, PAP was shown to bind to the peptidoglycan of Gram positive bacteria and exert a direct bactericidal effect. However, the role of N-terminal processing in the antib...

  17. Complete Genome Sequence of Gluconacetobacter hansenii Strain NQ5 (ATCC 53582), an Efficient Producer of Bacterial Cellulose.

    Science.gov (United States)

    Pfeffer, Sarah; Mehta, Kalpa; Brown, R Malcolm

    2016-01-01

    This study reports the release of the complete nucleotide sequence of Gluconacetobacter hansenii strain NQ5 (ATCC 53582). This strain was isolated by R. Malcolm Brown, Jr. in a sugar mill in North Queensland, Australia, and is an efficient producer of bacterial cellulose. The elucidation of the genome will contribute to the study of the molecular mechanisms necessary for cellulose biosynthesis. PMID:27516505

  18. Essential bacterial helicases that counteract the toxicity of recombination proteins

    OpenAIRE

    Petit, Marie-Agnès; Ehrlich, Dusko

    2002-01-01

    PcrA, Rep and UvrD are three closely related bacterial helicases with a DExx signature. PcrA is encoded by Gram-positive bacteria and is essential for cell growth. Rep and UvrD are encoded by Gram-negative bacteria, and mutants lacking both helicases are also not viable. To understand the non-viability of the helicase mutants, we characterized spontaneous extragenic suppressors of a Bacillus subtilis pcrA null mutation. Here we report that one of these suppressors maps in recF and that previo...

  19. Bacterial S-layer protein coupling to lipids

    DEFF Research Database (Denmark)

    Weygand, M.; Wetzer, B.; Pum, D.;

    1999-01-01

    -filled cavities near its center. The protein volume fraction reaches maxima of >60% in two horizontal sections of the S-layer, close to the lipid monolayer and close to the free subphase. In between it drops to similar to 20%. Four S-layer protein monomers are located within the unit cell of a square lattice with...

  20. BACTERIAL SOLUTE TRANSPORT PROTEINS IN THEIR LIPID ENVIRONMENT

    NARCIS (Netherlands)

    TVELD, GI; DRIESSEN, AJM; KONINGS, WN; Veld, Gerda in 't

    1993-01-01

    The cytoplasmic membrane of bacteria is a selective barrier that restricts entry and exit of solutes. Transport of solutes across this membrane is catalyzed by specific membrane proteins. Integral membrane proteins usually require specific lipids for optimal activity and are inhibited by other lipid

  1. Functional properties of proteins isolated from industrially produced sunflower meal

    Directory of Open Access Journals (Sweden)

    Petia Ivanova

    2014-10-01

    Full Text Available Protein isolate 1 (PI1 and protein isolate 2 (PI2 were prepared from industrially produced sunflower meal by using isoelectric and ethanol precipitation respectively. The water absorption capacity of PI1 was 6 times higher than that of PI2 and was significantly reduced by the presence of 0.03 M and 0.25 M NaCl. Oil absorption capacity of both protein isolates was not influenced by NaCl supplementation. Foam capacity of PI1 and PI2 was pH-dependent. While the foam capacity of both isolates was improved by either 0.03 M or 0.25 M NaCl, the foam stability was negatively influenced by the addition of NaCl at all pH values with except for pH 4. Emulsifying activity of PI1 and PI2 was lowest at pH 4. The emulsions exhibited relatively high stability (> 90% under all studied conditions. Knowledge of the influence of pH and boundary concentrations of NaCl on the functionality of sunflower meal protein isolates could be beneficial for their future potential application in food industry.

  2. Photorhabdus adhesion modification protein (Pam) binds extracellular polysaccharide and alters bacterial attachment

    LENUS (Irish Health Repository)

    Jones, Robert T

    2010-05-12

    Abstract Background Photorhabdus are Gram-negative nematode-symbiotic and insect-pathogenic bacteria. The species Photorhabdus asymbiotica is able to infect humans as well as insects. We investigated the secreted proteome of a clinical isolate of P. asymbiotica at different temperatures in order to identify proteins relevant to the infection of the two different hosts. Results A comparison of the proteins secreted by a clinical isolate of P. asymbiotica at simulated insect (28°C) and human (37°C) temperatures led to the identification of a small and highly abundant protein, designated Pam, that is only secreted at the lower temperature. The pam gene is present in all Photorhabdus strains tested and shows a high level of conservation across the whole genus, suggesting it is both ancestral to the genus and probably important to the biology of the bacterium. The Pam protein shows limited sequence similarity to the 13.6 kDa component of a binary toxin of Bacillus thuringiensis. Nevertheless, injection or feeding of heterologously produced Pam showed no insecticidal activity to either Galleria mellonella or Manduca sexta larvae. In bacterial colonies, Pam is associated with an extracellular polysaccharide (EPS)-like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. Interestingly, Surface Plasmon Resonance (SPR) binding studies revealed that the Pam protein itself has adhesive properties. Although Pam is produced throughout insect infection, genetic knockout does not affect either insect virulence or the ability of P. luminescens to form a symbiotic association with its host nematode, Heterorhabditis bacteriophora. Conclusions We studied a highly abundant protein, Pam, which is secreted in a temperature-dependent manner in P. asymbiotica. Our findings indicate that Pam plays an important role in enhancing surface attachment in insect blood. Its association with exopolysaccharide suggests it may exert its effect through mediation of

  3. Photorhabdus adhesion modification protein (Pam binds extracellular polysaccharide and alters bacterial attachment

    Directory of Open Access Journals (Sweden)

    Joyce Susan A

    2010-05-01

    Full Text Available Abstract Background Photorhabdus are Gram-negative nematode-symbiotic and insect-pathogenic bacteria. The species Photorhabdus asymbiotica is able to infect humans as well as insects. We investigated the secreted proteome of a clinical isolate of P. asymbiotica at different temperatures in order to identify proteins relevant to the infection of the two different hosts. Results A comparison of the proteins secreted by a clinical isolate of P. asymbiotica at simulated insect (28°C and human (37°C temperatures led to the identification of a small and highly abundant protein, designated Pam, that is only secreted at the lower temperature. The pam gene is present in all Photorhabdus strains tested and shows a high level of conservation across the whole genus, suggesting it is both ancestral to the genus and probably important to the biology of the bacterium. The Pam protein shows limited sequence similarity to the 13.6 kDa component of a binary toxin of Bacillus thuringiensis. Nevertheless, injection or feeding of heterologously produced Pam showed no insecticidal activity to either Galleria mellonella or Manduca sexta larvae. In bacterial colonies, Pam is associated with an extracellular polysaccharide (EPS-like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. Interestingly, Surface Plasmon Resonance (SPR binding studies revealed that the Pam protein itself has adhesive properties. Although Pam is produced throughout insect infection, genetic knockout does not affect either insect virulence or the ability of P. luminescens to form a symbiotic association with its host nematode, Heterorhabditis bacteriophora. Conclusions We studied a highly abundant protein, Pam, which is secreted in a temperature-dependent manner in P. asymbiotica. Our findings indicate that Pam plays an important role in enhancing surface attachment in insect blood. Its association with exopolysaccharide suggests it may exert its effect

  4. Horizontal gene transfer of zinc and non-zinc forms of bacterial ribosomal protein S4

    Directory of Open Access Journals (Sweden)

    Luthey-Schulten Zaida

    2009-07-01

    Full Text Available Abstract Background The universal ribosomal protein S4 is essential for the initiation of small subunit ribosomal assembly and translational accuracy. Being part of the information processing machinery of the cell, the gene for S4 is generally thought of as being inherited vertically and has been used in concatenated gene phylogenies. Here we report the evolution of ribosomal protein S4 in relation to a broad sharing of zinc/non-zinc forms of the gene and study the scope of horizontal gene transfer (HGT of S4 during bacterial evolution. Results In this study we present the complex evolutionary history of ribosomal protein S4 using 660 bacterial genomes from 16 major bacterial phyla. According to conserved characteristics in the sequences, S4 can be classified into C+ (zinc-binding and C- (zinc-free variants, with 26 genomes (mainly from the class Clostridia containing genes for both. A maximum likelihood phylogenetic tree of the S4 sequences was incongruent with the standard bacterial phylogeny, indicating a departure from strict vertical inheritance. Further analysis using the genome content near the S4 genes, which are usually located in a conserved gene cluster, showed not only that HGT of the C- gene had occurred at various stages of bacterial evolution, but also that both the C- and C+ genes were present before the individual phyla diverged. To explain the latter, we theorize that a gene pool existed early in bacterial evolution from which bacteria could sample S4 gene variants, according to environmental conditions. The distribution of the C+/- variants for seven other zinc-binding ribosomal proteins in these 660 bacterial genomes is consistent with that seen for S4 and may shed light on the evolutionary pressures involved. Conclusion The complex history presented for "core" protein S4 suggests the existence of a gene pool before the emergence of bacterial lineages and reflects the pervasive nature of HGT in subsequent bacterial evolution

  5. Utilization of 15N-Diammonium Phosphate by Ruminants to Produce Milk and Meat Proteins

    International Nuclear Information System (INIS)

    The authors investigated the alimentary role of diammonium phosphate (DAP) in ruminants. For this study DAP labelled with 15N was used; analysis of the 15N atomic per cent excess was made with an Italelettronica mass spectrophotometer (model SP 21 F) and the amino acid determination by a Beckman-Spinco amino acid analyser (model 120B) fitted with a preparative column. For the experiment 7 g of DAP at 15 and 20 at. % excess 15N were administered once to mature lactating and non-lactating sheep, respectively. The measurement of 15N in the protein and isolated amino acids of milk and meat showed: (1) The milk protein produced in the first 24 h contained the highest atomic per cent excess of 15SN, 0.093; (2) That the supplemental 15N was found in all the amino acids of milk proteins except tryptophane. The atomic per cent excess of 15N was observed to vary between the various amino acids. These results confirmed previous observations on bacterial protein synthesized from DAP. (3) Muscle protein 15N maximized on the third day after administration of the 15N-DAP, with an atomic per cent excess of 0.040; (4) The atomic per cent excess of 15N in the individual amino acids of muscle protein is significant in only two amino' acids, serine and cystine; and (5) That after 8 d of adaptation there are no traces of DAP in milk or meat proteins, urine or faeces. The authors conclude that the ruminant, after a period of adaptation and through the mediation of ruminant microorganisms, is able to use the nitrogen of diammonium phosphate for the synthesis of milk and meat proteins. (author)

  6. Electrically conductive bacterial cellulose composite membranes produced by the incorporation of graphite nanoplatelets in pristine bacterial cellulose membranes

    Directory of Open Access Journals (Sweden)

    T. Zhou

    2013-09-01

    Full Text Available Graphite nanoplatelets (GNPs were utilized to improve the electrical conductivity of pristine bacterial cellulose (BC membranes. By physical and chemical methods, flake-shaped GNPs, weaving through the surface layer of web-like cellulose nanofibrils, were indeed fixed or trapped by the adjacent nanofibrils in the BC surface network, for comparison, rod-shaped multi-walled carbon nanotubes (MWCNTs were homogeneously inserted into BC membrane through the pore structures and tunnels within the BC membrane. Strong physical and chemical interaction exists between the BC nanofibrils and the particles of GNP or MWCNT even after 15 h sonication. BC membrane with 8.7 wt% incorporated GNPs reached the maximum electrical conductivity of 4.5 S/cm, while 13.9 wt% MWCNT/BC composite membrane achieved the maximum electrical conductivity of 1.2 S/cm. Compared with one dimensional (1-D MWCNTs, as long as GNPs inserted into BC membranes, the 2-D reinforcement of GNPs was proven to be more effective in improving the electrical conductivity of BC membranes thus not only break the bottleneck of further improvement of the electrical conductivity of BC-based composite membranes but also broaden the applications of BC and GNPs.

  7. High-Throughput Screening of Bacterial Protein Localization

    OpenAIRE

    Werner, John N.; Gitai, Zemer

    2010-01-01

    The ever-increasing number of sequenced genomes and subsequent sequence-based analysis has provided tremendous insight into cellular processes; however, the ability to experimentally manipulate this genomic information in the laboratory requires the development of new high-throughput methods. To translate this genomic information into information on protein function, molecular and cell biological techniques are required. One strategy to gain insight into protein function is to observe where e...

  8. 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. PMID:21091448

  9. A simple yeast-based strategy to identify host cellular processes targeted by bacterial effector proteins.

    Directory of Open Access Journals (Sweden)

    Eran Bosis

    Full Text Available Bacterial effector proteins, which are delivered into the host cell via the type III secretion system, play a key role in the pathogenicity of gram-negative bacteria by modulating various host cellular processes to the benefit of the pathogen. To identify cellular processes targeted by bacterial effectors, we developed a simple strategy that uses an array of yeast deletion strains fitted into a single 96-well plate. The array is unique in that it was optimized computationally such that despite the small number of deletion strains, it covers the majority of genes in the yeast synthetic lethal interaction network. The deletion strains in the array are screened for hypersensitivity to the expression of a bacterial effector of interest. The hypersensitive deletion strains are then analyzed for their synthetic lethal interactions to identify potential targets of the bacterial effector. We describe the identification, using this approach, of a cellular process targeted by the Xanthomonas campestris type III effector XopE2. Interestingly, we discover that XopE2 affects the yeast cell wall and the endoplasmic reticulum stress response. More generally, the use of a single 96-well plate makes the screening process accessible to any laboratory and facilitates the analysis of a large number of bacterial effectors in a short period of time. It therefore provides a promising platform for studying the functions and cellular targets of bacterial effectors and other virulence proteins.

  10. Bacterial Inhibition and Antioxidant Activity of Kefir Produced from Thai Jasmine Rice Milk

    OpenAIRE

    Deeseenthum Sirirat; Pejovic Jelena

    2010-01-01

    The aim of this study was to investigate the bacterial inhibition and antioxidant activity of 24 and 48 h of rice milk-kefir and cow milk-kefir. Bacterial inhibition activity of kefir was investigated against Staphylococcus aureus, Bacillus subtillis, Escherichia coli and Pseudomonas fluorescens by using the disk diffusion method. Kefir showed some activity against all organisms tested. Antioxidant activity of kefir was measured using three different methods: DPPH radical scavenging activity ...

  11. Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain

    Science.gov (United States)

    Florea, Michael; Hagemann, Henrik; Santosa, Gabriella; Micklem, Chris N.; Spencer-Milnes, Xenia; de Arroyo Garcia, Laura; Paschou, Despoina; Lazenbatt, Christopher; Kong, Deze; Chughtai, Haroon; Jensen, Kirsten; Freemont, Paul S.; Kitney, Richard; Reeve, Benjamin; Ellis, Tom

    2016-01-01

    Bacterial cellulose is a strong and ultrapure form of cellulose produced naturally by several species of the Acetobacteraceae. Its high strength, purity, and biocompatibility make it of great interest to materials science; however, precise control of its biosynthesis has remained a challenge for biotechnology. Here we isolate a strain of Komagataeibacter rhaeticus (K. rhaeticus iGEM) that can produce cellulose at high yields, grow in low-nitrogen conditions, and is highly resistant to toxic chemicals. We achieved external control over its bacterial cellulose production through development of a modular genetic toolkit that enables rational reprogramming of the cell. To further its use as an organism for biotechnology, we sequenced its genome and demonstrate genetic circuits that enable functionalization and patterning of heterologous gene expression within the cellulose matrix. This work lays the foundations for using genetic engineering to produce cellulose-based materials, with numerous applications in basic science, materials engineering, and biotechnology. PMID:27247386

  12. Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain.

    Science.gov (United States)

    Florea, Michael; Hagemann, Henrik; Santosa, Gabriella; Abbott, James; Micklem, Chris N; Spencer-Milnes, Xenia; de Arroyo Garcia, Laura; Paschou, Despoina; Lazenbatt, Christopher; Kong, Deze; Chughtai, Haroon; Jensen, Kirsten; Freemont, Paul S; Kitney, Richard; Reeve, Benjamin; Ellis, Tom

    2016-06-14

    Bacterial cellulose is a strong and ultrapure form of cellulose produced naturally by several species of the Acetobacteraceae Its high strength, purity, and biocompatibility make it of great interest to materials science; however, precise control of its biosynthesis has remained a challenge for biotechnology. Here we isolate a strain of Komagataeibacter rhaeticus (K. rhaeticus iGEM) that can produce cellulose at high yields, grow in low-nitrogen conditions, and is highly resistant to toxic chemicals. We achieved external control over its bacterial cellulose production through development of a modular genetic toolkit that enables rational reprogramming of the cell. To further its use as an organism for biotechnology, we sequenced its genome and demonstrate genetic circuits that enable functionalization and patterning of heterologous gene expression within the cellulose matrix. This work lays the foundations for using genetic engineering to produce cellulose-based materials, with numerous applications in basic science, materials engineering, and biotechnology. PMID:27247386

  13. Genome sequence and plasmid transformation of the model high-yield bacterial cellulose producer Gluconacetobacter hansenii ATCC 53582

    Science.gov (United States)

    Florea, Michael; Reeve, Benjamin; Abbott, James; Freemont, Paul S.; Ellis, Tom

    2016-03-01

    Bacterial cellulose is a strong, highly pure form of cellulose that is used in a range of applications in industry, consumer goods and medicine. Gluconacetobacter hansenii ATCC 53582 is one of the highest reported bacterial cellulose producing strains and has been used as a model organism in numerous studies of bacterial cellulose production and studies aiming to increased cellulose productivity. Here we present a high-quality draft genome sequence for G. hansenii ATCC 53582 and find that in addition to the previously described cellulose synthase operon, ATCC 53582 contains two additional cellulose synthase operons and several previously undescribed genes associated with cellulose production. In parallel, we also develop optimized protocols and identify plasmid backbones suitable for transformation of ATCC 53582, albeit with low efficiencies. Together, these results provide important information for further studies into cellulose synthesis and for future studies aiming to genetically engineer G. hansenii ATCC 53582 for increased cellulose productivity.

  14. Identification of Dominant Immunogenic Bacteria and Bacterial Proteins in Periodontitis

    DEFF Research Database (Denmark)

    Agerbæk, Mette Rylev; Haubek, Dorte; Birkelund, Svend;

    Marginal periodontitis is considered an infectious disease that triggers host inflammatory responses resulting in destruction of the periodontium. A complex biofilm of bacteria is associated with periodontitis. Some species have been identified as putative pathogens such as Porphyromonas gingivalis...... (P.g) and Actinobacillus actinomycetemcomitans (A.a), but the identity of dominate immunogens of these bacteria is poorly elucidated. The aim of the study was to identify dominant immunogenic proteins of P.g and A.a in patients suffering from chronic and aggressive periodontitis by proteomic analysis...... will be able to identify immunodominant proteins and potentially important virulence factors of putative periodontal pathogens....

  15. Low protein diets produce divergent effects on energy balance

    OpenAIRE

    Adel Pezeshki; Rizaldy C. Zapata; Arashdeep Singh; Yee, Nicholas J.; Chelikani, Prasanth K.

    2016-01-01

    Diets deficient in protein often increase food consumption, body weight and fat mass; however, the underlying mechanisms remain poorly understood. We compared the effects of diets varying in protein concentrations on energy balance in obesity-prone rats. We demonstrate that protein-free (0% protein calories) diets decreased energy intake and increased energy expenditure, very low protein (5% protein) diets increased energy intake and expenditure, whereas moderately low protein (10% protein) d...

  16. Bacterial Protein Characterization of Streptococcus agalactiae by SDS-page Method for Subclinical Mastitis Irradiated Vaccine Materials in Dairy Cattle

    Directory of Open Access Journals (Sweden)

    B.J. Tuasikal

    2012-08-01

    Full Text Available A study have been conducted to isolate and characterize bacterial protein S. agalactiae, which is antigenic and can be used to test immunogenicity of vaccine in order to manufacture irradiated mastitis (inflammation of the udder vaccine in ruminant. The study aims to determine the Molecular Weight (MW bacterial protein S. agalactiae irradiation, which can be used to test the nature of its antigenic caharacteristic. The character of S. agalactiae antigenic stimulates antibody induction of the immune system, in which case is the body's defense system against mastitis disease in cattle. In this study, irradiation of gamma ray is used to attenuate the pathogenicity of bacteria by reducing S. agalactiae antigenic caharacteristic. Previous research, in irradiation dose orientation before antigenic protein isolation of S. agalactiae, indicated that irradiation lethal dose to 50% (LD50 is 17 Gy. The characterization of S. agalactiae bacteria isolate using SDS-page method results in no significance different between irradiated and non-irradiated group, which indicated by MW range 75 – 100 kDa base on marker standard which used, or 99 kDa by the linier equation of Y = 11,60 – 0.05X (where Y = bands distance; X = MW standard protein; r2 = 0.99. In conclusion, 17 Gy irradiation dose does not impair antigenic property of S. agalactiae and therefore, can be applied to produce base material of irradiated vaccine for mastitis

  17. Bacterial Protein Characterization of Streptococcus agalactiae by SDS-page Method for Subclinical Mastitis Irradiated Vaccine Materials in Dairy Cattle

    International Nuclear Information System (INIS)

    A study have been conducted to isolate and characterize bacterial protein S. agalactiae, which is antigenic and can be used to test immunogenicity of vaccine in order to manufacture irradiated mastitis (inflammation of the udder) vaccine in ruminant. The study aims to determine the Molecular Weight (MW) bacterial protein S. agalactiae irradiation, which can be used to test the nature of its antigenic caharacteristic. The character of S. agalactiae antigenic stimulates antibody induction of the immune system, in which case is the body's defense system against mastitis disease in cattle. In this study, irradiation of gamma ray is used to attenuate the pathogenicity of bacteria by reducing S. agalactiae antigenic characteristic. Previous research, in irradiation dose orientation before antigenic protein isolation of S. agalactiae, indicated that irradiation lethal dose to 50% (LD50) is 17 Gy. The characterization of S. agalactiae bacteria isolate using SDS-page method results in no significance different between irradiated and non-irradiated group, which indicated by MW range 75 - 100 kDa base on marker standard which used, or 99 kDa by the linier equation of Y = 11,60 - 0.05X (where Y = bands distance; X = MW standard protein); r2 = 0.99. In conclusion, 17 Gy irradiation dose does not impair antigenic property of S. agalactiae and therefore, can be applied to produce base material of irradiated vaccine for mastitis. (author)

  18. Prediction of Bacterial Virulent Proteins with Composition Moment Vector Feature Encoding Method

    Directory of Open Access Journals (Sweden)

    Gök Murat

    2016-01-01

    Full Text Available Prediction of bacterial virulent proteins is critical for vaccine development and understanding of virulence mechanisms in pathogens. For this purpose, a number of feature encoding methods based on sequences and evolutionary information of a given protein have been proposed and applied with some classifier algorithms so far. In this paper, we performed composition moment vector (CMV, which includes information about both composition and position of amino acid in the protein sequence to predict bacterial virulent proteins. The tests were validated in three different independent datasets. Experimental results show that CMV feature encoding method leads to better classification performance in terms of accuracy, sensitivity, f-measure and the Matthews correlation coefficient (MCC scores on diverse classifiers.

  19. Polyacrylamide Slab Gel Electrophoresis of Soluble Proteins for Studies of Bacterial Floras

    OpenAIRE

    Moore, W. E. C.; Hash, D E; Holdeman, Lillian V.; Cato, Elizabeth P.

    1980-01-01

    A polyacrylamide slab gel electrophoresis procedure was used to compare cellular proteins from bacterial isolates of gingival crevice floras. Isolates with identical protein patterns consistently were shown to be members of the same species. When used to screen isolates, the procedure reduced total analytical time and expense without sacrificing accuracy, and it provided additional verification of the identity of strains characterized by conventional phenotypic tests.

  20. Rapid and widely disseminated acute phase protein response after experimental bacterial infection of pigs

    OpenAIRE

    Skovgaard, Kerstin; Mortensen, Shila; Boye, Mette; Poulsen, Karin T.; Campbell, Fiona M; Eckersall, P. David; Heegaard, Peter M.H.

    2009-01-01

    International audience The acute phase protein response is a well-described generalized early host response to tissue injury, inflammation and infection, observed as pronounced changes in the concentrations of a number of circulating serum proteins. The biological function of this response and its interplay with other parts of innate host defence reactions remain somewhat elusive. In order to gain new insight into this early host defence response in the context of bacterial infection we st...

  1. Procalcitonin and C-reactive protein as markers of bacterial infection in patients with solid tumours

    DEFF Research Database (Denmark)

    Diness, Laura V; Maraldo, Maja V; Mortensen, Christiane E;

    2014-01-01

    infection. In this prospective study, we wanted to investigate the value of procalcitonin (PCT) compared with C-reactive protein (CRP) as an indicator of bacterial infection in adult patients with solid tumours. METHODS: A total of 41 patients with solid tumours admitted to hospital due to fever or clinical...

  2. Niobium Uptake and Release by Bacterial Ferric Ion Binding Protein

    Directory of Open Access Journals (Sweden)

    Yanbo Shi

    2010-01-01

    Full Text Available Ferric ion binding proteins (Fbps transport FeIII across the periplasm and are vital for the virulence of many Gram negative bacteria. Iron(III is tightly bound in a hinged binding cleft with octahedral coordination geometry involving binding to protein side chains (including tyrosinate residues together with a synergistic anion such as phosphate. Niobium compounds are of interest for their potential biological activity, which has been little explored. We have studied the binding of cyclopentadienyl and nitrilotriacetato NbV complexes to the Fbp from Neisseria gonorrhoeae by UV-vis spectroscopy, chromatography, ICP-OES, mass spectrometry, and Nb K-edge X-ray absorption spectroscopy. These data suggest that NbV binds strongly to Fbp and that a dinuclear NbV centre can be readily accommodated in the interdomain binding cleft. The possibility of designing niobium-based antibiotics which block iron uptake by pathogenic bacteria is discussed.

  3. TRPA1 channels mediate acute neurogenic inflammation and pain produced by bacterial endotoxins

    Science.gov (United States)

    Meseguer, Victor; Alpizar, Yeranddy A.; Luis, Enoch; Tajada, Sendoa; Denlinger, Bristol; Fajardo, Otto; Manenschijn, Jan-Albert; Fernández-Peña, Carlos; Talavera, Arturo; Kichko, Tatiana; Navia, Belén; Sánchez, Alicia; Señarís, Rosa; Reeh, Peter; Pérez-García, María Teresa; López-López, José Ramón; Voets, Thomas; Belmonte, Carlos; Talavera, Karel; Viana, Félix

    2014-01-01

    Gram-negative bacterial infections are accompanied by inflammation and somatic or visceral pain. These symptoms are generally attributed to sensitization of nociceptors by inflammatory mediators released by immune cells. Nociceptor sensitization during inflammation occurs through activation of the Toll-like receptor 4 (TLR4) signalling pathway by lipopolysaccharide (LPS), a toxic by-product of bacterial lysis. Here we show that LPS exerts fast, membrane delimited, excitatory actions via TRPA1, a transient receptor potential cation channel that is critical for transducing environmental irritant stimuli into nociceptor activity. Moreover, we find that pain and acute vascular reactions, including neurogenic inflammation (CGRP release) caused by LPS are primarily dependent on TRPA1 channel activation in nociceptive sensory neurons, and develop independently of TLR4 activation. The identification of TRPA1 as a molecular determinant of direct LPS effects on nociceptors offers new insights into the pathogenesis of pain and neurovascular responses during bacterial infections and opens novel avenues for their treatment.

  4. Proteinaceous determinants of surface colonization in bacteria: Bacterial adhesion and biofilm formation from a protein secretion perspective

    Directory of Open Access Journals (Sweden)

    MickaelDesvaux

    2013-10-01

    Full Text Available Bacterial colonization of biotic or abiotic surfaces results from two quite distinct physiological processes, namely bacterial adhesion and biofilm formation. Broadly speaking, a biofilm is defined as the sessile development of microbial cells. Biofilm formation arises following bacterial adhesion but not all single bacterial cells adhering reversibly or irreversibly engage inexorably into a sessile mode of growth. Among molecular determinants promoting bacterial colonization, surface proteins are the most functionally diverse active components. To be present on the bacterial cell surface, though, a protein must be secreted in the first place. Considering the close association of secreted proteins with their cognate secretion systems, the secretome (which refers both to the secretion systems and their protein substrates is a key concept to apprehend the protein secretion and related physiological functions. The protein secretion systems are here considered in light of the differences in the cell-envelope architecture between diderm-LPS (archetypal Gram-negative, monoderm (archetypal Gram-positive and diderm-mycolate (archetypal acid-fast bacteria. Besides, their cognate secreted proteins engaged in the bacterial colonization process are regarded from single protein to supramolecular protein structure as well as the non-classical protein secretion. This state-of-the-art on the complement of the secretome (the secretion systems and their cognate effectors involved in the surface colonization process in diderm-LPS and monoderm bacteria paves the way for future research directions in the field.

  5. The Population Structure of Antibiotic-Producing Bacterial Symbionts of Apterostigma dentigerum Ants: Impacts of Coevolution and Multipartite Symbiosis

    OpenAIRE

    Caldera, Eric J.; Currie, Cameron R

    2012-01-01

    Fungus-growing ants (Attini) are part of a complex symbiosis with Basidiomycetous fungi, which the ants cultivate for food, Ascomycetous fungal pathogens (Escovopsis), which parasitize cultivars, and Actinobacteria, which produce antibiotic compounds that suppress pathogen growth. Earlier studies that have characterized the association between attine ants and their bacterial symbionts have employed broad phylogenetic approaches, with conclusions ranging from a diffuse coevolved mutualism to n...

  6. Characterization of Geographically Distinct Bacterial Communities Associated with Coral Mucus Produced by Acropora spp. and Porites spp.

    OpenAIRE

    McKew, B.A.; Dumbrell, A.J.; Daud, S. D.; Hepburn, L; Thorpe, E.; Mogensen, L.; Whitby, C.

    2012-01-01

    Acropora and Porites corals are important reef builders in the Indo-Pacific and Caribbean. Bacteria associated with mucus produced by Porites spp. and Acropora spp. from Caribbean (Punta Maroma, Mexico) and Indo-Pacific (Hoga and Sampela, Indonesia) reefs were determined. Analysis of pyrosequencing libraries showed that bacterial communities from Caribbean corals were significantly more diverse (H′, 3.18 to 4.25) than their Indonesian counterparts (H′, 2.54 to 3.25). Dominant taxa were Gammap...

  7. Exploration and conservation of bacterial genetic resources as bacteriocin producing inhibitory microorganisms to pathogen bacteria in livestock

    OpenAIRE

    2013-01-01

    Exploration and conservation of microorganisms producing bacteriocin was done as the primary study towards the collection of potential bacteria and its application in improving livestock health condition and inhibit food borne pathogens. Diferent kinds of samples such as beef cattle rectal swab, rumen fluids, cow’s milk, chicken gut content, goat’s milk were collected at Bogor cattle slaughter houses, poultry slaughter houses, dairy cattle and goat farms. A total of 452 bacterial isolates con...

  8. Effects of Interactions of Auxin-Producing Bacteria and Bacterial-Feeding Nematodes on Regulation of Peanut Growths

    OpenAIRE

    Li Xu; Wensi Xu; Ying Jiang; Feng Hu; Huixin Li

    2015-01-01

    The influences of an IAA (indole-3-acetic acid)-producing bacterium (Bacillus megaterium) and two bacterial-feeding nematodes (Cephalobus sp. or Mesorhabditis sp.) on the growth of peanut (Arachis hypogaea L. cv. Haihua 1) after various durations of time were investigated in natural soils. The addition of bacteria and nematodes and incubation time all significantly affected plant growth, plant root growth, plant nutrient concentrations, soil nutrient concentrations, soil microorganisms and so...

  9. Identification of a New Marine Bacterial Strain SD8 and Optimization of Its Culture Conditions for Producing Alkaline Protease

    OpenAIRE

    Hongxia Cui; Muyang Yang; Liping Wang; Xian, Cory J.

    2015-01-01

    While much attention has been given to marine microorganisms for production of enzymes, which in general are relatively more stable and active compared to those from plants and animals, studies on alkaline protease production from marine microorganisms have been very limited. In the present study, the alkaline protease producing marine bacterial strain SD8 isolated from sea muds in the Geziwo Qinhuangdao sea area of China was characterized and its optimal culture conditions were investigated....

  10. Alkylpyrazines produced by bacterial spoilage of heat-treated and gamma-irradiated coconut

    International Nuclear Information System (INIS)

    This paper reports the sterilisation of coconut by autoclaving or gamma irradiation, followed by storage in water at 250 C for 8 weeks. Bacillus subtilis developed after storage in water. The volatile compounds formed as a result of bacterial activity were extracted and identified. (U.K.)

  11. Bacterial effector binding to ribosomal protein s3 subverts NF-kappaB function.

    Directory of Open Access Journals (Sweden)

    Xiaofei Gao

    2009-12-01

    Full Text Available Enteric bacterial pathogens cause food borne disease, which constitutes an enormous economic and health burden. Enterohemorrhagic Escherichia coli (EHEC causes a severe bloody diarrhea following transmission to humans through various means, including contaminated beef and vegetable products, water, or through contact with animals. EHEC also causes a potentially fatal kidney disease (hemolytic uremic syndrome for which there is no effective treatment or prophylaxis. EHEC and other enteric pathogens (e.g., enteropathogenic E. coli (EPEC, Salmonella, Shigella, Yersinia utilize a type III secretion system (T3SS to inject virulence proteins (effectors into host cells. While it is known that T3SS effectors subvert host cell function to promote diarrheal disease and bacterial transmission, in many cases, the mechanisms by which these effectors bind to host proteins and disrupt the normal function of intestinal epithelial cells have not been completely characterized. In this study, we present evidence that the E. coli O157:H7 nleH1 and nleH2 genes encode T3SS effectors that bind to the human ribosomal protein S3 (RPS3, a subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB transcriptional complexes. NleH1 and NleH2 co-localized with RPS3 in the cytoplasm, but not in cell nuclei. The N-terminal region of both NleH1 and NleH2 was required for binding to the N-terminus of RPS3. NleH1 and NleH2 are autophosphorylated Ser/Thr protein kinases, but their binding to RPS3 is independent of kinase activity. NleH1, but not NleH2, reduced the nuclear abundance of RPS3 without altering the p50 or p65 NF-kappaB subunits or affecting the phosphorylation state or abundance of the inhibitory NF-kappaB chaperone IkappaBalpha NleH1 repressed the transcription of a RPS3/NF-kappaB-dependent reporter plasmid, but did not inhibit the transcription of RPS3-independent reporters. In contrast, NleH2 stimulated RPS3-dependent transcription, as well

  12. Mutations in the bacterial ribosomal protein l3 and their association with antibiotic resistance

    DEFF Research Database (Denmark)

    Klitgaard, Rasmus N; Ntokou, Eleni; Nørgaard, Katrine;

    2015-01-01

    Different groups of antibiotics bind to the peptidyl transferase center (PTC) in the large subunit of the bacterial ribosome. Resistance to these groups of antibiotics has often been linked with mutations or methylations of the 23S rRNA. In recent years, there has been a rise in the number of...... studies where mutations have been found in the ribosomal protein L3 in bacterial strains resistant to PTC-targeting antibiotics but there is often no evidence that these mutations actually confer antibiotic resistance. In this study, a plasmid exchange system was used to replace plasmid-carried wild...

  13. Effect of Dietary Protein Levels on Composition of Odorous Compounds and Bacterial Ecology in Pig Manure

    OpenAIRE

    Cho, Sungback; Hwang, Okhwa; Park, Sungkwon

    2015-01-01

    This study was performed to investigate the effect of different levels of dietary crude protein (CP) on composition of odorous compounds and bacterial communities in pig manure. A total of 48 male pigs (average initial body weight 45 kg) fed diets containing three levels of dietary CP (20%, 17.5%, and 15%) and their slurry samples were collected from the pits under the floor every week for one month. Changes in composition of odorous compounds and bacterial communities were analyzed by gas ch...

  14. Excretion of purine base derivatives after intake of bacterial protein meal in pigs

    DEFF Research Database (Denmark)

    Hellwing, Anne Louise Frydendahl; Tauson, Anne-Helene; Skrede, A.

    2007-01-01

    Bacterial protein meal has a high content ofprotein but also of RNA and DNA. Sixteen barrows were allocated to four diets containing increasing levels of bacterial protein meal (BPM), from weaning to 80 kg live weight, to evaluate whether the RNA and DNA contents of BPM influenced the retention...... of nitrogen. It was hypothesised that an increased intake of RNA and DNA would lead to an increased urinary excretion of purine base derivatives and increased plasma concentrations. Retention of nitrogen was unaffected by dietary content of BPM (P=0.08) and the urinary excretion of purine base derivatives...... increased with increasing dietary content of BPM. No differences in fasting plasma concentration of uric acid, xanthine and hypoxanthine were observed. It can therefore be concluded that increasing levels of dietary BPM maintained protein accretion and led to changes in excretion of purine detrivatices...

  15. Effect of sulfur analogue of lysine on bacterial protein biosynthesis

    International Nuclear Information System (INIS)

    S-(beta-Aminoethyl)-L-cysteine, a sulfur analogue of lysine inhibited strongly growth of Escherichia coli A-19, and weakly that of Corynebacterium sp. isolated from soil, but did not inhibit growth of Aerobacter aerogenes. In Corynebacterium sp. the inhibitory effect was markedly enhanced in the presence of L-threonine. The inhibition of growth by S-(beta-aminoethyl)-L-cysteine was rapidly reversed by the addition of L-lysine. S-(beta-Aminoethyl)-L-cysteine inhibited protein synthesis and the activity of lysyl-tRNA synthetase from E. coli and A. aerogenes. All the other lysine analogues tested inhibited the activity of enzyme, but S-(beta-aminoethyl)-L-cysteine derivatives, S-(beta-N-acetyl-aminoethyl)-L-cysteine and S-(beta-aminoethyl)-alpha-N-acetyl-L-cysteine were not effective. (auth.)

  16. Flexibility in targeting and insertion during bacterial membrane protein biogenesis

    International Nuclear Information System (INIS)

    The biogenesis of Escherichia coli inner membrane proteins (IMPs) is assisted by targeting and insertion factors such as the signal recognition particle (SRP), the Sec-translocon and YidC with translocation of (large) periplasmic domains energized by SecA and the proton motive force (pmf). The use of these factors and forces is probably primarily determined by specific structural features of an IMP. To analyze these features we have engineered a set of model IMPs based on endogenous E. coli IMPs known to follow distinct targeting and insertion pathways. The modified model IMPs were analyzed for altered routing using an in vivo protease mapping approach. The data suggest a facultative use of different combinations of factors

  17. Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily.

    Science.gov (United States)

    Matsunaga, James; Barocchi, Michele A; Croda, Julio; Young, Tracy A; Sanchez, Yolanda; Siqueira, Isadora; Bolin, Carole A; Reis, Mitermayer G; Riley, Lee W; Haake, David A; Ko, Albert I

    2003-08-01

    Proteins with bacterial immunoglobulin-like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig-like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudogene, ligC, were identified. The ligA and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy-terminal non-repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High-pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis. PMID:12890019

  18. Effects of Iron on Hydrogen-producing Capacity,Hydrogenase and NADH-fd Reductase Activities of a Fermentative Hydrogen-producing Bacterial Strain B49

    Institute of Scientific and Technical Information of China (English)

    Wang Xiangjing(王相晶); Ren Nanqi; Xiang Wensheng

    2004-01-01

    Iron plays an important role in hydrogen production, cell growth, hydrogenase and NADH-fd reductase activities of hydrogen-producing bacterial strain B49 (AF481148 in EMBL). At the end of fermentation from 10 g/L glucose, for the culture containing 10 mg/L FeSO4*7H2O the cell growth in terms of optical density (OD) at 600nm was 1.13, the ratio of ethanol amount (mg/L) to acetate amount (mg/L) was 1.55, and the accumulated hydrogen volume was 1816.3 ml H2/L culture; whereas for the culture of 80 mg/L FeSO4*7H2O OD600nm was increased to 1.34, the accumulated hydrogen volume was increased to 2360.5 ml H2/L culture, and the ratio of ethanol amount (mg/L) to acetate amount (mg/L) decreased to 1.31. Moreover, the iron addition to the medium at different fermentation time could affect hydrogen-producing ability. However, the later the addition time of FeSO4*7H2O was postponed, the less the effect on hydrogen evolution was. In the course of fermentation, the specific activities of hydrogenase and NADH-fd reductase of hydrogen-producing bacterial strain B49 decreased with the consumption of iron.

  19. No evidence for a culturable bacterial tetrodotoxin producer in Pleurobranchaea maculata (Gastropoda: Pleurobranchidae) and Stylochoplana sp. (Platyhelminthes: Polycladida).

    Science.gov (United States)

    Salvitti, Lauren R; Wood, Susanna A; McNabb, Paul; Cary, Stephen Craig

    2015-02-01

    Tetrodotoxin (TTX) is a potent neurotoxin found in the tissues of many taxonomically diverse organisms. Its origin has been the topic of much debate, with suggestions including endogenous production, acquisition through diet, and symbiotic bacterial synthesis. Bacterial production of TTX has been reported in isolates from marine biota, but at lower than expected concentrations. In this study, 102 strains were isolated from Pleurobranchaea maculata (Opisthobranchia) and Stylochoplana sp. (Platyhelminthes). Tetrodotoxin production was tested utilizing a recently developed sensitive method to detect the C9 base of TTX via liquid chromatography-mass spectrometry. Bacterial strains were characterized by sequencing a region of the 16S ribosomal RNA gene. To account for the possibility that TTX is produced by a consortium of bacteria, a series of experiments using marine broth spiked with various P. maculata tissues were undertaken. Sixteen unique strains from P. maculata and one from Stylochoplana sp. were isolated, representing eight different genera; Pseudomonadales, Actinomycetales, Oceanospirillales, Thiotrichales, Rhodobacterales, Sphingomonadales, Bacillales, and Vibrionales. Molecular fingerprinting of bacterial communities from broth experiments showed little change over the first four days. No C9 base or TTX was detected in isolates or broth experiments (past day 0), suggesting a culturable microbial source of TTX in P. maculata and Stylochoplana sp. is unlikely. PMID:25635464

  20. No Evidence for a Culturable Bacterial Tetrodotoxin Producer in Pleurobranchaea maculata (Gastropoda: Pleurobranchidae and Stylochoplana sp. (Platyhelminthes: Polycladida

    Directory of Open Access Journals (Sweden)

    Lauren R. Salvitti

    2015-01-01

    Full Text Available Tetrodotoxin (TTX is a potent neurotoxin found in the tissues of many taxonomically diverse organisms. Its origin has been the topic of much debate, with suggestions including endogenous production, acquisition through diet, and symbiotic bacterial synthesis. Bacterial production of TTX has been reported in isolates from marine biota, but at lower than expected concentrations. In this study, 102 strains were isolated from Pleurobranchaea maculata (Opisthobranchia and Stylochoplana sp. (Platyhelminthes. Tetrodotoxin production was tested utilizing a recently developed sensitive method to detect the C9 base of TTX via liquid chromatography—mass spectrometry. Bacterial strains were characterized by sequencing a region of the 16S ribosomal RNA gene. To account for the possibility that TTX is produced by a consortium of bacteria, a series of experiments using marine broth spiked with various P. maculata tissues were undertaken. Sixteen unique strains from P. maculata and one from Stylochoplana sp. were isolated, representing eight different genera; Pseudomonadales, Actinomycetales, Oceanospirillales, Thiotrichales, Rhodobacterales, Sphingomonadales, Bacillales, and Vibrionales. Molecular fingerprinting of bacterial communities from broth experiments showed little change over the first four days. No C9 base or TTX was detected in isolates or broth experiments (past day 0, suggesting a culturable microbial source of TTX in P. maculata and Stylochoplana sp. is unlikely.

  1. Chemical Changes in Proteins Produced by Thermal Processing.

    Science.gov (United States)

    Dutson, T. R.; Orcutt, M. W.

    1984-01-01

    Discusses effects of thermal processing on proteins, focusing on (1) the Maillard reaction; (2) heat denaturation of proteins; (3) aggregation, precipitation, gelation, and degradation; and (4) other thermally induced protein reactions. Also discusses effects of thermal processing on muscle foods, egg proteins, fruits and vegetables, and cereal…

  2. Biodegradation of Leonardite by an alkali-producing bacterial community and characterization of the degraded products.

    Science.gov (United States)

    Gao, Tong-Guo; Jiang, Feng; Yang, Jin-Shui; Li, Bao-Zhen; Yuan, Hong-Li

    2012-03-01

    In this study, three bacterial communities were obtained from 12 Leonardite samples with the aim of identifying a clean, effective, and economic technique for the dissolution of Leonardite, a type of low-grade coal, in the production of humic acid (HA). The biodegradation ability and characteristics of the degraded products of the most effective bacterial community (MCSL-2), which degraded 50% of the Leonardite within 21 days, were further investigated. Analyses of elemental composition, (13)C NMR, and Fourier transform infrared revealed that the contents of C, O, and aliphatic carbon were similar in biodegraded humic acid (bHA) and chemically (alkali) extracted humic acid (cHA). However, the N and carboxyl carbon contents of bHA was higher than that of cHA. Furthermore, a positive correlation was identified between the degradation efficiency and the increasing pH of the culture medium, while increases of manganese peroxidase and esterase activities were also observed. These data demonstrated that both alkali production and enzyme reactions were involved in Leonardite solubilization by MCSL-2, although the former mechanism predominated. No fungus was observed by microscopy. Only four bacterial phylotypes were recognized, and Bacillus licheniformis-related bacteria were identified as the main group in MCSL-2 by analysis of amplified 16S rRNA genes, thus demonstrating that Leonardite degradation ability has a limited distribution in bacteria. Hormone-like bioactivities of bHA were also detected. In this study, a bacterial community capable of Leonardite degradation was identified and the products characterized. These data implicate the use of such bacteria for the exploitation of Leonardite as a biofertilizer. PMID:22075634

  3. Identification of beta-subunit of bacterial RNA-polymerase--a non-species-specific bacterial protein--as target of antibodies in primary biliary cirrhosis.

    Science.gov (United States)

    Roesler, Kai-Wolfgang; Schmider, Wolfgang; Kist, Manfred; Batsford, Stephen; Schiltz, Emile; Oelke, Mathias; Tuczek, Anja; Dettenborn, Therese; Behringer, Dirk; Kreisel, Wolfgang

    2003-03-01

    Several observations suggest that bacteria induce autoimmunity in primary biliary cirrhosis (PBC). Since no PBC-specific bacterial species could be identified, it can be speculated that the triggers are non-species-specific bacterial proteins. This hypothesis would imply that several or even all bacterial species can trigger PBC. Therefore, we investigated whether PBC exhibits immune reactions to non-species-specific bacterial antigens. Yersinia enterocolitica O3 was screened for the presence of proteins that were labeled by immunoblotting using PBC sera. We focused our investigations on a 160-kDa protein, which was further enriched and characterized by partial N-terminal amino acid sequencing. The prevalence of antibodies to this protein was determined by immunoblotting in a variety of diseases. The 160-kDa protein was identified as the beta-subunit of bacterial RNA-polymerase, a highly conserved bacterial protein with a very high degree of sequence identity among all bacterial species. Antibodies to the beta-subunit of bacterial RNA polymerase were specific for this protein. Until now no mammalian protein could be found that cross-reacts with these antibodies. The prevalence of antibodies to the beta-subunit of bacterial RNA polymerase (ARPA) using the protein from Yersinia enterocolitica O3 (serum dilution 1:1000) was: healthy controls (HC, N = 101) 7.9%, primary biliary cirrhosis (PBC, N = 61) 32.8%, autoimmune hepatitis type 1 (AIH, N = 46) 26.1%, alcoholic liver cirrhosis (ALC, N = 44) 9.1%, Crohn's disease (CD, N = 38) 7.9%, ulcerative colitis (UC, N = 24) 8.3%, primary sclerosing cholangitis + UC (PSC/UC, N = 11) 0%, acute yersiniosis (Yers, N = 36) 19.4%, acute infection with Campylobacter jejuni (Camp, N = 10) 0%, acute Q-fever (QF, N = 16) 6.25%, chronic hepatitis C (HCV, N = 39) 7.7%, c-ANCA-positive vasculitis (Vasc, N = 40) 15%, systemic lupus erythematosus (SLE, N = 28) 10.7%, and malaria tropica (MT, N = 24) 16.7%. There was no significant

  4. Resistance to ketolide antibiotics by coordinated expression of rRNA methyltransferases in a bacterial producer of natural ketolides

    DEFF Research Database (Denmark)

    Almutairi, Mashal M; Park, Sung Ryeol; Rose, Simon;

    2015-01-01

    Ketolides are promising new antimicrobials effective against a broad range of Gram-positive pathogens, in part because of the low propensity of these drugs to trigger the expression of resistance genes. A natural ketolide pikromycin and a related compound methymycin are produced by Streptomyces...... activation by ketolide antibiotics. The resistance genes and the induction mechanism remain fully functional when transferred to heterologous bacterial hosts. The anticipated wide use of ketolide antibiotics could promote horizontal transfer of these highly efficient resistance genes to pathogens. Taken...... together, these findings emphasized the need for surveillance of pikR1/pikR2-based bacterial resistance and the preemptive development of drugs that can remain effective against the ketolide-specific resistance mechanism....

  5. The use of fed batch approaches to maximise yields in bacterial fermentation and protein expression

    International Nuclear Information System (INIS)

    A fermentation facility for the scale up of bacterial and yeast fermentations has been set up at the University of Queensland under the auspices of the ARC Special Research Centre for Functional and Applied Genomics. A major application is the production of recombinant proteins for determination of tertiary structures by X-ray crystallography or nuclear magnetic resonance. For this purpose, large amounts of protein arc needed and the yield from a single fermentation run is crucial to success within constrained laboratory budgets. To achieve maximal yields we are optimising fed batch approaches in bacterial fermentation. Fed batch offers many advantages over batch cultures. Coupled with the ability to monitor online the internal conditions of the fermentation including pH and dissolved oxygen and stirrer cascading functions it is possible to ensure that the nutritional environment of the microorganism is optimised for its growth and or for optimal protein expression. The poster will describe some of our experience in setting up fed batch fermentations and successful applications of fed batches to increasing protein yield. It will also outline services that are available to academic groups outside the University of Queensland For structure determination and functional studies, the production of radiolabelled proteins can also be an advantage. We will describe initial experiments aimed at coupling the principles of fed batch fermentation to the introduction of carbon or nitrogen isotopes into the recombinant protein

  6. Phase variation of Opa proteins of Neisseria meningitidis and the effects of bacterial transformation

    Indian Academy of Sciences (India)

    Manish Sadarangani; J Claire Hoe; Katherine Makepeace; Peter Van Der Ley; Andrew J Pollard

    2016-03-01

    Opa proteins are major proteins involved in meningococcal colonization of the nasopharynx and immune interactions. Opa proteins undergo phase variation (PV) due to the presence of the 5′-CTCTT-3′ coding repeat (CR) sequence. The dynamics of PV of meningococcal Opa proteins is unknown. Opa PV, including the effect of transformation on PV, was assessed using a panel of Opa-deficient strains of Neisseria meningitidis. Analysis of Opa expression from UK disease-causing isolates was undertaken. Different opagenes demonstrated variable rates of PV, between 6.4 ×10–4 and 6.9 ×10–3 per cell per generation. opa genes with a longer CR tract had a higher rate of PV (r2=0.77, p=0.1212). Bacterial transformation resulted in a 180-fold increase in PV rate. The majority of opagenes in UK disease isolates (315/463, 68.0%) were in the ‘on’ phase, suggesting the importance of Opa proteins during invasive disease. These data provide valuable information for the first time regarding meningococcal Opa PV. The presence of Opa PV in meningococcal populations and high expression of Opa among invasive strains likely indicates the importance of this protein in bacterial colonization in the human nasopharynx. These findings have potential implications for development of vaccines derived from meningococcal outer membranes.

  7. Zoosporic plant pathogens produce bacterial autoinducer-2 that affects Vibrio harveyi quorum sensing

    Science.gov (United States)

    Kong, Ping; Lee, Bobby W.K.; Zhou, Zhaohui Sunny; Hong, Chuanxue

    2009-01-01

    The frequent co-isolation of bacteria with Phytophthora and Pythium species suggests possible interspecies communication. Zoospore free fluids (ZFF) from bacteria-free and nutrient-depleted zoospore suspensions were examined to investigate production of autoinducer-2 (AI-2), a bacterial interspecies signal molecule, by zoosporic oomycetes. ZFF from P. nicotianae, P. sojae and Py. aphanidermatum triggered luminescence of Vibrio harveyi AI-2 reporter, indicating the presence of AI-2 in zoospore extracellular products and the potential of cross-kingdom communication between oomycetes and bacteria. Production of AI-2 by zoospores was confirmed by chemical assays. These results provide new insight into the physiology and ecology of oomycetes. PMID:20002192

  8. Crystal structure of the Campylobacter jejuni Cj0090 protein reveals a novel variant of the immunoglobulin fold among bacterial lipoproteins.

    Science.gov (United States)

    Paek, Seonghee; Kawai, Fumihiro; Choi, Kyoung-Jae; Yeo, Hye-Jeong

    2012-12-01

    Bacterial lipoproteins play an important role in bacterial pathogenesis and physiology. The genome of Campylobacter jejuni, a major foodborn pathogen, is predicted to contain over 20 lipoproteins. However, the functions of the majority of C. jejuni lipoproteins remain unknown. The Cj0090 protein is encoded by a lipoprotein operon composed of cj0089, cj0090, and cj0091. Here, we report the crystal structure of Cj0090 at 1.9 Å resolution, revealing a novel variant of the immunoglobulin fold with β-sandwich architecture. The structure suggests that Cj0090 may be involved in protein-protein interactions, consistent with a possible role for bacterial lipoproteins. PMID:22987763

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

    OpenAIRE

    Cashman Kathleen A; Ferro Philip J; Sampey Darryl B; Goba Augustine; Fair Joseph N; Matschiner Alex; Branco Luis M; Schoepp Randal J; Tesh Robert B; Bausch Daniel G; Garry Robert F; Guttieri Mary C

    2008-01-01

    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 ...

  10. In vitro estimation of rumen protein degradability using 35S to label the bacterial mass

    International Nuclear Information System (INIS)

    An experiment was carried out in order to simplify a previously developed 15N-method for in vitro estimation of rumen protein degradability. Casein (Cas), whole soybeans (Sb) heated at 120oC for 20 min (SbTherm) and sunflower (Sfl) were incubated at 39oC for 4 hours in a water bathshaker with the following media: McDougall's buffer, strained and enriched with particle associated bacteria rumen fluid (2:1), rapidly (maltose, sucrose, glucose) and more slowly (pectin, soluble starch) degradable carbohydrates with final concentration of 815 mg/100 ml and 21.7 μCi/100 ml of35S (from Na235SO4). After the incubation had been ceased, a bacterial fraction was isolated through differential centrifugation and specific activity of bacterial (Bac) and high speed total solids (TS) nitrogen was measured. The ratio was used to calculate bacterial mass in TS and through the Kjeldahl nitrogen concentration in TS - the net bacterial growth (against control vessels without protein). The level of ammonia-N in the supernate after blank correction was used to find the ammonia-N released from protein degradation. The data showed that the rate (and extend) of degradation for the Cas (as a standard protein) was lower compared to those obtained through the 15N-method but it was higher than the rate derived through another in vitro method. The Cas equivalent of the Sb was higher than the figure we found in a previous experiment with solvent extracted soybean meal suggesting that the 35S-method underestimated the degradability of the Cas. After being tested on a wider range of foodstuffs, the proposed 35S-method might be considered as an alternative procedure which is less laborous than the 15N-method. (author)

  11. Dependence of Bacterial Protein Adhesins on Toll-Like Receptors for Proinflammatory Cytokine Induction

    OpenAIRE

    Hajishengallis, George; Martin, Michael; Sojar, Hakimuddin T.; Sharma, Ashu; Schifferle, Robert E.; DeNardin, Ernesto; Russell, Michael W.; Genco, Robert J.

    2002-01-01

    Toll-like receptors (TLRs) are important signal transducers that mediate inflammatory reactions induced by microbes through pattern recognition of virulence molecules such as lipopolysaccharide (LPS) and lipoproteins. We investigated whether proinflammatory cytokine responses induced by certain bacterial protein adhesins may also depend on TLRs. In differentiated THP-1 mononuclear cells stimulated by LPS-free recombinant fimbrillin (rFimA) from Porphyromonas gingivalis, cytokine release was a...

  12. Manipulating the glycosylation pathway in bacterial and lower eukaryotes for production of therapeutic proteins

    DEFF Research Database (Denmark)

    Anyaogu, Diana Chinyere; Mortensen, Uffe Hasbro

    2015-01-01

    The medical use of pharmaceutical proteins is rapidly increasing and cheap, fast and efficient production is therefore attractive. Microbial production hosts are promising candidates for development and production of pharmaceutical proteins. However, as most therapeutic proteins are secreted prot...... to produce proteins with humanlike glycan structures setting the stage for production of pharmaceutical proteins in bacteria, yeasts and algae.......The medical use of pharmaceutical proteins is rapidly increasing and cheap, fast and efficient production is therefore attractive. Microbial production hosts are promising candidates for development and production of pharmaceutical proteins. However, as most therapeutic proteins are secreted...... proteins, they are frequently N-glycosylated. This hampers production in microbes as these hosts glycosylate proteins differently. The resulting products may therefore be immunogenic, unstable and show reduced efficacy. Recently, successful glycoengineering of microbes has demonstrated that it is possible...

  13. Structural basis of a rationally rewired protein-protein interface critical to bacterial signaling.

    Science.gov (United States)

    Podgornaia, Anna I; Casino, Patricia; Marina, Alberto; Laub, Michael T

    2013-09-01

    Two-component signal transduction systems typically involve a sensor histidine kinase that specifically phosphorylates a single, cognate response regulator. This protein-protein interaction relies on molecular recognition via a small set of residues in each protein. To better understand how these residues determine the specificity of kinase-substrate interactions, we rationally rewired the interaction interface of a Thermotoga maritima two-component system, HK853-RR468, to match that found in a different two-component system, Escherichia coli PhoR-PhoB. The rewired proteins interacted robustly with each other, but no longer interacted with the parent proteins. Analysis of the crystal structures of the wild-type and mutant protein complexes and a systematic mutagenesis study reveal how individual mutations contribute to the rewiring of interaction specificity. Our approach and conclusions have implications for studies of other protein-protein interactions and protein evolution and for the design of novel protein interfaces. PMID:23954504

  14. Recombinant expression and purification of "virus-like" bacterial encapsulin protein cages.

    Science.gov (United States)

    Rurup, W Frederik; Cornelissen, Jeroen J L M; Koay, Melissa S T

    2015-01-01

    Ultracentrifugation, particularly the use of sucrose or cesium chloride density gradients, is a highly reliable and efficient technique for the purification of virus-like particles and protein cages. Since virus-like particles and protein cages have a unique size compared to cellular macromolecules and organelles, the rate of migration can be used as a tool for purification. Here we describe a detailed protocol for the purification of recently discovered virus-like assemblies called bacterial encapsulins from Thermotoga maritima and Brevibacterium linens. PMID:25358773

  15. Cell-penetrating peptides mediated protein cross-membrane delivery and its use in bacterial vector vaccine.

    Science.gov (United States)

    Ma, Jimei; Xu, Jinmei; Guan, Lingyu; Hu, Tianjian; Liu, Qin; Xiao, Jingfan; Zhang, Yuanxing

    2014-07-01

    It is an attractive strategy to develop a recombinant bacterial vector vaccine by expressing exogenous protective antigen to induce the immune response, and the main concern is how to enhance the cellular internalization of antigen produced by bacterial vector. Cell-penetrating peptides (CPPs) are short cationic/amphipathic peptides which facilitate cellular uptake of various molecular cargoes and therefore have great potentials in vector vaccine design. In this work, eleven different CPPs were fused to the C-terminus of EGFP respectively, and the resultant EGFP-CPP fusion proteins were expressed and purified to assay their cross-membrane transport in macrophage J774 A.1 cells. Among the tested CPPs, TAT showed an excellent capability to deliver the cargo protein EGFP into cytoplasm. In order to establish an efficient antigen delivery system in Escherichia coli, the EGFP-TAT synthesis circuit was combined with an in vivo inducible lysis circuit PviuA-E in E. coli to form an integrated antigen delivery system, the resultant E. coli was proved to be able to lyse upon the induction of a mimic in vivo signal and thus release intracellular EGFP-TAT intensively, which were assumed to undergo a more efficient intracellular delivery by CPP to evoke protective immune responses. Based on the established antigen delivery system, the protective antigen gene flgD from an invasive intracellular fish pathogen Edwardsiella tarda EIB202, was applied to establish an E. coli recombinant vector vaccine. This E. coli vector vaccine presented superior immune protection (RPS = 63%) under the challenge with E. tarda EIB202, suggesting that the novel antigen delivery system had great potential in bacterial vector vaccine applications. PMID:24746937

  16. Preclinical test: bacterial reverse mutation test for {sup 18}F-fluorocholine produced in CDTN

    Energy Technology Data Exchange (ETDEWEB)

    Mendes, Bruno M.; Bispo, Ana Carolina A.; Campos, Danielle C.; Silva, Juliana B., E-mail: bmm@cdtn.br, E-mail: acab@cdtn.br, E-mail: dcc@cdtn.br, E-mail: silvajb@cdtn.b [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)

    2013-07-01

    The choline labeled with fluorine-18 (18FCH) is being considered as a great importance radiopharmaceutical due to its effective detection of many type of malignant neoplasm. The research related to {sup 18}F-fluorocholine synthesis in CDTN was initiated in 2010. In order to obtain clinical research approval, as well as to register {sup 18}FCH for marketing, safety and efficacy preclinical testing are required. The present work evaluated the {sup 18}FCH genotoxic potential through the bacterial reverse mutation test (Ames test) using Salmonella typhimurium TA-98, TA-100, TA-1535 and TA-1537 strains and Escherichia coli WP2 uvrA strain. The reverse mutation test in bacteria for fluorcolina was conducted in two stages. Initially the method was applied to 'cold' fluorocholine molecule (19FCH). Subsequently, the decayed product of {sup 18}FCH synthesis was evaluated. The first step was performed in order to examine the FCH molecule mutagenicity. The second was carried out to determine the mutagenic potential of final product. All strains were tested in triplicate for each exposure concentration, in the presence and absence of metabolic activation (S-9 mix - 10%). There were no statistically significant increases in revertant colonies rate for any strains tested after their exposure to decayed {sup 18}FCH or {sup 19}FCH. The number of revertant colonies in positive controls was significantly higher than that observed in significant increases in revertant colonies rate for any strains tested after their exposure to decayed {sup 18}FCH or {sup 19}FCH. The number of revertant colonies in positive controls was significantly higher than that observed in negative controls. Based on results of this assay, {sup 18}FCH and {sup 19}FCH, at tested doses, were found to be non-mutagenic in bacterial reverse mutation test. (author)

  17. Preclinical test: bacterial reverse mutation test for 18F-fluorocholine produced in CDTN

    International Nuclear Information System (INIS)

    The choline labeled with fluorine-18 (18FCH) is being considered as a great importance radiopharmaceutical due to its effective detection of many type of malignant neoplasm. The research related to 18F-fluorocholine synthesis in CDTN was initiated in 2010. In order to obtain clinical research approval, as well as to register 18FCH for marketing, safety and efficacy preclinical testing are required. The present work evaluated the 18FCH genotoxic potential through the bacterial reverse mutation test (Ames test) using Salmonella typhimurium TA-98, TA-100, TA-1535 and TA-1537 strains and Escherichia coli WP2 uvrA strain. The reverse mutation test in bacteria for fluorcolina was conducted in two stages. Initially the method was applied to 'cold' fluorocholine molecule (19FCH). Subsequently, the decayed product of 18FCH synthesis was evaluated. The first step was performed in order to examine the FCH molecule mutagenicity. The second was carried out to determine the mutagenic potential of final product. All strains were tested in triplicate for each exposure concentration, in the presence and absence of metabolic activation (S-9 mix - 10%). There were no statistically significant increases in revertant colonies rate for any strains tested after their exposure to decayed 18FCH or 19FCH. The number of revertant colonies in positive controls was significantly higher than that observed in significant increases in revertant colonies rate for any strains tested after their exposure to decayed 18FCH or 19FCH. The number of revertant colonies in positive controls was significantly higher than that observed in negative controls. Based on results of this assay, 18FCH and 19FCH, at tested doses, were found to be non-mutagenic in bacterial reverse mutation test. (author)

  18. Production of recombinant proteins and metabolites in yeasts: when are these systems better than bacterial production systems?

    Science.gov (United States)

    Porro, Danilo; Gasser, Brigitte; Fossati, Tiziana; Maurer, Michael; Branduardi, Paola; Sauer, Michael; Mattanovich, Diethard

    2011-02-01

    Recombinant DNA (rDNA) technologies allow the production of a wide range of peptides, proteins and metabolites from naturally non-producing cells. Since human insulin was the first heterologous compound produced in a laboratory in 1977, rDNA technology has become one of the most important technologies developed in the 20th century. Recombinant protein and metabolites production is a multi-billion dollar market. The development of a new product begins with the choice of the cell factory. The final application of the compound dictates the main criteria that should be taken into consideration: (1) quality, (2) quantity, (3) yield and (4) space time yield of the desired product. Quantity and quality are the most predominant requirements that must be considered for the commercial production of a protein. Quantity and yield are the requirements for the production of a metabolite. Finally, space time yield is crucial for any production process. It therefore becomes clear why the perfect host does not exist yet, and why-despite important advances in rDNA applications in higher eukaryotic cells-microbial biodiversity continues to represent a potential source of attractive cell factories. In this review, we compare the advantages and limitations of the principal yeast and bacterial workhorse systems. PMID:21125266

  19. The role of bacterial antizyme: From an inhibitory protein to AtoC transcriptional regulator

    Directory of Open Access Journals (Sweden)

    Kyriakidis Dimitrios A

    2004-06-01

    Full Text Available Abstract This review considers the role of bacterial antizyme in the regulation of polyamine biosynthesis and gives new perspectives on the involvement of antizyme in other significant cellular mechanisms. Antizyme is a protein molecule induced by the end product of the enzymic reaction that it inhibits, in a non-competitive manner. The bacterial ornithine decarboxylase is regulated by nucleotides, phosphorylation and antizyme. The inhibition of ornithine decarboxylase by antizyme can be relieved to different degrees by DNA or by a variety of synthetic nucleic acid polymers, attributed to a specific interaction between nucleic acid and antizyme. Recently, this interplay between bacterial antizyme and nucleic acid was determined by discerning an additional function to antizyme that proved to be the atoC gene product, encoding the response regulator of the bacterial two-component system AtoS-AtoC. The gene located just upstream of atoC encodes the sensor kinase, named AtoS, that modulates AtoC activity. AtoC regulates expression of atoDAEB operon which is involved in short-chain fatty acid metabolism. Antizyme is thus referred to as AtoC, functioning both as a post-translational and transcriptional regulator. Also, the AtoS-AtoC signal transduction system in E. coli has a positive regulatory role on poly-(R-3-hydroxybutyrate biosynthesis. The properties and gene structural similarities of antizymes from different organisms were compared. It was revealed that conserved domains are present mostly in the C-domain of all antizymes. BLAST analysis of the E. coli antizyme protein (AtoC showed similarities around 69–58% among proteobacteria, g-proteobacteria, enterobacteria and the thermophilic bacterium Thermus thermophilus. A working hypothesis is proposed for the metabolic role of antizyme (AtoC describing the significant biological implications of this protein molecule. Whether antizymes exist to other enzymes in different tissues, meeting the

  20. Textile Dye Removal from Wastewater Effluents Using Bioflocculants Produced by Indigenous Bacterial Isolates

    OpenAIRE

    Balakrishna Pillay; Ademola O Olaniran; Simphiwe P. Buthelezi

    2012-01-01

    Bioflocculant-producing bacteria were isolated from activated sludge of a wastewater treatment plant located in Durban, South Africa, and identified using standard biochemical tests as well as the analysis of their 16S rRNA gene sequences. The bioflocculants produced by these organisms were ethanol precipitated, purified using 2% (w/v) cetylpyridinium chloride solution and evaluated for removal of wastewater dyes under different pH, temperature and nutritional conditions. Bioflocculants from ...

  1. Isolation and characterization of rhamnolipid-producing bacterial strains from a biodiesel facility.

    Science.gov (United States)

    Rooney, Alejandro P; Price, Neil P J; Ray, Karen J; Kuo, Tsung-Min

    2009-06-01

    Novel strains of rhamnolipid-producing bacteria were isolated from soils at a biodiesel facility on the basis of their ability to grow on glycerol as a sole carbon source. Strains were identified as Acinetobacter calcoaceticus, Enterobacter asburiae, Enterobacter hormaechei, Pantoea stewartii, and Pseudomonas aeruginosa. The strains of the former five species were found to produce rhamnolipids in quantities the same as, or similar to, coisolated strains of P. aeruginosa. Measurements of surface tension revealed that that emulsifying properties of these strains were similar to levels displayed by rhamnolipids produced by P. aeruginosa. Results of matrix-assisted laser desorption/ionization time-of-flight MS analyses revealed that the predominant compounds made by all strains were C10-C10 mono- and dirhamnolipids. Notably, E. hormaechei and one strain of A. calcoaceticus produced rhamnolipids in amounts similar to the pseudomonads. As all strains examined were from the same taxonomic class of Proteobacteria, further examination of this group may reveal many additional species not previously known to produce rhamnolipids in addition to novel strains of species currently known to produce rhamnolipids. PMID:19473254

  2. Bacterial Protein Characterization of Streptococcus agalactiae by SDS-page Method for Subclinical Mastitis Irradiated Vaccine Materials in Dairy Cattle

    OpenAIRE

    B.J. Tuasikal; I.W.T. Wibawan2; F.H. Pasaribu2; S. Estuningsih2

    2012-01-01

    A study have been conducted to isolate and characterize bacterial protein S. agalactiae, which is antigenic and can be used to test immunogenicity of vaccine in order to manufacture irradiated mastitis (inflammation of the udder) vaccine in ruminant. The study aims to determine the Molecular Weight (MW) bacterial protein S. agalactiae irradiation, which can be used to test the nature of its antigenic caharacteristic. The character of S. agalactiae antigenic stimulates antibody induction of th...

  3. Biochemical characterization and bacterial expression of an odorant-binding protein from Locusta migratoria.

    Science.gov (United States)

    Ban, L; Scaloni, A; D'Ambrosio, C; Zhang, L; Yahn, Y; Pelosi, P

    2003-02-01

    Analysis of soluble proteins from different body parts of Locusta migratoria revealed a fast-migrating component in native electrophoresis, unique to antennae of both sexes. N-terminal sequence analysis and cloning identified this protein as a member of the insect odorant-binding proteins, carrying a well-conserved six-cysteine motif. Mass spectrometry analysis confirmed the occurrence of two distinct polypeptide species determined by nucleotide sequencing and demonstrated that the cysteine residues are paired in an interlocked fashion. The protein was expressed in a bacterial system with yields of about 10 mg/l of culture, mostly present as inclusion bodies. However, this recombinant product was solubilized after disulfide reduction. Air oxidation yielded a species with all disulfides spontaneously formed as in the native counterpart. Both native and recombinant proteins migrated as a dimer in gel filtration chromatography. Ligand binding was measured, using N-phenyl-1-naphthylamine as the fluorescent probe; the affinity of other ligands was measured in competitive binding assays. The protein exhibited great resistance to thermal denaturation even following prolonged treatment at 100 degrees C. A structural model for this dimeric species was generated on the basis of its sequence homology with Bombyx mori pheromone-binding protein, whose three-dimensional structure has been resolved as an unbound species and in complex with its physiological ligand. This is the first report of an odorant-binding protein identified and characterized from Orthoptera. PMID:12678502

  4. Object-adapted trapping and shape-tracking to probe a bacterial protein chain motor

    Science.gov (United States)

    Roth, Julian; Koch, Matthias; Rohrbach, Alexander

    2015-03-01

    The helical bacterium Spiroplasma is a motile plant and anthropod pathogen which swims by propagating pairs of kinks along its cell body. As a well suited model system for bacterial locomotion, understanding the cell's molecular motor is of vital interest also regarding the combat of bacterial diseases. The extensive deformations related to these kinks are caused by a contractile cytoskeletal protein ribbon representing a linear motor in contrast to common rotary motors as, e.g., flagella. We present new insights into the working of this motor through experiments with object-adapted optical traps and shape-tracking techniques. We use the given laser irradiation from the optical trap to hinder bacterial energy (ATP) production through the production of O2 radicals. The results are compared with experiments performed under the influence of an O2-Scavenger and ATP inhibitors, respectively. Our results show clear dependences of the kinking properties on the ATP concentration inside the bacterium. The experiments are supported by a theoretical model which we developed to describe the switching of the ribbon's protein subunits.

  5. Effect of Dietary Protein Levels on Composition of Odorous Compounds and Bacterial Ecology in Pig Manure.

    Science.gov (United States)

    Cho, Sungback; Hwang, Okhwa; Park, Sungkwon

    2015-09-01

    This study was performed to investigate the effect of different levels of dietary crude protein (CP) on composition of odorous compounds and bacterial communities in pig manure. A total of 48 male pigs (average initial body weight 45 kg) fed diets containing three levels of dietary CP (20%, 17.5%, and 15%) and their slurry samples were collected from the pits under the floor every week for one month. Changes in composition of odorous compounds and bacterial communities were analyzed by gas chromatography and 454 FLX titanium pyrosequencing systems, respectively. Levels of phenols, indoles, short chain fatty acid and branched chain fatty acid were lowest (pp-cresol and skatole with Bacteroides, acetic acid and butyric acid with AM982595_g of Porphyromonadaceae family, and propionic acid with Tissierella. Taken together, administration of 15% CP showed less production of odorous compounds than 20% CP group and this result might be associated with the changes in bacterial communities especially whose roles in protein metabolism. PMID:26194219

  6. Establishing a role for bacterial cellulose in environmental interactions: lessons learned from diverse biofilm-producing Proteobacteria

    Directory of Open Access Journals (Sweden)

    Richard Vincent Augimeri

    2015-11-01

    Full Text Available Bacterial cellulose (BC serves as a molecular glue to facilitate intra- and inter-domain interactions in nature. Biosynthesis of BC-containing biofilms occurs in a variety of Proteobacteria that inhabit diverse ecological niches. The enzymatic and regulatory systems responsible for the polymerization, exportation and regulation of BC are equally as diverse. Though the magnitude and environmental consequences of BC production are species-specific, the common role of BC containing biofilms is to establish close contact with a preferred host to facilitate efficient host-bacteria interactions. Universally, BC aids in attachment, adherence, and subsequent colonization of a substrate. Bi-directional interactions influence host physiology, bacterial physiology and regulation of BC biosynthesis, primarily through modulation of intracellular bis-(3’→5’-cyclic diguanylate (c-di-GMP levels. Depending on the circumstance, BC producers exhibit a pathogenic or symbiotic relationship with plant, animal or fungal hosts. Rhizobiaceae species colonize plant roots, Pseudomonadaceae inhabit the phyllosphere, Acetobacteriaceae associate with sugar-loving insects and inhabit the carposphere, Enterobacteriaceae use fresh produce as vehicles to infect animal hosts, and Vibrionaceae, particularly Aliivibrio fischeri, colonize the light organ of squid. This review will highlight the diversity of the biosynthesis and regulation of BC in nature by discussing various examples of Proteobacteria that use BC-containing biofilms to facilitate host-bacteria interactions. Through discussion of current data we will establish new directions for the elucidation of BC biosynthesis, regulation and ecophysiological roles.

  7. Establishing a Role for Bacterial Cellulose in Environmental Interactions: Lessons Learned from Diverse Biofilm-Producing Proteobacteria

    Science.gov (United States)

    Augimeri, Richard V.; Varley, Andrew J.; Strap, Janice L.

    2015-01-01

    Bacterial cellulose (BC) serves as a molecular glue to facilitate intra- and inter-domain interactions in nature. Biosynthesis of BC-containing biofilms occurs in a variety of Proteobacteria that inhabit diverse ecological niches. The enzymatic and regulatory systems responsible for the polymerization, exportation, and regulation of BC are equally as diverse. Though the magnitude and environmental consequences of BC production are species-specific, the common role of BC-containing biofilms is to establish close contact with a preferred host to facilitate efficient host–bacteria interactions. Universally, BC aids in attachment, adherence, and subsequent colonization of a substrate. Bi-directional interactions influence host physiology, bacterial physiology, and regulation of BC biosynthesis, primarily through modulation of intracellular bis-(3′→5′)-cyclic diguanylate (c-di-GMP) levels. Depending on the circumstance, BC producers exhibit a pathogenic or symbiotic relationship with plant, animal, or fungal hosts. Rhizobiaceae species colonize plant roots, Pseudomonadaceae inhabit the phyllosphere, Acetobacteriaceae associate with sugar-loving insects and inhabit the carposphere, Enterobacteriaceae use fresh produce as vehicles to infect animal hosts, and Vibrionaceae, particularly Aliivibrio fischeri, colonize the light organ of squid. This review will highlight the diversity of the biosynthesis and regulation of BC in nature by discussing various examples of Proteobacteria that use BC-containing biofilms to facilitate host–bacteria interactions. Through discussion of current data we will establish new directions for the elucidation of BC biosynthesis, its regulation and its ecophysiological roles. PMID:26635751

  8. Development of a Selective Medium for the Fungal Pathogen Fusarium graminearum Using Toxoflavin Produced by the Bacterial Pathogen Burkholderia glumae

    Directory of Open Access Journals (Sweden)

    Boknam Jung

    2013-12-01

    Full Text Available The ascomycete fungus Fusarium graminearum is a major causal agent for Fusarium head blight in cereals and produces mycotoxins such as trichothecenes and zearalenone. Isolation of the fungal strains from air or cereals can be hampered by various other airborne fungal pathogens and saprophytic fungi. In this study, we developed a selective medium specific to F. graminearum using toxoflavin produced by the bacterial pathogen Burkholderia glumae. F. graminearum was resistant to toxoflavin, while other fungi were sensitive to this toxin. Supplementing toxoflavin into medium enhanced the isolation of F. graminearum from rice grains by suppressing the growth of saprophytic fungal species. In addition, a medium with or without toxoflavin exposed to wheat fields for 1 h had 84% or 25%, respectively, of colonies identified as F. graminearum. This selection medium provides an efficient tool for isolating F. graminearum, and can be adopted by research groups working on genetics and disease forecasting.

  9. Caenorhabditis elegans bacterial pathogen resistant bus-4 mutants produce altered mucins.

    Directory of Open Access Journals (Sweden)

    Lisa M Parsons

    Full Text Available Caenorabditis elegans bus-4 glycosyltransferase mutants are resistant to infection by Microbacterium nematophilum, Yersinia pestis and Yersinia pseudotuberculosis and have altered susceptibility to two Leucobacter species Verde1 and Verde2. Our objective in this study was to define the glycosylation changes leading to this phenotype to better understand how these changes lead to pathogen resistance. We performed MALDI-TOF MS, tandem MS and GC/MS experiments to reveal fine structural detail for the bus-4 N- and O-glycan pools. We observed dramatic changes in O-glycans and moderate ones in N-glycan pools compared to the parent strain. Ce core-I glycans, the nematode's mucin glycan equivalent, were doubled in abundance, halved in charge and bore shifts in terminal substitutions. The fucosyl O-glycans, Ce core-II and neutral fucosyl forms, were also increased in abundance as were fucosyl N-glycans. Quantitative expression analysis revealed that two mucins, let-653 and osm-8, were upregulated nearly 40 fold and also revealed was a dramatic increase in GDP-Man 4,6 dehydratease expression. We performed detailed lectin binding studies that showed changes in glycoconjugates in the surface coat, cuticle surface and intestine. The combined changes in cell surface glycoconjugate distribution, increased abundance and altered properties of mucin provide an environment where likely the above pathogens are not exposed to normal glycoconjugate dependent cues leading to barriers to these bacterial infections.

  10. Lactobacillus rhamnosus GG-supplemented formula expands butyrate-producing bacterial strains in food allergic infants.

    Science.gov (United States)

    Berni Canani, Roberto; Sangwan, Naseer; Stefka, Andrew T; Nocerino, Rita; Paparo, Lorella; Aitoro, Rosita; Calignano, Antonio; Khan, Aly A; Gilbert, Jack A; Nagler, Cathryn R

    2016-03-01

    Dietary intervention with extensively hydrolyzed casein formula supplemented with Lactobacillus rhamnosus GG (EHCF+LGG) accelerates tolerance acquisition in infants with cow's milk allergy (CMA). We examined whether this effect is attributable, at least in part, to an influence on the gut microbiota. Fecal samples from healthy controls (n=20) and from CMA infants (n=19) before and after treatment with EHCF with (n=12) and without (n=7) supplementation with LGG were compared by 16S rRNA-based operational taxonomic unit clustering and oligotyping. Differential feature selection and generalized linear model fitting revealed that the CMA infants have a diverse gut microbial community structure dominated by Lachnospiraceae (20.5±9.7%) and Ruminococcaceae (16.2±9.1%). Blautia, Roseburia and Coprococcus were significantly enriched following treatment with EHCF and LGG, but only one genus, Oscillospira, was significantly different between infants that became tolerant and those that remained allergic. However, most tolerant infants showed a significant increase in fecal butyrate levels, and those taxa that were significantly enriched in these samples, Blautia and Roseburia, exhibited specific strain-level demarcations between tolerant and allergic infants. Our data suggest that EHCF+LGG promotes tolerance in infants with CMA, in part, by influencing the strain-level bacterial community structure of the infant gut. PMID:26394008

  11. Mechanisms of Host-Pathogen Protein Complex Formation and Bacterial Immune Evasion of Streptococcus suis Protein Fhb.

    Science.gov (United States)

    Li, Xueqin; Liu, Peng; Gan, Shuzhen; Zhang, Chunmao; Zheng, Yuling; Jiang, Yongqiang; Yuan, Yuan

    2016-08-12

    Streptococcus suis serotype 2 (S. suis 2)-induced sepsis and meningitis are often accompanied by bacteremia. The evasion of polymorphonuclear leukocyte-mediated phagocytic clearance is central to the establishment of bacteremia caused by S. suis 2 and is facilitated by the ability of factor H (FH)-binding protein (Fhb) to bind FH on the bacterial surface, thereby impeding alternative pathway complement activation and phagocytic clearance. Here, C3b/C3d was found to bind to Fhb, along with FH, forming a large immune complex. The formation of this immune complex was mediated by domain II of Fhb via electrostatic and hydrophobic interactions, which, to our knowledge, is a new type of interaction. Interestingly, Fhb was found to be associated with the cell envelope and also present in the culture supernatant, where secreted Fhb inhibited complement activation via interactions with domain II, thereby enhancing antiphagocytic clearance by polymorphonuclear leukocytes. Thus, Fhb is a multifunctional bacterial protein, which binds host complement component C3 as well as FH and interferes with innate immune recognition in a secret protein manner. S. suis 2 therefore appears to have developed a new strategy to combat host innate immunity and enhance survival in host blood. PMID:27342778

  12. Chirality Switching by Martensitic Transformation in Protein Cylindrical Crystals: Application to Bacterial Flagella

    Science.gov (United States)

    Komai, Ricardo Kiyohiro

    Martensitic transformations provide unique engineering properties that, when designed properly, become important parts of new technology. Martensitic transformations have been studied for many years in traditional alloys (iron, steel, titanium, etc.), however there is still much to be learned in regards to these transformations in biological materials. Olson and Hartman showed in 1982 that these transformations are also observed in bacterial flagella and T4 bacteriophage viral sheaths, allowing for propulsion of bacteria in a fluid environment and, for the virus, is responsible for the infection mechanism. This work demonstrates, using the bacterial flagella as an example, that these transformations can be modelled using thermodynamic methods that are also used to model the transformations in alloys. This thesis work attempts to explain the transformations that occur in bacterial flagella, which are capable of small strain, highly reversible martensitic transformations. The first stress/temperature phase diagrams of these flagella were created by adding the mechanical energy of the transformation of the flagella to limited chemical thermodynamics information of the transformation. Mechanical energy is critical to the transformation process because the bacterial body applies a torque to the radius of the flagella. Finally, work has begun and will be completed in regards to understanding the kinetics of the transformation of the flagella. The motion of the transformation interface can be predicted by using a Landau-Ginzburg model. The crystallography of the transformation in bacterial flagella is also being computed to determine the invariant lines of transformation that occur within this cylindrical crystal. This work has shown that it is possible to treat proteins in a similar manner that alloys are treated when using thermodynamic modelling. Much can be learned from translating what is known regarding phase transformations in hard material systems to soft, organic

  13. Lipidation of intact proteins produces highly immunogenic vaccine candidates.

    Science.gov (United States)

    Zeng, Weiguang; Eriksson, Emily M; Lew, Andrew; Jackson, David C

    2011-01-01

    In this study we investigate the feasibility of generating self-adjuvanting vaccines capable of inducing high titre antibody responses following the covalent attachment of the TLR2 agonist Pam(2)Cys to intact proteins. Three Pam(2)Cys-based lipid moieties were prepared which contain a solubilising spacer composed of either lysine residues or polyethyleneglycol. A model protein, hen egg white lysozyme (HEL), was lipidated individually with each of these lipid modules and the immunogenicity of the lipidated species studied in mice by measuring antibody responses. We found that lipidated HEL elicited antibodies which is much stronger than the responses obtained when the HEL was administered in Freund's adjuvant or in Alum. Little or no antibody was elicited by the lipidated HEL in CD4 T cell-deficient mice indicating that the antibody response is T cell dependent. Furthermore, the lipidated protein elicited similar antibody responses in two different strains of mice indicating that sufficient helper T cell epitopes are available to enable antibody production across the histocompatability barrier. In a similar way, lipidated bovine insulin was found to be highly immunogenic in mice despite the largely conserved sequences of bovine and murine insulin. The results provide evidence that lipidation of proteins provides a simple and safe method for the manufacture of soluble self-adjuvanting protein-based vaccines. PMID:21056473

  14. Cyclic enterobacterial common antigen: Potential contaminant of bacterially expressed protein preparations

    International Nuclear Information System (INIS)

    We have previously reported the identification of the cyclic enterobacterial common antigen (ECACYC) polysaccharide in E. coli strains commonly used for heterologous protein expression (PJA Erbel et al., J. Bacteriol.185 (2003): 1995). Following this initial report, interactions among several NMR groups established that characteristic N-acetyl signals of ECACYC have been observed in 15N-1H HSQC spectra of samples of various bacterially-expressed proteins suggesting that this water-soluble carbohydrate is a common contaminant. We provide NMR spectroscopic tools to recognize ECACYC in protein samples, as well as several methods to remove this contaminant. Early recognition of ECA-based NMR signals will prevent time-consuming analyses of this copurifying carbohydrate

  15. [Construction and evaluation of an engineered bacterial strain for producing lipopeptide under anoxic conditions].

    Science.gov (United States)

    Liang, Xiao-long; Zhao, Feng; Shi, Rong-jiu; Ban, Yun-he; Zhou, Ji-dong; Han, Si-qin; Zhang, Ying

    2015-08-01

    Biosurfactant-facilitated oil recovery is one of the most important aspects of microbial enhanced oil recovery (MEOR). However, the biosurfactant production by biosurfactant-producing microorganisms, most of which are aerobes, is severely suppressed due to the in-situ anoxic conditions within oil reservoirs. In this research, we successfully engineered a strain JD-3, which could grow rapidly and produce lipopeptide under anoxic conditions, by protoplast confusion using a Bacillus amyloliquefaciens strain BQ-2 which produces biosurfactant aerobically, and a facultative anaerobic Pseudomonas stutzeri strain DQ-1 as parent strains. The alignment of 16S rDNA sequence (99% similarity) and comparisons of cell colony morphology showed that fusant JD-3 was closer to the parental strain B. amyloliquefaciens BQ-2. The surface tension of culture broth of fusant JD-3, after 36-hour cultivation under anaerobic conditions, decreased from initially 63.0 to 32.5 mN · m(-1). The results of thin layer chromatography and infrared spectrum analysis demonstrated that the biosurfactant produced by JD-3 was lipopeptide. The surface-active lipopeptide had a low critical micelle concentration (CMC) of 90 mg · L(-1) and presented a good ability to emulsify various hydrocarbons such as crude oil, liquid paraffin, and kerosene. Strain JD-3 could utilize peptone as nitrogen source and sucrose, glucose, glycerin or other common organics as carbon sources for anaerobic lipopeptide synthesis. The subculture of fusant JD-3 showed a stable lipopeptide-producing ability even after ten serial passages. All these results indicated that fusant JD-3 holds a great potential to microbially enhance oil recovery under anoxic conditions. PMID:26685621

  16. Genome sequencing and systems biology analysis of a lipase-producing bacterial strain.

    Science.gov (United States)

    Li, N; Li, D D; Zhang, Y Z; Yuan, Y Z; Geng, H; Xiong, L; Liu, D L

    2016-01-01

    Lipase-producing bacteria are naturally-occurring, industrially-relevant microorganisms that produce lipases, which can be used to synthesize biodiesel from waste oils. The efficiency of lipase expression varies between various microbial strains. Therefore, strains that can produce lipases with high efficiency must be screened, and the conditions of lipase metabolism and optimization of the production process in a given environment must be thoroughly studied. A high efficiency lipase-producing strain was isolated from the sediments of Jinsha River, identified by 16S rRNA sequence analysis as Serratia marcescens, and designated as HS-L5. A schematic diagram of the genome sequence was constructed by high-throughput genome sequencing. A series of genes related to lipid degradation were identified by functional gene annotation through sequence homology analysis. A genome-scale metabolic model of HS-ML5 was constructed using systems biology techniques. The model consisted of 1722 genes and 1567 metabolic reactions. The topological graph of the genome-scale metabolic model was compared to that of conventional metabolic pathways using a visualization software and KEGG database. The basic components and boundaries of the tributyrin degradation subnetwork were determined, and its flux balance analyzed using Matlab and COBRA Toolbox to simulate the effects of different conditions on the catalytic efficiency of lipases produced by HS-ML5. We proved that the catalytic activity of microbial lipases was closely related to the carbon metabolic pathway. As production and catalytic efficiency of lipases varied greatly with the environment, the catalytic efficiency and environmental adaptability of microbial lipases can be improved by proper control of the production conditions. PMID:27050954

  17. Bacterial Ortholog of Mammalian Translocator Protein (TSPO) with Virulence Regulating Activity

    Science.gov (United States)

    Chapalain, Annelise; Chevalier, Sylvie; Orange, Nicole; Murillo, Laurence; Papadopoulos, Vassilios; Feuilloley, Marc G. J.

    2009-01-01

    The translocator protein (TSPO), previously designated as peripheral-type benzodiazepine receptor, is a protein mainly located in the outer mitochondrial membrane of eukaryotic cells. TSPO is implicated in major physiological functions and functionally associated with other proteins such as the voltage-dependent anionic channel, also designated as mitochondrial porin. Surprisingly, a TSPO-related protein was identified in the photosynthetic bacterium Rhodobacter sphaeroides but it was initially considered as a relict of evolution. In the present study we cloned a tspO gene in Pseudomonas fluorescens MF37, a non-photosynthetic eubacterium and we used bioinformatics tools to identify TSPO in the genome of 97 other bacteria. P. fluorescens TSPO was recognized by antibodies against mouse protein and by PK 11195, an artificial ligand of mitochondrial TSPO. As in eukaryotes, bacterial TSPO appears functionally organized as a dimer and the apparent Kd for PK 11195 is in the same range than for its eukaryotic counterpart. When P. fluorescens MF37 was treated with PK 11195 (10−5 M) adhesion to living or artificial surfaces and biofilm formation activity were increased. Conversely, the apoptotic potential of bacteria on eukaryotic cells was significantly reduced. This effect of PK11195 was abolished in a mutant of P. fluorescens MF37 deficient for its major outer membrane porin, OprF. The present results demonstrate the existence of a bacterial TSPO that shares common structural and functional characteristics with its mammalian counterpart. This protein, apparently involved in adhesion and virulence, reveals the existence of a possible new inter kingdom signalling system and suggests that the human microbiome should be involuntarily exposed to the evolutionary pressure of benzodiazepines and related molecules. This discovery also represents a promising opportunity for the development of alternative antibacterial strategies. PMID:19564920

  18. Bacterial ortholog of mammalian translocator protein (TSPO with virulence regulating activity.

    Directory of Open Access Journals (Sweden)

    Annelise Chapalain

    Full Text Available The translocator protein (TSPO, previously designated as peripheral-type benzodiazepine receptor, is a protein mainly located in the outer mitochondrial membrane of eukaryotic cells. TSPO is implicated in major physiological functions and functionally associated with other proteins such as the voltage-dependent anionic channel, also designated as mitochondrial porin. Surprisingly, a TSPO-related protein was identified in the photosynthetic bacterium Rhodobacter sphaeroides but it was initially considered as a relict of evolution. In the present study we cloned a tspO gene in Pseudomonas fluorescens MF37, a non-photosynthetic eubacterium and we used bioinformatics tools to identify TSPO in the genome of 97 other bacteria. P. fluorescens TSPO was recognized by antibodies against mouse protein and by PK 11195, an artificial ligand of mitochondrial TSPO. As in eukaryotes, bacterial TSPO appears functionally organized as a dimer and the apparent Kd for PK 11195 is in the same range than for its eukaryotic counterpart. When P. fluorescens MF37 was treated with PK 11195 (10(-5 M adhesion to living or artificial surfaces and biofilm formation activity were increased. Conversely, the apoptotic potential of bacteria on eukaryotic cells was significantly reduced. This effect of PK11195 was abolished in a mutant of P. fluorescens MF37 deficient for its major outer membrane porin, OprF. The present results demonstrate the existence of a bacterial TSPO that shares common structural and functional characteristics with its mammalian counterpart. This protein, apparently involved in adhesion and virulence, reveals the existence of a possible new inter kingdom signalling system and suggests that the human microbiome should be involuntarily exposed to the evolutionary pressure of benzodiazepines and related molecules. This discovery also represents a promising opportunity for the development of alternative antibacterial strategies.

  19. Bacterial ortholog of mammalian translocator protein (TSPO) with virulence regulating activity.

    Science.gov (United States)

    Chapalain, Annelise; Chevalier, Sylvie; Orange, Nicole; Murillo, Laurence; Papadopoulos, Vassilios; Feuilloley, Marc G J

    2009-01-01

    The translocator protein (TSPO), previously designated as peripheral-type benzodiazepine receptor, is a protein mainly located in the outer mitochondrial membrane of eukaryotic cells. TSPO is implicated in major physiological functions and functionally associated with other proteins such as the voltage-dependent anionic channel, also designated as mitochondrial porin. Surprisingly, a TSPO-related protein was identified in the photosynthetic bacterium Rhodobacter sphaeroides but it was initially considered as a relict of evolution. In the present study we cloned a tspO gene in Pseudomonas fluorescens MF37, a non-photosynthetic eubacterium and we used bioinformatics tools to identify TSPO in the genome of 97 other bacteria. P. fluorescens TSPO was recognized by antibodies against mouse protein and by PK 11195, an artificial ligand of mitochondrial TSPO. As in eukaryotes, bacterial TSPO appears functionally organized as a dimer and the apparent Kd for PK 11195 is in the same range than for its eukaryotic counterpart. When P. fluorescens MF37 was treated with PK 11195 (10(-5) M) adhesion to living or artificial surfaces and biofilm formation activity were increased. Conversely, the apoptotic potential of bacteria on eukaryotic cells was significantly reduced. This effect of PK11195 was abolished in a mutant of P. fluorescens MF37 deficient for its major outer membrane porin, OprF. The present results demonstrate the existence of a bacterial TSPO that shares common structural and functional characteristics with its mammalian counterpart. This protein, apparently involved in adhesion and virulence, reveals the existence of a possible new inter kingdom signalling system and suggests that the human microbiome should be involuntarily exposed to the evolutionary pressure of benzodiazepines and related molecules. This discovery also represents a promising opportunity for the development of alternative antibacterial strategies. PMID:19564920

  20. Primordial soup was edible: abiotically produced Miller-Urey mixture supports bacterial growth

    OpenAIRE

    Xueshu Xie; Daniel Backman; Albert T. Lebedev; Viatcheslav B. Artaev; Liying Jiang; Ilag, Leopold L.; Zubarev, Roman A.

    2015-01-01

    Sixty years after the seminal Miller-Urey experiment that abiotically produced a mixture of racemized amino acids, we provide a definite proof that this primordial soup, when properly cooked, was edible for primitive organisms. Direct admixture of even small amounts of Miller-Urey mixture strongly inhibits E. coli bacteria growth due to the toxicity of abundant components, such as cyanides. However, these toxic compounds are both volatile and extremely reactive, while bacteria are highly capa...

  1. Viability of adhered bacterial cells: tracking MinD protein oscillations

    Science.gov (United States)

    Barrett, Matt; Colville, Keegan; Schultz-Nielsen, Chris; Jericho, Manfred; Dutcher, John

    2010-03-01

    To study bacterial cells using atomic force microscopy, it is necessary to immobilize the cells on a substrate. Because bacterial cells and common substrates such as glass and mica have a net negative charge, positively charged polymers such as poly-L-lysine (PLL) and polyethyleneimine (PEI) are commonly used as adhesion layers. However, the use of adhesion polymers could stress the cell and even render it inviable. Viable E. coli cells use oscillations of Min proteins along the axis of the rod-shaped cells to ensure accurate cell division. By tagging MinD proteins with GFP, oscillations can be observed using fluorescence microscopy. For a healthy cell in an ideal environment, the oscillation period is measured to be ˜40 s. Prior experiments have shown that PLL increases the oscillation period significantly (up to 80%). In the present study, we have used epifluorescence and total internal reflection fluorescence (TIRF) to track MinD protein oscillations in E. coli bacteria adhered to a variety of positively charged polymers on mica as a function of polymer surface coverage.

  2. Role of acute-phase proteins in interleukin-1-induced nonspecific resistance to bacterial infections in mice.

    OpenAIRE

    Vogels, M.T.E.; L. Cantoni; Carelli, M.; Sironi, M; Ghezzi, P; van der Meer, J. W M

    1993-01-01

    Treatment with a single low dose (80 to 800 ng) of interleukin-1 (IL-1) 24 h before a lethal bacterial challenge of granulocytopenic and normal mice enhances nonspecific resistance. Since IL-1 induces secretion of acute-phase proteins, liver proteins which possess several detoxifying effects, we investigated the role of these proteins in the IL-1-induced protection. Inhibition of liver protein synthesis with D-galactosamine (GALN) completely inhibited the IL-1-induced synthesis of acute-phase...

  3. Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins

    OpenAIRE

    Lin Jin; Jong Hyun Ham; Rosemary Hage; Wanying Zhao; Jaricelis Soto-Hernández; Sang Yeol Lee; Seung-Mann Paek; Min Gab Kim; Charles Boone; Coplin, David L.; David Mackey

    2016-01-01

    Bacterial AvrE-family Type-III effector proteins (T3Es) contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, w...

  4. Structural and functional characterization of the bacterial ferrous homeostasis protein FeoA

    OpenAIRE

    Vieira, Vanessa Cristina de Carvalho

    2012-01-01

    O objectivo deste trabalho intitulado ““Structural and functional characterization of the bacterial ferrous homeostasis protein FeoA” consistiu na determinação da estrutura e função da proteína FeoA da bacteria E.coli. A principal via bacteriana de entrada do ferro ferroso é através do sistema Feo que deriva das palavras inglesas ferrous iron transport. O ferro é um elemento essencial para a maioria dos organismos participando em vias metabólicas essenciais. Os sistemas de importação ...

  5. Branched signal wiring of an essential bacterial cell-cycle phosphotransfer protein

    OpenAIRE

    Blair, Jimmy A.; Xu, Qingping; Childers, W. Seth; Mathews, Irimpan I.; Kern, Justin W.; Eckart, Michael; Deacon, Ashley M.; Shapiro, Lucy

    2013-01-01

    Vital to bacterial survival is the faithful propagation of cellular signals, and in Caulobacter crescentus ChpT is an essential mediator within the cell cycle circuit. ChpT functions as a histidine-containing phosphotransfer protein (HPt) that shuttles a phosphoryl group from the receiver domain of CckA, the upstream hybrid histidine kinase (HK), to one of two downstream response regulators (RRs)—CtrA or CpdR—that controls cell cycle progression. To understand how ChpT interacts with multiple...

  6. Heterologously expressed bacterial and human multidrug resistance proteins confer cadmium resistance to Escherichia coli

    OpenAIRE

    Achard-Joris, M; van Saparoea, HBV; Driessen, AJM; Bourdineaud, JP; Bourdineaud, Jean-Paul

    2005-01-01

    The human MDR1 gene is induced by cadmium exposure although no resistance to this metal is observed in human cells overexpressing hMDR1. To access the role of MDR proteins in cadmium resistance, human MDR1, Lactococcus lactis lmrA, and Oenococcus oeni omrA were expressed in an Escherichia coli tolC mutant strain which proved to be hypersensitive to cadmium. Both the human and bacterial MDR genes conferred cadmium resistance to E. coli up to 0.4 mM concentration. Protection was abolished by 10...

  7. Rosmarinic acid is a homoserine lactone mimic produced by plants that activates a bacterial quorum-sensing regulator.

    Science.gov (United States)

    Corral-Lugo, Andrés; Daddaoua, Abdelali; Ortega, Alvaro; Espinosa-Urgel, Manuel; Krell, Tino

    2016-01-01

    Quorum sensing is a bacterial communication mechanism that controls genes, enabling bacteria to live as communities, such as biofilms. Homoserine lactone (HSL) molecules function as quorum-sensing signals for Gram-negative bacteria. Plants also produce previously unidentified compounds that affect quorum sensing. We identified rosmarinic acid as a plant-derived compound that functioned as an HSL mimic. In vitro assays showed that rosmarinic acid bound to the quorum-sensing regulator RhlR of Pseudomonas aeruginosa PAO1 and competed with the bacterial ligand N-butanoyl-homoserine lactone (C4-HSL). Furthermore, rosmarinic acid stimulated a greater increase in RhlR-mediated transcription in vitro than that of C4-HSL. In P. aeruginosa, rosmarinic acid induced quorum sensing-dependent gene expression and increased biofilm formation and the production of the virulence factors pyocyanin and elastase. Because P. aeruginosa PAO1 infection induces rosmarinic acid secretion from plant roots, our results indicate that rosmarinic acid secretion is a plant defense mechanism to stimulate a premature quorum-sensing response. P. aeruginosa is a ubiquitous pathogen that infects plants and animals; therefore, identification of rosmarinic acid as an inducer of premature quorum-sensing responses may be useful in agriculture and inform human therapeutic strategies. PMID:26732761

  8. Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family.

    Science.gov (United States)

    Broussard, Tyler C; Miller, Darcie J; Jackson, Pamela; Nourse, Amanda; White, Stephen W; Rock, Charles O

    2016-03-18

    Fatty acid kinase (Fak) is a ubiquitous Gram-positive bacterial enzyme consisting of an ATP-binding protein (FakA) that phosphorylates the fatty acid bound to FakB. In Staphylococcus aureus, Fak is a global regulator of virulence factor transcription and is essential for the activation of exogenous fatty acids for incorporation into phospholipids. The 1.2-Å x-ray structure of S. aureus FakB2, activity assays, solution studies, site-directed mutagenesis, and in vivo complementation were used to define the functions of the five conserved residues that define the FakB protein family (Pfam02645). The fatty acid tail is buried within the protein, and the exposed carboxyl group is bound by a Ser-93-fatty acid carboxyl-Thr-61-His-266 hydrogen bond network. The guanidinium of the invariant Arg-170 is positioned to potentially interact with a bound acylphosphate. The reduced thermal denaturation temperatures of the T61A, S93A, and H266A FakB2 mutants illustrate the importance of the hydrogen bond network in protein stability. The FakB2 T61A, S93A, and H266A mutants are 1000-fold less active in the Fak assay, and the R170A mutant is completely inactive. All FakB2 mutants form FakA(FakB2)2 complexes except FakB2(R202A), which is deficient in FakA binding. Allelic replacement shows that strains expressing FakB2 mutants are defective in fatty acid incorporation into phospholipids and virulence gene transcription. These conserved residues are likely to perform the same critical functions in all bacterial fatty acid-binding proteins. PMID:26774272

  9. Effect of PGR producing bacterial strains isolated from vermisources on germination and growth of Vigna unguiculata (L. Walp.

    Directory of Open Access Journals (Sweden)

    Anandharaj Marimuthu

    2014-12-01

    Full Text Available Nineteen bacterial strains were isolated from vermisources andscreened for Indole-3-acetic acid (IAA production among themonly nine strains produce IAA and they were identified asStreptococcus spp., Micrococcus spp., Klebsiella spp., Bacillus spp., Enterobacter spp., Escherichia spp., Alcaligenes spp., Erwinia spp., and Pseudomonas spp. Among all other strains Bacillus sp. showed the higher IAA production hence selected for further molecular analysis and confirmed as Bacillus cereus. The B. cereus was grown in nutrient broth supplemented with different concentrations (1, 2, 3, 4 and 5mg/ml of tryptophan for seven days at pH 7 and at 37ºC. Crude IAA was used for in vitro phytostimulatory studies using Vigna unguiculata (L. Walp. The plant growth parameters were analyzed at different day intervals (5, 10 and 15 days. Supplementation of 5 ml crude IAA (2mg/ml of tryptophan dynamically enhances the plant growth parameters after 15 days.

  10. Optimization of biohydrogen yield produced by bacterial consortia using residual glycerin from biodiesel production.

    Science.gov (United States)

    Faber, Mariana de Oliveira; Ferreira-Leitão, Viridiana Santana

    2016-11-01

    The aims of this study were to simplify the fermentation medium and to optimize the conditions of dark fermentation of residual glycerin to produce biohydrogen. It was possible to remove all micronutrients of fermentation medium and improve biohydrogen production by applying residual glycerin as feedstock. After statistical analysis of the following parameters pH, glycerin concentration and volatile suspended solids, the values of 5.5; 0.5g.L(-1) and 8.7g.L(-1), respectively, were defined as optimum condition for this process. It generated 2.44molH2/molglycerin, an expressive result when compared to previous results reported in literature and considering that theoretical yield of H2 from glycerol in dark fermentation process is 3molH2/molglycerol. This study allowed the improvement of yield and productivity by 68% and 67%, respectively. PMID:27501033

  11. Study on Screening and Cultivation Conditions of Xylanase-Producing Alkalophilic Bacterial

    Institute of Scientific and Technical Information of China (English)

    Han Xiao-fang; Zheng Lian-shuang; Xie Yi-min

    2004-01-01

    An xylanase producting alkalophilic Bacillus NT-9 was obtaind by the screening method of transparent zone on the selective medium, and the effects of carbon source and nitrogen source on xylanase production were studied. The medium composed of xylose 1.5%, (NH4)2SO4 0.25%, K2HPO4 0.1%, MgSO4·7H2O 0.02%, with the initial pH of 10, was suggested to be optimal for the enzyme production in this study. When cultivatied at 37 ℃ for 72 h, the enzyme activity elaborated by the strain may reach as high as 10.5 U/mL. The xylanase produced by Bacillus NT-9 was a constituent enzyme.

  12. Characterization of novel extracellular protease produced by marine bacterial isolate from the Indian Ocean

    Directory of Open Access Journals (Sweden)

    Rachana Fulzele

    2011-12-01

    Full Text Available Out of the vast pool of enzymes, proteolytic enzymes from microorganisms are the most widely used in different industries such as detergent, food, peptide production etc. Several marine microorganisms are known to produce proteases with commercially desirable characteristics. We have isolated nine different cultures from marine samples of the Indian Ocean. All of them were i motile ii rod shaped iii non spore forming iv catalase and amylase positive v able to grow in presence of 10 % NaCl. They produced acid from glucose, fructose and maltose and grew optimally at 30 0C temperature and pH 7.0-8.0. None of them could grow above 45 0C and below 15 0C. Only one of them (MBRI 7 exhibited extracellular protease activity on skim milk agar plates. Based on 16S rDNA sequencing, it belonged to the genus Marinobacter (98% sequence similarity, 1201 bp. The cell free extract was used to study effects of temperature and pH on protease activity. The optimum temperature and pH for activity were found to be 40 0C and 7.0 respectively. The crude enzyme was stable at temperature range of 30-80 0C and pH 5.0-9.0. It retained 60 % activity at 80 0C after 4 h and more than 70 % activity at 70 0C after 1 h. D value was found to be 342 minutes and 78 minutes for 40 0C and 80 0C respectively. Interestingly the enzyme remained 50 % active at pH 9.0 after 1 h. Comparison with other proteases from different microbial sources indicated that the neutral protease from the halotolerant marine isolate MBRI 7 is a novel enzyme with high thermostability.

  13. The pneumococcal serine-rich repeat protein is an intra-species bacterial adhesin that promotes bacterial aggregation in vivo and in biofilms.

    Directory of Open Access Journals (Sweden)

    Carlos J Sanchez

    Full Text Available The Pneumococcal serine-rich repeat protein (PsrP is a pathogenicity island encoded adhesin that has been positively correlated with the ability of Streptococcus pneumoniae to cause invasive disease. Previous studies have shown that PsrP mediates bacterial attachment to Keratin 10 (K10 on the surface of lung cells through amino acids 273-341 located in the Basic Region (BR domain. In this study we determined that the BR domain of PsrP also mediates an intra-species interaction that promotes the formation of large bacterial aggregates in the nasopharynx and lungs of infected mice as well as in continuous flow-through models of mature biofilms. Using numerous methods, including complementation of mutants with BR domain deficient constructs, fluorescent microscopy with Cy3-labeled recombinant (rBR, Far Western blotting of bacterial lysates, co-immunoprecipitation with rBR, and growth of biofilms in the presence of antibodies and competitive peptides, we determined that the BR domain, in particular amino acids 122-166 of PsrP, promoted bacterial aggregation and that antibodies against the BR domain were neutralizing. Using similar methodologies, we also determined that SraP and GspB, the Serine-rich repeat proteins (SRRPs of Staphylococcus aureus and Streptococcus gordonii, respectively, also promoted bacterial aggregation and that their Non-repeat domains bound to their respective SRRPs. This is the first report to show the presence of biofilm-like structures in the lungs of animals infected with S. pneumoniae and show that SRRPs have dual roles as host and bacterial adhesins. These studies suggest that recombinant Non-repeat domains of SRRPs (i.e. BR for S. pneumoniae may be useful as vaccine antigens to protect against Gram-positive bacteria that cause infection.

  14. Anti-bacterial selenium nanoparticles produced by UV/VIS/NIR pulsed nanosecond laser ablation in liquids

    International Nuclear Information System (INIS)

    The ability to produce nanoparticles free of any surface contamination is very challenging especially for bio-medical applications. Using a pulsed nanosecond Nd-YAG laser, pure selenium nanoparticles have been synthesized by irradiating selenium powder (99.999%) immerged in de-ionized water and ethanol. The wavelength of the laser beam has been varied from the UV to NIR (355, 532 and 1064 nm) and its effect on the particle size distribution has been studied by dynamic light scattering (DLS) and transmission electronic microscopy (TEM), revealing then the production of selenium quantum dots (size < 4 nm) by photo-fragmentation. It has been found that the crystallinity of the nanoparticles depends on their size. The zeta-potential measurement reveals that the colloidal solutions produced in de-ionized water were stable while the ones synthesized in ethanol agglomerate. The concentration of selenium has been measured using inductively coupled plasma mass spectrometry (ICP-MS). The anti-bacterial effect of selenium nanostructures has been analyzed on E. Coli bacteria. Finally, selenium quantum dots produced by this method can also be useful for quantum dot solar cells. (letter)

  15. Producing Fish Protein Hydrolysates from Mackerel By-Products

    OpenAIRE

    Ana Luísa De Sousa Augusto

    2014-01-01

    Portugal is one of the largest consumers of fishery products in Europe. This consumption involves a large amount of discarded raw material, such as rejected fish in selling auctions and the generation of by-products in industrial production processes. The by-products in the canning industry alone reach 40% of the raw material, while the frozen fish industries may reach 10-50% of the raw material (INE, 2013). Fish protein hydrolysates (FPH) are one of the most promising technologies for th...

  16. Blood parameters in growing pigs fed increasing levels of bacterial protein meal

    DEFF Research Database (Denmark)

    Hellwing, Anne Louise Frydendahl; Tauson, Anne-Helene; Skrede, Anders

    2007-01-01

    The experiment investigated the effects of increasing dietary levels of bacterial protein meal (BPM) on various blood parameters reflecting protein and fat metabolism, liver function, and purine base metabolism in growing pigs. Sixteen barrows were allocated to four different experimental diets....... The control diet was based on soybean meal. In the other three diets soybean meal was replaced with increasing levels of BPM, approximately 17%, 35%, and 50% of the nitrogen being derived from BPM. Blood samples from the jugular vein were taken when the body weights of the pigs were approximately 10 kg, 21 kg......, 45 kg, and 77 kg. The blood parameters reflecting fat metabolism and liver funtion were not affected by diet. Both the plasma albumin and uric acid concentrations tended to decrease (P = 0.07 and 0.01, respectively) with increasing dietary BPM content, whereas the plasma glucose concentration tended...

  17. Reconstitution of nanomachine driving the assembly of proteins into bacterial outer membranes

    International Nuclear Information System (INIS)

    Over 9.5 million people die each year due to infectious diseases caused by pathogens. Many species of pathogenic bacteria require nanomachines acting like a molecular pump that shuttle key disease-causing molecules (proteins) from inside bacteria cells to the outside surface, priming the bacteria for infections. How such proteins are assembled remains an important question in biology. If we can inhibit the nanomachines function in transporting specific violence factors, it would disable the disease process. Therefore it is crucial to understand how the proteins are transported through the nanomachines from the periplasm to the extracellular space. Measuring the activity of the component parts of membrane-embedded nanomachines in solution is a major technological challenge. The translocation assembly module (the TAM) is a nanomachine required for virulence of bacterial pathogens. We have reconstituted a membrane containing the TAM onto a gold surface for characterization by Quartz Crystal Microbalance with Dissipation (QCM-D) and Magnetic Contrast Neutron Reflectrometry (MCNR). We show that dynamic movements within the TamA component of the TAM are initiated in the presence of a substrate protein, Ag43, and that these movements recapitulate an initial stage in membrane protein assembly. The reconstituted system provides a powerful new means to study molecular movements in biological membranes, and the technology is widely applicable to studying the dynamics of diverse cellular nanomachines.

  18. Pestalone, a new antibiotic produced by a marine fungus in response to bacterial challenge.

    Science.gov (United States)

    Cueto, M; Jensen, P R; Kauffman, C; Fenical, W; Lobkovsky, E; Clardy, J

    2001-11-01

    The isolation and structure determination of a new chlorinated benzophenone antibiotic, pestalone (1), is described. The new compound was produced by a cultured marine fungus only when a unicellular marine bacterium, strain CNJ-328, was co-cultured in the fungal fermentation. The fungus, isolated from the surface of the brown alga Rosenvingea sp. collected in the Bahamas Islands, was identified as an undescribed member of the genus Pestalotia. The structure of 1, initially assigned with only modest confidence by combined spectral and chemical data, was confirmed by single-crystal X-ray analysis. Pestalone (1) exhibits moderate in vitro cytotoxicity in the National Cancer Institute's 60 human tumor cell line screen (mean GI(50) = 6.0 microM). More importantly, pestalone shows potent antibiotic activity against methicillin-resistant Staphylococcus aureus (MIC = 37 ng/mL) and vancomycin-resistant Enterococcus faecium (MIC = 78 ng/mL), indicating that pestalone should be evaluated in advanced models of infectious disease. PMID:11720529

  19. Agitation down-regulates immunoglobulin binding protein EibG expression in Shiga toxin-producing Escherichia coli (STEC.

    Directory of Open Access Journals (Sweden)

    Thorsten Kuczius

    Full Text Available Shiga toxin (Stx-producing Escherichia coli (STEC carrying eibG synthesize Escherichia coli immunoglobulin binding protein (EibG. EibG nonspecifically binds to immunoglobulins and tends to aggregate in multimers but is poorly expressed in wild-type strains. To study synthesis of the proteins and their regulation in the pathogens, we identified natural growth conditions that increased EibG synthesis. EibG proteins as well as corresponding mRNA were highly expressed under static growth conditions while shearing stress created by agitation during growth repressed protein synthesis. Further regulation effects were driven by reduced oxygen tension, and pH up-regulated EibG expression, but to a lesser extent than growth conditions while decreased temperature down-regulated EibG. Bacteria with increased EibG expression during static growth conditions showed a distinct phenotype with chain formation and biofilm generation, which disappeared with motion. High and low EibG expression was reversible indicating a process with up- and down-regulation of the protein expression. Our findings indicate that shear stress represses EibG expression and might reduce bacterial attachments to cells and surfaces.

  20. Identification of the interactome between fish plasma proteins and Edwardsiella tarda reveals tissue-specific strategies against bacterial infection.

    Science.gov (United States)

    Li, Hui; Huang, Xiaoyan; Zeng, Zaohai; Peng, Xuan-Xian; Peng, Bo

    2016-09-01

    Elucidating the complex pathogen-host interaction is essential for a comprehensive understanding of how these remarkable agents invade their hosts and how the hosts defend against these invaders. During the infection, pathogens interact intensively with host to enable their survival, which can be revealed through their interactome. Edwardsiella tarda is a Gram-negative bacterial pathogen causing huge economic loss in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. E. tarda is an ideal model for host-pathogen investigation as it infects fish in three distinct steps: entering the host, circulating through the blood and establishing infection. We adopted a previous established proteomic approach that inactivated E. tarda cells and covalent crosslink fish plasma proteins were used to capture plasma proteins and bacterial outer membrane proteins, respectively. By the combinatorial use of proteomic and biochemical approaches, six plasma proteins and seven outer membrane proteins (OMPs) were identified. Interactions among these proteins were validated with protein-array, far-Western blotting and co-immunoprecipitation. At last, seventeen plasma protein-bacteria protein-protein interaction were confirmed to be involved in the interaction network, forming a complex interactome. Compared to our previous results, different host proteins were detected, whereas some of the bacterial proteins were similar, which indicates that hosts adopt tissue-specific strategies to cope with the same pathogen during infection. Thus, our results provide a robust demonstration of both bacterial initiators and host receptors or interacting proteins to further explore infection and anti-infective mechanisms between hosts and microbes. PMID:27458055

  1. Crystal structure of the Campylobacter jejuni Cj0090 protein reveals a novel variant of the immunoglobulin fold among bacterial lipoproteins

    OpenAIRE

    Paek, Seonghee; Kawai, Fumihiro; Choi, Kyoung-Jae; Yeo, Hye-Jeong

    2012-01-01

    Bacterial lipoproteins play an important role in bacterial pathogenesis and physiology. The genome of Campylobacter jejuni, a major foodborn pathogen, is predicted to contain over 20 lipoproteins. However, the functions of the majority of C. jejuni lipoproteins remain unknown. The Cj0090 protein is encoded by a lipoprotein operon composed of cj0089, cj0090, and cj0091. Here, we report the crystal structure of Cj0090 at 1.9 Å resolution, revealing a novel variant of the immunoglobulin fold wit...

  2. The use of C-reactive protein in predicting bacterial co-Infection in children with bronchiolitis

    OpenAIRE

    Mohamad Fares; Sawsan Mourad; Mariam Rajab; Nahida Rifai

    2011-01-01

    Background: Bronchiolitis is a potentially life-threatening respiratory illness commonly affecting children who are less than two years of age. Patients with viral lower respiratory tract infection are at risk for co-bacterial infection. Aim: The aim of our study was to evaluate the use of C-reactive protein (CRP) in predicting bacterial co-infection in patients hospitalized for bronchiolitis and to correlate the results with the use of antibiotics. Patients and Methods: This is a prospective...

  3. Urokinase-targeted recombinant bacterial protein toxins-a rationally designed and engineered anticancer agent for cancer therapy

    Institute of Scientific and Technical Information of China (English)

    Yizhen LIU; Shi-Yan LI

    2009-01-01

    Urokinase-targeted recombinant bacterial protein toxins are a sort of rationally designed and engineered anticancer recombinant fusion proteins representing a novel class of agents for cancer therapy.Bacterial protein toxins have long been known as the primary virulence factor(s) for a variety of pathogenic bacteria and are the most powerful human poisons.On the other hand,it has been well documented that urokinase-type plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR),making up the uPA system,are overexpressed in a variety of human tumors and tumor cell lines.The expression of uPA system is highly correlated with tumor invasion and metastasis.To exploit these characteristics in the design of tumor cell-selective cytotoxins,two prominent bacterial protein toxins,i.e.,the diphtheria toxin and anthrax toxin are deliberately engineered through placing a sequence targeted specifically by the uPA system to form anticancer recombinant fusion proteins.These uPA system-targeted bacterial protein toxins are activated selectively on the surface of uPA systemexpressing tumor cells,thereby killing these cells.This article provides a review on the latest progress in the exploitation of these recombinant fusion proteins as potent tumoricidal agents.It is perceptible that the strategies for cancer therapy are being innovated by this novel therapeutic approach.

  4. 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.

  5. Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins.

    Science.gov (United States)

    Jin, Lin; Ham, Jong Hyun; Hage, Rosemary; Zhao, Wanying; Soto-Hernández, Jaricelis; Lee, Sang Yeol; Paek, Seung-Mann; Kim, Min Gab; Boone, Charles; Coplin, David L; Mackey, David

    2016-05-01

    Bacterial AvrE-family Type-III effector proteins (T3Es) contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, we employed a yeast-two-hybrid screen with non-lethal fragments of WtsE and a synthetic genetic array with full-length WtsE. Together these screens indicate that WtsE targets maize protein phosphatase 2A (PP2A) heterotrimeric enzyme complexes via direct interaction with B' regulatory subunits. AvrE1, another AvrE-family T3E from Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), associates with specific PP2A B' subunit proteins from its susceptible host Arabidopsis that are homologous to the maize B' subunits shown to interact with WtsE. Additionally, AvrE1 was observed to associate with the WtsE-interacting maize proteins, indicating that PP2A B' subunits are likely conserved targets of AvrE-family T3Es. Notably, the ability of AvrE1 to promote bacterial growth and/or suppress callose deposition was compromised in Arabidopsis plants with mutations of PP2A genes. Also, chemical inhibition of PP2A activity blocked the virulence activity of both WtsE and AvrE1 in planta. The function of HopM1, a Pto DC3000 T3E that is functionally redundant to AvrE1, was also impaired in specific PP2A mutant lines, although no direct interaction with B' subunits was observed. These results indicate that sub-component specific PP2A complexes are targeted by bacterial T3Es, including direct targeting by members of the widely conserved AvrE-family. PMID:27191168

  6. Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins.

    Directory of Open Access Journals (Sweden)

    Lin Jin

    2016-05-01

    Full Text Available Bacterial AvrE-family Type-III effector proteins (T3Es contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, we employed a yeast-two-hybrid screen with non-lethal fragments of WtsE and a synthetic genetic array with full-length WtsE. Together these screens indicate that WtsE targets maize protein phosphatase 2A (PP2A heterotrimeric enzyme complexes via direct interaction with B' regulatory subunits. AvrE1, another AvrE-family T3E from Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000, associates with specific PP2A B' subunit proteins from its susceptible host Arabidopsis that are homologous to the maize B' subunits shown to interact with WtsE. Additionally, AvrE1 was observed to associate with the WtsE-interacting maize proteins, indicating that PP2A B' subunits are likely conserved targets of AvrE-family T3Es. Notably, the ability of AvrE1 to promote bacterial growth and/or suppress callose deposition was compromised in Arabidopsis plants with mutations of PP2A genes. Also, chemical inhibition of PP2A activity blocked the virulence activity of both WtsE and AvrE1 in planta. The function of HopM1, a Pto DC3000 T3E that is functionally redundant to AvrE1, was also impaired in specific PP2A mutant lines, although no direct interaction with B' subunits was observed. These results indicate that sub-component specific PP2A complexes are targeted by bacterial T3Es, including direct targeting by members of the widely conserved AvrE-family.

  7. Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins

    Science.gov (United States)

    Jin, Lin; Ham, Jong Hyun; Hage, Rosemary; Zhao, Wanying; Soto-Hernández, Jaricelis; Lee, Sang Yeol; Paek, Seung-Mann; Kim, Min Gab; Boone, Charles; Coplin, David L.; Mackey, David

    2016-01-01

    Bacterial AvrE-family Type-III effector proteins (T3Es) contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, we employed a yeast-two-hybrid screen with non-lethal fragments of WtsE and a synthetic genetic array with full-length WtsE. Together these screens indicate that WtsE targets maize protein phosphatase 2A (PP2A) heterotrimeric enzyme complexes via direct interaction with B’ regulatory subunits. AvrE1, another AvrE-family T3E from Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), associates with specific PP2A B’ subunit proteins from its susceptible host Arabidopsis that are homologous to the maize B’ subunits shown to interact with WtsE. Additionally, AvrE1 was observed to associate with the WtsE-interacting maize proteins, indicating that PP2A B’ subunits are likely conserved targets of AvrE-family T3Es. Notably, the ability of AvrE1 to promote bacterial growth and/or suppress callose deposition was compromised in Arabidopsis plants with mutations of PP2A genes. Also, chemical inhibition of PP2A activity blocked the virulence activity of both WtsE and AvrE1 in planta. The function of HopM1, a Pto DC3000 T3E that is functionally redundant to AvrE1, was also impaired in specific PP2A mutant lines, although no direct interaction with B’ subunits was observed. These results indicate that sub-component specific PP2A complexes are targeted by bacterial T3Es, including direct targeting by members of the widely conserved AvrE-family. PMID:27191168

  8. Exploration and conservation of bacterial genetic resources as bacteriocin producing inhibitory microorganisms to pathogen bacteria in livestock

    Directory of Open Access Journals (Sweden)

    Chotiah S

    2013-06-01

    Full Text Available Exploration and conservation of microorganisms producing bacteriocin was done as the primary study towards the collection of potential bacteria and its application in improving livestock health condition and inhibit food borne pathogens. Diferent kinds of samples such as beef cattle rectal swab, rumen fluids, cow’s milk, chicken gut content, goat’s milk were collected at Bogor cattle slaughter houses, poultry slaughter houses, dairy cattle and goat farms. A total of 452 bacterial isolates consisted of 73 Gram negative bacteria and 379 Gram positive bacteria were isolated from samples collected and screened for bacteriocin activity. Determination of bacteriocin activity with bioassay using agar spot tests were carried out on liquid and semisolid medium assessing 8 kins of indicators of pathogenic bacteria and food borne pathogens. A total of 51 bacteriocin producing strains were collected and some of the strains had high inhibitory zone such as Lactobacillus casei SS14C (26 mm, Enterobacter cloacae SRUT (24mm, Enterococcus faecalis SK39 (21mm and Bifidobacterium dentium SS14T (20mm respectively, to Salmonella typhimurium BCC B0046/ATCC 13311, E. coli O157 hemolytic BCC B2717, Listeria monocytogenes BCC B2767/ATCC 7764 and Escherichia coli VTEC O157 BCC B2687. Evaluation after conservation ex situ to all bacterocin producing strain at 5oC for 1 year in freeze drying ampoules in vacuum and dry condition revealed the decreasing viability starting from log 0.8 CFU/ml for Lactococcus and Leuconostoc to log 2.2. CFU/ml for Streptococcus. Result of the study showed that the bacteriocin producing strains obtained were offered a potential resource for preventing disease of livestock and food borne diseases.

  9. Two Proteins Form a Heteromeric Bacterial Self-Recognition Complex in Which Variable Subdomains Determine Allele-Restricted Binding

    OpenAIRE

    Cardarelli, Lia; Saak, Christina; Gibbs, Karine A

    2015-01-01

    ABSTRACT Self- versus nonself-recognition in bacteria has been described recently through genetic analyses in multiple systems; however, understanding of the biochemical properties and mechanisms of recognition-determinant proteins remains limited. Here we extend the molecular and biochemical understanding of two recognition-determinant proteins in bacteria. We have found that a heterotypic complex is formed between two bacterial self-recognition proteins, IdsD and IdsE, the genes of which ha...

  10. Identification of multiple physicochemical and structural properties associated with soluble expression of eukaryotic proteins in cell-free bacterial extracts

    OpenAIRE

    AlexanderA.Tokmakov

    2014-01-01

    Bacterial extracts are widely used to synthesize recombinant proteins. Vast data volumes have been accumulated in cell-free expression databases, covering a whole range of existing proteins. It makes possible comprehensive bioinformatics analysis and identification of multiple features associated with protein solubility and aggregation. In the present paper, an approach to identify the multiple physicochemical and structural properties of amino acid sequences associated with soluble expressio...

  11. Allosteric effects of chromophore interaction with dimeric near-infrared fluorescent proteins engineered from bacterial phytochromes.

    Science.gov (United States)

    Stepanenko, Olesya V; Baloban, Mikhail; Bublikov, Grigory S; Shcherbakova, Daria M; Stepanenko, Olga V; Turoverov, Konstantin K; Kuznetsova, Irina M; Verkhusha, Vladislav V

    2016-01-01

    Fluorescent proteins (FPs) engineered from bacterial phytochromes attract attention as probes for in vivo imaging due to their near-infrared (NIR) spectra and use of available in mammalian cells biliverdin (BV) as chromophore. We studied spectral properties of the iRFP670, iRFP682 and iRFP713 proteins and their mutants having Cys residues able to bind BV either in both PAS (Cys15) and GAF (Cys256) domains, in one of these domains, or without these Cys residues. We show that the absorption and fluorescence spectra and the chromophore binding depend on the location of the Cys residues. Compared with NIR FPs in which BV covalently binds to Cys15 or those that incorporate BV noncovalently, the proteins with BV covalently bound to Cys256 have blue-shifted spectra and higher quantum yield. In dimeric NIR FPs without Cys15, the covalent binding of BV to Сys256 in one monomer allosterically inhibits the covalent binding of BV to the other monomer, whereas the presence of Cys15 allosterically promotes BV binding to Cys256 in both monomers. The NIR FPs with both Cys residues have the narrowest blue-shifted spectra and the highest quantum yield. Our analysis resulted in the iRFP713/Val256Cys protein with the highest brightness in mammalian cells among available NIR FPs. PMID:26725513

  12. Re-evaluation of a bacterial antifreeze protein as an adhesin with ice-binding activity.

    Directory of Open Access Journals (Sweden)

    Shuaiqi Guo

    Full Text Available A novel role for antifreeze proteins (AFPs may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, Marinomonas primoryensis. MpAFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII and region IV (RIV, divide MpAFP into five distinct regions, all of which require mM Ca(2+ levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca(2+-bound beta-helix containing thirteen Repeats-In-Toxin (RTX-like repeats. RII accounts for approximately 90% of the mass of MpAFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2 server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice.

  13. Effects of bacterially produced precipitates on the metabolism of sulfate reducing bacteria during the bio-treatment process of copper-containing wastewater

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    A large volume of bacterially produced precipitates are generated during the bio-treatment of heavy metal wastewater.The composition of the bacterially produced precipitates and its effects on sulfate reducing bacteria (SRB) in copper-containing waste stream were evaluated in this study.The elemental composition of the microbial precipitate was studied using electrodispersive X-ray spectroscopy (EDX),and it was found that the ratio of S:Cu was 1.12.Combining with the results of copper distribution in the SRB metabolism culture,which was analyzed by the sequential extraction procedure,copper in the precipitates was determined as covellite (CuS).The bacterially produced precipitates caused a decrease of the sulfate reduction rate,and the more precipitates were generated,the lower the sulfate reduction rate was.The particle sizes of bacterially generated covellite were ranging from 0.03 to 2 m by particles size distribution (PSD) analysis,which was smaller than that of the SRB cells.Transmission electron microscopy (TEM) analysis showed that the microbial covellite was deposited on the surface of the cell.The effects of the microbial precipitate on SRB metabolism were found to be weakened by increasing the precipitation time and adding microbial polymeric substances in later experiments.These results provided direct evidence that the SRB activity was inhibited by the bacterially produced covellite,which enveloped the bacterium and thus affected the metabolism of SRB on mass transfer.

  14. Stealth Proteins: In Silico Identification of a Novel Protein Family Rendering Bacterial Pathogens Invisible to Host Immune Defense.

    Directory of Open Access Journals (Sweden)

    2005-11-01

    Full Text Available There are a variety of bacterial defense strategies to survive in a hostile environment. Generation of extracellular polysaccharides has proved to be a simple but effective strategy against the host's innate immune system. A comparative genomics approach led us to identify a new protein family termed Stealth, most likely involved in the synthesis of extracellular polysaccharides. This protein family is characterized by a series of domains conserved across phylogeny from bacteria to eukaryotes. In bacteria, Stealth (previously characterized as SacB, XcbA, or WefC is encoded by subsets of strains mainly colonizing multicellular organisms, with evidence for a protective effect against the host innate immune defense. More specifically, integrating all the available information about Stealth proteins in bacteria, we propose that Stealth is a D-hexose-1-phosphoryl transferase involved in the synthesis of polysaccharides. In the animal kingdom, Stealth is strongly conserved across evolution from social amoebas to simple and complex multicellular organisms, such as Dictyostelium discoideum, hydra, and human. Based on the occurrence of Stealth in most Eukaryotes and a subset of Prokaryotes together with its potential role in extracellular polysaccharide synthesis, we propose that metazoan Stealth functions to regulate the innate immune system. Moreover, there is good reason to speculate that the acquisition and spread of Stealth could be responsible for future epidemic outbreaks of infectious diseases caused by a large variety of eubacterial pathogens. Our in silico identification of a homologous protein in the human host will help to elucidate the causes of Stealth-dependent virulence. At a more basic level, the characterization of the molecular and cellular function of Stealth proteins may shed light on fundamental mechanisms of innate immune defense against microbial invasion.

  15. Stealth proteins: in silico identification of a novel protein family rendering bacterial pathogens invisible to host immune defense.

    Directory of Open Access Journals (Sweden)

    Peter Sperisen

    2005-11-01

    Full Text Available There are a variety of bacterial defense strategies to survive in a hostile environment. Generation of extracellular polysaccharides has proved to be a simple but effective strategy against the host's innate immune system. A comparative genomics approach led us to identify a new protein family termed Stealth, most likely involved in the synthesis of extracellular polysaccharides. This protein family is characterized by a series of domains conserved across phylogeny from bacteria to eukaryotes. In bacteria, Stealth (previously characterized as SacB, XcbA, or WefC is encoded by subsets of strains mainly colonizing multicellular organisms, with evidence for a protective effect against the host innate immune defense. More specifically, integrating all the available information about Stealth proteins in bacteria, we propose that Stealth is a D-hexose-1-phosphoryl transferase involved in the synthesis of polysaccharides. In the animal kingdom, Stealth is strongly conserved across evolution from social amoebas to simple and complex multicellular organisms, such as Dictyostelium discoideum, hydra, and human. Based on the occurrence of Stealth in most Eukaryotes and a subset of Prokaryotes together with its potential role in extracellular polysaccharide synthesis, we propose that metazoan Stealth functions to regulate the innate immune system. Moreover, there is good reason to speculate that the acquisition and spread of Stealth could be responsible for future epidemic outbreaks of infectious diseases caused by a large variety of eubacterial pathogens. Our in silico identification of a homologous protein in the human host will help to elucidate the causes of Stealth-dependent virulence. At a more basic level, the characterization of the molecular and cellular function of Stealth proteins may shed light on fundamental mechanisms of innate immune defense against microbial invasion.

  16. Quantitative Mass Spectrometry for Bacterial Protein Toxins — A Sensitive, Specific, High-Throughput Tool for Detection and Diagnosis

    Directory of Open Access Journals (Sweden)

    Suzanne Kalb

    2011-03-01

    Full Text Available Matrix-assisted laser-desorption time-of-flight (MALDI-TOF mass spectrometry (MS is a valuable high-throughput tool for peptide analysis. Liquid chromatography electrospray ionization (LC-ESI tandem-MS provides sensitive and specific quantification of small molecules and peptides. The high analytic power of MS coupled with high-specificity substrates is ideally suited for detection and quantification of bacterial enzymatic activities. As specific examples of the MS applications in disease diagnosis and select agent detection, we describe recent advances in the analyses of two high profile protein toxin groups, the Bacillus anthracis toxins and the Clostridium botulinum neurotoxins. The two binary toxins produced by B. anthracis consist of protective antigen (PA which combines with lethal factor (LF and edema factor (EF, forming lethal toxin and edema toxin respectively. LF is a zinc-dependent endoprotease which hydrolyzes specific proteins involved in inflammation and immunity. EF is an adenylyl cyclase which converts ATP to cyclic-AMP. Toxin-specific enzyme activity for a strategically designed substrate, amplifies reaction products which are detected by MALDI-TOF-MS and LC-ESI-MS/MS. Pre-concentration/purification with toxin specific monoclonal antibodies provides additional specificity. These combined technologies have achieved high specificity, ultrasensitive detection and quantification of the anthrax toxins. We also describe potential applications to diseases of high public health impact, including Clostridium difficile glucosylating toxins and the Bordetella pertussis adenylyl cyclase.

  17. LocateP: Genome-scale subcellular-location predictor for bacterial proteins

    Directory of Open Access Journals (Sweden)

    Zhou Miaomiao

    2008-03-01

    current tools especially where the N-terminally anchored and the SPIase-cleaved secreted proteins are concerned. Overall, the accuracy of LocateP was always higher than 90%. LocateP was then used to predict the SCLs of all proteins encoded by completed Gram-positive bacterial genomes. The results are stored in the database LocateP-DB http://www.cmbi.ru.nl/locatep-db1. Conclusion LocateP is by far the most accurate and detailed protein SCL predictor for Gram-positive bacteria currently available.

  18. IN VITRO CO-STIMULATORY ACTIVITY OF HUMAN B7.2(IgV+C)PROTEIN PRODUCED BY ENGINEERED BACTERIA

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To express human B7.2 extracellular domain with prokaryote expression system and to evaluate its biological activity in vitro. Methods PCR was used to amplify the extracellular region of human B7. 2which contained both the IgV and IgC domains. The recombinant PGEX-4T-3/hB7. 2 (IgV+C) was obtained by cloning the PCR product into a prokaryote expression plasmid PGEX-4T-3 and was transformed into the host strain of DH5-α. The fusion protein consisted of GST and hB7.2(IgV+C) was identified by SDS-PAGE and Western blotting.T cell activation was observed by exposing purified T lymphocytes to the fusion protein and [3H]-TdR incorporation with the presence of the first signal imitated by anti-CD3 antibody. Results The fusion protein GST-hB7.2 (IgV+C) was produced and detected in inclusive body form from engineered bacterial cells. With the first signal existed,T lymphocytes proliferated when it was co-stimulated by the fusion protein. Conclusion These results indicated that the functional human B7.2(IgV+C) fusion protein can be produced in bacterial cells and the fusion protein displays the co-stimulatory activity in T lymphocytes activation.

  19. Some chemical and physical properties of nisin, a small-protein antibiotic produced by Lactococcus lactis.

    Science.gov (United States)

    Liu, W; Hansen, J N

    1990-08-01

    Nisin is a small gene-encoded antimicrobial protein produced by Lactococcus lactis that contains unusual dehydroalanine and dehydrobutyrine residues. The reactivity of these residues toward nucleophiles was explored by reacting nisin with a variety of mercaptans. The kinetics of reaction with 2-mercaptoethane-sulfonate and thioglycolate indicated that the reaction pathway includes a binding step. Reaction of nisin at high pH resulted in the formation of multimeric products, apparently as a result of intramolecular and intermolecular reactions between nucleophilic groups and the dehydro residues. One of the nucleophiles had a pKa of about 9.8. The unique vinyl protons of the dehydro residues that give readily identifiable proton nuclear magnetic resonances were used to observe the addition of nucleophiles to the dehydro moiety. After reaction with nucleophiles, nisin lost its antibiotic activity and no longer showed the dehydro resonances, indicating that the dehydro groups had been modified. The effect of pH on the solubility of nisin was determined; the solubility was quite high at low pH (57 mg/ml at pH 2) and was much lower at high pH (0.25 mg/ml at pH 8 to 12), as measured before significant pH-induced chemical modification had occurred. High-performance liquid chromatography on a C18 column was an effective technique for separating unmodified nisin from its reaction products. The cyanogen bromide cleavage products of nisin were about 90% less active toward inhibition of bacterial spore outgrowth than was native nisin. These results are consistent with earlier observations, which suggested that the dehydro residues of nisin have a role in the mechanism of antibiotic action, in which they act as electrophilic Michael acceptors toward nucleophiles in the cellular target. PMID:2119570

  20. Communication: Microsecond dynamics of the protein and water affect electron transfer in a bacterial bc{sub 1} complex

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Daniel R.; Matyushov, Dmitry V., E-mail: dmitrym@asu.edu [Department of Physics and Department of Chemistry and Biochemistry, Arizona State University, P.O. Box 871504, Tempe, Arizona 85287 (United States)

    2015-04-28

    Cross-membrane electron transport between cofactors localized in proteins of mitochondrial respiration and bacterial photosynthesis is the source of all biological energy. The statistics and dynamics of nuclear fluctuations in these protein/membrane/water heterogeneous systems are critical for their energetic efficiency. The results of 13 μs of atomistic molecular dynamics simulations of the membrane-bound bc{sub 1} bacterial complex are analyzed here. The reaction is affected by a broad spectrum of nuclear modes, with the slowest dynamics in the range of time-scales ∼0.1-1.6 μs contributing half of the reaction reorganization energy. Two reorganization energies are required to describe protein electron transfer due to dynamical arrest of protein conformations on the observation window. This mechanistic distinction allows significant lowering of activation barriers for reactions in proteins.

  1. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

    Directory of Open Access Journals (Sweden)

    Lei eShi

    2014-09-01

    Full Text Available Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

  2. Antiadhesive Properties of Arabinogalactan Protein from Ribes nigrum Seeds against Bacterial Adhesion of Helicobacter pylori

    Directory of Open Access Journals (Sweden)

    Jutta Messing

    2014-03-01

    Full Text Available Fruit extracts from black currants (Ribes nigrum L. are traditionally used for treatment of gastritis based on seed polysaccharides that inhibit the adhesion of Helicobacter pylori to stomach cells. For detailed investigations an arabinogalactan protein (F2 was isolated from seeds and characterized concerning molecular weight, carbohydrate, amino acid composition, linkage, configuration and reaction with β-glucosyl Yariv. Functional testing of F2 was performed by semiquantitative in situ adhesion assay on sections of human gastric mucosa and by quantitative in vitro adhesion assay with FITC-labled H. pylori strain J99 and human stomach AGS cells. Bacterial adhesins affected were identified by overlay assay with immobilized ligands. 125I-radiolabeled F2 served for binding studies to H. pylori and interaction experiments with BabA and SabA. F2 had no cytotoxic effects against H. pylori and AGS cells; but inhibited bacterial binding to human gastric cells. F2 inhibited the binding of BabA and fibronectin-binding adhesin to its specific ligands. Radiolabeled F2 bound non-specifically to different strains of H. pylori; and to BabA deficient mutant. F2 did not lead to subsequent feedback regulation or increased expression of adhesins or virulence factors. From these data the non-specific interactions between F2 and the H. pylori lead to moderate antiadhesive effects.

  3. Antiadhesive properties of arabinogalactan protein from ribes nigrum seeds against bacterial adhesion of Helicobacter pylori.

    Science.gov (United States)

    Messing, Jutta; Niehues, Michael; Shevtsova, Anna; Borén, Thomas; Hensel, Andreas

    2014-01-01

    Fruit extracts from black currants (Ribes nigrum L.) are traditionally used for treatment of gastritis based on seed polysaccharides that inhibit the adhesion of Helicobacter pylori to stomach cells. For detailed investigations an arabinogalactan protein (F2) was isolated from seeds and characterized concerning molecular weight, carbohydrate, amino acid composition, linkage, configuration and reaction with β-glucosyl Yariv. Functional testing of F2 was performed by semiquantitative in situ adhesion assay on sections of human gastric mucosa and by quantitative in vitro adhesion assay with FITC-labled H. pylori strain J99 and human stomach AGS cells. Bacterial adhesins affected were identified by overlay assay with immobilized ligands. ¹²⁵I-radiolabeled F2 served for binding studies to H. pylori and interaction experiments with BabA and SabA. F2 had no cytotoxic effects against H. pylori and AGS cells; but inhibited bacterial binding to human gastric cells. F2 inhibited the binding of BabA and fibronectin-binding adhesin to its specific ligands. Radiolabeled F2 bound non-specifically to different strains of H. pylori; and to BabA deficient mutant. F2 did not lead to subsequent feedback regulation or increased expression of adhesins or virulence factors. From these data the non-specific interactions between F2 and the H. pylori lead to moderate antiadhesive effects. PMID:24662083

  4. Acute phase proteins in serum and cerebrospinal fluid in the course of bacterial meningitis.

    Science.gov (United States)

    Paradowski, M; Lobos, M; Kuydowicz, J; Krakowiak, M; Kubasiewicz-Ujma, B

    1995-08-01

    We carried out estimations of the following acute phase proteins: C-reactive protein (CRP), alpha-1-antitrypsin (AAT), alpha-1-acid glycoprotein (AAG), alpha-2-ceruloplasmin (CER), and alpha-2-haptoglobin (HPT) in serum and in cerebrospinal fluid (CSF) in patients with bacterial meningitis (BM, n = 30) and viral meningitis (VM, n = 30). We have shown that determinations of concentrations of AAG and CRP in serum and CER in CSF are useful in differentiation between BM and VM. The diagnostic power of these three tests (the areas under their ROC curves equal 0.942, 0.929, and 0.931, respectively) is bigger, though statistically not significantly, than that of traditional parameters of BM in CSF, i.e., total protein concentration and white blood cell count. Determination of AAG, CRP, and AAT in serum is a valuable monitoring marker in the course of BM treatment. Convenience of serum sampling constitutes an advantage over traditional BM parameters in CSF. PMID:8521602

  5. Optimized localization of bacterial infections with technetium-99m labelled human immunoglobulin after protein charge selection

    International Nuclear Information System (INIS)

    To improve the scintigraphic detection of bacterial infections a protein charge-purified fraction of polyclonal human immunoglobulin was applied as a radiopharmaceutical. This purification was achieved by attaching the immunoglobulin to an anion-exchanger column and by obtaining the column-bound fraction with buffer. The binding to bacteria in vitro and the target to non-target ratios of an experimental thigh infection with Staphylococcus aureus or Klebsiella pneumoniae in mice were evaluated to compare the purified and the unpurified immunoglobulin. The percentage of binding to all gram-positive and gram-negative bacteria used in this study was significantly (P99mTc-labelled protein charge-purified polyclonal human immunoglobulin was administered intravenously. At all time intervals the target (infected thighs) to non-target (non-infected thighs) ratios for both infections were significantly higher (P99mTc-labelled protein charge-purified immunoglobulin localizes both a gram-positive and a gram-negative thigh infection more intensely and faster than 99mTc-labelled unpurified immunoglobulin. (orig.)

  6. Symmetry and scale orient Min protein patterns in shaped bacterial sculptures

    Science.gov (United States)

    Wu, Fabai; van Schie, Bas G. C.; Keymer, Juan E.; Dekker, Cees

    2015-08-01

    The boundary of a cell defines the shape and scale of its subcellular organization. However, the effects of the cell's spatial boundaries as well as the geometry sensing and scale adaptation of intracellular molecular networks remain largely unexplored. Here, we show that living bacterial cells can be ‘sculpted’ into defined shapes, such as squares and rectangles, which are used to explore the spatial adaptation of Min proteins that oscillate pole-to-pole in rod-shaped Escherichia coli to assist cell division. In a wide geometric parameter space, ranging from 2 × 1 × 1 to 11 × 6 × 1 μm3, Min proteins exhibit versatile oscillation patterns, sustaining rotational, longitudinal, diagonal, stripe and even transversal modes. These patterns are found to directly capture the symmetry and scale of the cell boundary, and the Min concentration gradients scale with the cell size within a characteristic length range of 3-6 μm. Numerical simulations reveal that local microscopic Turing kinetics of Min proteins can yield global symmetry selection, gradient scaling and an adaptive range, when and only when facilitated by the three-dimensional confinement of the cell boundary. These findings cannot be explained by previous geometry-sensing models based on the longest distance, membrane area or curvature, and reveal that spatial boundaries can facilitate simple molecular interactions to result in far more versatile functions than previously understood.

  7. Structure of the Bacterial Cytoskeleton Protein Bactofilin by NMR Chemical Shifts and Sequence Variation.

    Science.gov (United States)

    Kassem, Maher M; Wang, Yong; Boomsma, Wouter; Lindorff-Larsen, Kresten

    2016-06-01

    Bactofilins constitute a recently discovered class of bacterial proteins that form cytoskeletal filaments. They share a highly conserved domain (DUF583) of which the structure remains unknown, in part due to the large size and noncrystalline nature of the filaments. Here, we describe the atomic structure of a bactofilin domain from Caulobacter crescentus. To determine the structure, we developed an approach that combines a biophysical model for proteins with recently obtained solid-state NMR spectroscopy data and amino acid contacts predicted from a detailed analysis of the evolutionary history of bactofilins. Our structure reveals a triangular β-helical (solenoid) conformation with conserved residues forming the tightly packed core and polar residues lining the surface. The repetitive structure explains the presence of internal repeats as well as strongly conserved positions, and is reminiscent of other fibrillar proteins. Our work provides a structural basis for future studies of bactofilin biology and for designing molecules that target them, as well as a starting point for determining the organization of the entire bactofilin filament. Finally, our approach presents new avenues for determining structures that are difficult to obtain by traditional means. PMID:27276252

  8. EXPRESSION OF BACTERIAL PROTEIN-A IN TOBACCO LEADS TO ENHANCED RESISTANCE TO STRESS CONDITIONS

    Directory of Open Access Journals (Sweden)

    Chaitali Roy

    2014-08-01

    Full Text Available Tobacco is the most commonly used plant for expression of transgenes from a variety of organisms because it can be easily grown and transformed, it provides abundant amounts of fresh tissue and has a well-established cell culture system. As bacterial enzymes can be synthesized in tobacco, here we explore the possibility of in planta expression of staphylococcal protein-A(PA which is an antibody, an important group among biopharmaceuticals. In our study we have shown that the tobacco plants harboring PA gene could combat the crown gall infection and also effective in resisting abiotic stress conditions. Transgenic plants when subjected to interact with wild variety of Agrobacterium shows its enhanced capability to resist the gall formation. And when transgenic tobacco plants were grown in presence of 200mM NaCl and/or MG(Methylglyoxal solution, shows their increased tolerance towards salinity stress and high MG stress. So far transgenic tobacco plants are concerned, improvements in the expression of recombinant proteins and their recovery from tobacco may also enhance production and commercial use of this protein.

  9. Temporal expression of bacterial proteins instructs host CD4 T cell expansion and Th17 development.

    Directory of Open Access Journals (Sweden)

    Seung-Joo Lee

    2012-01-01

    Full Text Available Pathogens can substantially alter gene expression within an infected host depending on metabolic or virulence requirements in different tissues, however, the effect of these alterations on host immunity are unclear. Here we visualized multiple CD4 T cell responses to temporally expressed proteins in Salmonella-infected mice. Flagellin-specific CD4 T cells expanded and contracted early, differentiated into Th1 and Th17 lineages, and were enriched in mucosal tissues after oral infection. In contrast, CD4 T cells responding to Salmonella Type-III Secretion System (TTSS effectors steadily accumulated until bacterial clearance was achieved, primarily differentiated into Th1 cells, and were predominantly detected in systemic tissues. Thus, pathogen regulation of antigen expression plays a major role in orchestrating the expansion, differentiation, and location of antigen-specific CD4 T cells in vivo.

  10. Characterization of geographically distinct bacterial communities associated with coral mucus produced by Acropora spp. and Porites spp.

    Science.gov (United States)

    McKew, B A; Dumbrell, A J; Daud, S D; Hepburn, L; Thorpe, E; Mogensen, L; Whitby, C

    2012-08-01

    Acropora and Porites corals are important reef builders in the Indo-Pacific and Caribbean. Bacteria associated with mucus produced by Porites spp. and Acropora spp. from Caribbean (Punta Maroma, Mexico) and Indo-Pacific (Hoga and Sampela, Indonesia) reefs were determined. Analysis of pyrosequencing libraries showed that bacterial communities from Caribbean corals were significantly more diverse (H', 3.18 to 4.25) than their Indonesian counterparts (H', 2.54 to 3.25). Dominant taxa were Gammaproteobacteria, Alphaproteobacteria, Firmicutes, and Cyanobacteria, which varied in relative abundance between coral genera and region. Distinct coral host-specific communities were also found; for example, Clostridiales were dominant on Acropora spp. (at Hoga and the Mexican Caribbean) compared to Porites spp. and seawater. Within the Gammproteobacteria, Halomonas spp. dominated sequence libraries from Porites spp. (49%) and Acropora spp. (5.6%) from the Mexican Caribbean, compared to the corresponding Indonesian coral libraries (coral mucus. In addition, the predominance of Clostridiales associated with Acropora spp. provided additional evidence for coral host-specific microorganisms. PMID:22636010

  11. Effects of interactions of auxin-producing bacteria and bacterial-feeding nematodes on regulation of peanut growths.

    Science.gov (United States)

    Xu, Li; Xu, Wensi; Jiang, Ying; Hu, Feng; Li, Huixin

    2015-01-01

    The influences of an IAA (indole-3-acetic acid)-producing bacterium (Bacillus megaterium) and two bacterial-feeding nematodes (Cephalobus sp. or Mesorhabditis sp.) on the growth of peanut (Arachis hypogaea L. cv. Haihua 1) after various durations of time were investigated in natural soils. The addition of bacteria and nematodes and incubation time all significantly affected plant growth, plant root growth, plant nutrient concentrations, soil nutrient concentrations, soil microorganisms and soil auxin concentration. The addition of nematodes caused greater increases in these indices than those of bacteria, while the addition of the combination of bacteria and nematodes caused further increases. After 42-day growth, the increases in soil respiration differed between the additions of two kinds of nematodes because of differences in their life strategies. The effects of the bacteria and nematodes on the nutrient and hormone concentrations were responsible for the increases in plant growth. These results indicate the potential for promoting plant growth via the addition of nematodes and bacteria to soil. PMID:25867954

  12. Effects of interactions of auxin-producing bacteria and bacterial-feeding nematodes on regulation of peanut growths.

    Directory of Open Access Journals (Sweden)

    Li Xu

    Full Text Available The influences of an IAA (indole-3-acetic acid-producing bacterium (Bacillus megaterium and two bacterial-feeding nematodes (Cephalobus sp. or Mesorhabditis sp. on the growth of peanut (Arachis hypogaea L. cv. Haihua 1 after various durations of time were investigated in natural soils. The addition of bacteria and nematodes and incubation time all significantly affected plant growth, plant root growth, plant nutrient concentrations, soil nutrient concentrations, soil microorganisms and soil auxin concentration. The addition of nematodes caused greater increases in these indices than those of bacteria, while the addition of the combination of bacteria and nematodes caused further increases. After 42-day growth, the increases in soil respiration differed between the additions of two kinds of nematodes because of differences in their life strategies. The effects of the bacteria and nematodes on the nutrient and hormone concentrations were responsible for the increases in plant growth. These results indicate the potential for promoting plant growth via the addition of nematodes and bacteria to soil.

  13. Identification of a New Marine Bacterial Strain SD8 and Optimization of Its Culture Conditions for Producing Alkaline Protease.

    Directory of Open Access Journals (Sweden)

    Hongxia Cui

    Full Text Available While much attention has been given to marine microorganisms for production of enzymes, which in general are relatively more stable and active compared to those from plants and animals, studies on alkaline protease production from marine microorganisms have been very limited. In the present study, the alkaline protease producing marine bacterial strain SD8 isolated from sea muds in the Geziwo Qinhuangdao sea area of China was characterized and its optimal culture conditions were investigated. Strain SD8 was initially classified to belong to genus Pseudomonas by morphological, physiological and biochemical characterizations, and then through 16S rDNA sequence it was identified to be likely Pseudomonas hibiscicola. In addition, the culture mediums, carbon sources and culture conditions of strain SD8 were optimized for maximum production of alkaline protease. Optimum enzyme production (236U/mL when cultured bacteria being at 0.75 mg dry weight/mL fermentation broth was obtained when the isolate at a 3% inoculum size was grown in LB medium at 20 mL medium/100mL Erlenmeyer flask for 48h culture at 30°C with an initial of pH 7.5. This was the first report of strain Pseudomonas hibiscicola secreting alkaline protease, and the data for its optimal cultural conditions for alkaline protease production has laid a foundation for future exploration for the potential use of SD8 strain for alkaline protease production.

  14. Identification of a New Marine Bacterial Strain SD8 and Optimization of Its Culture Conditions for Producing Alkaline Protease.

    Science.gov (United States)

    Cui, Hongxia; Yang, Muyang; Wang, Liping; Xian, Cory J

    2015-01-01

    While much attention has been given to marine microorganisms for production of enzymes, which in general are relatively more stable and active compared to those from plants and animals, studies on alkaline protease production from marine microorganisms have been very limited. In the present study, the alkaline protease producing marine bacterial strain SD8 isolated from sea muds in the Geziwo Qinhuangdao sea area of China was characterized and its optimal culture conditions were investigated. Strain SD8 was initially classified to belong to genus Pseudomonas by morphological, physiological and biochemical characterizations, and then through 16S rDNA sequence it was identified to be likely Pseudomonas hibiscicola. In addition, the culture mediums, carbon sources and culture conditions of strain SD8 were optimized for maximum production of alkaline protease. Optimum enzyme production (236U/mL when cultured bacteria being at 0.75 mg dry weight/mL fermentation broth) was obtained when the isolate at a 3% inoculum size was grown in LB medium at 20 mL medium/100mL Erlenmeyer flask for 48h culture at 30°C with an initial of pH 7.5. This was the first report of strain Pseudomonas hibiscicola secreting alkaline protease, and the data for its optimal cultural conditions for alkaline protease production has laid a foundation for future exploration for the potential use of SD8 strain for alkaline protease production. PMID:26716833

  15. Crystalline bacterial biofilm formation on urinary catheters by urease-producing urinary tract pathogens: a simple method of control.

    Science.gov (United States)

    Broomfield, Robert J; Morgan, Sheridan D; Khan, Azhar; Stickler, David J

    2009-10-01

    The problem of catheter encrustation stems from infection by urease-producing bacteria. These organisms generate ammonia from urea, elevate the pH of urine and cause crystals of calcium and magnesium phosphates to form in the urine and the biofilm that develops on the catheter. In this study, a laboratory model was used to compare the ability of 12 urease-positive species of urinary tract pathogens to encrust and block catheters. Proteus mirabilis, Proteus vulgaris and Providencia rettgeri were able to raise the urinary pH above 8.3 and produce catheter-blocking crystalline biofilms within 40 h. Morganella morganii and Staphylococcus aureus elevated the pH of urine to 7.4 and 6.9, respectively, and caused some crystal deposition in the biofilms but did not block catheters in the 96 h experimental period. Isolates of Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Serratia marcescens, Pseudomonas aeruginosa and Providencia stuartii were only capable of raising the pH of urine to a maximum of 6.4 and failed to cause crystal deposition in the biofilm. The most effective way to prevent catheter encrustation was shown to be diluting urine and increasing its citrate concentration. This strategy raises the nucleation pH (pH(n)) at which calcium and magnesium phosphates crystallize from urine. Increasing the fluid intake of a healthy volunteer with citrated drinks resulted in urine with a pH(n) of >8.0 in which catheter encrustation was inhibited. It is suggested that this dietary strategy will be an effective means of controlling catheter encrustation, whichever bacterial species is causing the problem. PMID:19556373

  16. Quantitative polymerase chain reaction (PCR) assays for a bacterial thiaminase I gene and the thiaminase-producing bacterium Paenibacillus thiaminolyticus.

    Science.gov (United States)

    Richter, C.A.; Wright-Osment, Maureen K.; Zajicek, J.L.; Honeyfield, D.C.; Tillitt, D.E.

    2009-01-01

    The thiaminase I enzyme produced by the gram-positive bacterium Paenibacillus thiaminolyticus isolated from the viscera of Lake Michigan alewives Alosa pseudoharengus is currently the only defined source of the thiaminase activity linked to thiamine (vitamin B1) deficiency in early mortality syndrome (EMS) in the larvae of Great Lakes salmonines. Diets of alewife or isolated strains of P. thiaminolyticus mixed in a semipurified diet and fed to lake trout Salvelinus namaycush have been shown to produce EMS in fry. We utilized quantitative polymerase chain reaction (Q-PCR) to aid in studies of the sources of P. thiaminolyticus and thiaminase I. Quantitative PCR assays were established to detect the thiaminase I gene of P. thiaminolyticus, the 16S rRNA gene from most species of bacteria, and the 16S rRNA gene specifically from P. thiaminolyticus and a few closely related taxa. The Q-PCR assays are linear over at least six orders of magnitude and can detect the thiaminase I gene of P. thiaminolyticus from as few as 1,000 P. thiaminolyticus cells/g of sample or the Paenibacillus 16S rRNA gene from as few as 100 P. thiaminolyticus cells/g of sample. The initial results from alewife viscera samples with high thiaminase activity yielded unexpectedly low densities of P. thiaminolyticus cells; Paenibacillus thiaminolyticus was detectable in 2 of 6 alewife viscera tested at densities on the order of 100 cells/g out of 100,000,000 total bacterial cells/g. The low numbers of P. thiaminolyticus detected suggest that alewives contain additional non-P. thiaminolyticus sources of thiaminase activity.

  17. Dependence of Bacterial Protein Adhesins on Toll-Like Receptors for Proinflammatory Cytokine Induction

    Science.gov (United States)

    Hajishengallis, George; Martin, Michael; Sojar, Hakimuddin T.; Sharma, Ashu; Schifferle, Robert E.; DeNardin, Ernesto; Russell, Michael W.; Genco, Robert J.

    2002-01-01

    Toll-like receptors (TLRs) are important signal transducers that mediate inflammatory reactions induced by microbes through pattern recognition of virulence molecules such as lipopolysaccharide (LPS) and lipoproteins. We investigated whether proinflammatory cytokine responses induced by certain bacterial protein adhesins may also depend on TLRs. In differentiated THP-1 mononuclear cells stimulated by LPS-free recombinant fimbrillin (rFimA) from Porphyromonas gingivalis, cytokine release was abrogated by monoclonal antibodies (MAbs) to CD14 and TLR4 but not to TLR2. Similar experiments using anti-β2 integrin MAbs suggested that β2 integrins (CD11/CD18) also play a role in cytokine induction by rFimA or native fimbriae. Minor fimbriae (distinct from the fimA-encoded major fimbriae) of P. gingivalis induced proinflammatory cytokine release in a CD14- and TLR2-dependent mode. Cytokine induction by BspA, a leucine-rich repeat protein from Bacteroides forsythus, depended heavily on CD14 and TLR2. We also found that the ability of the streptococcal protein AgI/II to stimulate cytokine release depended partially on CD14 and TLR4, and the AgI/II segment that possibly interacts with these receptors was identified as its N-terminal saliva-binding region. When THP-1 cells were exposed to rFimA for 24 h, surface expression of CD14 and CD18 was decreased and the cells became hyporesponsive to cytokine induction by a second challenge with rFimA. However, tolerance induction was abolished when the THP-1 cells were pretreated with rFimA in the presence of either anti-CD14 MAb or anti-TLR4 MAb. Induction of cross-tolerance between rFimA and LPS correlated with downregulation of the pattern recognition receptors involved. Our data suggest that the CD14-TLR2/4 system is involved in cytokine production and tolerance induction upon interaction with certain proinflammatory bacterial protein adhesins. PMID:11874886

  18. A robust and rapid method of producing soluble, stable, and functional G-protein coupled receptors.

    Directory of Open Access Journals (Sweden)

    Karolina Corin

    Full Text Available Membrane proteins, particularly G-protein coupled receptors (GPCRs, are notoriously difficult to express. Using commercial E. coli cell-free systems with the detergent Brij-35, we could rapidly produce milligram quantities of 13 unique GPCRs. Immunoaffinity purification yielded receptors at >90% purity. Secondary structure analysis using circular dichroism indicated that the purified receptors were properly folded. Microscale thermophoresis, a novel label-free and surface-free detection technique that uses thermal gradients, showed that these receptors bound their ligands. The secondary structure and ligand-binding results from cell-free produced proteins were comparable to those expressed and purified from HEK293 cells. Our study demonstrates that cell-free protein production using commercially available kits and optimal detergents is a robust technology that can be used to produce sufficient GPCRs for biochemical, structural, and functional analyses. This robust and simple method may further stimulate others to study the structure and function of membrane proteins.

  19. Immunotoxicity of nucleic acid reduced BioProtein - a bacterial derived single cell protein - in Wistar rats

    DEFF Research Database (Denmark)

    Mølck, Anne-marie; Poulsen, Morten; Christensen, Hanne Risager;

    2002-01-01

    NABP induced a humoral immune response and proliferation of phagocytic cell lines, mainly macrophages. The humoral response involved induction of NABP specific IgM and IgG. The proliferation of phagocytic cells involved increase of the white blood cell count of all dosed female groups. Males showed......BioProtein is a single cell protein produced by a mixed methanotrophic and heterotrophic bacteria culture using natural gas as energy source, which has been approved for animal feed. BioProtein contains a large amount of nucleic acids making the product less suitable for human consumption......, hyperplasia of Kupffer cells in the liver, increased granulopoiesis in spleen and bone marrow, and infiltration of lamina propria of the large intestine with eosinophilic granulocytes. The most consistent and pronounced changes were observed in the highest dose group, but even at the lowest dose level some...

  20. Mixed biofilm formation by Shiga toxin-producing Escherichia coli and Salmonella enterica serovar typhimurium enhanced bacterial resistance to sanitization due to extracellular polymeric substances

    Science.gov (United States)

    Shiga toxin–producing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium are important foodborne pathogens capable of forming single-species biofilms or coexisting in multispecies biofilm communities. Bacterial biofilm cells are usually more resistant to sanitization than their pla...

  1. Optimized localization of bacterial infections with technetium-99m labelled human immunoglobulin after protein charge selection

    Energy Technology Data Exchange (ETDEWEB)

    Welling, M. (Dept. of Diagnostic Radiology and Nuclear Medicine, University Hospital, Leiden (Netherlands)); Feitsma, H.I.J. (Dept. of Diagnostic Radiology and Nuclear Medicine, University Hospital, Leiden (Netherlands)); Calame, W. (Dept. of Diagnostic Radiology and Nuclear Medicine, University Hospital, Leiden (Netherlands)); Ensing, G.J. (Mallinckrodt Medical, Petten (Netherlands)); Goedemans, W. (Mallinckrodt Medical, Petten (Netherlands)); Pauwels, E.K.J. (Dept. of Diagnostic Radiology and Nuclear Medicine, University Hospital, Leiden (Netherlands))

    1994-10-01

    To improve the scintigraphic detection of bacterial infections a protein charge-purified fraction of polyclonal human immunoglobulin was applied as a radiopharmaceutical. This purification was achieved by attaching the immunoglobulin to an anion-exchanger column and by obtaining the column-bound fraction with buffer. The binding to bacteria in vitro and the target to non-target ratios of an experimental thigh infection with Staphylococcus aureus or Klebsiella pneumoniae in mice were evaluated to compare the purified and the unpurified immunoglobulin. The percentage of binding to all gram-positive and gram-negative bacteria used in this study was significantly (P<0.03) higher for the purified than for the unpurified immunoglobulin. For the in vivo study, mice were infected in the thigh muscle with Staph. aureus or K. pneumoniae. After 18 h 0.1 mg of technetium-99m labelled polyclonal immunoglobulin or [sup 99m]Tc-labelled protein charge-purified polyclonal human immunoglobulin was administered intravenously. At all time intervals the target (infected thighs) to non-target (non-infected thighs) ratios for both infections were significantly higher (P<0.03) for protein charge-purified polyclonal immunoglobulin than for unpurified polyclonal human immunoglobulin. Already within 1 h the infected tissues could be detected by the purified immunoglobulin. It is concluded that [sup 99m]Tc-labelled protein charge-purified immunoglobulin localizes both a gram-positive and a gram-negative thigh infection more intensely and faster than [sup 99m]Tc-labelled unpurified immunoglobulin. (orig.)

  2. Isolation and Identification of a New Tetrodotoxin-Producing Bacterial Species, Raoultella terrigena, from Hong Kong Marine Puffer Fish Takifugu niphobles

    Directory of Open Access Journals (Sweden)

    Fred Wang-Fat Lee

    2011-11-01

    Full Text Available Puffer fish, Takifugu niphobles, collected from the Hong Kong coastal waters were screened for tetrodotoxin-producing bacteria. A Gram-negative, non-acid-fast, non-sporing and rod shaped bacterial strain (designated as gutB01 was isolated from the intestine of the puffer fish and was shown to produce tetrodotoxin (TTX. Based on the Microbial Identification (MIDI and 16S-23S rDNA internal transcribed spacer (ITS phylogenetic analysis, the strain was identified as Raoultella terrigena. The TTX production ability of the strain was confirmed by mouse bioassay, ELISA and mass spectrometry (MALDI-TOF. Our results reiterate that the TTX found in puffer fish was likely produced by the associated bacteria and TTX are widely produced amongst a diversity of bacterial species.

  3. Secreted and immunogenic proteins produced by the honey bee bacterial pathogen, Paenibacillus larvae

    Science.gov (United States)

    American Foulbrood is a severe disease affecting larvae of honeybee Apis mellifera, causing significant decrease in the honeybee population, beekeeping industries and agricultural production. In spite of its importance, little is known about the virulence factors secreted by Paenibacillus larvae dur...

  4. Method for producing limited and extensive protein hydrol yzates from agroindustrial waste

    OpenAIRE

    Millán, Francisco; Pedroche, Justo; Yust, María del Mar; Alcaide-Hidalgo, J. M.; Millán-Linares, María del Carmen; Villanueva, Álvaro; Tejedor, José Luis

    2010-01-01

    [EN] The present invention relates to a method for producing limited and extensive protein hydrolyzates that comprises perfonning enzymatic hydrolysis on agroindustrial protein waste. Said method leads to the production, in a single hydrolytic step without prior treatment of the starting substrate, of two different protein hydrolyzates that differ in tenns of the degree of hydrolysis thereof, instead of a single product as in the case of other similar methods. These two products that...

  5. Surface-modified nanoparticles as a new, versatile, and mechanically robust nonadhesive coating : Suppression of protein adsorption and bacterial adhesion

    NARCIS (Netherlands)

    Holmes, P. F.; Currie, E. P. K.; Thies, J. C.; van der Mei, H. C.; Busscher, H. J.; Norde, W.

    2009-01-01

    The synthesis of surface-modified silica nanoparticles, chemically grafted with acrylate and poly(ethylene glycol) (PEG) groups, and the ability of the resulting crosslinked coatings to inhibit protein adsorption and bacterial adhesion are explored. Water contact angles, nanoindentation, and atomic

  6. Bacterial pathogen gene regulation: a DNA-structure-centred view of a protein-dominated domain.

    Science.gov (United States)

    Dorman, Charles J; Colgan, Aoife; Dorman, Matthew J

    2016-07-01

    The mechanisms used by bacterial pathogens to regulate the expression of their genes, especially their virulence genes, have been the subject of intense investigation for several decades. Whole genome sequencing projects, together with more targeted studies, have identified hundreds of DNA-binding proteins that contribute to the patterns of gene expression observed during infection as well as providing important insights into the nature of the gene products whose expression is being controlled by these proteins. Themes that have emerged include the importance of horizontal gene transfer to the evolution of pathogens, the need to impose regulatory discipline upon these imported genes and the important roles played by factors normally associated with the organization of genome architecture as regulatory principles in the control of virulence gene expression. Among these architectural elements is the structure of DNA itself, its variable nature at a topological rather than just at a base-sequence level and its ability to play an active (as well as a passive) part in the gene regulation process. PMID:27252403

  7. Optimization of Mutation Pressure in Relation to Properties of Protein-Coding Sequences in Bacterial Genomes.

    Directory of Open Access Journals (Sweden)

    Paweł Błażej

    Full Text Available Most mutations are deleterious and require energetically costly repairs. Therefore, it seems that any minimization of mutation rate is beneficial. On the other hand, mutations generate genetic diversity indispensable for evolution and adaptation of organisms to changing environmental conditions. Thus, it is expected that a spontaneous mutational pressure should be an optimal compromise between these two extremes. In order to study the optimization of the pressure, we compared mutational transition probability matrices from bacterial genomes with artificial matrices fulfilling the same general features as the real ones, e.g., the stationary distribution and the speed of convergence to the stationarity. The artificial matrices were optimized on real protein-coding sequences based on Evolutionary Strategies approach to minimize or maximize the probability of non-synonymous substitutions and costs of amino acid replacements depending on their physicochemical properties. The results show that the empirical matrices have a tendency to minimize the effects of mutations rather than maximize their costs on the amino acid level. They were also similar to the optimized artificial matrices in the nucleotide substitution pattern, especially the high transitions/transversions ratio. We observed no substantial differences between the effects of mutational matrices on protein-coding sequences in genomes under study in respect of differently replicated DNA strands, mutational cost types and properties of the referenced artificial matrices. The findings indicate that the empirical mutational matrices are rather adapted to minimize mutational costs in the studied organisms in comparison to other matrices with similar mathematical constraints.

  8. Blood parameters in growing pigs fed increasing levels of bacterial protein meal

    Directory of Open Access Journals (Sweden)

    Tauson Anne-Helene

    2007-11-01

    Full Text Available Abstract The experiment investigated the effects of increasing dietary levels of bacterial protein meal (BPM on various blood parameters reflecting protein and fat metabolism, liver function, and purine base metabolism in growing pigs. Sixteen barrows were allocated to four different experimental diets. The control diet was based on soybean meal. In the other three diets soybean meal was replaced with increasing levels of BPM, approximately 17%, 35%, and 50% of the nitrogen being derived from BPM. Blood samples from the jugular vein were taken when the body weights of the pigs were approximately 10 kg, 21 kg, 45 kg, and 77 kg. The blood parameters reflecting fat metabolism and liver function were not affected by diet. Both the plasma albumin and uric acid concentrations tended to decrease (P = 0.07 and 0.01, respectively with increasing dietary BPM content, whereas the plasma glucose concentration tended to increase (P = 0.07 with increasing dietary BPM content. It was concluded that up to 50% of the nitrogen could be derived from BPM without affecting metabolic function, as reflected in the measured blood parameters.

  9. Intramammary Immunization of Pregnant Mice with Staphylococcal Protein A Reduces the Post-Challenge Mammary Gland Bacterial Load but Not Pathology.

    Directory of Open Access Journals (Sweden)

    Jully Gogoi-Tiwari

    Full Text Available Protein A, encoded by the spa gene, is one of the major immune evading MSCRAMM of S. aureus, demonstrated to be prevalent in a significant percentage of clinical bovine mastitis isolates in Australia. Given its' reported significance in biofilm formation and the superior performance of S. aureus biofilm versus planktonic vaccine in the mouse mastitis model, it was of interest to determine the immunogenicity and protective potential of Protein A as a potential vaccine candidate against bovine mastitis using the mouse mastitis model. Pregnant Balb/c mice were immunised with Protein A emulsified in an alum-based adjuvant by subcutaneous (s/c or intramammary (i/mam routes. While humoral immune response of mice post-immunization were determined using indirect ELISA, cell-mediated immune response was assessed by estimation of interferon-gamma (IFN-γ produced by protein A-stimulated splenocyte supernatants. Protective potential of Protein A against experimental mastitis was determined by challenge of immunized versus sham-vaccinated mice by i/mam route, based upon manifestation of clinical symptoms, total bacterial load and histopathological damage to mammary glands. Significantly (p<0.05 higher levels of IgG1 isotype were produced in mice immunized by the s/c route. In contrast, significantly higher levels of the antibody isotype IgG2a were produced in mice immunized by the i/mam route (p<0.05. There was significant reduction (p<0.05 in bacterial loads of the mammary glands of mice immunized by Protein A regardless of the route of immunization, with medium level of clinical symptoms observed up to day 3 post-challenge. However, Protein A vaccine failed to protect immunized mice post-challenge with biofilm producing encapsulated S. aureus via i/mam route, regardless of the route of immunization, as measured by the level of mammary tissue damage. It was concluded that, Protein A in its' native state was apparently not a suitable candidate for inclusion

  10. Binary Bacterial Toxins: Biochemistry, Biology, and Applications of Common Clostridium and Bacillus Proteins

    OpenAIRE

    Barth, Holger; Aktories, Klaus; Popoff, Michel R.; Stiles, Bradley G.

    2004-01-01

    Certain pathogenic species of Bacillus and Clostridium have developed unique methods for intoxicating cells that employ the classic enzymatic “A-B” paradigm for protein toxins. The binary toxins produced by B. anthracis, B. cereus, C. botulinum, C. difficile, C. perfringens, and C. spiroforme consist of components not physically associated in solution that are linked to various diseases in humans, animals, or insects. The “B” components are synthesized as precursors that are subsequently acti...

  11. Purification and functional analysis of the recombinant protein isolated from E. coli by employing three different methods of bacterial lysis

    Directory of Open Access Journals (Sweden)

    MARIJA MOJSIN

    2005-07-01

    Full Text Available In this paper, the purification of the human recombinant protein expressed in E. coli using the GSTGene Fusion System, by applying various methods of bacterial lysis: sonication, freeze/thaw and beadbeating, is presented. The study was an attempt to compare the properties of the proteins obtained by the sonication method, recommended by manufacturers but inaccessible for many researchers, with those obtained using two other readily available lysis methods. The data show that all purified proteins were soluble and intact with the highest protein yield being obtained via the freeze/thaw method. The results of functional analysis indicate that the proteins purified using the sonication and freeze/thaw methods of lysis exhibited similar DNA binding affinity, while the protein purified by beadbeating was also functional but with a lower binding affinity. The conclusion of this study is that all three lysis methods could be successfully employed for protein purification.

  12. Leaching and heating process as alternative to produce fish protein powder from Kilka (Clupeonella cultiventris caspia

    Directory of Open Access Journals (Sweden)

    KAVEH RAHMANIFARAH

    2014-05-01

    Full Text Available Rahmanifarah K, Shabanpour B, Shaviklo AR, Aalami M. 2014. Leaching and heating process as alternative to produce fish protein powder from Kilka (Clupeonella cultiventris caspia. Nusantara Bioscience 6: 1-6. The effect of protein extraction procedures (leached mince and heated suspension on selected properties of fish protein powder (proximate composition, pH, color, density, viscosity, fat adsorption, emulsifying capacity, emulsifying stability, foaming capacity, foaming stability, WBC, protein solubility in water, hygroscopicity, Trichloroacetic acid (TCA-soluble peptides and free sulfhydryl groups was investigated. Results showed that Fish protein powder (FPP produced by leaching mince (LM have higher protein, moisture, ash, pH, L*, viscosity, emulsion capacity, emulsion stability, foam capacity, foam stability, water binding capacity (WBC, protein solubility, hygroscopicity, TCA soluble peptides and free sulfhydryl group content than heated suspension (HS (P0.05. Overall, it was observed that high temperature during heating of suspension in HS method makes possible protein denaturation and aggregation. Consequently, based on functional, chemical and physical properties, extraction of fish protein by leaching process was found to be suitable for the production of fish protein powder.

  13. The population structure of antibiotic-producing bacterial symbionts of Apterostigma dentigerum ants: impacts of coevolution and multipartite symbiosis.

    Science.gov (United States)

    Caldera, Eric J; Currie, Cameron R

    2012-11-01

    Fungus-growing ants (Attini) are part of a complex symbiosis with Basidiomycetous fungi, which the ants cultivate for food, Ascomycetous fungal pathogens (Escovopsis), which parasitize cultivars, and Actinobacteria, which produce antibiotic compounds that suppress pathogen growth. Earlier studies that have characterized the association between attine ants and their bacterial symbionts have employed broad phylogenetic approaches, with conclusions ranging from a diffuse coevolved mutualism to no specificity being reported. However, the geographic mosaic theory of coevolution proposes that coevolved interactions likely occur at a level above local populations but within species. Moreover, the scale of population subdivision is likely to impact coevolutionary dynamics. Here, we describe the population structure of bacteria associated with the attine Apterostigma dentigerum across Central America using multilocus sequence typing (MLST) of six housekeeping genes. The majority (90%) of bacteria that were isolated grouped into a single clade within the genus Pseudonocardia. In contrast to studies that have suggested that Pseudonocardia dispersal is high and therefore unconstrained by ant associations, we found highly structured ([Formula: see text]) and dispersal-limited (i.e., significant isolation by distance; [Formula: see text], [Formula: see text]) populations over even a relatively small scale (e.g., within the Panama Canal Zone). Estimates of recombination versus mutation were uncharacteristically low compared with estimates for free-living Actinobacteria (e.g., [Formula: see text] in La Selva, Costa Rica), which suggests that recombination is constrained by association with ant hosts. Furthermore, Pseudonocardia population structure was correlated with that of Escovopsis species ([Formula: see text], [Formula: see text]), supporting the bacteria's role in disease suppression. Overall, the population dynamics of symbiotic Pseudonocardia are more consistent with a

  14. Feasibility of EPS-producing bacterial inoculation to speed up the sand aggregation in the Gurbantunggut Desert, Northwestern China.

    Science.gov (United States)

    Wu, Nan; Pan, Hui-Xia; Qiu, Dong; Zhang, Yuan-Ming

    2014-12-01

    Exopolymers are known to be useful in improving sand aggregation and the development of biological soil crusts (BSCs). A facultative bacterium KLBB0001 was isolated from BSCs in the Gurbantunggut Desert in northwestern China. With the strong effective production of extracellular polymeric substances (EPS), this strain exhibits a multifunctional role for sand stabilization and maintenance of water under laboratory conditions. Practical testing of the feasibility of its inoculation to speed up BSC recovery in the field was also conducted in this experiment. This strain stimulated the heterotrophic community assembly in the topsoil layer (0-2 cm) before the commencement of autotrophic cyanobacteria, while also significantly increasing the number of bacteria, actinomycetes, and content of total phosphorus, available nitrogen, and available phosphorus. However, the low nitrogenase activity (NA) (0.57 µmol/h) that was observed caused us to doubt the previous identification as Azotobacter Beijerinck that was based on physiological and biochemical properties. A phylogenetic analysis based on 16S rRNA gene sequences revealed that this strain was a member of the genus Paenibacillus. It exhibited the closest phylogenetic affinity and highest sequence similarity to the strain Paenibacillus mucilaginosus VKPM B-7519 (sequence similarity 99.698%), which is well known as a typical silicate-weathering bacteria that releases lots of nutritional ions from minerals and the soil. Because P. mucilaginosus can excrete carbonic anhydrase (CA) to capture atmospheric CO2 through hydration of CO2 , it is possible that KLBB0001 might use a similar strategy for heterotrophs in the BSCs to sequester CO2 from the air. Because of its potential role in the reestablishment of the BSC ecosystem due to its ability to improve water relations, sand stabilization, and chemical erosion, EPS-producing bacterial inoculation was concluded to be a suitable and effective treatment for BSC recovery

  15. The use of C-reactive protein in predicting bacterial co-Infection in children with bronchiolitis

    Directory of Open Access Journals (Sweden)

    Mohamad Fares

    2011-03-01

    Full Text Available Background: Bronchiolitis is a potentially life-threatening respiratory illness commonly affecting children who are less than two years of age. Patients with viral lower respiratory tract infection are at risk for co-bacterial infection. Aim: The aim of our study was to evaluate the use of C-reactive protein (CRP in predicting bacterial co-infection in patients hospitalized for bronchiolitis and to correlate the results with the use of antibiotics. Patients and Methods: This is a prospective study that included patients diagnosed with bronchiolitis admitted to Makassed General Hospital in Beirut from October 2008 to April 2009. A tracheal aspirate culture was taken from all patients with bronchiolitis on admission to the hospital. Blood was drawn to test C-reactive protein level, white cell count, transaminases level, and blood sugar level. Results: Forty-nine patients were enrolled in the study and were divided into two groups. Group 1 included patients with positive tracheal aspirate culture and Group 2 included those with negative culture. All patients with a CRP level ≥2 mg/dL have had bacterial co-infection. White cell count, transaminases and blood sugar levels were not predictive for bacterial co-infection. The presence of bacterial co-infection increased the length of hospital stay in the first group by 2 days compared to those in the second group. Conclusion: Bacterial co-infection is frequent in infants with moderate to severe bronchiolitis and requires admission. Our data showed that a CRP level greater than 1.1 mg/dL raised suspicion for bacterial co-infection. Thus, a tracheal aspirate should be investigated microbiologically in all hospitalized patients in order to avoid unnecessary antimicrobial therapy and to shorten the duration of the hospital stay.

  16. Host and bacterial proteins that repress recruitment of LC3 to Shigella early during infection.

    Directory of Open Access Journals (Sweden)

    Leigh A Baxt

    Full Text Available Shigella spp. are intracytosolic gram-negative pathogens that cause disease by invasion and spread through the colonic mucosa, utilizing host cytoskeletal components to form propulsive actin tails. We have previously identified the host factor Toca-1 as being recruited to intracellular S. flexneri and being required for efficient bacterial actin tail formation. We show that at early times during infection (40 min., the type three-secreted effector protein IcsB recruits Toca-1 to intracellular bacteria and that recruitment of Toca-1 is associated with repression of recruitment of LC3, as well as with repression of recruitment of the autophagy marker NDP52, around these intracellular bacteria. LC3 is best characterized as a marker of autophagosomes, but also marks phagosomal membranes in the process LC3-associated phagocytosis. IcsB has previously been demonstrated to be required for S. flexneri evasion of autophagy at late times during infection (4-6 hr by inhibiting binding of the autophagy protein Atg5 to the Shigella surface protein IcsA (VirG. Our results suggest that IcsB and Toca-1 modulation of LC3 recruitment restricts LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants. Together with published results, our findings suggest that IcsB inhibits innate immune responses in two distinct ways, first, by inhibiting LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants early during infection, and second, by inhibiting autophagy late during infection.

  17. Effects of soybean fortification on protein quality of tortilla-based diets produced from regular and quality protein maize.

    Science.gov (United States)

    Amaya-Guerra, Carlos A; Alanis-Guzman, Maria Guadalupe; Serna Saldívar, Sergio O

    2004-01-01

    The physiological development of laboratory rats fed with a typical indigenous tortilla diet was studied for two generations. The experiment compared casein control diet and five different types of diets: (1) a diet of tortillas obtained from fresh masa (FM); (2) regular tortillas produced from enriched dry masa flour containing vitamins B1, B2, niacin, folic acid, and the microminerals iron and zinc (REDMF); (3) tortillas produced from enriched dry masa flour fortified with 6% defatted soybean meal (FEDMF); (4) tortillas produced from enriched quality protein maize flour (EQPM); and (5) and, tortillas produced from enriched quality protein maize flour fortified with 3% defatted soybean meal (FEQPM). The growth of rats fed FEDMF and FEQPM diets was significantly higher (P tortilla had the highest protein digestibility, but the FEQPM and FEDMF diets had the highest biological value (BV), net protein utilization (NPU), and protein-digestibility-corrected EAA scores (PDCEAAS). The difference among treatments was more evident in the second-generation rats. The pregnancy rate, number of newborns/litter, litter weight, and newborn survival rate was also higher for rats fed FEDMF, EQPM, and FEQPM diets than their counterparts fed REDMF and FM. PMID:15678750

  18. Surfactant protein D augments bacterial association but attenuates major histocompatibility complex class II presentation of bacterial antigens

    DEFF Research Database (Denmark)

    Hansen, Søren; Lo, Bernice; Evans, Kathy;

    2006-01-01

    Development of dementia, including Alzheimer's disease (AD), is associated with lipid dysregulation and inflammation. As the host defense lectin surfactant protein D (SP-D) has multiple effects in lipid homeostasis and inflammation, the correlation between SP-D concentrations and development of d...

  19. Nucleotide and partner-protein control of bacterial replicative helicase structure and function.

    Science.gov (United States)

    Strycharska, Melania S; Arias-Palomo, Ernesto; Lyubimov, Artem Y; Erzberger, Jan P; O'Shea, Valerie L; Bustamante, Carlos J; Berger, James M

    2013-12-26

    Cellular replication forks are powered by ring-shaped, hexameric helicases that encircle and unwind DNA. To better understand the molecular mechanisms and control of these enzymes, we used multiple methods to investigate the bacterial replicative helicase, DnaB. A 3.3 Å crystal structure of Aquifex aeolicus DnaB, complexed with nucleotide, reveals a newly discovered conformational state for this motor protein. Electron microscopy and small angle X-ray scattering studies confirm the state seen crystallographically, showing that the DnaB ATPase domains and an associated N-terminal collar transition between two physical states in a nucleotide-dependent manner. Mutant helicases locked in either collar state are active but display different capacities to support critical activities such as duplex translocation and primase-dependent RNA synthesis. Our findings establish the DnaB collar as an autoregulatory hub that controls the ability of the helicase to transition between different functional states in response to both nucleotide and replication initiation/elongation factors. PMID:24373746

  20. Bacterial cocaine esterase: a protein-based therapy for cocaine overdose and addiction

    Science.gov (United States)

    Narasimhan, Diwahar; Woods, James H; Sunahara, Roger K

    2012-01-01

    Cocaine is highly addictive and there are no pharmacotherapeutic drugs available to treat acute cocaine toxicity or chronic abuse. Antagonizing an inhibitor such as cocaine using a small molecule has proven difficult. The alternative approach is to modify cocaine’s pharmacokinetic properties by sequestering or hydrolyzing it in serum and limiting access to its sites of action. We took advantage of a bacterial esterase (CocE) that has evolved to hydrolyze cocaine and have developed it as a therapeutic that rapidly and specifically clears cocaine from the subject. Native enzyme was unstable at 37°C, thus limiting CocE’s potential. Innovative computational methods based on the protein’s structure helped elucidate its mechanism of destabilization. Novel protein engineering methodologies were applied to substantially improve its stability in vitro and in vivo. These improvements rendered CocE as a powerful and efficacious therapeutic to treat cocaine intoxication and lead the way towards developing a therapy for addiction. PMID:22300094

  1. Conformation of protein secreted across bacterial outer membranes: a study of enterotoxin translocation from Vibrio cholerae

    International Nuclear Information System (INIS)

    The secretion of enterotoxin by Vibrio cholerae is punctuated by the transient entry of the toxin subunits into the periplasm. In this paper, the authors show that the subunits oligomerize into an assembled holotoxin within the periplasm prior to their secretion across the outer membrane. The rate of toxin assembly was studied by pulse-labeling cells with [35S]-methionine and then monitoring the turnover of radiolabeled subunits as they assembled within the periplasm. The subunits entered the periplasm as monomers and assembled into oligomers with a half-time of ≅ 1 min. Since assembly was a rapid event compared to the rate of toxin efflux from the periplasm, which had a half-time of ≅ 13 min, they conclude that all of the subunits that pass through the periplasm assemble before they traverse the outer membrane. The average concentration of subunit monomers and assembled holotoxin within the periplasm was calculated to be ≅ 20 and ≅ 260 μg/ml, respectively. This indicates that the periplasm is a suitably concentrated milieu where spontaneous toxin assembly can occur. These findings suggest that protein movement across bacterial outer membranes, in apparent contrast to export across other biological membranes, involves translocation of polypeptides that have already folded into tertiary and even quaternary conformations

  2. Immunological study of the outer membrane proteins of Vibrio harveyi: insights that link immunoprotectivity to interference with bacterial infection.

    Science.gov (United States)

    Yu, Lan-ping; Hu, Yong-hua; Sun, Bo-guang; Sun, Li

    2013-10-01

    Vibrio harveyi is a bacterial pathogen that affects marine vertebrates and invertebrates. In this study, we identified 13 outer membrane proteins (OMPs) from a pathogenic V. harveyi strain and analyzed their immunological properties. In vivo immunogenicity analysis showed that antibodies specific to recombinant proteins of the 13 OMPs were detected in the antiserum of V. harveyi-infected rat. When used as subunit vaccines to immunize Japanese flounder (Paralichthys olivaceus), all OMPs were able to elicit specific serum antibody production in the vaccinated fish; however, only two OMPs (OMP173 and OMP214) induced high levels (>70%) of relative percent survival. Pre-incubation of V. harveyi with the antisera of protective OMPs significantly impaired bacterial infectivity against peripheral blood leukocytes (PBL), whereas the antisera of non-protective OMPs had no apparent effect on infection. OMP173 antibodies could bind whole V. harveyi cells and exhibit bactericidal effect in a complement-dependent manner. Passive immunization showed that fish received OMP173 antiserum before being infected with V. harveyi exhibited significantly reduced mortality rate and lower bacterial loads in liver, spleen, and kidney. Finally, treatment of FG cells with OMP173 prior to V. harveyi infection protected the cells from bacterial invasion to a significant extent. Take together, these results indicate that two of the examined OMPs induce protective immunity through production of specific antibodies that block bacterial invasion, and that one OMP is likely to be involved in host cell interaction during the infection process. Thus, the immunoprotectivity of the OMPs is probably associated with functional participations of the OMPs in bacterial infection. PMID:23932987

  3. Selected lactic acid-producing bacterial isolates with the capacity to reduce Salmonella translocation and virulence gene expression in chickens.

    Directory of Open Access Journals (Sweden)

    Xiaojian Yang

    Full Text Available BACKGROUND: Probiotics have been used to control Salmonella colonization/infection in chickens. Yet the mechanisms of probiotic effects are not fully understood. This study has characterized our previously-selected lactic acid-producing bacterial (LAB isolates for controlling Salmonella infection in chickens, particularly the mechanism underlying the control. METHODOLOGY/PRINCIPAL FINDINGS: In vitro studies were conducted to characterize 14 LAB isolates for their tolerance to low pH (2.0 and high bile salt (0.3-1.5% and susceptibility to antibiotics. Three chicken infection trials were subsequently carried out to evaluate four of the isolates for reducing the burden of Salmonella enterica serovar Typhimurium in the broiler cecum. Chicks were gavaged with LAB cultures (10(6-7 CFU/chick or phosphate-buffered saline (PBS at 1 day of age followed by Salmonella challenge (10(4 CFU/chick next day. Samples of cecal digesta, spleen, and liver were examined for Salmonella counts on days 1, 3, or 4 post-challenge. Salmonella in the cecum from Trial 3 was also assessed for the expression of ten virulence genes located in its pathogenicity island-1 (SPI-1. These genes play a role in Salmonella intestinal invasion. Tested LAB isolates (individuals or mixed cultures were unable to lower Salmonella burden in the chicken cecum, but able to attenuate Salmonella infection in the spleen and liver. The LAB treatments also reduced almost all SPI-1 virulence gene expression (9 out of 10 in the chicken cecum, particularly at the low dose. In vitro treatment with the extracellular culture fluid from a LAB culture also down-regulated most SPI-1 virulence gene expression. CONCLUSIONS/SIGNIFICANCE: The possible correlation between attenuation of Salmonella infection in the chicken spleen and liver and reduction of Salmonella SPI-1 virulence gene expression in the chicken cecum by LAB isolates is a new observation. Suppression of Salmonella virulence gene expression in

  4. How do marine bacteria produce light, why are they luminescent, and can we employ bacterial bioluminescence in aquatic biotechnology?

    Directory of Open Access Journals (Sweden)

    Grzegorz Wêgrzyn

    2002-09-01

    Full Text Available Bioluminescence, the phenomenon of light production by living organisms, occurs in forms of life as various as bacteria, fungi and animals. Nevertheless, light-emitting bacteria are the most abundant and widespread of luminescent organisms. Interestingly, most species of such bacteria live in marine environments. In this article, the biochemical mechanism of bacterial luminescence and its genetic regulation are summarized. Although the biochemistry and genetics of light emission by cells have been investigated in detail, the biological role of bacterial luminescence has remained obscure. Here, we discuss recent discoveries that shed new light on this problem. Finally, we provide examples of how bacterial luminescence can be employed in marine biotechnology, especially in the detection of toxic and mutagenic pollution in aquatic environments.

  5. Visualizing the Translocation and Localization of Bacterial Type III Effector Proteins by Using a Genetically Encoded Reporter System.

    Science.gov (United States)

    Gawthorne, Jayde A; Audry, Laurent; McQuitty, Claire; Dean, Paul; Christie, John M; Enninga, Jost; Roe, Andrew J

    2016-05-01

    Bacterial type III secretion system (T3SS) effector proteins are critical determinants of infection for many animal and plant pathogens. However, monitoring of the translocation and delivery of these important virulence determinants has proved to be technically challenging. Here, we used a genetically engineered LOV (light-oxygen-voltage) sensing domain derivative to monitor the expression, translocation, and localization of bacterial T3SS effectors. We found theEscherichia coliO157:H7 bacterial effector fusion Tir-LOV was functional following its translocation and localized to the host cell membrane in discrete foci, demonstrating that LOV-based reporters can be used to visualize the effector translocation with minimal manipulation and interference. Further evidence for the versatility of the reporter was demonstrated by fusing LOV to the C terminus of theShigella flexnerieffector IpaB. IpaB-LOV localized preferentially at bacterial poles before translocation. We observed the rapid translocation of IpaB-LOV in a T3SS-dependent manner into host cells, where it localized at the bacterial entry site within membrane ruffles. PMID:26921426

  6. Stainless steel modified with poly(ethylene glycol) can prevent protein adsorption but not bacterial adhesion

    DEFF Research Database (Denmark)

    Wei, Jiang; Bagge, Dorthe; Gram, Lone;

    2003-01-01

    . The chemical composition and uniformity of the surfaces were determined using X-ray photoelectron spectroscopy (XPS) and time-of-flight static secondary ion mass spectrometry (ToF-SSIMS) in the imaging mode. The effects of PEI concentration and different substrate pre-cleaning methods on the structure....... The surface density of PEI was shown to increase with increasing PEI concentration (up to 30 mg/ml), as determined from XPS measurements, and subsequently produced the PEG layer with the highest density of attached chains. In model experiments using beta-lactoglobulin no protein adsorption was detected...

  7. Structure of the complex between teicoplanin and a bacterial cell-wall peptide: use of a carrier-protein approach

    Energy Technology Data Exchange (ETDEWEB)

    Economou, Nicoleta J.; Zentner, Isaac J. [Drexel University College of Medicine, 245 North 15th Street, Philadelphia, PA 19102 (United States); Lazo, Edwin; Jakoncic, Jean; Stojanoff, Vivian [Brookhaven National Laboratory, Upton, NY 11973 (United States); Weeks, Stephen D.; Grasty, Kimberly C.; Cocklin, Simon; Loll, Patrick J. [Drexel University College of Medicine, 245 North 15th Street, Philadelphia, PA 19102 (United States)

    2013-04-01

    Using a carrier-protein strategy, the structure of teicoplanin bound to its bacterial cell-wall target has been determined. The structure reveals the molecular determinants of target recognition, flexibility in the antibiotic backbone and intrinsic radiation sensitivity of teicoplanin. Multidrug-resistant bacterial infections are commonly treated with glycopeptide antibiotics such as teicoplanin. This drug inhibits bacterial cell-wall biosynthesis by binding and sequestering a cell-wall precursor: a d-alanine-containing peptide. A carrier-protein strategy was used to crystallize the complex of teicoplanin and its target peptide by fusing the cell-wall peptide to either MBP or ubiquitin via native chemical ligation and subsequently crystallizing the protein–peptide–antibiotic complex. The 2.05 Å resolution MBP–peptide–teicoplanin structure shows that teicoplanin recognizes its ligand through a combination of five hydrogen bonds and multiple van der Waals interactions. Comparison of this teicoplanin structure with that of unliganded teicoplanin reveals a flexibility in the antibiotic peptide backbone that has significant implications for ligand recognition. Diffraction experiments revealed an X-ray-induced dechlorination of the sixth amino acid of the antibiotic; it is shown that teicoplanin is significantly more radiation-sensitive than other similar antibiotics and that ligand binding increases radiosensitivity. Insights derived from this new teicoplanin structure may contribute to the development of next-generation antibacterials designed to overcome bacterial resistance.

  8. Structure of the complex between teicoplanin and a bacterial cell-wall peptide: use of a carrier-protein approach

    International Nuclear Information System (INIS)

    Using a carrier-protein strategy, the structure of teicoplanin bound to its bacterial cell-wall target has been determined. The structure reveals the molecular determinants of target recognition, flexibility in the antibiotic backbone and intrinsic radiation sensitivity of teicoplanin. Multidrug-resistant bacterial infections are commonly treated with glycopeptide antibiotics such as teicoplanin. This drug inhibits bacterial cell-wall biosynthesis by binding and sequestering a cell-wall precursor: a d-alanine-containing peptide. A carrier-protein strategy was used to crystallize the complex of teicoplanin and its target peptide by fusing the cell-wall peptide to either MBP or ubiquitin via native chemical ligation and subsequently crystallizing the protein–peptide–antibiotic complex. The 2.05 Å resolution MBP–peptide–teicoplanin structure shows that teicoplanin recognizes its ligand through a combination of five hydrogen bonds and multiple van der Waals interactions. Comparison of this teicoplanin structure with that of unliganded teicoplanin reveals a flexibility in the antibiotic peptide backbone that has significant implications for ligand recognition. Diffraction experiments revealed an X-ray-induced dechlorination of the sixth amino acid of the antibiotic; it is shown that teicoplanin is significantly more radiation-sensitive than other similar antibiotics and that ligand binding increases radiosensitivity. Insights derived from this new teicoplanin structure may contribute to the development of next-generation antibacterials designed to overcome bacterial resistance

  9. New Supercharging Reagents Produce Highly Charged Protein Ions in Native Mass Spectrometry

    OpenAIRE

    Going, Catherine C; Xia, Zijie; Williams, Evan R.

    2015-01-01

    The effectiveness of two new supercharging reagents for producing highly charged ions by electrospray ionization (ESI) from aqueous solutions in which proteins have native structures and reactivities were investigated. In aqueous solution, 2-thiophenone and 4-hydroxymethyl-1,3-dioxolan-2-one (HD) at a concentration of 2% by volume can increase the average charge of cytochrome c and myoglobin by up to 163%, resulting in even higher charge states than those that are produced from water/methanol...

  10. Ras GTPase-like protein MglA, a controller of bacterial social-motility in Myxobacteria, has evolved to control bacterial predation by Bdellovibrio.

    Directory of Open Access Journals (Sweden)

    David S Milner

    2014-04-01

    Full Text Available Bdellovibrio bacteriovorus invade Gram-negative bacteria in a predatory process requiring Type IV pili (T4P at a single invasive pole, and also glide on surfaces to locate prey. Ras-like G-protein MglA, working with MglB and RomR in the deltaproteobacterium Myxococcus xanthus, regulates adventurous gliding and T4P-mediated social motility at both M. xanthus cell poles. Our bioinformatic analyses suggested that the GTPase activating protein (GAP-encoding gene mglB was lost in Bdellovibrio, but critical residues for MglA(Bd GTP-binding are conserved. Deletion of mglA(Bd abolished prey-invasion, but not gliding, and reduced T4P formation. MglA(Bd interacted with a previously uncharacterised tetratricopeptide repeat (TPR domain protein Bd2492, which we show localises at the single invasive pole and is required for predation. Bd2492 and RomR also interacted with cyclic-di-GMP-binding receptor CdgA, required for rapid prey-invasion. Bd2492, RomR(Bd and CdgA localize to the invasive pole and may facilitate MglA-docking. Bd2492 was encoded from an operon encoding a TamAB-like secretion system. The TamA protein and RomR were found, by gene deletion tests, to be essential for viability in both predatory and non-predatory modes. Control proteins, which regulate bipolar T4P-mediated social motility in swarming groups of deltaproteobacteria, have adapted in evolution to regulate the anti-social process of unipolar prey-invasion in the "lone-hunter" Bdellovibrio. Thus GTP-binding proteins and cyclic-di-GMP inputs combine at a regulatory hub, turning on prey-invasion and allowing invasion and killing of bacterial pathogens and consequent predatory growth of Bdellovibrio.

  11. Structure and Function of a Bacterial Microcompartment Shell Protein Engineered to Bind a [4Fe-4S] Cluster.

    Science.gov (United States)

    Aussignargues, Clément; Pandelia, Maria-Eirini; Sutter, Markus; Plegaria, Jefferson S; Zarzycki, Jan; Turmo, Aiko; Huang, Jingcheng; Ducat, Daniel C; Hegg, Eric L; Gibney, Brian R; Kerfeld, Cheryl A

    2016-04-27

    Bacterial microcompartments (BMCs) are self-assembling organelles composed of a selectively permeable protein shell and encapsulated enzymes. They are considered promising templates for the engineering of designed bionanoreactors for biotechnology. In particular, encapsulation of oxidoreductive reactions requiring electron transfer between the lumen of the BMC and the cytosol relies on the ability to conduct electrons across the shell. We determined the crystal structure of a component protein of a synthetic BMC shell, which informed the rational design of a [4Fe-4S] cluster-binding site in its pore. We also solved the structure of the [4Fe-4S] cluster-bound, engineered protein to 1.8 Å resolution, providing the first structure of a BMC shell protein containing a metal center. The [4Fe-4S] cluster was characterized by optical and EPR spectroscopies; it has a reduction potential of -370 mV vs the standard hydrogen electrode (SHE) and is stable through redox cycling. This remarkable stability may be attributable to the hydrogen-bonding network provided by the main chain of the protein scaffold. The properties of the [4Fe-4S] cluster resemble those in low-potential bacterial ferredoxins, while its ligation to three cysteine residues is reminiscent of enzymes such as aconitase and radical S-adenosymethionine (SAM) enzymes. This engineered shell protein provides the foundation for conferring electron-transfer functionality to BMC shells. PMID:26704697

  12. Metabolism of DMSP, DMS and DMSO by the cultivable bacterial community associated with the DMSP-producing dinoflagellate Scrippsiella trochoidea

    Digital Repository Service at National Institute of Oceanography (India)

    Hatton, A.D.; Shenoy, D.M.; Hart, M.C.; Mogg, A.; Green, D.H.

    Howard EC, Henriksen JR, Buchan A, Reisch CR, Burgmann H, Welsh R, Ye W, Gonzalez JM, Mace K, Joye SB, Kiene RP, Whitman WB, Moran MA (2006) Bacterial taxa that limit sulfur flux from the ocean. Science 314(5799): 649–652 Howard EC, Sun S, Biers EJ...

  13. Improving protein delivery of fibroblast growth factor-2 from bacterial inclusion bodies used as cell culture substrates

    OpenAIRE

    Seras Franzoso, Joaquin; Peebo, Karl; Garcia Fruitós, Elena; Vázquez Gómez, Esther; Rinas, Ursula; Villaverde Corrales, Antonio

    2014-01-01

    Altres ajuts: We are indebted CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN, Spain) for funding our research on inclusion bodies. Bacterial inclusion bodies (IBs) have recently been used to generate biocompatible cell culture interfaces, with diverse effects on cultured cells such as cell adhesion enhancement, stimulation of cell growth or induction of mesenchymal stem cell differentiation. Additionally, novel applications of IBs as sustained protein delivery systems with...

  14. Comparative proteome analysis of psychrophilic versus mesophilic bacterial species: Insights into the molecular basis of cold adaptation of proteins

    OpenAIRE

    Metpally, Raghu Prasad Rao; Reddy, Boojala Vijay B.

    2009-01-01

    Background Cold adapted or psychrophilic organisms grow at low temperatures, where most of other organisms cannot grow. This adaptation requires a vast array of sequence, structural and physiological adjustments. To understand the molecular basis of cold adaptation of proteins, we analyzed proteomes of psychrophilic and mesophilic bacterial species and compared the differences in amino acid composition and substitution patterns to investigate their likely association with growth temperatures....

  15. Evaluation of antibacterial activity of crude protein extracts from seeds of six different medical plants against standard bacterial strains

    OpenAIRE

    Al Akeel, Raid; Al-Sheikh, Yazeed; Mateen, Ayesha; Syed, Rabbani; Janardhan, K.; V C Gupta

    2013-01-01

    A huge group of natural antimicrobial compounds are active against a large spectrum of bacterial strains causing infectious threat. The present study was conducted to investigate the crude extracts of antimicrobial protein and peptide efficacy from six medicinal plant seeds. Extraction was carried out in Sodium phosphate citrate buffer, and Sodium acetate buffer using different pH. Antimicrobial activities of these plants were determined by the microbiological technique using Agar well diffus...

  16. Alteration of intracellular protein expressions as a key mechanism of the deterioration of bacterial denitrification caused by copper oxide nanoparticles

    OpenAIRE

    Yinglong Su; Xiong Zheng; Yinguang Chen; Mu Li; Kun Liu

    2015-01-01

    The increasing production and utilization of copper oxide nanoparticles (CuO NPs) result in the releases into the environment. However, the influence of CuO NPs on bacterial denitrification, one of the most important pathways to transform nitrate to dinitrogen in environment, has seldom been studied. Here we reported that CuO NPs caused a significant alteration of key protein expressions of a model denitrifier, Paracoccus denitrificans, leading to severe inhibition to denitrification. Total n...

  17. Linkage of bacterial protein synthesis and presentation of MHC class I-restricted Listeria monocytogenes-derived antigenic peptides.

    Directory of Open Access Journals (Sweden)

    Silke Grauling-Halama

    Full Text Available The processing and MHC class I-restricted presentation of antigenic peptides derived from the p60 protein of the facultative intracellular bacterium Listeria monocytogenes is tightly linked to bacterial protein synthesis. We used non-linear regression analysis to fit a mathematical model of bacterial antigen processing to a published experimental data set showing the accumulation and decay of p60-derived antigenic peptides in L. monocytogenes-infected cells. Two alternative models equally describe the experimental data. The simulation accounting for a stable and a hypothetical rapidly degraded form of antigen predicts that the antigenic peptides p60 217-225 and p60 449-457 are derived from a putative instable form of p60 with an average intracellular half-life of approximately 3 minutes accounting for approximately 31% of all p60 molecules synthesized. The alternative model predicts that both antigenic peptides are processed from p60 degraded intracellularly with a half-life of 109 min and that antigen processing only occurs as long as bacterial protein synthesis is not inhibited. In order to decide between both models the intracellular accumulation of p60 in infected cells was studied experimentally and compared with model predictions. Inhibition of p60 degradation by the proteasome inhibitor epoxomicin revealed that during the first 3 h post infection approximately 30% of synthesized p60 molecules were degraded. This value is significantly lower than the approximately 50% degradation of p60 that would be expected in the presence of the predicted putative short-lived state of p60 and also fits precisely with the predictions of the alternative model, indicating that the tight connection of bacterial protein biosynthesis and antigen processing and presentation of L. monocyctogenes-derived antigenic peptides is not caused by the presence of a highly instable antigenic substrate.

  18. Protein induced by vitamin K absence or antagonist II-producing gastric cancer

    OpenAIRE

    Takahashi, Yoshihisa; Inoue, Tohru; Fukusato, Toshio

    2010-01-01

    Protein induced by vitamin K absence or antagonist II (PIVKA-II) is a putative specific marker of hepatocellular carcinoma (HCC), but it may also be produced by a small number of gastric cancers. To date, 16 cases of PIVKA-II-producing gastric cancer have been reported, 2 of which were reported by us and all of which were identified in Japan. There are no symptoms specific to PIVKA-II-producing gastric cancer, and the representative clinical symptoms are general fatigue, appetite loss, and up...

  19. Unique natural-protein hollow-nanofiber membranes produced by weaver ants for medical applications.

    Science.gov (United States)

    Reddy, Narendra; Xu, Helan; Yang, Yiqi

    2011-07-01

    We report the properties of unique natural-protein hollow-nanofiber membranes produced by weaver ants (Oecophylla smaragdina) and the potential of using the nanofiber membranes for medical applications. Although natural proteins such as silk and collagen have been used to produce electrospun nanofibers for medical applications, there are no reports on producing hollow nanofibers from proteins. Hollow nanofibers are expected to have unique properties such as high drug loading. Weaver ant larvae extrude proteins in the form of nanofibers that are hollow and the adult ants build the nests using the hollow nanofibers. It was found that the nanofiber membranes are composed of fibers with average diameters of 450 nm. The membranes have tensile strength of about 4 MPa, high elongation of about 31% and modulus of 31 MPa, better than any protein nanofiber membrane reported so far. The membranes withstand rigorous boiling in weak alkali, show good attachment and proliferation of osteoblasts and can load up to 4.7 times higher drugs compared to common silk. These features make ant nanofiber membranes unique and preferable for medical and biotechnology industries. PMID:21337323

  20. Isolation and Identification of a New Tetrodotoxin-Producing Bacterial Species, Raoultella terrigena, from Hong Kong Marine Puffer Fish Takifugu niphobles

    OpenAIRE

    Fred Wang-Fat Lee; Peter Hoi-Fu Yu; Vincent Chung-Him Yu; Kin-Chung Ho

    2011-01-01

    Puffer fish, Takifugu niphobles, collected from the Hong Kong coastal waters were screened for tetrodotoxin-producing bacteria. A Gram-negative, non-acid-fast, non-sporing and rod shaped bacterial strain (designated as gutB01) was isolated from the intestine of the puffer fish and was shown to produce tetrodotoxin (TTX). Based on the Microbial Identification (MIDI) and 16S-23S rDNA internal transcribed spacer (ITS) phylogenetic analysis, the strain was identified as Raoultella terrigena. The ...

  1. Unusual Heme Binding in the Bacterial Iron Response Regulator Protein (Irr): Spectral Characterization of Heme Binding to Heme Regulatory Motif

    OpenAIRE

    Ishikawa, Haruto; Nakagaki, Megumi; Bamba, Ai; Uchida, Takeshi; Hori, Hiroshi; O'Brian, Mark R.; Iwai, Kazuhiro; Ishimori, Koichiro

    2011-01-01

    We characterized heme binding in the bacterial iron response regulator (Irr) protein, which is a simple heme-regulated protein having a single “heme-regulatory motif”, HRM, and plays a key role in the iron homeostasis of a nitrogen fixing bacterium. The heme titration to wild-type and mutant Irr clearly showed that Irr has two heme binding sites: one of the heme binding sites is in the HRM, where 29Cys is the axial ligand, and the other one, the secondary heme binding site, is located outside...

  2. Stable nano-sized emulsions produced from soy protein and soy polysaccharide complexes.

    Science.gov (United States)

    Yin, Baoru; Deng, Wei; Xu, Keke; Huang, Luwen; Yao, Ping

    2012-08-15

    In this research, a simple, green and effective strategy was developed to produce long-term stable oil in water emulsion from soy protein and soy polysaccharide. Soy protein and soy polysaccharide formed dispersible complexes at pH around 3.25 aqueous solution through electrostatic and hydrophobic interactions. A high pressure homogenization produced the protein/polysaccharide complex emulsion having a droplet size about 250 nm. A heat treatment of the emulsion resulted in the protein denaturation, forming irreversible oil-water interfacial films composed of soy protein/soy polysaccharide complexes. The droplets of the emulsion were characterized by dynamic light scattering, ζ-potential, transmission electron microscopy, polysaccharide digestion via pectinase, and confocal laser scanning microscopy observation via dual fluorescence probes. As a result of the polysaccharide being fixed on the droplet surface, the emulsions exhibited long-term stability in the media containing pH values of 2-8 and 0.2 mol/L NaCl. The stable soy protein/soy polysaccharide complex emulsion is a suitable food-grade delivery system in which lipophilic bioactive compounds can be encapsulated. PMID:22682324

  3. Protein and Bacterial Antifouling Behavior of Melt-Coextruded Nanofiber Mats.

    Science.gov (United States)

    Kim, Si-Eun; Zhang, Cong; Advincula, Abigail A; Baer, Eric; Pokorski, Jonathan K

    2016-04-13

    Antifouling surfaces are important for biomedical devices to prevent secondary infections and mitigate the effects of the foreign body response. Herein, we describe melt-coextruded poly(ε-caprolactone) (PCL) nanofiber mats grafted with antifouling polymers. Nonwoven PCL fiber mats are produced using a multilayered melt coextrusion process followed by high-pressure hydroentanglement to yield porous patches. The resulting fiber mats show submicrometer cross-sectional fiber dimensions and yield pore sizes that were nearly uniform, with a mean pore size of 1.6 ± 0.9 μm. Several antifouling polymers, including hydrophilic, zwitterionic, and amphipathic molecules, are grafted to the surface of the mats using a two-step procedure that includes photochemistry followed by the copper-catalyzed azide-alkyne cycloaddition reaction. Fiber mats are evaluated using separate adsorption tests for serum proteins and E. coli. The results indicate that poly(oligo(ethylene glycol) methyl ether methacrylate)-co-(trifluoroethyl methacrylate) (poly(OEGMEMA-co-TFEMA)) grafted mats exhibit approximately 85% less protein adhesion and 97% less E. coli adsorption when compared to unmodified PCL fibermats. In dynamic antifouling testing, the amphiphilic fluorous polymer surface shows the highest flux and highest rejection value of foulants. The work presented within has implications on the high-throughput production of antifouling microporous patches for medical applications. PMID:27043205

  4. Assessment of Relationship Between Bacterial Stripe Resistance And Leaf Protein Bands In Rice (Oryza sativa L.) Varieties.

    Science.gov (United States)

    Talei, D.; Fotokian, M. H.

    2008-01-01

    Bacterial stripe as a new rice disease in Iran is more frequent nowadays. The objective of this study was to assessment of resistance in rice varieties together with evaluating of zymogram bands resulted from SDS PAGE electrophoresis of leaf proteins. For this purpose, 30 lines were tested in a randomized complete block design with three replications. The analysis of variance showed that there was significant difference between genotypes for resistance. Mean compare based on field results revealed that Domsiyah had the lowest resistance while Nemat and 7162 demonstrated the highest resistance. Laboratory results showed that there were significant difference between protein bands resulted from sensitive and resistance verities. Twenty bands were observed through SDS PAGE electrophoresis of leaf proteins. The 9th and 12th bands were found in sensitive varieties while were not in resistance genotypes. According to the results of this study, 7162 variety can be considered as the sources of resistance in breeding programs. Meanwhile attending to existence of 9th and 12th bands in sensitive varieties, resistance against bacterial stripe of rice maybe influenced by absence of these proteins.

  5. Alteration of intracellular protein expressions as a key mechanism of the deterioration of bacterial denitrification caused by copper oxide nanoparticles

    Science.gov (United States)

    Su, Yinglong; Zheng, Xiong; Chen, Yinguang; Li, Mu; Liu, Kun

    2015-10-01

    The increasing production and utilization of copper oxide nanoparticles (CuO NPs) result in the releases into the environment. However, the influence of CuO NPs on bacterial denitrification, one of the most important pathways to transform nitrate to dinitrogen in environment, has seldom been studied. Here we reported that CuO NPs caused a significant alteration of key protein expressions of a model denitrifier, Paracoccus denitrificans, leading to severe inhibition to denitrification. Total nitrogen removal efficiency was decreased from 98.3% to 62.1% with the increase of CuO NPs from 0.05 to 0.25 mg/L. Cellular morphology and integrity studies indicated that nanoparticles entered the cells. The proteomic bioinformatics analysis showed that CuO NPs caused regulation of proteins involved in nitrogen metabolism, electron transfer and substance transport. The down-regulation of GtsB protein (responsible for glucose transport) decreased the production of NADH (electron donor for denitrification). Also, the expressions of key electron-transfer proteins (including NADH dehydrogenase and cytochrome) were suppressed by CuO NPs, which adversely affected electrons transfer for denitrification. Further investigation revealed that CuO NPs significantly inhibited the expressions and catalytic activities of nitrate reductase and nitrite reductase. These results provided a fundamental understanding of the negative influences of CuO NPs on bacterial denitrification.

  6. Bacterial S-layer protein coupling to lipids: x-ray reflectivity and grazing incidence diffraction studies.

    Science.gov (United States)

    Weygand, M; Wetzer, B; Pum, D; Sleytr, U B; Cuvillier, N; Kjaer, K; Howes, P B; Lösche, M

    1999-01-01

    The coupling of bacterial surface (S)-layer proteins to lipid membranes is studied in molecular detail for proteins from Bacillus sphaericus CCM2177 and B. coagulans E38-66 recrystallized at dipalmitoylphosphatidylethanolamine (DPPE) monolayers on aqueous buffer. A comparison of the monolayer structure before and after protein recrystallization shows minimal reorganization of the lipid chains. By contrast, the lipid headgroups show major rearrangements. For the B. sphaericus CCM2177 protein underneath DPPE monolayers, x-ray reflectivity data suggest that amino acid side chains intercalate the lipid headgroups at least to the phosphate moieties, and probably further beyond. The number of electrons in the headgroup region increases by more than four per lipid. Analysis of the changes of the deduced electron density profiles in terms of a molecular interpretation shows that the phosphatidylethanolamine headgroups must reorient toward the surface normal to accommodate such changes. In terms of the protein structure (which is as yet unknown in three dimensions), the electron density profile reveals a thickness lz approximately 90 A of the recrystallized S-layer and shows water-filled cavities near its center. The protein volume fraction reaches maxima of >60% in two horizontal sections of the S-layer, close to the lipid monolayer and close to the free subphase. In between it drops to approximately 20%. Four S-layer protein monomers are located within the unit cell of a square lattice with a spacing of approximately 131 A. PMID:9876158

  7. Some chemical and physical properties of nisin, a small-protein antibiotic produced by Lactococcus lactis.

    OpenAIRE

    Liu, W.; Hansen, J N

    1990-01-01

    Nisin is a small gene-encoded antimicrobial protein produced by Lactococcus lactis that contains unusual dehydroalanine and dehydrobutyrine residues. The reactivity of these residues toward nucleophiles was explored by reacting nisin with a variety of mercaptans. The kinetics of reaction with 2-mercaptoethane-sulfonate and thioglycolate indicated that the reaction pathway includes a binding step. Reaction of nisin at high pH resulted in the formation of multimeric products, apparently as a re...

  8. Use of Extrusion Technology and Fat Replacers to Produce High Protein, Low Fat Cheese

    OpenAIRE

    Dubey, Amrita

    2011-01-01

    This study investigated the use of extrusion technology and fat replacers to produce high protein, low fat Cheddar cheese. In chapter 3, four different fat replacers were tested at the highest concentration level of each, as recommended by the manufacturers for low fat cheese, to investigate the change in cheese texture and optimize extruder conditions. In addition, the press time/pressure combinations of the extruded cheeses were optimized. The fat replacers and extruder conditions that w...

  9. A census of membrane-bound and intracellular signal transduction proteins in bacteria: Bacterial IQ, extroverts and introverts

    Directory of Open Access Journals (Sweden)

    Galperin Michael Y

    2005-06-01

    Full Text Available Abstract Background Analysis of complete microbial genomes showed that intracellular parasites and other microorganisms that inhabit stable ecological niches encode relatively primitive signaling systems, whereas environmental microorganisms typically have sophisticated systems of environmental sensing and signal transduction. Results This paper presents results of a comprehensive census of signal transduction proteins – histidine kinases, methyl-accepting chemotaxis receptors, Ser/Thr/Tyr protein kinases, adenylate and diguanylate cyclases and c-di-GMP phosphodiesterases – encoded in 167 bacterial and archaeal genomes, sequenced by the end of 2004. The data have been manually checked to avoid false-negative and false-positive hits that commonly arise during large-scale automated analyses and compared against other available resources. The census data show uneven distribution of most signaling proteins among bacterial and archaeal phyla. The total number of signal transduction proteins grows approximately as a square of genome size. While histidine kinases are found in representatives of all phyla and are distributed according to the power law, other signal transducers are abundant in certain phylogenetic groups but virtually absent in others. Conclusion The complexity of signaling systems differs even among closely related organisms. Still, it usually can be correlated with the phylogenetic position of the organism, its lifestyle, and typical environmental challenges it encounters. The number of encoded signal transducers (or their fraction in the total protein set can be used as a measure of the organism's ability to adapt to diverse conditions, the 'bacterial IQ', while the ratio of transmembrane receptors to intracellular sensors can be used to define whether the organism is an 'extrovert', actively sensing the environmental parameters, or an 'introvert', more concerned about its internal homeostasis. Some of the microorganisms with the

  10. A simple synthesis method to produce metal oxide loaded carbon paper using bacterial cellulose gel and characterization of its electrochemical behavior in an aqueous electrolyte

    Science.gov (United States)

    Miyajima, Naoya; Jinguji, Ken; Matsumura, Taiyu; Matsubara, Toshihiro; Sakane, Hideto; Akatsu, Takashi; Tanaike, Osamu

    2016-04-01

    A simple synthetic chemical process to produce metal oxide loaded carbon papers was developed using bacterial cellulose gel, which consisted of nanometer-sized fibrous cellulose and water. Metal ions were successfully impregnated into the gel via aqueous solution media before drying and carbonization methods resulting in metal oxide contents that were easy to control through variations in the concentration of aqueous solutions. The papers loaded by molybdenum oxides were characterized as pseudocapacitor electrodes preliminary, and the large redox capacitance of the oxides was followed by a conductive fibrous carbon substrate, suggesting that a binder and carbon black additive-free electrode consisting of metal oxides and carbon paper was formed.

  11. Oral mucosal lipids are antibacterial against Porphyromonas gingivalis, induce ultrastructural damage, and alter bacterial lipid and protein compositions

    Institute of Scientific and Technical Information of China (English)

    Carol L Fischer; Katherine S Walters; David R Drake; Deborah V Dawson; Derek R Blanchette; Kim A Brogden; Philip W Wertz

    2013-01-01

    Oral mucosal and salivary lipids exhibit potent antimicrobial activity for a variety of Gram-positive and Gram-negative bacteria;however, little is known about their spectrum of antimicrobial activity or mechanisms of action against oral bacteria. In this study, we examine the activity of two fatty acids and three sphingoid bases against Porphyromonas gingivalis, an important colonizer of the oral cavity implicated in periodontitis. Minimal inhibitory concentrations, minimal bactericidal concentrations, and kill kinetics revealed variable, but potent, activity of oral mucosal and salivary lipids against P. gingivalis, indicating that lipid structure may be an important determinant in lipid mechanisms of activity against bacteria, although specific components of bacterial membranes are also likely important. Electron micrographs showed ultrastructural damage induced by sapienic acid and phytosphingosine and confirmed disruption of the bacterial plasma membrane. This information, coupled with the association of treatment lipids with P. gingivalis lipids revealed via thin layer chromatography, suggests that the plasma membrane is a likely target of lipid antibacterial activity. Utilizing a combination of two-dimensional in-gel electrophoresis and Western blot followed by mass spectroscopy and N-terminus degradation sequencing we also show that treatment with sapienic acid induces upregulation of a set of proteins comprising a unique P. gingivalis stress response, including proteins important in fatty acid biosynthesis, metabolism and energy production, protein processing, cell adhesion and virulence. Prophylactic or therapeutic lipid treatments may be beneficial for intervention of infection by supplementing the natural immune function of endogenous lipids on mucosal surfaces.

  12. Properties of a new gasoline oxygenate blend component: 3-Hydroxybutyrate methyl ester produced from bacterial poly-3-hydroxybutyrate

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Shen Yu.; Wang, Zhen; Liu, Ming Ming; Zhang, Xiao Jun [Multidisciplinary Research Center, Shantou University, Shantou 515063, Guangdong (China); Xu, Yu. [Oilcare Oil Test Laboratory of China National Analytical Center, 6th Floor, Building 34, No. 100, Xian Lie Zhong Road, Guangzhou 510000 (China); Chen, Guo-Qiang [Dept Biological Sciences and Biotechnology, School of Life Science, Tsinghua University, Beijing 100084 (China)

    2010-08-15

    3-Hydroxybutyrate methyl ester (HBME) was prepared from hydrolysis of bacterial poly-3-hydroxybutyrate (PHB) using methanol as an esterification agent in the presence of sulfuric acid. Physicochemical and fuel related properties of HBME were studied for the possibility of using HBME as a gasoline additive. When HBME was blended with 97 gasoline in volume ratios of 5%, 8.5%, 10%, 15% and 20%, respectively, it was found that HBME had similar or better properties as a fuel additive compared with ethanol in terms of oxygen content, dynamic viscosity, flash point and boiling point. The blending of HBME and gasoline showed only little difference compared with the 97 gasoline in terms of octane number (RON) and distillation ranges, especially for the HBME 8.5% and 10% blends, which demonstrated an over 93% combustion heat of gasoline with less than 5% reduction in RON. (author)

  13. Protein secretion by hybrid bacterial ABC-transporters: specific functions of the membrane ATPase and the membrane fusion protein.

    OpenAIRE

    Binet, R; Wandersman, C

    1995-01-01

    The Erwinia chrysanthemi metalloprotease C and the Serratia marcescens haem acquisition protein HasA are both secreted from Gram-negative bacteria by a signal peptide-independent pathway which requires a C-terminal secretion signal and a specific ABC-transporter made up of three proteins: a membrane ATPase (the ABC-protein), a second inner membrane component belonging to the membrane fusion protein family and an outer membrane polypeptide. HasA and protease C transporters are homologous altho...

  14. A Teleost Bactericidal Permeability-Increasing Protein Kills Gram-Negative Bacteria, Modulates Innate Immune Response, and Enhances Resistance against Bacterial and Viral Infection.

    Science.gov (United States)

    Sun, Yuan-Yuan; Sun, Li

    2016-01-01

    Bactericidal/permeability-increasing protein (BPI) is an important factor of innate immunity that in mammals is known to take part in the clearance of invading Gram-negative bacteria. In teleost, the function of BPI is unknown. In the present work, we studied the function of tongue sole (Cynoglossus semilaevis) BPI, CsBPI. We found that CsBPI was produced extracellularly by peripheral blood leukocytes (PBL). Recombinant CsBPI (rCsBPI) was able to bind to a number of Gram-negative bacteria but not Gram-positive bacteria. Binding to bacteria led to bacterial death through membrane permeabilization and structural destruction, and the bound bacteria were more readily taken up by PBL. In vivo, rCsBPI augmented the expression of a wide arrange of genes involved in antibacterial and antiviral immunity. Furthermore, rCsBPI enhanced the resistance of tongue sole against bacterial as well as viral infection. These results indicate for the first time that a teleost BPI possesses immunoregulatory effect and plays a significant role in antibacterial and antiviral defense. PMID:27105425

  15. A LytM Domain Dictates the Localization of Proteins to the Mother Cell-Forespore Interface during Bacterial Endospore Formation▿ †

    OpenAIRE

    Meisner, Jeffrey; Moran, Charles P.

    2010-01-01

    A large number of proteins are known to reside at specific subcellular locations in bacterial cells. However, the molecular mechanisms by which many of these proteins are anchored at these locations remains unclear. During endospore formation in Bacillus subtilis, several integral membrane proteins are located specifically at the interface of the two adjacent cells of the developing sporangium, the mother cell and forespore. The mother cell membrane protein SpoIIIAH recognizes the cell-cell i...

  16. Antioxidant activity of pea protein hydrolysates produced by batch fermentation with lactic acid bacteria

    Directory of Open Access Journals (Sweden)

    Stanisavljević Nemanja S.

    2015-01-01

    Full Text Available Nine Lactobacillus strains known for surface proteinase activity were chosen from our collection and tested for their ability to grow in pea seed protein-based medium, and to hydrolyze purified pea proteins in order to produce peptides with antioxidant (AO activity. Two strains, Lactobacillus rhamnosus BGT10 and Lactobacillus zeae LMG17315, exhibited strong proteolytic activity against pea proteins. The AO activity of the pea hydrolysate fraction, MW <10 kDa, obtained by the fermentation of purified pea proteins with Lactobacillus rhamnosus BGT10, was tested by standard spectrophotometric assays (DPPH, ABTS, Fe3+-reducing capacity and the recently developed direct current (DC polarographic assay. The low molecular weight fraction of the obtained hydrolysate was separated using ion exchange chromatography, while the AO activity of eluted fractions was determined by means of a sensitive DC polarographic assay without previous concentration of samples. Results revealed that the fraction present in low abundance that contained basic peptides possessed the highest antioxidant activity. Based on the obtained results, it can be concluded that Lactobacillus rhamnosus BGT10 should be further investigated as a candidate strain for large-scale production of bioactive peptides from legume proteins. [Projekat Ministartsva nauke Republike Srbije, br. 173005 i br. 173026

  17. The topology of the bacterial co-conserved protein network and its implications for predicting protein function

    Directory of Open Access Journals (Sweden)

    Leach Sonia M

    2008-06-01

    Full Text Available Abstract Background Protein-protein interactions networks are most often generated from physical protein-protein interaction data. Co-conservation, also known as phylogenetic profiles, is an alternative source of information for generating protein interaction networks. Co-conservation methods generate interaction networks among proteins that are gained or lost together through evolution. Co-conservation is a particularly useful technique in the compact bacteria genomes. Prior studies in yeast suggest that the topology of protein-protein interaction networks generated from physical interaction assays can offer important insight into protein function. Here, we hypothesize that in bacteria, the topology of protein interaction networks derived via co-conservation information could similarly improve methods for predicting protein function. Since the topology of bacteria co-conservation protein-protein interaction networks has not previously been studied in depth, we first perform such an analysis for co-conservation networks in E. coli K12. Next, we demonstrate one way in which network connectivity measures and global and local function distribution can be exploited to predict protein function for previously uncharacterized proteins. Results Our results showed, like most biological networks, our bacteria co-conserved protein-protein interaction networks had scale-free topologies. Our results indicated that some properties of the physical yeast interaction network hold in our bacteria co-conservation networks, such as high connectivity for essential proteins. However, the high connectivity among protein complexes in the yeast physical network was not seen in the co-conservation network which uses all bacteria as the reference set. We found that the distribution of node connectivity varied by functional category and could be informative for function prediction. By integrating of functional information from different annotation sources and using the

  18. A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level.

    Science.gov (United States)

    Binder, Stephan; Schendzielorz, Georg; Stäbler, Norma; Krumbach, Karin; Hoffmann, Kristina; Bott, Michael; Eggeling, Lothar

    2012-01-01

    We present a novel method for visualizing intracellular metabolite concentrations within single cells of Escherichia coli and Corynebacterium glutamicum that expedites the screening process of producers. It is based on transcription factors and we used it to isolate new L-lysine producing mutants of C. glutamicum from a large library of mutagenized cells using fluorescence-activated cell sorting (FACS). This high-throughput method fills the gap between existing high-throughput methods for mutant generation and genome analysis. The technology has diverse applications in the analysis of producer populations and screening of mutant libraries that carry mutations in plasmids or genomes. PMID:22640862

  19. Effect of Bacterial Flora on Postimmunization Gastritis following Oral Vaccination of Mice with Helicobacter pylori Heat Shock Protein 60

    OpenAIRE

    Yamaguchi, Hiroyuki; Osaki, Takako; Taguchi, Haruhiko; Sato, Noriko; Toyoda, Atushi; Takahashi, Motomichi; Kai, Masanori; Nakata, Noboru; Komatsu, Akio; Atomi, Yutaka; Kamiya, Shigeru

    2003-01-01

    In order to assess the efficacy of oral Helicobacter pylori heat shock protein 60 (HSP60) as a vaccine, protection against H. pylori infection in specific-pathogen-free (SPF) C57BL/6 and germfree (GF) IQI mice was examined. Prophylactic oral vaccination of these two strains of mice with either H. pylori HSP60 or Escherichia coli GroEL inhibited H. pylori colonization by 90 to 95% at 3 weeks postinfection (p.i.). However, these mice were only partially protected because bacterial loads increas...

  20. Protein Modification: Bacterial Effectors Rewrite the Rules of Ubiquitylation.

    Science.gov (United States)

    Berk, Jason M; Hochstrasser, Mark

    2016-07-11

    A family of virulence factors from the bacterial pathogen Legionella pneumophila has been discovered to modify human Rab GTPases with ubiquitin. Surprisingly, this modification occurs via a non-canonical mechanism that uses nicotinamide adenine dinucleotide as a cofactor. PMID:27404243

  1. A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level

    OpenAIRE

    Binder, Stephan; Schendzielorz, Georg; Stäbler, Norma; Krumbach, Karin; Hoffmann, Kristina; Bott, Michael; Eggeling, Lothar

    2012-01-01

    We present a novel method for visualizing intracellular metabolite concentrations within single cells of Escherichia coli and Corynebacterium glutamicum that expedites the screening process of producers. It is based on transcription factors and we used it to isolate new L-lysine producing mutants of C. glutamicum from a large library of mutagenized cells using fluorescence-activated cell sorting (FACS). This high-throughput method fills the gap between existing high-throughput methods for mut...

  2. Assessment of heavy metal bioavailability in contaminated sediments and soils using green fluorescent protein-based bacterial biosensors

    International Nuclear Information System (INIS)

    A green fluorescent protein (GFP)-based bacterial biosensor Escherichia coli DH5α (pVLCD1) was developed based on the expression of gfp under the control of the cad promoter and the cadC gene of Staphylococcus aureus plasmid pI258. DH5α (pVLCD1) mainly responded to Cd(II), Pb(II), and Sb(III), the lowest detectable concentrations being 0.1 nmol L-1, 10 nmol L-1, and 0.1 nmol L-1, respectively, with 2 h exposure. The biosensor was field-tested to measure the relative bioavailability of the heavy metals in contaminated sediments and soil samples. The results showed that the majority of heavy metals remained adsorbed to soil particles: Cd(II)/Pb(II) was only partially available to the biosensor in soil-water extracts. Our results demonstrate that the GFP-based bacterial biosensor is useful and applicable in determining the bioavailability of heavy metals with high sensitivity in contaminated sediment and soil samples and suggests a potential for its inexpensive application in environmentally relevant sample tests. - Nonpathogenic GFP-based bacterial biosensor is applicable in determining the bioavailability of heavy metals in environmental samples

  3. Bacterial curli protein promotes the conversion of PAP248-286 into the amyloid SEVI: cross-seeding of dissimilar amyloid sequences

    Directory of Open Access Journals (Sweden)

    Kevin Hartman

    2013-02-01

    Full Text Available Fragments of prostatic acid phosphatase (PAP248-286 in human semen dramatically increase HIV infection efficiency by increasing virus adhesion to target cells. PAP248-286 only enhances HIV infection in the form of amyloid aggregates termed SEVI (Semen Enhancer of Viral Infection, however monomeric PAP248-286 aggregates very slowly in isolation. It has therefore been suggested that SEVI fiber formation in vivo may be promoted by exogenous factors. We show here that a bacterially-produced extracellular amyloid (curli or Csg acts as a catalytic agent for SEVI formation from PAP248-286 at low concentrations in vitro, producing fibers that retain the ability to enhance HIV (Human Immunodeficiency Virus infection. Kinetic analysis of the cross-seeding effect shows an unusual pattern. Cross-seeding PAP248-286 with curli only moderately affects the nucleation rate while significantly enhancing the growth of fibers from existing nuclei. This pattern is in contrast to most previous observations of cross-seeding, which show cross-seeding partially bypasses the nucleation step but has little effect on fiber elongation. Seeding other amyloidogenic proteins (IAPP (islet amyloid polypeptide and Aβ1−40 with curli showed varied results. Curli cross-seeding decreased the lag-time of IAPP amyloid formation but strongly inhibited IAPP elongation. Curli cross-seeding exerted a complicated concentration dependent effect on Aβ1−40 fibrillogenesis kinetics. Combined, these results suggest that the interaction of amyloidogenic proteins with preformed fibers of a different type can take a variety of forms and is not limited to epitaxial nucleation between proteins of similar sequence. The ability of curli fibers to interact with proteins of dissimilar sequences suggests cross-seeding may be a more general phenomenon than previously supposed.

  4. 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...... improve the usefulness of the E. coli expression platform we have investigated combinations of promoters and selected N-terminal fusion tags for the extracellular expression of human target proteins. A comparative study was conducted on 24 target proteins fused to outer membrane protein A (OmpA), outer...... 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...

  5. Expression of nucleolar-related proteins in porcine preimplantation embryos produced in vivo and in vitro

    DEFF Research Database (Denmark)

    Bjerregaard, Bolette; Wrenzycki, Christine; Strejcek, Frantisek;

    2004-01-01

    The expression of nucleolar-related proteins was studied as an indirect marker of the ribosomal RNA (rRNA) gene activation in porcine embryos up to the blastocyst stage produced in vivo and in vitro. A group of the in vivo-developed embryos were cultured with alpha-amanitin to block the de novo...... pocket proteins pRb and p130, which are involved in cell-cycle regulation, was assessed by semiquantitative RT-PCR up to the blastocyst stage. Toward the end of third cell cycle, the nuclei in non-alpha-amanitin-treated, in vivo-produced embryos displayed different stages of transformation of the nuclear...... was delayed in porcine embryos produced in vitro compared to the in vivo-derived counterparts with respect to mRNAs encoding PAF53 and UBF. Moreover, differences existed in the mRNA expression patterns of pRb between in vivo- and in vitro-developed embryos. These findings show, to our knowledge for...

  6. NEW EMBO MEMBER’S REVIEW: Viral and bacterial proteins regulating apoptosis at the mitochondrial level

    OpenAIRE

    Boya, Patricia; Roques, Bernard,; Kroemer, Guido

    2001-01-01

    Mitochondrial membrane permeabilization (MMP) is a critical step of several apoptotic pathways. Some infectious intracellular pathogens can regulate (induce or inhibit) apoptosis of their host cells at the mitochondrial level, by targeting proteins to mitochondrial membranes that either induce or inhibit MMP. Pathogen-encoded mitochondrion-targeted proteins may or may not show amino acid sequence homology to Bcl-2-like proteins. Among the Bcl-2-unrelated, mitochondrion-targeted proteins, seve...

  7. Bacterial Ortholog of Mammalian Translocator Protein (TSPO) with Virulence Regulating Activity

    OpenAIRE

    Chapalain, Annelise; Chevalier, Sylvie; Orange, Nicole; Murillo, Laurence; Papadopoulos, Vassilios; Feuilloley, Marc G J

    2009-01-01

    The translocator protein (TSPO), previously designated as peripheral-type benzodiazepine receptor, is a protein mainly located in the outer mitochondrial membrane of eukaryotic cells. TSPO is implicated in major physiological functions and functionally associated with other proteins such as the voltage-dependent anionic channel, also designated as mitochondrial porin. Surprisingly, a TSPO-related protein was identified in the photosynthetic bacterium Rhodobacter sphaeroides but it was initial...

  8. Endocytosis-inducer adhesins produced by enteropathogenic serogroups of Escherichia coli participate on bacterial attachment to infant enterocytes

    Directory of Open Access Journals (Sweden)

    João Ramos Costa Andrade

    1987-03-01

    Full Text Available Enteropathogenic E. coli (EPEC infection of Hep-2 cells preoceeds through bacterial attachment to cell surface and internalization of adhered bacteria. EPEC attachment is a prerequisite for cell infection and is mediated by adhesins that recognize carbohydrate-containing receptors on cell membrane. Such endocytosis-inducer adhesins (EIA also promote EPEC binding to infant enterocytes, suggesting that EIA may have an important role on EPEC gastroenteritis.A infecção de células Hep-2 por E. coli enteropatogênicas (ECEP implica na aderência bacteriana e posterior interiorização dos microrganismos aderidos por um mecanismo de endocitose. A aderência das ECEP é pré-requisito para a infecção e é mediada por adesinas que reconhecem receptores inibidos por certas oses na membrana celular. Tais "adesinas indutoras da endocitose" (AIE também promovem a ligação bacteriana a enterócitos obtidos do intestino delgado de lactente, sugerindo que as AIE possam desempenhar algum papel nas diarréias causadas por ECEP.

  9. Construction and evaluation of an exopolysaccharide-producing engineered bacterial strain by protoplast fusion for microbial enhanced oil recovery.

    Science.gov (United States)

    Sun, Shanshan; Luo, Yijing; Cao, Siyuan; Li, Wenhong; Zhang, Zhongzhi; Jiang, Lingxi; Dong, Hanping; Yu, Li; Wu, Wei-Min

    2013-09-01

    Enterobacter cloacae strain JD, which produces water-insoluble biopolymers at optimal temperature of 30°C, and a thermophilic Geobacillus strain were used to construct an engineered strain for exopolysaccharide production at high temperatures by protoplast fusion. The obtained fusant strain ZR3 produced exopolysaccharides at up to 45°C with optimal growth temperature at 35°C. The fusant produced exopolysaccharides of approximately 7.5 g/L or more at pH between 7.0 and 9.0. The feasibility of the enhancement of crude oil recovery with the fusant was tested in a sand-packed column at 40°C. The results demonstrated that bioaugmentation of the fusant was promising approach for MEOR. Mass growth of the fusant was confirmed in fermentor tests. PMID:23856587

  10. Identification of the Zinc Finger Protein ZRANB2 as a Novel Maternal Lipopolysaccharide-binding Protein That Protects Embryos of Zebrafish against Gram-negative Bacterial Infections.

    Science.gov (United States)

    Wang, Xia; Du, Xiaoyuan; Li, Hongyan; Zhang, Shicui

    2016-02-19

    Zinc finger ZRANB2 proteins are widespread in animals, but their functions and mechanisms remain poorly defined. Here we clearly demonstrate that ZRANB2 is a newly identified LPS-binding protein present abundantly in the eggs/embryos of zebrafish. We also show that recombinant ZRANB2 (rZRANB2) acts as a pattern recognition receptor capable of identifying the bacterial signature molecule LPS as well as binding the Gram-negative bacteria Escherichia coli, Vibrio anguilarum, and Aeromonas hydrophila and functions as an antibacterial effector molecule capable of directly killing the bacteria. Furthermore, we reveal that N-terminal residues 11-37 consisting of the first ZnF_RBZ domain are indispensable for ZRANB2 antimicrobial activity. Importantly, microinjection of rZRANB2 into early embryos significantly enhanced the resistance of the embryos against pathogenic A. hydrophila challenge, and this enhanced bacterial resistance was markedly reduced by co-injection of anti-ZRANB2 antibody. Moreover, precipitation of ZRANB2 in the embryo extracts by preincubation with anti-ZRANB2 antibody caused a marked decrease in the antibacterial activity of the extracts against the bacteria tested. In addition, the N-terminal peptide Z1/37 or Z11/37 with in vitro antibacterial activity also promoted the resistance of embryos against A. hydrophila, but the peptide Z38/198 without in vitro antibacterial activity did not. Collectively, these results indicate that ZRANB2 is a maternal LPS-binding protein that can protect the early embryos of zebrafish against pathogenic attacks, a novel role ever assigned to ZRANB2 proteins. This work also provides new insights into the immunological function of the zinc finger proteins that are widely distributed in various animals. PMID:26740623

  11. Surf1, associated with Leigh syndrome in humans, is a heme-binding protein in bacterial oxidase biogenesis.

    Science.gov (United States)

    Bundschuh, Freya A; Hannappel, Achim; Anderka, Oliver; Ludwig, Bernd

    2009-09-18

    Biogenesis of mitochondrial cytochrome c oxidase (COX) relies on a large number of assembly factors, among them the transmembrane protein Surf1. The loss of human Surf1 function is associated with Leigh syndrome, a fatal neurodegenerative disorder caused by severe COX deficiency. In the bacterium Paracoccus denitrificans, two homologous proteins, Surf1c and Surf1q, were identified, which we characterize in the present study. When coexpressed in Escherichia coli together with enzymes for heme a synthesis, the bacterial Surf1 proteins bind heme a in vivo. Using redox difference spectroscopy and isothermal titration calorimetry, the binding of the heme cofactor to purified apo-Surf1c and apo-Surf1q is quantified: Each of the Paracoccus proteins binds heme a in a 1:1 stoichiometry and with Kd values in the submicromolar range. In addition, we identify a conserved histidine as a residue crucial for heme binding. Contrary to most earlier concepts, these data support a direct role of Surf1 in heme a cofactor insertion into COX subunit I by providing a protein-bound heme a pool. PMID:19625251

  12. Mass-spectrometry data for Rhizoctonia solani proteins produced during infection of wheat and vegetative growth.

    Science.gov (United States)

    Anderson, Jonathan P; Hane, James K; Stoll, Thomas; Pain, Nicholas; Hastie, Marcus L; Kaur, Parwinder; Hoogland, Christine; Gorman, Jeffrey J; Singh, Karam B

    2016-09-01

    Rhizoctonia solani is an important root infecting pathogen of a range of food staples worldwide including wheat, rice, maize, soybean, potato, legumes and others. Conventional resistance breeding strategies are hindered by the absence of tractable genetic resistance in any crop host. Understanding the biology and pathogenicity mechanisms of this fungus is important for addressing these disease issues, however, little is known about how R. solani causes disease. The data described in this article is derived from applying mass spectrometry based proteomics to identify soluble, membrane-bound and culture filtrate proteins produced under wheat infection and vegetative growth conditions. Comparisons of the data for sample types in this set will be useful to identify metabolic pathway changes as the fungus switches from saprophytic to a pathogenic lifestyle or pathogenicity related proteins contributing to the ability to cause disease on wheat. The data set is deposited in the PRIDE archive under identifier PRIDE: PXD002806. PMID:27331100

  13. Bacterial heme-transport proteins and their heme-coordination modes

    OpenAIRE

    Tong, Yong; Guo, Maolin

    2008-01-01

    Efficient iron acquisition is critical for an invading microbe’s survival and virulence. Most of the iron in mammals is incorporated into heme, which can be plundered by certain bacterial pathogens as a nutritional iron source. Utilization of exogenous heme by bacteria involves the binding of heme or hemoproteins to the cell surface receptors, followed by the transport of heme into cells. Once taken into the cytosol, heme is presented to heme oxygenases where the tetrapyrrole ring is cleaved ...

  14. Bacterial cocaine esterase: a protein-based therapy for cocaine overdose and addiction

    OpenAIRE

    Narasimhan, Diwahar; Woods, James H.; Sunahara, Roger K

    2012-01-01

    Cocaine is highly addictive and there are no pharmacotherapeutic drugs available to treat acute cocaine toxicity or chronic abuse. Antagonizing an inhibitor such as cocaine using a small molecule has proven difficult. The alternative approach is to modify cocaine’s pharmacokinetic properties by sequestering or hydrolyzing it in serum and limiting access to its sites of action. We took advantage of a bacterial esterase (CocE) that has evolved to hydrolyze cocaine and have developed it as a the...

  15. New supercharging reagents produce highly charged protein ions in native mass spectrometry.

    Science.gov (United States)

    Going, Catherine C; Xia, Zijie; Williams, Evan R

    2015-11-01

    The effectiveness of two new supercharging reagents for producing highly charged ions by electrospray ionization (ESI) from aqueous solutions in which proteins have native structures and reactivities were investigated. In aqueous solution, 2-thiophenone and 4-hydroxymethyl-1,3-dioxolan-2-one (HD) at a concentration of 2% by volume can increase the average charge of cytochrome c and myoglobin by up to 163%, resulting in even higher charge states than those that are produced from water/methanol/acid solutions in which these proteins are denatured. The greatest extent of supercharging occurs in pure water, but these supercharging reagents are also highly effective in aqueous solutions containing 200 mM ammonium acetate buffer commonly used in native mass spectrometry (MS). These reagents are less effective supercharging reagents than m-nitrobenzyl alcohol (m-NBA) and propylene carbonate (PC) when ions are formed from water/methanol/acid. The extent to which loss of the heme group from myoglobin occurs is related to the extent of supercharging. Results from guanidine melts of cytochrome c monitored with tryptophan fluorescence show that the supercharging reagents PC, sulfolane and HD are effective chemical denaturants in solution. These results provide additional evidence for the role of protein structural changes in the electrospray droplet as the primary mechanism for supercharging with these reagents in native MS. These results also demonstrate that for at least some proteins, the formation of highly charged ions from native MS is no longer a significant barrier for obtaining structural information using conventional tandem MS methods. PMID:26421324

  16. Rapid and widely disseminated acute phase protein response after experimental bacterial infection of pigs

    DEFF Research Database (Denmark)

    Skovgaard, Kerstin; Mortensen, Shila; Boye, Mette;

    2009-01-01

    The acute phase protein response is a well-described generalized early host response to tissue injury, inflammation and infection, observed as pronounced changes in the concentrations of a number of circulating serum proteins. The biological function of this response and its interplay with other...... measurements of interleukin-6 and selected acute phase proteins in serum. C-reactive protein and serum amyloid A were clearly induced 14-18 h after infection. Extrahepatic expression of acute phase proteins was found to be dramatically altered as a result of the lung infection with an extrahepatic acute phase...... protein response occurring concomitantly with the hepatic response. This suggests that the acute phase protein response is a more disseminated systemic response than previously thought. The current study provides to our knowledge the first example of porcine extrahepatic expression and regulation of C...

  17. C-reactive protein is produced by a small number of normal human peripheral blood lymphocytes

    OpenAIRE

    1986-01-01

    Biosynthetic labeling with [35S]met and immunoprecipitation with anti-C- reactive protein (CRP) antibodies and Staphylococcus aureus indicate that cell surface CRP is produced by lymphocytes. The ability of anti- CRP to reduce NK activity, and the demonstration that 125I-anti-CRP- labeled PBL are found in low-density Percoll fractions associated with large granular lymphocyte (LGL) and NK activity suggest that S-CRP- bearing cells are NK effectors. The production of S-CRP by LGL supports this...

  18. Mediastinal Yolk Sac Tumor Producing Protein Induced by Vitamin K Absence or Antagonist-II.

    Science.gov (United States)

    Akutsu, Noriyuki; Adachi, Yasushi; Isosaka, Mai; Mita, Hiroaki; Takagi, Hideyasu; Sasaki, Shigeru; Yamamoto, Hiroyuki; Arimura, Yoshiaki; Ishii, Yoshifumi; Masumori, Naoya; Endo, Takao; Shinomura, Yasuhisa

    2015-01-01

    Extragonadal yolk sac tumors (YSTs) are rare. We herein report the case of a 66-year-old man with mediastinal, lung and liver tumors. The largest mass was located in the liver and contained a high concentration of protein induced by vitamin K absence or antagonist-II (PIVKA-II) and alpha-fetoprotein. Therefore, the lesion was difficult to distinguish from hepatocellular carcinoma. Finally, YST was diagnosed based on the results of a liver biopsy. Although chemotherapy was effective, the patient died of respiratory failure. The autopsy revealed primary mediastinal YST. In the current report, we describe this case of PIVKA-II-producing YST and review previous cases of PIVKA-II-producing tumors other than hepatoma. PMID:26073245

  19. Enzymes produced by halotolerant spore-forming gram-positive bacterial strains isolated from a resting habitat (Restinga de Jurubatiba) in Rio de Janeiro, Brazil: focus on proteases.

    Science.gov (United States)

    D Santos, Anderson Fragoso; Pacheco, Clarissa Almeida; Valle, Roberta D Santos; Seldin, Lucy; D Santos, André Luis Souza

    2014-12-01

    The screening for hydrolases-producing, halotolerant, and spore-forming gram-positive bacteria from the root, rhizosphere, and non-rhizosphere soil of Blutaparon portulacoides, a plant found in the Restinga de Jurubatiba located at the northern region of Rio de Janeiro State, Brazil, resulted in the isolation of 22 strains. These strains were identified as Halobacillus blutaparonensis (n = 2), Oceanobacillus picturae (n = 5), and Oceanobacillus iheyensis (n = 15), and all showed the ability to produce different extracellular enzymes. A total of 20 isolates (90.9 %) showed activity for protease, 5 (22.7 %) for phytase, 3 (13.6 %) for cellulase, and 2 (9.1 %) for amylase. Some bacterial strains were capable of producing three (13.6 %) or two (9.1 %) distinct hydrolytic enzymes. However, no bacterial strain with ability to produce esterase and DNase was observed. The isolate designated M9, belonging to the species H. blutaparonensis, was the best producer of protease and also yielded amylase and phytase. This strain was chosen for further studies regarding its protease activity. The M9 strain produced similar amounts of protease when grown either without or with different NaCl concentrations (from 0.5 to 10 %). A simple inspection of the cell-free culture supernatant by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of three major alkaline proteases of 40, 50, and 70 kDa, which were fully inhibited by phenylmethylsulfonyl fluoride (PMSF) and tosyl-L-phenylalanine chloromethyl ketone (TPCK) (two classical serine protease inhibitors). The secreted proteases were detected in a wide range of temperature (from 4 to 45 °C) and their hydrolytic activities were stimulated by NaCl (up to 10 %). The serine proteases produced by the M9 strain cleaved gelatin, casein, albumin, and hemoglobin, however, in different extensions. Collectively, these results suggest the potential use of the M9 strain in biotechnological

  20. Structural and functional features of self-assembling protein nanoparticles produced in endotoxin-free Escherichia coli

    OpenAIRE

    Rueda, Fabián; Céspedes, María Virtudes; Sánchez-Chardi, Alejandro; Seras-Franzoso, Joaquin; Pesarrodona, Mireia; Ferrer-Miralles, Neus; Vázquez, Esther; Rinas, Ursula; Unzueta, Ugutz; Mamat, Uwe; Mangues, Ramón; García-Fruitós, Elena; Villaverde, Antonio

    2016-01-01

    Background Production of recombinant drugs in process-friendly endotoxin-free bacterial factories targets to a lessened complexity of the purification process combined with minimized biological hazards during product application. The development of nanostructured recombinant materials in innovative nanomedical activities expands such a need beyond plain functional polypeptides to complex protein assemblies. While Escherichia coli has been recently modified for the production of endotoxin-free...

  1. Heat Shock Protein 47: A Novel Biomarker of Phenotypically Altered Collagen-Producing Cells

    International Nuclear Information System (INIS)

    Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that helps the molecular maturation of various types of collagens. A close association between increased expression of HSP47 and the excessive accumulation of collagens is found in various human and experimental fibrotic diseases. Increased levels of HSP47 in fibrotic diseases are thought to assist in the increased assembly of procollagen, and thereby contribute to the excessive deposition of collagens in fibrotic areas. Currently, there is not a good universal histological marker to identify collagen-producing cells. Identifying phenotypically altered collagen-producing cells is essential for the development of cell-based therapies to reduce the progression of fibrotic diseases. Since HSP47 has a single substrate, which is collagen, the HSP47 cellular expression provides a novel universal biomarker to identify phenotypically altered collagen-producing cells during wound healing and fibrosis. In this brief article, we explained why HSP47 could be used as a universal marker for identifying phenotypically altered collagen-producing cells

  2. GPR109A is a G-protein-coupled receptor for the bacterial fermentation product butyrate and functions as a tumor suppressor in colon

    OpenAIRE

    Thangaraju, Muthusamy; Cresci, Gail A.; Liu, Kebin; Ananth, Sudha; Gnanaprakasam, Jaya P.; Browning, Darren D.; Mellinger, John D.; Smith, Sylvia B.; Digby, Gregory J.; Lambert, Nevin A.; Prasad, Puttur D.; Ganapathy, Vadivel

    2009-01-01

    Short-chain fatty acids, generated in colon by bacterial fermentation of dietary fiber, protect against colorectal cancer and inflammatory bowel disease. Among these bacterial metabolites, butyrate is biologically most relevant. GPR109A is a G-protein-coupled receptor for nicotinate, but recognizes butyrate with low affinity. Millimolar concentrations of butyrate are needed to activate the receptor. Although concentrations of butyrate in colonic lumen are sufficient to activate the receptor m...

  3. Comparative effects of dietary nucleoside-nucleotide mixture and its components on endotoxin induced bacterial translocation and small intestinal injury in protein deficient mice.

    OpenAIRE

    Adjei, A A; Yamauchi, K.; Chan, Y. C.; Konishi, M; Yamamoto, S.

    1996-01-01

    BACKGROUND--Nucleoside-nucleotide mixture has been shown to improve gut morphology and reduce the incidence of bacterial translocation in protein deficient mice. AIMS--To compare the reparative effect of nucleoside-nucleotide mixture and their individual components on maintenance of gut integrity and bacterial translocation based on their differential metabolism and utilisation. METHODS--ICR (CD-1) mice were randomised into eight groups of 10 animals each and fed 20% casein diet (control), pr...

  4. 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

  5. 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). PMID:18411272

  6. Protein complexes in bacterial and yeast mitochondrial membranes differ in their sensitivity towards dissociation by SDS.

    Science.gov (United States)

    Gubbens, Jacob; Slijper, Monique; de Kruijff, Ben; de Kroon, Anton I P M

    2008-12-01

    Previously, a 2D gel electrophoresis approach was developed for the Escherichia coli inner membrane, which detects membrane protein complexes that are stable in sodium dodecyl sulfate (SDS) at room temperature, and dissociate under the influence of trifluoroethanol [R. E. Spelbrink et al., J. Biol. Chem. 280 (2005), 28742-8]. Here, the method was applied to the evolutionarily related mitochondrial inner membrane that was isolated from the yeast Saccharomyces cerevisiae. Surprisingly, only very few proteins were found to be dissociated by trifluoroethanol of which Lpd1p, a component of multiple protein complexes localized in the mitochondrial matrix, is the most prominent. Usage of either milder or more stringent conditions did not yield any additional proteins that were released by fluorinated alcohols. This strongly suggests that membrane protein complexes in yeast are less stable in SDS solution than their E. coli counterparts, which might be due to the overall reduced hydrophobicity of mitochondrial transmembrane proteins. PMID:18817900

  7. A secretory system for bacterial production of high-profile protein targets

    OpenAIRE

    Kotzsch, Alexander; Vernet, Erik; Hammarström, Martin; Berthelsen, Jens; Weigelt, Johan; Gräslund, Susanne; Sundström, Michael

    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 improve the usefulness of the E. coli expression platform we have investigated combinations of promoters and selected N-terminal fusion tags for the extracellular expression of human target proteins. A co...

  8. A protein residing at the subunit interface of the bacterial ribosome.

    Science.gov (United States)

    Agafonov, D E; Kolb, V A; Nazimov, I V; Spirin, A S

    1999-10-26

    Surface labeling of Escherichia coli ribosomes with the use of the tritium bombardment technique has revealed a minor unidentified ribosome-bound protein (spot Y) that is hidden in the 70S ribosome and becomes highly labeled on dissociation of the 70S ribosome into subunits. In the present work, the N-terminal sequence of the protein Y was determined and its gene was identified as yfia, an ORF located upstream the phe operon of E. coli. This 12.7-kDa protein was isolated and characterized. An affinity of the purified protein Y for the 30S subunit, but not for the 50S ribosomal subunit, was shown. The protein proved to be exposed on the surface of the 30S subunit. The attachment of the 50S subunit resulted in hiding the protein Y, thus suggesting the protein location at the subunit interface in the 70S ribosome. The protein was shown to stabilize ribosomes against dissociation. The possible role of the protein Y as ribosome association factor in translation is discussed. PMID:10535924

  9. Procalcitonin and C-reactive protein cannot differentiate bacterial or viral infection in COPD exacerbation requiring emergency department visits

    Directory of Open Access Journals (Sweden)

    Chang CH

    2015-04-01

    Full Text Available Chih-Hao Chang,1 Kuo-Chien Tsao,2,3 Han-Chung Hu,1,4 Chung-Chi Huang,1,4 Kuo-Chin Kao,1,4 Ning-Hung Chen,1,4 Cheng-Ta Yang,1,4 Ying-Huang Tsai,4,5 Meng-Jer Hsieh4,51Department of Pulmonary and Critical Care Medicine, Linkou Chang-Gung Memorial Hospital, Chang-Gung Medical Foundation, Chang-Gung University College of Medicine, Taoyuan, Taiwan; 2Department of Laboratory Medicine, Linkou Chang-Gung Memorial Hospital, Chang-Gung Medical Foundation; 3Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Taoyuan, Taiwan; 4Department of Respiratory Therapy, Chang-Gung University, Taoyuan, Taiwan; 5Department of Pulmonary and Critical Care Medicine, Chiayi Chang-Gung Memorial Hospital, Chang-Gung Medical Foundation, Puzi City, TaiwanBackground: Viral and bacterial infections are the most common causes of chronic obstructive pulmonary disease (COPD exacerbations. Whether serum inflammatory markers can differentiate bacterial from virus infection in patients with COPD exacerbation requiring emergency department (ED visits remains controversial.Methods: Viral culture and polymerase chain reaction (PCR were used to identify the viruses in the oropharynx of patients with COPD exacerbations. The bacteria were identified by the semiquantitative culture of the expectorated sputum. The peripheral blood white blood cell (WBC counts, serum C-reactive protein (CRP, procalcitonin (PCT, and clinical symptoms were compared among patients with different types of infections.Results: Viruses were isolated from 16 (22.2% of the 72 patients enrolled. The most commonly identified viruses were parainfluenza type 3, influenza A, and rhinovirus. A total of 30 (41.7% patients had positive bacterial cultures, with the most commonly found bacteria being Haemophilus influenzae and Haemophilus parainfluenzae. Five patients (6.9% had both positive sputum cultures and virus identification. The WBC, CRP, and PCT levels of the bacteria-positive and bacteria

  10. EXTRACELLULAR PROTEINS PRODUCED BY DIFFERENT SPECIES OF THE FUNGUS TRICHODERMA ON SECONDARY PAPER MILL SLUDGE SUBSTRATE

    Directory of Open Access Journals (Sweden)

    Ida Vaskova,

    2012-01-01

    Full Text Available Kraft pulping is the most commonly used pulping process in the pulp and paper industry. In this process wood chips are chemically delignified using sodium sulfide and sodium hydroxide. Delignification is usually followed by mechanical fiberization and a bleaching process of the resulting wood pulp. In addition to lignin-free wood pulp, this process also produces waste that contains residues of used chemicals, lignin, cellulose, hemicelluloses, and small amounts of other wood components. Because of the worldwide large-scale production of paper, the sludge from paper mills contributes significantly to environmental pollution. Although there have been great efforts being made to utilize this lignin-rich material, sludge is mostly disposed in landfills or incinerated in a boiler. This research project used secondary sludge as a substrate for 7 wood-decay fungi taxonomically belonging to the genus Trichoderma. The examined fungi expressed the capability of consuming sludge components as a carbon source to produce extracellular proteins. The proteins were separated by gel electrophoresis. Before and after fungi cultivation, the sludge was analyzed by Fourier transform infrared spectroscopy (FTIR.

  11. A novel human tectonin protein with multivalent beta-propeller folds interacts with ficolin and binds bacterial LPS.

    Directory of Open Access Journals (Sweden)

    Diana Hooi Ping Low

    Full Text Available BACKGROUND: Although the human genome database has been completed a decade ago, approximately 50% of the proteome remains hypothetical as their functions are unknown. The elucidation of the functions of these hypothetical proteins can lead to additional protein pathways and revelation of new cascades. However, many of these inferences are limited to proteins with substantial sequence similarity. Of particular interest here is the Tectonin domain-containing family of proteins. METHODOLOGY/PRINCIPAL FINDINGS: We have identified hTectonin, a hypothetical protein in the human genome database, as a distant ortholog of the limulus galactose binding protein (GBP. Phylogenetic analysis revealed strong evolutionary conservation of hTectonin homologues from parasite to human. By computational analysis, we showed that both the hTectonin and GBP form beta-propeller structures with multiple Tectonin domains, each containing beta-sheets of 4 strands per beta-sheet. hTectonin is present in the human leukocyte cDNA library and immune-related cell lines. It interacts with M-ficolin, a known human complement protein whose ancient homolog, carcinolectin (CL5, is the functional protein partner of GBP during infection. Yeast 2-hybrid assay showed that only the Tectonin domains of hTectonin recognize the fibrinogen-like domain of the M-ficolin. Surface plasmon resonance analysis showed real-time interaction between the Tectonin domains 6 & 11 and bacterial LPS, indicating that despite forming 2 beta-propellers with its different Tectonin domains, the hTectonin molecule could precisely employ domains 6 & 11 to recognise bacteria. CONCLUSIONS/SIGNIFICANCE: By virtue of a recent finding of another Tectonin protein, leukolectin, in the human leukocyte, and our structure-function analysis of the hypothetical hTectonin, we propose that Tectonin domains of proteins could play a vital role in innate immune defense, and that this function has been conserved over several

  12. Avoiding acidic region streaking in two-dimensional gel electrophoresis: Case study with two bacterial whole cell protein extracts

    Indian Academy of Sciences (India)

    Arnab Roy; Umesh Varshney; Debnath Pal

    2014-09-01

    Acidic region streaking (ARS) is one of the lacunae in two-dimensional gel electrophoresis (2DE) of bacterial proteome. This streaking is primarily caused by nucleic acid (NuA) contamination and poses major problem in the downstream processes like image analysis and protein identification. Although cleanup and nuclease digestion are practiced as remedial options, these strategies may incur loss in protein recovery and perform incomplete removal of NuA. As a result, ARS has remained a common observation across publications, including the recent ones. In this work, we demonstrate how ultrasound wave can be used to shear NuA in plain ice-cooled water, facilitating the elimination of ARS in the 2DE gels without the need for any additional sample cleanup tasks. In combination with a suitable buffer recipe, IEF program and frequent paper-wick changing approach, we are able to reproducibly demonstrate the production of clean 2DE gels with improved protein recovery and negligible or no ARS. We illustrate our procedure using whole cell protein extracts from two diverse organisms, Escherichia coli and Mycobacterium smegmatis. Our designed protocols are straightforward and expected to provide good 2DE gels without ARS, with comparable times and significantly lower cost.

  13. Recombinant expression and purification of 'virus-like' bacterial encapsulin protein cages

    NARCIS (Netherlands)

    Rurup, W.F.; Cornelissen, J.J.L.M.; Koay, M.S.T.; Orner, Brendan P.

    2014-01-01

    Ultracentrifugation, particularly the use of sucrose or cesium chloride density gradients, is a highly reliable and efficient technique for the purification of virus-like particles and protein cages. Since virus-like particles and protein cages have a unique size compared to cellular macromolecules

  14. The F-box protein MAX2 contributes to resistance to bacterial phytopathogens in Arabidopsis thaliana

    OpenAIRE

    PiisilÀ, Maria; Keceli, Mehmet A; Brader, GÌnter; Jakobson, Liina; Jõesaar, Indrek; Sipari, Nina; Kollist, Hannes; Palva, E. T.; Kariola, Tarja

    2015-01-01

    Abstract Background The Arabidopsis thaliana F-box protein MORE AXILLARY GROWTH2 (MAX2) has previously been characterized for its role in plant development. MAX2 appears essential for the perception of the newly characterized phytohormone strigolactone, a negative regulator of polar auxin transport in Arabidopsis. Results A reverse genetic screen for F-box protein ...

  15. Efficacious recombinant influenza vaccines produced by high yield bacterial expression: a solution to global pandemic and seasonal needs.

    Directory of Open Access Journals (Sweden)

    Langzhou Song

    Full Text Available It is known that physical linkage of TLR ligands and vaccine antigens significantly enhances the immunopotency of the linked antigens. We have used this approach to generate novel influenza vaccines that fuse the globular head domain of the protective hemagglutinin (HA antigen with the potent TLR5 ligand, flagellin. These fusion proteins are efficiently expressed in standard E. coli fermentation systems and the HA moiety can be faithfully refolded to take on the native conformation of the globular head. In mouse models of influenza infection, the vaccines elicit robust antibody responses that mitigate disease and protect mice from lethal challenge. These immunologically potent vaccines can be efficiently manufactured to support pandemic response, pre-pandemic and seasonal vaccines.

  16. Mutant bacterial sodium channels as models for local anesthetic block of eukaryotic proteins.

    Science.gov (United States)

    Smith, Natalie E; Corry, Ben

    2016-05-01

    Voltage gated sodium channels are the target of a range of local anesthetic, anti-epileptic and anti-arrhythmic compounds. But, gaining a molecular level understanding of their mode of action is difficult as we only have atomic resolution structures of bacterial sodium channels not their eukaryotic counterparts. In this study we used molecular dynamics simulations to demonstrate that the binding sites of both the local anesthetic benzocaine and the anti-epileptic phenytoin to the bacterial sodium channel NavAb can be altered significantly by the introduction of point mutations. Free energy techniques were applied to show that increased aromaticity in the pore of the channel, used to emulate the aromatic residues observed in eukaryotic Nav1.2, led to changes in the location of binding and dissociation constants of each drug relative to wild type NavAb. Further, binding locations and dissociation constants obtained for both benzocaine (660 μM) and phenytoin (1 μ M) in the mutant channels were within the range expected from experimental values obtained from drug binding to eukaryotic sodium channels, indicating that these mutant NavAb may be a better model for drug binding to eukaryotic channels than the wild type. PMID:26852716

  17. Kosmotropes enhance the yield of antibody purified by affinity chromatography using immobilized bacterial immunoglobulin binding proteins.

    Science.gov (United States)

    Ngo, That T; Narinesingh, Dyer

    2008-01-01

    The yield of antibody purified using affinity chromatography on immobilized Protein A or Protein G was increased up to 5-fold (500%) by including kosmotropic salts in the binding buffer. The binding buffer is used to equilibrate the affinity column before applying a sample to the column and also to dilute the sample prior to loading onto the affinity column to optimize conditions for a maximal binding of antibodies to affinity gels. In this study, the kosmotropic salts that were effective in greatly increasing antibody binding to Protein A included both inorganic and organic salts of ammonium; sodium; or potassium sulfate, phosphate, polycarboxylates; for example, succinate, citrate, isocitrate, N-(2-hydroxyethylene diamine triacetate (HEDTA), ethylene diamine tetraacetate (EDTA), and ethylene glycol-O,O'-bis(2-aminoethyl)-N,N,N'N'-tetra acetate(EGTA). On an equal-molar basis, the greater the number of carboxylic groups within the polycarboxylate molecule, the greater the increase in the yield of the purified antibody that was observed. The data show that kosmotropes can be used as effective additives to enhance the binding of immunoglobulins to Protein A or Protein G gels with a resultant increase in the yield of the purified antibodies. Thus, it appears that strongly hydrated anions (citrate, sulfate, and phosphate) and weakly hydrated cations (ammonium, potassium) increase the yield of antibody purified on either Protein A or Protein G affinity gels. PMID:18080884

  18. Bacterial lipases

    NARCIS (Netherlands)

    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, mea

  19. A bacterial type III secretion assay for delivery of fungal effector proteins into wheat.

    Science.gov (United States)

    Upadhyaya, Narayana M; Mago, Rohit; Staskawicz, Brian J; Ayliffe, Michael A; Ellis, Jeffrey G; Dodds, Peter N

    2014-03-01

    Large numbers of candidate effectors from fungal pathogens are being identified through whole-genome sequencing and in planta expression studies. Although Agrobacterium-mediated transient expression has enabled high-throughput functional analysis of effectors in dicot plants, this assay is not effective in cereal leaves. Here, we show that a nonpathogenic Pseudomonas fluorescens engineered to express the type III secretion system (T3SS) of P. syringae and the wheat pathogen Xanthomonas translucens can deliver fusion proteins containing T3SS signals from P. syringae (AvrRpm1) and X. campestris (AvrBs2) avirulence (Avr) proteins, respectively, into wheat leaf cells. A calmodulin-dependent adenylate cyclase reporter protein was delivered effectively into wheat and barley by both bacteria. Absence of any disease symptoms with P. fluorescens makes it more suitable than X. translucens for detecting a hypersensitive response (HR) induced by an effector protein with avirulence activity. We further modified the delivery system by removal of the myristoylation site from the AvrRpm1 fusion to prevent its localization to the plasma membrane which could inhibit recognition of an Avr protein. Delivery of the flax rust AvrM protein by the modified delivery system into transgenic tobacco leaves expressing the corresponding M resistance protein induced a strong HR, indicating that the system is capable of delivering a functional rust Avr protein. In a preliminary screen of effectors from the stem rust fungus Puccinia graminis f. sp. tritici, we identified one effector that induced a host genotype-specific HR in wheat. Thus, the modified AvrRpm1:effector-Pseudomonas fluorescens system is an effective tool for large-scale screening of pathogen effectors for recognition in wheat. PMID:24156769

  20. Inhibition of Salmonella typhimurium Invasion by Host Cell Expression of Secreted Bacterial Invasion Proteins

    OpenAIRE

    Carlson, Steve A.; Jones, Bradley D.

    1998-01-01

    Pathogenic Salmonella species initiate infection of a host by inducing their own uptake into intestinal epithelial cells. An invasive phenotype is conferred to this pathogen by a number of proteins that are components of a type III secretion system. During the invasion process, the bacteria utilize this secretion system to release proteins that enter the host cell and apparently interact with unknown host cell components that induce alterations in the actin cytoskeleton. To investigate the ro...

  1. Inhibition of bacterial aggregation by serum- and blood-derived proteins.

    OpenAIRE

    Malamud, D; Brown, C; Goldman, R

    1984-01-01

    Human and animal sera contain potent inhibitors of saliva-mediated aggregation of oral streptococci. The inhibitors consist of a high-molecular-weight heat-labile factor and a lower-molecular-weight heat-activated factor. The latter appears to be serum albumin. Analyses of purified blood-derived proteins indicated that several high-molecular-weight proteins (fibrinogen, fibronectin, and ferritin) were able to inhibit aggregation at low concentrations. These data suggest that high-molecular-we...

  2. A Bacterial Virulence Protein Promotes Pathogenicity by Inhibiting the Bacterium's Own F1Fo ATP Synthase

    OpenAIRE

    Lee, Eun-Jin; Pontes, Mauricio H.; Groisman, Eduardo A.

    2013-01-01

    Several intracellular pathogens including Salmonella enterica and Mycobacterium tuberculosis require the virulence protein MgtC to survive within macrophages and to cause a lethal infection in mice. We now report that, unlike secreted virulence factors that target the host vacuolar ATPase to withstand phagosomal acidity, the MgtC protein acts on Salmonella's own F1Fo ATP synthase. This complex couples proton translocation to ATP synthesis/ hydrolysis and is required for virulence. We establis...

  3. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    Energy Technology Data Exchange (ETDEWEB)

    Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  4. The role of lipids in membrane insertion and translocation of bacterial proteins.

    Science.gov (United States)

    van Dalen, Annemieke; de Kruijff, Ben

    2004-11-11

    Phospholipids are essential building blocks of membranes and maintain the membrane permeability barrier of cells and organelles. They provide not only the bilayer matrix in which the functional membrane proteins reside, but they also can play direct roles in many essential cellular processes. In this review, we give an overview of the lipid involvement in protein translocation across and insertion into the Escherichia coli inner membrane. We describe the key and general roles that lipids play in these processes in conjunction with the protein components involved. We focus on the Sec-mediated insertion of leader peptidase. We describe as well the more direct roles that lipids play in insertion of the small coat proteins Pf3 and M13. Finally, we focus on the role of lipids in membrane assembly of oligomeric membrane proteins, using the potassium channel KcsA as model protein. In all cases, the anionic lipids and lipids with small headgroups play important roles in either determining the efficiency of the insertion and assembly process or contributing to the directionality of the insertion process. PMID:15546660

  5. Detergent disruption of bacterial inner membranes and recovery of protein translocation activity

    International Nuclear Information System (INIS)

    Isolation of the integral membrane components of protein translocation requires methods for fractionation and functional reconstitution. The authors treated inner-membrane vesicles of Escherichia coli with mixtures of octyl β-D-glucoside, phospholipids, and an integral membrane carrier protein under conditions that extract most of the membrane proteins into micellar solution. Upon dialysis, proteoliposomes were reconstituted that supported translocation of radiochemically pure [35S]pro-OmpA (the precursor of outer membrane protein A). Translocation into these proteoliposomes required ATP hydrolysis and membrane proteins, indicating that the reaction is that of the inner membrane. The suspension of membranes in detergent was separated into supernatant and pellet fractions by ultracentrifugation. After reconstitution, translocation activity was observed in both fractions, but processing by leader peptidase of translocated pro-OmpA to OmpA was not detectable in the reconstituted pellet fraction. Processing activity was restored by addition of pure leader peptidase as long as this enzyme was added before detergent removal, indicating that the translocation activity is not associated with detergent-resistant membrane vesicles. These results show that protein translocation activity can be recovered from detergent-disrupted membrane vesicles, providing a first step towards the goal of isolating the solubilized components

  6. ParB Partition Proteins: Complex Formation and Spreading at Bacterial and Plasmid Centromeres.

    Science.gov (United States)

    Funnell, Barbara E

    2016-01-01

    In bacteria, active partition systems contribute to the faithful segregation of both chromosomes and low-copy-number plasmids. Each system depends on a site-specific DNA binding protein to recognize and assemble a partition complex at a centromere-like site, commonly called parS. Many plasmid, and all chromosomal centromere-binding proteins are dimeric helix-turn-helix DNA binding proteins, which are commonly named ParB. Although the overall sequence conservation among ParBs is not high, the proteins share similar domain and functional organization, and they assemble into similar higher-order complexes. In vivo, ParBs "spread," that is, DNA binding extends away from the parS site into the surrounding non-specific DNA, a feature that reflects higher-order complex assembly. ParBs bridge and pair DNA at parS and non-specific DNA sites. ParB dimers interact with each other via flexible conformations of an N-terminal region. This review will focus on the properties of the HTH centromere-binding protein, in light of recent experimental evidence and models that are adding to our understanding of how these proteins assemble into large and dynamic partition complexes at and around their specific DNA sites. PMID:27622187

  7. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    International Nuclear Information System (INIS)

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states

  8. Structural and functional similarity between the bacterial type III secretion system needle protein PrgI and the eukaryotic apoptosis Bcl-2 proteins.

    Directory of Open Access Journals (Sweden)

    Matthew D Shortridge

    Full Text Available BACKGROUND: Functional similarity is challenging to identify when global sequence and structure similarity is low. Active-sites or functionally relevant regions are evolutionarily more stable relative to the remainder of a protein structure and provide an alternative means to identify potential functional similarity between proteins. We recently developed the FAST-NMR methodology to discover biochemical functions or functional hypotheses of proteins of unknown function by experimentally identifying ligand binding sites. FAST-NMR utilizes our CPASS software and database to assign a function based on a similarity in the structure and sequence of ligand binding sites between proteins of known and unknown function. METHODOLOGY/PRINCIPAL FINDINGS: The PrgI protein from Salmonella typhimurium forms the needle complex in the type III secretion system (T3SS. A FAST-NMR screen identified a similarity between the ligand binding sites of PrgI and the Bcl-2 apoptosis protein Bcl-xL. These ligand binding sites correlate with known protein-protein binding interfaces required for oligomerization. Both proteins form membrane pores through this oligomerization to release effector proteins to stimulate cell death. Structural analysis indicates an overlap between the PrgI structure and the pore forming motif of Bcl-xL. A sequence alignment indicates conservation between the PrgI and Bcl-xL ligand binding sites and pore formation regions. This active-site similarity was then used to verify that chelerythrine, a known Bcl-xL inhibitor, also binds PrgI. CONCLUSIONS/SIGNIFICANCE: A structural and functional relationship between the bacterial T3SS and eukaryotic apoptosis was identified using our FAST-NMR ligand affinity screen in combination with a bioinformatic analysis based on our CPASS program. A similarity between PrgI and Bcl-xL is not readily apparent using traditional global sequence and structure analysis, but was only identified because of conservation in

  9. Functional characterization of Kaposi's sarcoma-associated herpesvirus small capsid protein by bacterial artificial chromosome-based mutagenesis

    International Nuclear Information System (INIS)

    A systematic investigation of interactions amongst KSHV capsid proteins was undertaken in this study to comprehend lesser known KSHV capsid assembly mechanisms. Interestingly the interaction patterns of the KSHV small capsid protein, ORF65 suggested its plausible role in viral capsid assembly pathways. Towards further understanding this, ORF65-null recombinant mutants (BAC-Δ65 and BAC-stop65) employing a bacterial artificial chromosome (BAC) system were generated. No significant difference was found in both overall viral gene expression and lytic DNA replication between stable monolayers of 293T-BAC36 (wild-type) and 293T-BAC-ORF65-null upon induction with 12-O-tetradecanoylphorbol-13-acetate, though the latter released 30-fold fewer virions to the medium than 293T-BAC36 cells. Sedimentation profiles of capsid proteins of ORF65-null recombinant mutants were non-reflective of their organization into the KSHV capsids and were also undetectable in cytoplasmic extracts compared to noticeable levels in nuclear extracts. These observations collectively suggested the pivotal role of ORF65 in the KSHV capsid assembly processes.

  10. PROTEIN QUALITY CONTROL IN BACTERIAL CELLS: INTEGRATED NETWORKS OF CHAPERONES AND ATP-DEPENDENT PROTEASES.

    Energy Technology Data Exchange (ETDEWEB)

    FLANAGAN,J.M.BEWLEY,M.C.

    2002-10-01

    It is generally accepted that the information necessary to specify the native, functional, three-dimensional structure of a protein is encoded entirely within its amino acid sequence; however, efficient reversible folding and unfolding is observed only with a subset of small single-domain proteins. Refolding experiments often lead to the formation of kinetically-trapped, misfolded species that aggregate, even in dilute solution. In the cellular environment, the barriers to efficient protein folding and maintenance of native structure are even larger due to the nature of this process. First, nascent polypeptides must fold in an extremely crowded environment where the concentration of macromolecules approaches 300-400 mg/mL and on average, each ribosome is within its own diameter of another ribosome (1-3). These conditions of severe molecular crowding, coupled with high concentrations of nascent polypeptide chains, favor nonspecific aggregation over productive folding (3). Second, folding of newly-translated polypeptides occurs in the context of their vehtorial synthesis process. Amino acids are added to a growing nascent chain at the rate of {approx}5 residues per set, which means that for a 300 residue protein its N-terminus will be exposed to the cytosol {approx}1 min before its C-terminus and be free to begin the folding process. However, because protein folding is highly cooperative, the nascent polypeptide cannot reach its native state until a complete folding domain (50-250 residues) has emerged from the ribosome. Thus, for a single-domain protein, the final steps in ffolding are only completed post-translationally since {approx}40 residues of a nascent chain are sequestered within the exit channel of the ribosome and are not available for folding (4). A direct consequence of this limitation in cellular folding is that during translation incomplete domains will exist in partially-folded states that tend to expose hydrophobic residues that are prone to

  11. PROTEIN QUALITY CONTROL IN BACTERIAL CELLS: INTEGRATED NETWORKS OF CHAPERONES AND ATP-DEPENDENT PROTEASES.

    Energy Technology Data Exchange (ETDEWEB)

    FLANAGAN,J.M.; BEWLEY,M.C.

    2001-12-03

    It is generally accepted that the information necessary to specify the native, functional, three-dimensional structure of a protein is encoded entirely within its amino acid sequence; however, efficient reversible folding and unfolding is observed only with a subset of small single-domain proteins. Refolding experiments often lead to the formation of kinetically-trapped, misfolded species that aggregate, even in dilute solution. In the cellular environment, the barriers to efficient protein folding and maintenance of native structure are even larger due to the nature of this process. First, nascent polypeptides must fold in an extremely crowded environment where the concentration of macromolecules approaches 300-400 mg/mL and on average, each ribosome is within its own diameter of another ribosome (1-3). These conditions of severe molecular crowding, coupled with high concentrations of nascent polypeptide chains, favor nonspecific aggregation over productive folding (3). Second, folding of newly-translated polypeptides occurs in the context of their vehtorial synthesis process. Amino acids are added to a growing nascent chain at the rate of -5 residues per set, which means that for a 300 residue protein its N-terminus will be exposed to the cytosol {approx}1 min before its C-terminus and be free to begin the folding process. However, because protein folding is highly cooperative, the nascent polypeptide cannot reach its native state until a complete folding domain (50-250 residues) has emerged from the ribosome. Thus, for a single-domain protein, the final steps in folding are only completed post-translationally since {approx}40 residues of a nascent chain are sequestered within the exit channel of the ribosome and are not available for folding (4). A direct consequence of this limitation in cellular folding is that during translation incomplete domains will exist in partially-folded states that tend to expose hydrophobic residues that are prone to aggregation and

  12. PROTEIN QUALITY CONTROL IN BACTERIAL CELLS: INTEGRATED NETWORKS OF CHAPERONES AND ATP-DEPENDENT PROTEASES

    International Nuclear Information System (INIS)

    It is generally accepted that the information necessary to specify the native, functional, three-dimensional structure of a protein is encoded entirely within its amino acid sequence; however, efficient reversible folding and unfolding is observed only with a subset of small single-domain proteins. Refolding experiments often lead to the formation of kinetically-trapped, misfolded species that aggregate, even in dilute solution. In the cellular environment, the barriers to efficient protein folding and maintenance of native structure are even larger due to the nature of this process. First, nascent polypeptides must fold in an extremely crowded environment where the concentration of macromolecules approaches 300-400 mg/mL and on average, each ribosome is within its own diameter of another ribosome (1-3). These conditions of severe molecular crowding, coupled with high concentrations of nascent polypeptide chains, favor nonspecific aggregation over productive folding (3). Second, folding of newly-translated polypeptides occurs in the context of their vehtorial synthesis process. Amino acids are added to a growing nascent chain at the rate of -5 residues per set, which means that for a 300 residue protein its N-terminus will be exposed to the cytosol(approx)1 min before its C-terminus and be free to begin the folding process. However, because protein folding is highly cooperative, the nascent polypeptide cannot reach its native state until a complete folding domain (50-250 residues) has emerged from the ribosome. Thus, for a single-domain protein, the final steps in folding are only completed post-translationally since(approx)40 residues of a nascent chain are sequestered within the exit channel of the ribosome and are not available for folding (4). A direct consequence of this limitation in cellular folding is that during translation incomplete domains will exist in partially-folded states that tend to expose hydrophobic residues that are prone to aggregation and

  13. Monitoring Dynamic Protein Expression in Single Living E. Coli. Bacterial Cells by Laser Tweezers Raman Spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Chan, J W; Winhold, H; Corzett, M H; Ulloa, J M; Cosman, M; Balhorn, R; Huser, T

    2007-01-09

    Laser tweezers Raman spectroscopy (LTRS) is a novel, nondestructive, and label-free method that can be used to quantitatively measure changes in cellular activity in single living cells. Here, we demonstrate its use to monitor changes in a population of E. coli cells that occur during overexpression of a protein, the extracellular domain of myelin oligodendrocyte glycoprotein (MOG(1-120)) Raman spectra were acquired of individual E. coli cells suspended in solution and trapped by a single tightly focused laser beam. Overexpression of MOG(1-120) in transformed E. coli Rosetta-Gami (DE3)pLysS cells was induced by addition of isopropyl thiogalactoside (IPTG). Changes in the peak intensities of the Raman spectra from a population of cells were monitored and analyzed over a total duration of three hours. Data was also collected for concentrated purified MOG(1-120) protein in solution, and the spectra compared with that obtained for the MOG(1-120) expressing cells. Raman spectra of individual, living E. coli cells exhibit signatures due to DNA and protein molecular vibrations. Characteristic Raman markers associated with protein vibrations, such as 1257 cm{sup -1}, 1340 cm{sup -1}, 1453 cm{sup -1} and 1660 cm{sup -1}, are shown to increase as a function of time following the addition of IPTG. Comparison of these spectra and the spectra of purified MOG protein indicates that the changes are predominantly due to the induction of MOG protein expression. Protein expression was found to occur mostly within the second hour, with a 470% increase relative to the protein expressed in the first hour. A 230% relative increase between the second and third hour indicates that protein expression begins to level off within the third hour. It is demonstrated that LTRS has sufficient sensitivity for real-time, nondestructive, and quantitative monitoring of biological processes, such as protein expression, in single living cells. Such capabilities, which are not currently available in

  14. Cloning, expression, purification and preliminary X-ray diffraction studies of a mycobacterial protein implicated in bacterial survival in macrophages

    International Nuclear Information System (INIS)

    The cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of MSMEG-5817 is described. Mycobacterium species have developed numerous strategies to avoid the antimycobacterial actions of macrophages, enabling them to survive within the generally inhospitable environment of the cell. The recently identified MSMEG-5817 protein from M. smegmatis is highly conserved in Mycobacterium spp. and is required for bacterial survival in macrophages. Here, the cloning, expression, purification and crystallization of MSMEG-5817 is reported. Crystals of MSMEG-5817 were grown in 1.42 M Li2SO4, 0.1 M Tris–HCl pH 7.7, 0.1 M sodium citrate tribasic dihydrate. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress

  15. Bacterial carbonatogenesis

    International Nuclear Information System (INIS)

    Several series of experiments in the laboratory as well as in natural conditions teach that the production of carbonate particles by heterotrophic bacteria follows different ways. The 'passive' carbonatogenesis is generated by modifications of the medium that lead to the accumulation of carbonate and bicarbonate ions and to the precipitation of solid particles. The 'active' carbonatogenesis is independent of the metabolic pathways. The carbonate particles are produced by ionic exchanges through the cell membrane following still poorly known mechanisms. Carbonatogenesis appears to be the response of heterotrophic bacterial communities to an enrichment of the milieu in organic matter. The active carbonatogenesis seems to start first. It is followed by the passive one which induces the growth of initially produced particles. The yield of heterotrophic bacterial carbonatogenesis and the amounts of solid carbonates production by bacteria are potentially very high as compared to autotrophic or chemical sedimentation from marine, paralic or continental waters. Furthermore, the bacterial processes are environmentally very ubiquitous; they just require organic matter enrichment. Thus, apart from purely evaporite and autotrophic ones, all Ca and/or Mg carbonates must be considered as from heterotrophic bacterial origin. By the way, the carbon of carbonates comes from primary organic matter. Such considerations ask questions about some interpretations from isotopic data on carbonates. Finally, bacterial heterotrophic carbonatogenesis appears as a fundamental phase in the relationships between atmosphere and lithosphere and in the geo-biological evolution of Earth. (author)

  16. Bacteriophage Tailspikes and Bacterial O-Antigens as a Model System to Study Weak-Affinity Protein-Polysaccharide Interactions.

    Science.gov (United States)

    Kang, Yu; Gohlke, Ulrich; Engström, Olof; Hamark, Christoffer; Scheidt, Tom; Kunstmann, Sonja; Heinemann, Udo; Widmalm, Göran; Santer, Mark; Barbirz, Stefanie

    2016-07-27

    Understanding interactions of bacterial surface polysaccharides with receptor protein scaffolds is important for the development of antibiotic therapies. The corresponding protein recognition domains frequently form low-affinity complexes with polysaccharides that are difficult to address with experimental techniques due to the conformational flexibility of the polysaccharide. In this work, we studied the tailspike protein (TSP) of the bacteriophage Sf6. Sf6TSP binds and hydrolyzes the high-rhamnose, serotype Y O-antigen polysaccharide of the Gram-negative bacterium Shigella flexneri (S. flexneri) as a first step of bacteriophage infection. Spectroscopic analyses and enzymatic cleavage assays confirmed that Sf6TSP binds long stretches of this polysaccharide. Crystal structure analysis and saturation transfer difference (STD) NMR spectroscopy using an enhanced method to interpret the data permitted the detailed description of affinity contributions and flexibility in an Sf6TSP-octasaccharide complex. Dodecasaccharide fragments corresponding to three repeating units of the O-antigen in complex with Sf6TSP were studied computationally by molecular dynamics simulations. They showed that distortion away from the low-energy solution conformation found in the octasaccharide complex is necessary for ligand binding. This is in agreement with a weak-affinity functional polysaccharide-protein contact that facilitates correct placement and thus hydrolysis of the polysaccharide close to the catalytic residues. Our simulations stress that the flexibility of glycan epitopes together with a small number of specific protein contacts provide the driving force for Sf6TSP-polysaccharide complex formation in an overall weak-affinity interaction system. PMID:27045683

  17. Proteomic analysis of growth phase-dependent expression of Legionella pneumophila proteins which involves regulation of bacterial virulence traits.

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Hayashi

    Full Text Available Legionella pneumophila, which is a causative pathogen of Legionnaires' disease, expresses its virulent traits in response to growth conditions. In particular, it is known to become virulent at a post-exponential phase in vitro culture. In this study, we performed a proteomic analysis of differences in expression between the exponential phase and post-exponential phase to identify candidates associated with L. pneumophila virulence using 2-Dimentional Fluorescence Difference Gel Electrophoresis (2D-DIGE combined with Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry (MALDI-TOF-MS. Of 68 identified proteins that significantly differed in expression between the two growth phases, 64 were up-regulated at a post-exponential phase. The up-regulated proteins included enzymes related to glycolysis, ketone body biogenesis and poly-3-hydroxybutyrate (PHB biogenesis, suggesting that L. pneumophila may utilize sugars and lipids as energy sources, when amino acids become scarce. Proteins related to motility (flagella components and twitching motility-associated proteins were also up-regulated, predicting that they enhance infectivity of the bacteria in host cells under certain conditions. Furthermore, 9 up-regulated proteins of unknown function were found. Two of them were identified as novel bacterial factors associated with hemolysis of sheep red blood cells (SRBCs. Another 2 were found to be translocated into macrophages via the Icm/Dot type IV secretion apparatus as effector candidates in a reporter assay with Bordetella pertussis adenylate cyclase. The study will be helpful for virulent analysis of L. pneumophila from the viewpoint of physiological or metabolic modulation dependent on growth phase.

  18. Oxygen and sulfur isotope systematics of sulfate produced during abiotic and bacterial oxidation of sphalerite and elemental sulfur

    Science.gov (United States)

    Balci, N.; Mayer, B.; Shanks, Wayne C.; Mandernack, K.W.

    2012-01-01

    Studies of metal sulfide oxidation in acid mine drainage (AMD) systems have primarily focused on pyrite oxidation, although acid soluble sulfides (e.g., ZnS) are predominantly responsible for the release of toxic metals. We conducted a series of biological and abiotic laboratory oxidation experiments with pure and Fe-bearing sphalerite (ZnS & Zn 0.88Fe 0.12S), respectively, in order to better understand the effects of sulfide mineralogy and associated biogeochemical controls of oxidation on the resultant ?? 34S and ?? 18O values of the sulfate produced. The minerals were incubated in the presence and absence of Acidithiobacillus ferrooxidans at an initial solution pH of 3 and with water of varying ?? 18O values to determine the relative contributions of H 2O-derived and O 2-derived oxygen in the newly formed sulfate. Experiments were conducted under aerobic and anaerobic conditions using O 2 and Fe(III) aq as the oxidants, respectively. Aerobic incubations with A. ferrooxidans, and S o as the sole energy source were also conducted. The ??34SSO4 values from both the biological and abiotic oxidation of ZnS and ZnS Fe by Fe(III) aq produced sulfur isotope fractionations (??34SSO4-ZnS) of up to -2.6???, suggesting the accumulation of sulfur intermediates during incomplete oxidation of the sulfide. No significant sulfur isotope fractionation was observed from any of the aerobic experiments. Negative sulfur isotope enrichment factors (??34SSO4-ZnS) in AMD systems could reflect anaerobic, rather than aerobic pathways of oxidation. During the biological and abiotic oxidation of ZnS and ZnS Fe by Fe(III) aq all of the sulfate oxygen was derived from water, with measured ?? 18OSO 4-H 2O values of 8.2??0.2??? and 7.5??0.1???, respectively. Also, during the aerobic oxidation of ZnS Fe and S o by A. ferrooxidans, all of the sulfate oxygen was derived from water with similar measured ?? 18OSO 4-H 2O values of 8.1??0.1??? and 8.3??0.3???, respectively. During biological oxidation

  19. An exciton-polariton laser based on biologically produced fluorescent protein

    Science.gov (United States)

    Dietrich, Christof P.; Steude, Anja; Tropf, Laura; Schubert, Marcel; Kronenberg, Nils M.; Ostermann, Kai; Höfling, Sven; Gather, Malte C.

    2016-01-01

    Under adequate conditions, cavity polaritons form a macroscopic coherent quantum state, known as polariton condensate. Compared to Wannier-Mott excitons in inorganic semiconductors, the localized Frenkel excitons in organic emitter materials show weaker interaction with each other but stronger coupling to light, which recently enabled the first realization of a polariton condensate at room temperature. However, this required ultrafast optical pumping, which limits the applications of organic polariton condensates. We demonstrate room temperature polariton condensates of cavity polaritons in simple laminated microcavities filled with biologically produced enhanced green fluorescent protein (eGFP). The unique molecular structure of eGFP prevents exciton annihilation even at high excitation densities, thus facilitating polariton condensation under conventional nanosecond pumping. Condensation is clearly evidenced by a distinct threshold, an interaction-induced blueshift of the condensate, long-range coherence, and the presence of a second threshold at higher excitation density that is associated with the onset of photon lasing. PMID:27551686

  20. An exciton-polariton laser based on biologically produced fluorescent protein.

    Science.gov (United States)

    Dietrich, Christof P; Steude, Anja; Tropf, Laura; Schubert, Marcel; Kronenberg, Nils M; Ostermann, Kai; Höfling, Sven; Gather, Malte C

    2016-08-01

    Under adequate conditions, cavity polaritons form a macroscopic coherent quantum state, known as polariton condensate. Compared to Wannier-Mott excitons in inorganic semiconductors, the localized Frenkel excitons in organic emitter materials show weaker interaction with each other but stronger coupling to light, which recently enabled the first realization of a polariton condensate at room temperature. However, this required ultrafast optical pumping, which limits the applications of organic polariton condensates. We demonstrate room temperature polariton condensates of cavity polaritons in simple laminated microcavities filled with biologically produced enhanced green fluorescent protein (eGFP). The unique molecular structure of eGFP prevents exciton annihilation even at high excitation densities, thus facilitating polariton condensation under conventional nanosecond pumping. Condensation is clearly evidenced by a distinct threshold, an interaction-induced blueshift of the condensate, long-range coherence, and the presence of a second threshold at higher excitation density that is associated with the onset of photon lasing. PMID:27551686

  1. Application of ionizing radiations to produce new polysaccharides and proteins with enhanced functionality

    Energy Technology Data Exchange (ETDEWEB)

    Al-Assaf, S. [Glyn O. Phillips Hydrocolloids Research Centre, North East Wales Institute, Mold Road, Wrexham LL11 2AW, North Wales (United Kingdom)], E-mail: alassafs@newi.ac.uk; Phillips, G.O. [Glyn O. Phillips Hydrocolloids Research Centre, North East Wales Institute, Mold Road, Wrexham LL11 2AW, North Wales (United Kingdom); Phillips Hydrocolloid Research Ltd., 45 Old Bond Street, London W1S 4AQ (United Kingdom); Williams, P.A. [Centre for Water Soluble Polymers, North East Wales Institute, Mold Road, Wrexham LL11 2AW, North Wales (United Kingdom); Plessis, T.A. du [Gammatron (PTY) Ltd., P.O. Box 26378, Monument Part 0105 (South Africa)

    2007-12-15

    Treatment of polysaccharides with ionizing radiation either in the solid state or in aqueous solution leads to degradation. Using a mediating alkyne gas during the radiation treatment prevents the degradation of natural and synthetic polysaccharides and enables the introduction of different molecular and functional characteristics, as previously achieved with synthetic polymers. The molecular weight can be increased and hydrogel forms of the polysaccharides or proteins can be produced, probably by a cross-linking mechanism involving addition of C-C bond between two chains. The product has higher viscosity and is more elastic than the original material and, therefore, gives enhanced functionality. Gum Arabic (Acacia senegal) irradiated for 6.2 kGy is used here to demonstrate the enhanced emulsification performance and stability compared with the original material.

  2. Characterisation of the bacterial microbiota of the vagina of dairy cows and isolation of pediocin-producing Pediococcus acidilactici

    Directory of Open Access Journals (Sweden)

    Wang Yvonne

    2013-01-01

    Full Text Available Abstract Background Uterine infections in dairy cows lower profitability of dairy operations. Infections of the reproductive tract are related to the overgrowth of pathogenic bacteria during the first three weeks after parturition. However, alterations in the vaginal microbiota composition in the first weeks after parturition remain poorly documented. Results In this study, bacteria isolated from the vagina of healthy pregnant, and infected postpartum cows were characterised by random amplification of polymorphic DNA (RAPD analysis and partial 16S ribosomal RNA (rDNA gene sequencing. Populations of bacilli and lactic acid bacteria of the genera Enterococcus, Lactobacillus, and Pediococcus were present in both healthy and infected cows. Infected cows had a significant increase in the vaginal enteric bacteria population which consisted mainly of Escherichia coli. Three E. coli isolates harboured the gene coding for Shiga-like-toxin (SLT I or II. Several isolates of the Pediococcus acidilactici were found to produce the bacteriocin pediocin AcH/PA-1. Quantitative PCR analyses of vaginal mucus samples collected from ten metritic cows before and after parturition confirmed the presence of the Lactobacillus group (Lactobacillus spp., Pediococcus spp., Leuconostoc spp., and Weissella spp.; Enterobacteriaceae, E. coli, and bacilli. The presence of the pediocin AcH/PA-1 structural gene and SLT genes were also confirmed with qPCR. Conclusions In conclusion, overgrowth of pathogenic bacteria, particularly E. coli, after parturition likely contributes to the development of metritis. Our microbiota analysis extends the information related to the composition of commensal bacteria in the bovine female reproductive tract and may facilitate the development of novel intervention strategies for prevention of uterine infections in dairy cows.

  3. BacHbpred: Support Vector Machine Methods for the Prediction of Bacterial Hemoglobin-Like Proteins.

    Science.gov (United States)

    Selvaraj, MuthuKrishnan; Puri, Munish; Dikshit, Kanak L; Lefevre, Christophe

    2016-01-01

    The recent upsurge in microbial genome data has revealed that hemoglobin-like (HbL) proteins may be widely distributed among bacteria and that some organisms may carry more than one HbL encoding gene. However, the discovery of HbL proteins has been limited to a small number of bacteria only. This study describes the prediction of HbL proteins and their domain classification using a machine learning approach. Support vector machine (SVM) models were developed for predicting HbL proteins based upon amino acid composition (AC), dipeptide composition (DC), hybrid method (AC + DC), and position specific scoring matrix (PSSM). In addition, we introduce for the first time a new prediction method based on max to min amino acid residue (MM) profiles. The average accuracy, standard deviation (SD), false positive rate (FPR), confusion matrix, and receiver operating characteristic (ROC) were analyzed. We also compared the performance of our proposed models in homology detection databases. The performance of the different approaches was estimated using fivefold cross-validation techniques. Prediction accuracy was further investigated through confusion matrix and ROC curve analysis. All experimental results indicate that the proposed BacHbpred can be a perspective predictor for determination of HbL related proteins. BacHbpred, a web tool, has been developed for HbL prediction. PMID:27034664

  4. Activation of Neutrophils via IP3 Pathway Following Exposure to Demodex-Associated Bacterial Proteins.

    Science.gov (United States)

    McMahon, Fred; Banville, Nessa; Bergin, David A; Smedman, Christian; Paulie, Staffan; Reeves, Emer; Kavanagh, Kevin

    2016-02-01

    Rosacea is a chronic inflammatory condition that predominantly affects the skin of the face. Sera from rosacea patients display elevated reactivity to proteins from a bacterium (Bacillus oleronius) originally isolated from a Demodex mite from a rosacea patient suggesting a possible role for bacteria in the induction and persistence of this condition. This work investigated the ability of B. oleronius proteins to activate neutrophils and demonstrated activation via the IP3 pathway. Activated neutrophils displayed increased levels of IP1 production, F-actin formation, chemotaxis, and production of the pro-inflammatory cytokines IL-1β and IL-6 following stimulation by pure and crude B. oleronius protein preparations (2 μg/ml), respectively. In addition, neutrophils exposed to pure and crude B. oleronius proteins (2 μg/ml) demonstrated increased release of internally stored calcium (Ca(2+)), a hallmark of the IP3 pathway of neutrophil activation. Neutrophils play a significant role in the inflammation associated with rosacea, and this work demonstrates how B. oleronius proteins can induce neutrophil recruitment and activation. PMID:26433579

  5. Systematic protein interactome analysis of glycosaminoglycans revealed YcbS as a novel bacterial virulence factor.

    Science.gov (United States)

    Hsiao, Felix Shih-Hsiang; Sutandy, Fx Reymond; Syu, Guan-Da; Chen, Yi-Wen; Lin, Jun-Mu; Chen, Chien-Sheng

    2016-01-01

    Microbial pathogens have evolved several strategies for interacting with host cell components, such as glycosaminoglycans (GAGs). Some microbial proteins involved in host-GAG binding have been described; however, a systematic study on microbial proteome-mammalian GAG interactions has not been conducted. Here, we used Escherichia coli proteome chips to probe four typical mammalian GAGs, heparin, heparan sulphate (HS), chondroitin sulphate B (CSB), and chondroitin sulphate C (CSC), and identified 185 heparin-, 62 HS-, 98 CSB-, and 101 CSC-interacting proteins. Bioinformatics analyses revealed the unique functions of heparin- and HS-specific interacting proteins in glycine, serine, and threonine metabolism. Among all the GAG-interacting proteins, three were outer membrane proteins (MbhA, YcbS, and YmgH). Invasion assays confirmed that mutant E. coli lacking ycbS could not invade the epithelial cells. Introducing plasmid carrying ycbS complemented the invading defects at ycbS lacking E. coli mutant, that can be further improved by overexpressing ycbS. Preblocking epithelial cells with YcbS reduced the percentage of E. coli invasions. Moreover, we observed that whole components of the ycb operon were crucial for invasion. The displacement assay revealed that YcbS binds to the laminin-binding site of heparin and might affect the host extracellular matrix structure by displacing heparin from laminin. PMID:27323865

  6. Trans-splicing as a novel method to rapidly produce antibody fusion proteins

    Energy Technology Data Exchange (ETDEWEB)

    Iwasaki, Ryohei; Kiuchi, Hiroki [Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Ihara, Masaki [Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Mori, Toshihiro; Kawakami, Masayuki [Lifescience Lab. R and D, Fujifilm Co., 577 Ushijima, Kaisei-machi, Ashigarakami-gun, Kanagawa 258-8577 (Japan); Ueda, Hiroshi, E-mail: hueda@chembio.t.u-tokyo.ac.jp [Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan)

    2009-07-03

    To cultivate the use of trans-splicing as a novel means to rapidly express various antibody fusion proteins, we tried to express antibody-reporter enzyme fusions in a COS-1 co-transfection model. When a vector designed to induce trans-splicing with IgH pre-mRNA was co-transfected with a vector encoding the mouse IgM locus, the expression of V{sub H}-secreted human placental alkaline phosphatase (SEAP) as well as Fab-SEAP were successfully expressed both in mRNA and protein levels. Especially, the vectors encoding complementary sequence to S{mu} as a binding domain was accurate and efficient, producing trans-spliced mRNA of up to 2% of cis-spliced one. Since S{mu} sequence should exist in every IgH pre-mRNA, our finding will lead to the rapid production and analysis of various antibody-enzyme fusions suitable for enzyme-linked immunosorbent assay (ELISA) or antibody-dependent enzyme prodrug therapy (ADEPT).

  7. Trans-splicing as a novel method to rapidly produce antibody fusion proteins

    International Nuclear Information System (INIS)

    To cultivate the use of trans-splicing as a novel means to rapidly express various antibody fusion proteins, we tried to express antibody-reporter enzyme fusions in a COS-1 co-transfection model. When a vector designed to induce trans-splicing with IgH pre-mRNA was co-transfected with a vector encoding the mouse IgM locus, the expression of VH-secreted human placental alkaline phosphatase (SEAP) as well as Fab-SEAP were successfully expressed both in mRNA and protein levels. Especially, the vectors encoding complementary sequence to Sμ as a binding domain was accurate and efficient, producing trans-spliced mRNA of up to 2% of cis-spliced one. Since Sμ sequence should exist in every IgH pre-mRNA, our finding will lead to the rapid production and analysis of various antibody-enzyme fusions suitable for enzyme-linked immunosorbent assay (ELISA) or antibody-dependent enzyme prodrug therapy (ADEPT).

  8. Recombinant GDNF: Tetanus toxin fragment C fusion protein produced from insect cells

    International Nuclear Information System (INIS)

    Glial cell line-derived neurotrophic factor (GDNF) has potent survival-promoting effects on CNS motor neurons in experimental animals. Its therapeutic efficacy in humans, however, may have been limited by poor bioavailability to the brain and spinal cord. With a view toward improving delivery of GDNF to CNS motor neurons in vivo, we generated a recombinant fusion protein comprised of rat GDNF linked to the non-toxic, neuron-binding fragment of tetanus toxin. Recombinant GDNF:TTC produced from insect cells was a soluble homodimer like wild-type GDNF and was bi-functional with respect to GDNF and TTC activity. Like recombinant rat GDNF, the fusion protein increased levels of immunoreactive phosphoAkt in treated NB41A3-hGFRα-1 neuroblastoma cells. Like TTC, GDNF:TTC bound to immobilized ganglioside GT1b in vitro with high affinity and selectivity. These results support further testing of recombinant GDNF:TTC as a non-viral vector to improve delivery of GDNF to brain and spinal cord in vivo.

  9. Chitin Degradation Proteins Produced by the Marine Bacterium Vibrio harveyi Growing on Different Forms of Chitin

    Science.gov (United States)

    Svitil, A. L.; Chadhain, S.; Moore, J. A.; Kirchman, D. L.

    1997-01-01

    Relatively little is known about the number, diversity, and function of chitinases produced by bacteria, even though chitin is one of the most abundant polymers in nature. Because of the importance of chitin, especially in marine environments, we examined chitin-degrading proteins in the marine bacterium Vibrio harveyi. This bacterium had a higher growth rate and more chitinase activity when grown on (beta)-chitin (isolated from squid pen) than on (alpha)-chitin (isolated from snow crab), probably because of the more open structure of (beta)-chitin. When exposed to different types of chitin, V. harveyi excreted several chitin-degrading proteins into the culture media. Some chitinases were present with all of the tested chitins, while others were unique to a particular chitin. We cloned and identified six separate chitinase genes from V. harveyi. These chitinases appear to be unique based on DNA restriction patterns, immunological data, and enzyme activity. This marine bacterium and probably others appear to synthesize separate chitinases for efficient utilization of different forms of chitin and chitin by-products. PMID:16535505

  10. Recombinant GDNF: Tetanus toxin fragment C fusion protein produced from insect cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jianhong; Chian, Ru-Ju; Ay, Ilknur; Celia, Samuel A.; Kashi, Brenda B.; Tamrazian, Eric; Matthews, Jonathan C. [Cecil B. Day Laboratory for Neuromuscular Research, Department of Neurology, Massachusetts General Hospital, Charlestown, MA 02129 (United States); Remington, Mary P. [Research Service, Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201 (United States); Pepinsky, R. Blake [BiogenIdec, Inc., 14 Cambridge Center, Cambridge, MA 02142 (United States); Fishman, Paul S. [Research Service, Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201 (United States); Department of Neurology, University of Maryland School of Medicine, Baltimore, MD 21201 (United States); Brown, Robert H. [Cecil B. Day Laboratory for Neuromuscular Research, Department of Neurology, Massachusetts General Hospital, Charlestown, MA 02129 (United States); Francis, Jonathan W., E-mail: jwfrancisby@gmail.com [Cecil B. Day Laboratory for Neuromuscular Research, Department of Neurology, Massachusetts General Hospital, Charlestown, MA 02129 (United States)

    2009-07-31

    Glial cell line-derived neurotrophic factor (GDNF) has potent survival-promoting effects on CNS motor neurons in experimental animals. Its therapeutic efficacy in humans, however, may have been limited by poor bioavailability to the brain and spinal cord. With a view toward improving delivery of GDNF to CNS motor neurons in vivo, we generated a recombinant fusion protein comprised of rat GDNF linked to the non-toxic, neuron-binding fragment of tetanus toxin. Recombinant GDNF:TTC produced from insect cells was a soluble homodimer like wild-type GDNF and was bi-functional with respect to GDNF and TTC activity. Like recombinant rat GDNF, the fusion protein increased levels of immunoreactive phosphoAkt in treated NB41A3-hGFR{alpha}-1 neuroblastoma cells. Like TTC, GDNF:TTC bound to immobilized ganglioside GT1b in vitro with high affinity and selectivity. These results support further testing of recombinant GDNF:TTC as a non-viral vector to improve delivery of GDNF to brain and spinal cord in vivo.

  11. Evaluation of antibacterial activity of crude protein extracts from seeds of six different medical plants against standard bacterial strains.

    Science.gov (United States)

    Al Akeel, Raid; Al-Sheikh, Yazeed; Mateen, Ayesha; Syed, Rabbani; Janardhan, K; Gupta, V C

    2014-04-01

    A huge group of natural antimicrobial compounds are active against a large spectrum of bacterial strains causing infectious threat. The present study was conducted to investigate the crude extracts of antimicrobial protein and peptide efficacy from six medicinal plant seeds. Extraction was carried out in Sodium phosphate citrate buffer, and Sodium acetate buffer using different pH. Antimicrobial activities of these plants were determined by the microbiological technique using Agar well diffusion Assay. Extremely strong activity was observed in the seed extracts of Allium ascolinicum extracted in sodium phosphate citrate buffer at pH (5.8) against Proteus vulgaris, Escherichia coli and Staphylococcus aureus with zone of inhibition 17 mm, 17 mm and 15 mm and Rumex vesicarius at pH (7.6), Ammi majus at pH (6.8), Cichorium intybus at pH (7.4) and Cucumis sativus at pH (7.8) also showed better sensitivity against the bacterial strains with zone of inhibition ranges 16-10 mm and some of the strains were found to be resistant. Antibacterial activity pattern of different plant extracts prepared in sodium acetate buffer pH (6.5), among all the plant seed extracts used Foeniculum vulgare had shown good inhibition in all the bacterial strains used, with zone of inhibition ranges 11-12.5 mm, The extracts of C. intybus and C. sativus were found to be effective with zone of inhibition 11-6 mm and some of the strains were found to be resistant. Most of the strains found to have shown better sensitivity compared with the standard antibiotic Chloramphenicol (25 mcg). Our results showed that the plants used for our study are the richest source for antimicrobial proteins and peptides and they may be used for industrial extraction and isolation of antimicrobial compounds which may find a place in medicine industry as constituents of antibiotics. PMID:24600307

  12. Activation of phagocytic cells by Staphylococcus epidermidis biofilms: effects of extracellular matrix proteins and the bacterial stress protein GroEL on netosis and MRP-14 release.

    Science.gov (United States)

    Dapunt, Ulrike; Gaida, Matthias M; Meyle, Eva; Prior, Birgit; Hänsch, Gertrud M

    2016-07-01

    The recognition and phagocytosis of free-swimming (planktonic) bacteria by polymorphonuclear neutrophils have been investigated in depth. However, less is known about the neutrophil response towards bacterial biofilms. Our previous work demonstrated that neutrophils recognize activating entities within the extracellular polymeric substance (EPS) of biofilms (the bacterial heat shock protein GroEL) and that this process does not require opsonization. Aim of this study was to evaluate the release of DNA by neutrophils in response to biofilms, as well as the release of the inflammatory cytokine MRP-14. Neutrophils were stimulated with Staphylococcus epidermidis biofilms, planktonic bacteria, extracted EPS and GroEL. Release of DNA and of MRP-14 was evaluated. Furthermore, tissue samples from patients suffering from biofilm infections were collected and evaluated by histology. MRP-14 concentration in blood samples was measured. We were able to show that biofilms, the EPS and GroEL induce DNA release. MRP-14 was only released after stimulation with EPS, not GroEL. Histology of tissue samples revealed MRP-14 positive cells in association with neutrophil infiltration and MRP-14 concentration was elevated in blood samples of patients suffering from biofilm infections. Our data demonstrate that neutrophil-activating entities are present in the EPS and that GroEL induces DNA release by neutrophils. PMID:27109773

  13. Impacts of pH-mediated EPS structure on probiotic bacterial pili-whey proteins interactions.

    Science.gov (United States)

    Burgain, Jennifer; Scher, Joel; Lebeer, Sarah; Vanderleyden, Jos; Corgneau, Magda; Guerin, Justine; Caillet, Céline; Duval, Jérôme F L; Francius, Gregory; Gaiani, Claire

    2015-10-01

    Probiotic bacteria are routinely incorporated into dairy foods because of the health benefits they can provide when consumed. In this work, the marked pH-dependence of the pili/EPS organization at the outer surface of Lactobacillus rhamnosus GG is characterized in detail by Single Cell Force Microscopy and cell electrophoretic mobility measurements analyzed according to formalisms for nanomechanical contact and soft particle electrokinetics, respectively. At pH 6.8, LGG pili are easily accessible by AFM tips functionalized with whey proteins for specific binding, while at pH 4.8 the collapsed EPS surface layer significantly immobilized the LGG pili. This resulted in their reduced accessibility to the specific whey-coated AFM tip, and to stronger whey protein-pili rupture forces. Thus, pili interactions with whey proteins are screened to an extent that depends on the pH-mediated embedment of the pili within the EPS layer. PMID:26209966

  14. A bacterial ATP-dependent, enhancer binding protein that activates the housekeeping RNA polymerase

    Science.gov (United States)

    Bowman, William C.; Kranz, Robert G.

    1998-01-01

    A commonly accepted view of gene regulation in bacteria that has emerged over the last decade is that promoters are transcriptionally activated by one of two general mechanisms. The major type involves activator proteins that bind to DNA adjacent to where the RNA polymerase (RNAP) holoenzyme binds, usually assisting in recruitment of the RNAP to the promoter. This holoenzyme uses the housekeeping ς70 or a related factor, which directs the core RNAP to the promoter and assists in melting the DNA near the RNA start site. A second type of mechanism involves the alternative sigma factor (called ς54 or ςN) that directs RNAP to highly conserved promoters. In these cases, an activator protein with an ATPase function oligomerizes at tandem sites far upstream from the promoter. The nitrogen regulatory protein (NtrC) from enteric bacteria has been the model for this family of activators. Activation of the RNAP/ς54 holoenzyme to form the open complex is mediated by the activator, which is tethered upstream. Hence, this class of protein is sometimes called the enhancer binding protein family or the NtrC class. We describe here a third system that has properties of each of these two types. The NtrC enhancer binding protein from the photosynthetic bacterium, Rhodobacter capsulatus, is shown in vitro to activate the housekeeping RNAP/ς70 holoenzyme. Transcriptional activation by this NtrC requires ATP binding but not hydrolysis. Oligomerization at distant tandem binding sites on a supercoiled template is also necessary. Mechanistic and evolutionary questions of these systems are discussed. PMID:9637689

  15. THE EUKARYOTIC EXPRESSION OF HUMAN PERFORIN PROTEIN AND THE ESTABLISHMENT OF HYBRIDOMAS PRODUCING ANTI-HUMAN PERFORIN PROTEIN

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To prepare the monoclonal antibody against human perforin(HP). Methods Recombinant eukaryotic expression plasmid pCDM8-HP was extracted and purified, and the BALB/C mice were immunized with the plasmid. The hybridomas producing anti-HP McAbs were established by using hybridoma technique, then the specificity of the McAbs was identified by using immunocytochemical technique and Western blot. Results Three hybridoma cell lines secreting McAbs against human perforin were established, and the three McAbs showed positive only with LAK cells containing human perforin protein, and showed negative with inactive human peripheral blood lymphocytes (PBLs). The subclasses of the three McAbs were determined as lgG2bK. Western blot results showed that the three McAbs recognized a specific band of LAK cell lysateds with molecular weight of 70.0Kd. Conclusion The three hybridoma cell lines secreting McAbs against human perforin were established and the secreted McAbs were specific.

  16. Effects of volatile organic compounds produced by Bacillus amyloliquefaciens on the growth and virulence traits of tomato bacterial wilt pathogen Ralstonia solanacearum.

    Science.gov (United States)

    Raza, Waseem; Wang, Jichen; Wu, Yuncheng; Ling, Ning; Wei, Zhong; Huang, Qiwei; Shen, Qirong

    2016-09-01

    The production of volatile organic compounds (VOCs) by microbes is an important characteristic for their selection as biocontrol agents against plant pathogens. In this study, we identified the VOCs produced by the biocontrol strain Bacillus amyloliquefaciens T-5 and evaluated their impact on the growth and virulence traits of tomato bacterial wilt pathogen Ralstonia solanacearum. The results showed that the VOCs of strain T-5 significantly inhibited the growth of R. solanacearum in agar medium and in soil. In addition, VOCs significantly inhibited the motility traits, root colonization, biofilm formation, and production of antioxidant enzymes and exopolysaccharides by R. solanacearum. However, no effect of VOCs on the production of hydrolytic enzymes by R. solanacearum was observed. The strain T-5 produced VOCs, including benzenes, ketones, aldehydes, alkanes, acids, and one furan and naphthalene compound; among those, 13 VOCs showed 1-10 % antibacterial activity against R. solanacearum in their produced amounts by T-5; however, the consortium of all VOCs produced on agar medium, in sterilized soil, and in natural soil showed 75, 62, and 85 % growth inhibition of R. solanacearum, respectively. The real-time PCR analysis further confirmed the results when the expression of different virulence- and metabolism-related genes in R. solanacearum cells was decreased after exposure to the VOCs of strain T-5. The results of this study clearly revealed the significance of VOCs in the control of plant pathogens. This information would help to better comprehend the microbial interactions mediated by VOCs in nature and to develop safer strategies to control plant disease. PMID:27183998

  17. Horizontal gene transfer of zinc and non-zinc forms of bacterial ribosomal protein S4

    OpenAIRE

    Luthey-Schulten Zaida; Roberts Elijah; Chen Ke

    2009-01-01

    Abstract Background The universal ribosomal protein S4 is essential for the initiation of small subunit ribosomal assembly and translational accuracy. Being part of the information processing machinery of the cell, the gene for S4 is generally thought of as being inherited vertically and has been used in concatenated gene phylogenies. Here we report the evolution of ribosomal protein S4 in relation to a broad sharing of zinc/non-zinc forms of the gene and study the scope of horizontal gene tr...

  18. A bacterial symbiont is converted from an inedible producer of beneficial molecules into food by a single mutation in the gacA gene.

    Science.gov (United States)

    Stallforth, Pierre; Brock, Debra A; Cantley, Alexandra M; Tian, Xiangjun; Queller, David C; Strassmann, Joan E; Clardy, Jon

    2013-09-01

    Stable multipartite mutualistic associations require that all partners benefit. We show that a single mutational step is sufficient to turn a symbiotic bacterium from an inedible but host-beneficial secondary metabolite producer into a host food source. The bacteria's host is a "farmer" clone of the social amoeba Dictyostelium discoideum that carries and disperses bacteria during its spore stage. Associated with the farmer are two strains of Pseudomonas fluorescens, only one of which serves as a food source. The other strain produces diffusible small molecules: pyrrolnitrin, a known antifungal agent, and a chromene that potently enhances the farmer's spore production and depresses a nonfarmer's spore production. Genome sequence and phylogenetic analyses identify a derived point mutation in the food strain that generates a premature stop codon in a global activator (gacA), encoding the response regulator of a two-component regulatory system. Generation of a knockout mutant of this regulatory gene in the nonfood bacterial strain altered its secondary metabolite profile to match that of the food strain, and also, independently, converted it into a food source. These results suggest that a single mutation in an inedible ancestral strain that served a protective role converted it to a "domesticated" food source. PMID:23898207

  19. Phosphoproteome analysis of streptomyces development reveals extensive protein phosphorylation accompanying bacterial differentiation

    DEFF Research Database (Denmark)

    Manteca, Angel; Ye, Juanying; Sánchez, Jesús;

    2011-01-01

    bacteria encoding the largest number of eukaryotic type kinases, the biological role of protein phosphorylation in this bacterium has not been extensively studied before. In this issue, the variations of the phosphoproteome of S. coelicolor were characterized. Most distinct Ser/Thr/Tyr phosphorylation...

  20. Heterologously expressed bacterial and human multidrug resistance proteins confer cadmium resistance to Escherichia coli

    NARCIS (Netherlands)

    Achard-Joris, M; van Saparoea, HBV; Driessen, AJM; Bourdineaud, JP; Bourdineaud, Jean-Paul

    2005-01-01

    The human MDR1 gene is induced by cadmium exposure although no resistance to this metal is observed in human cells overexpressing hMDR1. To access the role of MDR proteins in cadmium resistance, human MDR1, Lactococcus lactis lmrA, and Oenococcus oeni omrA were expressed in an Escherichia coli tolC

  1. Role of bacterial virulence proteins in Agrobacterium-mediated transformation of Aspergillus awamori

    NARCIS (Netherlands)

    Michielse, C.B.; Ram, A.F.J.; Hooykaas, P.J.J.; Hondel, C.A.M.J.J. van den

    2004-01-01

    The Agrobacterium-mediated transformation of Aspergillus awamori was optimized using defined co-cultivation conditions, which resulted in a reproducible and efficient transformation system. Optimal co-cultivation conditions were used to study the role of Agrobacterium tumefaciens virulence proteins

  2. EFSA Panel on Biological Hazards (BIOHAZ); Scientific Opinion on the public health risks of bacterial strains producing extended-spectrum β-lactamases and/or AmpC β-lactamases in food and food-producing animals

    DEFF Research Database (Denmark)

    Hald, Tine; Baggesen, Dorte Lau

    The potential contribution of food-producing animals or foods to public health risks by ESBL and/or AmpC-producing bacteria is related to specific plasmid-mediated ESBL and/or AmpC genes encoded by a number of organisms. The predominant ESBL families encountered are CTX-M, TEM, and SHV; the...... predominant AmpC-family is CMY. The most common genes associated with this resistance in animals are blaCTX-M-1 (the most commonly identified ESBL), and blaCTX-M-14, followed by blaTEM-52 and blaSHV-12. Among the genes encoding AmpC-type β-lactamases, blaCMY-2 is the most common.The bacterial species most...... commonly identified with these genes are Escherichia coli and non-typhoidal Salmonella. ESBL/AmpC transmission is mainly driven by integrons, insertion sequences, transposons and plasmids, some of which are homologous in isolates from both food-production animals and humans. Cefotaxime is used as the drug...

  3. Bacterial Adhesion & Blocking Bacterial Adhesion

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk

    2008-01-01

    parameters, which influence the transition from a planktonic lifestyle to a sessile lifestyle, have been studied. Protein conditioning film formation was found to influence bacterial adhesion and subsequent biofilm formation considerable, and an aqueous extract of fish muscle tissue was shown to...... tract to the microbial flocs in waste water treatment facilities. Microbial biofilms may however also cause a wide range of industrial and medical problems, and have been implicated in a wide range of persistent infectious diseases, including implantassociated microbial infections. Bacterial adhesion is...... the first committing step in biofilm formation, and has therefore been intensely scrutinized. Much however, still remains elusive. Bacterial adhesion is a highly complex process, which is influenced by a variety of factors. In this thesis, a range of physico-chemical, molecular and environmental...

  4. Possible evidence of amide bond formation between sinapinic acid and lysine-containing bacterial proteins by matrix-assisted laser desorption/ionization (MALDI) at 355 nm

    Science.gov (United States)

    We previously reported the apparent formation of matrix adducts of 3,5-dimethoxy-4-hydroxy-cinnamic acid (sinapinic acid or SA) via covalent attachment to disulfide bond-containing proteins (HdeA, HdeB and YbgS) from bacterial cell lysates ionized by matrix-assisted laser desorption/ionization (MALD...

  5. Expressing a bacterial mercuric ion binding protein in plant for phytoremediation of heavy metals.

    Science.gov (United States)

    Hsieh, Ju-Liang; Chen, Ching-Yi; Chiu, Meng-Hsuen; Chein, Mei-Fang; Chang, Jo-Shu; Endo, Ginro; Huang, Chieh-Chen

    2009-01-30

    A specific mercuric ion binding protein (MerP) originating from transposon TnMERI1 of Bacillus megaterium strain MB1 isolated from Minamata Bay displayed good adsorption capability for a variety of heavy metals. In this study, the Gram-positive MerP protein was expressed in transgenic Arabidopsis to create a model system for phytoremediation of heavy metals. Under control of an actin promoter, the transgenic Arabidpsis showed higher tolerance and accumulation capacity for mercury, cadium and lead when compared with the control plant. Results from confocal microscopy analysis also indicate that MerP was localized at the cell membrane and vesicles of plant cells. The developed transgenic plants possessing excellent metal-accumulative ability could have potential applications in decontamination of heavy metals. PMID:18538925

  6. A Gram-Negative Bacterial Secreted Protein Types Prediction Method Based on PSI-BLAST Profile

    Science.gov (United States)

    2016-01-01

    Prediction of secreted protein types based solely on sequence data remains to be a challenging problem. In this study, we extract the long-range correlation information and linear correlation information from position-specific score matrix (PSSM). A total of 6800 features are extracted at 17 different gaps; then, 309 features are selected by a filter feature selection method based on the training set. To verify the performance of our method, jackknife and independent dataset tests are performed on the test set and the reported overall accuracies are 93.60% and 100%, respectively. Comparison of our results with the existing method shows that our method provides the favorable performance for secreted protein type prediction.

  7. Influence of intestinal inflammation on bacterial protein expression in monoassociated mice

    OpenAIRE

    Schumann, Sara

    2013-01-01

    Background: Increased numbers of intestinal E. coli are observed in inflammatory bowel disease, but the reasons for this proliferation and it exact role in intestinal inflammation are unknown. Aim of this PhD-project was to identify E. coli proteins involved in E. coli’s adaptation to the inflammatory conditions in the gut and to investigate whether these factors affect the host. Furthermore, the molecular basis for strain-specific differences between probiotic and harmful E. coli in their re...

  8. Tyrosine protein kinase inhibitors block invasin-promoted bacterial uptake by epithelial cells.

    OpenAIRE

    Rosenshine, I.; Duronio, V; Finlay, B B

    1992-01-01

    The ability to enter into (invade) mammalian cells is an essential virulence determinant of many pathogenic bacteria and intracellular parasites. These organisms are internalized by host cells upon attachment to their surface. However, the mechanisms used by intracellular parasites to induce internalization into host cells have not been defined. We found that the protein kinase inhibitor staurosporine blocks invasion by some pathogenic bacteria, including Yersinia enterocolitica and Yersinia ...

  9. Antiadhesive Properties of Arabinogalactan Protein from Ribes nigrum Seeds against Bacterial Adhesion of Helicobacter pylori

    OpenAIRE

    Jutta Messing; Michael Niehues; Anna Shevtsova; Thomas Borén; Andreas Hensel

    2014-01-01

    Fruit extracts from black currants (Ribes nigrum L.) are traditionally used for treatment of gastritis based on seed polysaccharides that inhibit the adhesion of Helicobacter pylori to stomach cells. For detailed investigations an arabinogalactan protein (F2) was isolated from seeds and characterized concerning molecular weight, carbohydrate, amino acid composition, linkage, configuration and reaction with beta-glucosyl Yariv. Functional testing of F2 was performed by semiquantitative in situ...

  10. Bacterial protein translocation requires only one copy of the SecY complex in vivo

    OpenAIRE

    Park, Eunyong; Rapoport, Tom A.

    2012-01-01

    The transport of proteins across the plasma membrane in bacteria requires a channel formed from the SecY complex, which cooperates with either a translating ribosome in cotranslational translocation or the SecA ATPase in post-translational translocation. Whether translocation requires oligomers of the SecY complex is an important but controversial issue: it determines channel size, how the permeation of small molecules is prevented, and how the channel interacts with the ribosome and SecA. He...

  11. Bacterial protein translocation: kinetic and thermodynamic role of ATP and the protonmotive force

    OpenAIRE

    Driessen, Arnold J. M.

    1992-01-01

    The energetic mechanism of preprotein export in Escherichia coli has been a source of controversy for many years. In vitro studies of translocation reactions that use purified soluble and membrane components have now clarified the main features of this mechanism. Translocation occurs through consecutive steps which each have distinct energy requirements. Initiation of translocation requires ATP and the SecA protein. Most of the further steps can be driven by the protonmotive force (Δp).

  12. A bacterial ATP-dependent, enhancer binding protein that activates the housekeeping RNA polymerase

    OpenAIRE

    Bowman, William C.; Kranz, Robert G.

    1998-01-01

    A commonly accepted view of gene regulation in bacteria that has emerged over the last decade is that promoters are transcriptionally activated by one of two general mechanisms. The major type involves activator proteins that bind to DNA adjacent to where the RNA polymerase (RNAP) holoenzyme binds, usually assisting in recruitment of the RNAP to the promoter. This holoenzyme uses the housekeeping ς70 or a related factor, which directs the core RNAP to the promoter and assists in melting the D...

  13. Inhibition of bacterial conjugation by phage M13 and its protein g3p: quantitative analysis and model.

    Directory of Open Access Journals (Sweden)

    Abraham Lin

    Full Text Available Conjugation is the main mode of horizontal gene transfer that spreads antibiotic resistance among bacteria. Strategies for inhibiting conjugation may be useful for preserving the effectiveness of antibiotics and preventing the emergence of bacterial strains with multiple resistances. Filamentous bacteriophages were first observed to inhibit conjugation several decades ago. Here we investigate the mechanism of inhibition and find that the primary effect on conjugation is occlusion of the conjugative pilus by phage particles. This interaction is mediated primarily by phage coat protein g3p, and exogenous addition of the soluble fragment of g3p inhibited conjugation at low nanomolar concentrations. Our data are quantitatively consistent with a simple model in which association between the pili and phage particles or g3p prevents transmission of an F plasmid encoding tetracycline resistance. We also observe a decrease in the donor ability of infected cells, which is quantitatively consistent with a reduction in pili elaboration. Since many antibiotic-resistance factors confer susceptibility to phage infection through expression of conjugative pili (the receptor for filamentous phage, these results suggest that phage may be a source of soluble proteins that slow the spread of antibiotic resistance genes.

  14. Acyl-acyl carrier protein as a source of fatty acids for bacterial bioluminescence

    International Nuclear Information System (INIS)

    Pulse-chase experiments with [3H]tetradecanoic acid and ATP showed that the bioluminescence-related 32-kDa acyltransferase from Vibrio harveyi can specifically catalyze the deacylation of a 3H-labeled 18-kDa protein observed in extracts of this bacterium. The 18-kDa protein has been partially purified and its physical and chemical properties strongly indicate that it is fatty acyl-acyl carrier protein (acyl-ACP). Both this V. harveyi [3H]acylprotein and [3H]palmitoyl-ACP from Escherichia coli were substrates in vitro for either the V. harveyi 32-kDa acyltransferase or the analogous enzyme (34K) from Photobacterium phosphoreum. TLC analysis indicated that the hexane-soluble product of the reaction is fatty acid. No significant cleavage of either E. coli or V. harveyi tetradecanoyl-ACP was observed in extracts of these bacteria unless the 32-kDa or 34K acyltransferase was present. Since these enzymes are believed to be responsible for the supply of fatty acids for reduction to form the aldehyde substrate of luciferase, the above results suggest that long-chain acyl-ACP is the source of fatty acids for bioluminescence

  15. Bacterial surface layer proteins as a novel capillary coating material for capillary electrophoretic separations.

    Science.gov (United States)

    Moreno-Gordaliza, Estefanía; Stigter, Edwin C A; Lindenburg, Petrus W; Hankemeier, Thomas

    2016-06-01

    A novel concept for stable coating in capillary electrophoresis, based on recrystallization of surface layer proteins on hydrophobized fused silica capillaries, was demonstrated. Surface layer protein A (SlpA) from Lactobacillus acidophilus bacteria was extracted, purified and used for coating pre-silanized glass substrates presenting different surface wettabilities (either hydrophobic or hydrophilic). Contact angle determination on SlpA-coated hydrophobic silica slides showed that the surfaces turned to hydrophilic after coating (53 ± 5°), due to a protein monolayer formation by protein-surface hydrophobic interactions. Visualization by atomic force microscopy demonstrated the presence of a SlpA layer on methylated silica slides displaying a surface roughness of 0.44 ± 0.02 nm. Additionally, a protein layer was visualized by fluorescence microscopy in methylated silica capillaries coated with SlpA and fluorescein isothiocyanate-labeled. The SlpA-coating showed an outstanding stability, even after treatment with 20 mM NaOH (pH 12.3). The electroosmotic flow in coated capillaries showed a partial suppression at pH 7.50 (3.8 ± 0.5 10(-9) m(2) V(-1) s(-1)) when compared with unmodified fused silica (5.9 ± 0.1 10(-8) m(2) V(-1) s(-1)). To demonstrate the potential of this novel coating, the SlpA-coated capillaries were applied for the first time for electrophoretic separation, and proved to be very suitable for the isotachophoretic separation of lipoproteins in human serum. The separations showed a high degree of repeatability (absolute migration times with 1.1-1.8% coefficient-of-variation (CV) within a day) and 2-3% CV inter-capillary reproducibility. The capillaries were stable for more than 100 runs at pH 9.40, and showed to be an exceptional alternative for challenging electrophoretic separations at long-term use. PMID:27155306

  16. Cortical spreading depression produces a neuroprotective effect activating mitochondrial uncoupling protein-5

    Directory of Open Access Journals (Sweden)

    Viggiano E

    2016-07-01

    Full Text Available Emanuela Viggiano,1,2 Vincenzo Monda,1 Antonietta Messina,1 Fiorenzo Moscatelli,3 Anna Valenzano,3 Domenico Tafuri,4 Giuseppe Cibelli,3 Bruno De Luca,1 Giovanni Messina,1,3 Marcellino Monda1 1Department of Experimental Medicine, Section of Human Physiology and Unit of Dietetics and Sports Medicine, Second University of Naples, Naples, 2Department of Medicine, University of Padua, Padua, 3Department of Clinical and Experimental Medicine, University of Foggia, Foggia, 4Department of Motor Sciences and Wellness, University of Naples “Parthenope”, Naples, Italy Abstract: Depression of electrocorticogram propagating over the cortex surface results in cortical spreading depression (CSD, which is probably related to the pathophysiology of stroke, epilepsy, and migraine. However, preconditioning with CSD produces neuroprotection to subsequent ischemic episodes. Such effects require the expression or activation of several genes, including neuroprotective ones. Recently, it has been demonstrated that the expression of the uncoupling proteins (UCPs 2 and 5 is amplified during brain ischemia and their expression exerts a long-term effect upon neuron protection. To evaluate the neuroprotective consequence of CSD, the expression of UCP-5 in the brain cortex was measured following CSD induction. CSD was evoked in four samples of rats, which were sacrificed after 2 hours, 4 hours, 6 hours, and 24 hours. Western blot analyses were carried out to measure UCP-5 concentrations in the prefrontal cortices of both hemispheres, and immunohistochemistry was performed to determine the localization of UCP-5 in the brain cortex. The results showed a significant elevation in UCP-5 expression at 24 hours in all cortical strata. Moreover, UCP-5 was triggered by CSD, indicating that UCP-5 production can have a neuroprotective effect. Keywords: cortical spreading depression, neuroprotective effect, uncoupling protein-5

  17. Initiation of assembly and association of the structural elements of a bacterial pilus depend on two specialized tip proteins.

    OpenAIRE

    Jacob-Dubuisson, F; Heuser, J.; Dodson, K.; Normark, S; Hultgren, S.

    1993-01-01

    Uropathogenic Escherichia coli produce heteropolymeric surface fibers called P pili, which present an adhesin at their tip that specifically recognizes globoside receptors on the host uroepithelium. The initial attachment step is thought to be essential for pathogenesis. P pili are composite fibers consisting of a thin tip fibrillum joined end to end to a rigid helical rod. Here we show that the ordered assembly of these structures requires the activity of two proteins that are minor componen...

  18. Bacterial Type I Glutamine Synthetase of the Rifamycin SV Producing Actinomycete, Amycolatopsis mediterranei U32, is the Only Enzyme Responsible for Glutamine Synthesis under Physiological Conditions

    Institute of Scientific and Technical Information of China (English)

    Wen-Tao PENG; Jin WANG; Ting WU; Jian-Qiang HUANG; Jui-Shen CHIAO; Guo-Ping ZHAO

    2006-01-01

    The structural gene for glutamine synthetase, glnA, from Amycolatopsis mediterranei U32 was cloned via screening a genomic library using the analog gene from Streptomyces coelicolor. The clone was functionally verified by complementing for glutamine requirement of an Escherichia coli glnA null mutant under the control of a lac promoter. Sequence analysis showed an open reading frame encoding a protein of466 amino acid residues. The deduced amino acid sequence bears significant homologies to other bacterial type I glutamine synthetases, specifically, 71% and 72% identical to the enzymes of S. coelicolor and Mycobacterium tuberculosis, respectively. Disruption of this glnA gene in A. mediterranei U32 led to glutamine auxotrophy with no detectable glutamine synthetase activity in vivo. In contrast, the cloned glnA+ gene can complement for both phenotypes in trans. It thus suggested that in A. mediterranei U32, the glnA gene encoding glutamine synthetase is uniquely responsible for in vivo glutamine synthesis under our laboratory defined physiological conditions.

  19. Effects of insecticidal crystal proteins (Cry proteins) produced by genetically modified maize (Bt maize) on the nematode Caenorhabditis elegans

    International Nuclear Information System (INIS)

    The genetically modified maize MON89034 × MON88017 expresses different crystal (Cry) proteins with pesticidal activity against the European corn borer (Cry1.105; Cry2Ab2) and the Western corn root worm (Cry3Bb1). Non-target organisms, such as soil nematodes, might be exposed to the Cry proteins that enter the soil in course of crop growing. Therefore, the risk of those proteins for nematodes was assessed by testing their toxic effects on Caenorhabditis elegans. All three insecticidal Cry proteins showed dose-dependent inhibitory effects on C. elegans reproduction (EC50: 0.12–0.38 μmol L−1), however, at concentrations that were far above the expected soil concentrations. Moreover, a reduced toxicity was observed when Cry proteins were added jointly. A C. elegans mutant strain deficient for receptors for the nematicidal Cry5B was also resistant against Cry1.105 and Cry2Ab2, suggesting that these Cry proteins bound to the same or similar receptors as nematicidal Cry proteins and thereby affect the reproduction of C. elegans. -- Highlights: •Insecticidal Cry proteins dose-dependently inhibited the reproduction of C. elegans. •Mixture toxicity was lower than expected from concentration-additive single effects. •Genes for MAPK-defense-pathway were up-regulated in presence of Cry protein mixture. •Knock-out strains deficient for Cry5B-receptors showed lower susceptibility to insecticidal Cry proteins. •Toxicity of insecticidal Cry-proteins on C. elegans occurred at concentrations far above expected field concentrations. -- Insecticidal Cry proteins expressed by genetically modified maize act on nematodes via a similar mode of action as nematicidal Cry proteins, however, at concentrations far above expected soil levels

  20. Simple screening method for autoantigen proteins using the N-terminal biotinylated protein library produced by wheat cell-free synthesis.

    Science.gov (United States)

    Matsuoka, Kazuhiro; Komori, Hiroaki; Nose, Masato; Endo, Yaeta; Sawasaki, Tatsuya

    2010-08-01

    Autoimmune diseases are a heterogeneous group of diseases characterized by immune reactions against either a major or a limited number of the bodies own autoantigens, causing inflammation and damage to tissues and organs. Thus, identification of autoantigens is an important first step to understanding autoimmune diseases. Here we demonstrate a simple screening method for identification of autoantigens reacting with patient serum antibodies by combination of an N-terminal biotinylated protein library (BPL), produced using a wheat cell-free protein production system, and a commercially available luminescence system. Optimization studies using well-characterized autoantigens showed specific interactions between N-terminal biotinylated proteins and antibody that were sensitively detected under homogeneous reaction conditions. In this optimized assay, 1 microL of the translation mixture expressing the biotinylated proteins produced significant luminescence signal by addition of diluted serum between 1:500 and 1:10 000 in 25 microL of reaction volume. For the BPL construction, 214 mouse genes, consisting of 103 well-known autoantigens and 111 genes in the mouse autoimmune susceptibility loci, and the sera of MRL/lpr mouse were used as an autoimmune model. By this screening method, 25 well-known autoantigens and 71 proteins in the loci were identified as autoantigen proteins specifically reacting with sera antibodies. Cross-referencing with the Gene Ontology Database, 26 and 38 of autoantigen proteins were predicted to have nuclear localization and identified as membrane and/or extracellular proteins. The immune reaction of six randomly selected proteins was confirmed by immunoprecipitation and/or immunoblot analyses. Interestingly, three autoantigen proteins were recognized by immunoprecipitation but not by immunoblot analysis. These results suggest that the BPL-based method could provide a simple system for screening of autoantigen proteins and would help with

  1. Meat, dairy and plant proteins alter bacterial composition of rat gut bacteria

    OpenAIRE

    Yingying Zhu; Xisha Lin; Fan Zhao; Xuebin Shi; He Li; Yingqiu Li; Weiyun Zhu; Xinglian Xu; Chunbao Lu; Guanghong Zhou

    2015-01-01

    Long-term consumption of red meat has been considered a potential risk to gut health, but this is based on clinic investigations, excessive intake of fat, heme and some injurious compounds formed during cooking or additions to processed meat products. Whether intake of red meat protein affects gut bacteria and the health of the host remains unclear. In this work, we compared the composition of gut bacteria in the caecum, by sequencing the V4-V5 region of 16S ribosomal RNA gene, obtained from ...

  2. Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Petranovic, Dina; Macek, B;

    2006-01-01

    Single-stranded DNA-binding proteins (SSBs) are required for repair, recombination and replication in all organisms. Eukaryotic SSBs are regulated by phosphorylation on serine and threonine residues. To our knowledge, phosphorylation of SSBs in bacteria has not been reported. A systematic search...... antagonistically by kinase YwqD and phosphatase YwqE. Phosphorylation of B.subtilis SSB increased binding almost 200-fold to single-stranded DNA in vitro. Tyrosine phosphorylation of B.subtilis, S.coelicolor and Escherichia coli SSBs occured while they were expressed in E.coli, indicating that tyrosine...

  3. Immunogenicity of bacterial-expressed recombinant Plasmodium knowlesi merozoite surface protein-142 (MSP-142)

    OpenAIRE

    Cheong, Fei Wen; Fong, Mun Yik; Lau, Yee Ling; Mahmud, Rohela

    2013-01-01

    Background Plasmodium knowlesi is the fifth Plasmodium species that can infect humans. The Plasmodium merozoite surface protein-142 (MSP-142) is a potential candidate for malaria vaccine. However, limited studies have focused on P. knowlesi MSP-142. Methods A ~42 kDa recombinant P. knowlesi MSP-142 (pkMSP-142) was expressed using an Escherichia coli system. The purified pkMSP-142 was evaluated with malaria and non-malaria human patient sera (n = 189) using Western blots and ELISA. The immunog...

  4. Deletion of Invasion Protein B in Salmonella enterica Serovar Typhimurium Influences Bacterial Invasion and Virulence.

    Science.gov (United States)

    Chen, Songbiao; Zhang, Chunjie; Liao, Chengshui; Li, Jing; Yu, Chuan; Cheng, Xiangchao; Yu, Zuhua; Zhang, Mingliang; Wang, Yang

    2015-12-01

    Salmonella enterica serovar Typhimurium (S. Typhimurium) has a wide host range and causes infections ranging from severe gastroenteritis to systemic infections in human, as well as causing typhoid-like disease in murine models of infection. S. Typhimurium translocates its effector proteins through the Salmonella pathogenicity island-I (SPI-I)-encoded T3SS-I needle complex. This study focuses on invasion protein B (SipB) of S. Typhimurium, which plays an active role in SPI-I invasion efficiency. To test our hypothesis, a sipB deletion mutant was constructed through double-crossover allelic using the suicide vector pRE112ΔsipB, and its biological characteristics were analyzed. The results showed that the SipB does not affect the growth of Salmonella, but the adherence, invasion, and virulence of the mutant were significantly decreased compared with wild-type S. Typhimurium (SL1344). This research indicates that SipB is an important virulence factor in the pathogenicity of S. Typhimurium. PMID:26341924

  5. Secondary Structure Preferences of Mn2+ Binding Sites in Bacterial Proteins

    Directory of Open Access Journals (Sweden)

    Tatyana Aleksandrovna Khrustaleva

    2014-01-01

    Full Text Available 3D structures of proteins with coordinated Mn2+ ions from bacteria with low, average, and high genomic GC-content have been analyzed (149 PDB files were used. Major Mn2+ binders are aspartic acid (6.82% of Asp residues, histidine (14.76% of His residues, and glutamic acid (3.51% of Glu residues. We found out that the motif of secondary structure “beta strand-major binder-random coil” is overrepresented around all the three major Mn2+ binders. That motif may be followed by either alpha helix or beta strand. Beta strands near Mn2+ binding residues should be stable because they are enriched by such beta formers as valine and isoleucine, as well as by specific combinations of hydrophobic and hydrophilic amino acid residues characteristic to beta sheet. In the group of proteins from GC-rich bacteria glutamic acid residues situated in alpha helices frequently coordinate Mn2+ ions, probably, because of the decrease of Lys usage under the influence of mutational GC-pressure. On the other hand, the percentage of Mn2+ sites with at least one amino acid in the “beta strand-major binder-random coil” motif of secondary structure (77.88% does not depend on genomic GC-content.

  6. Green fluorescent protein (GFP) transgenic pig produced by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    LIU ZhongHua; SUN Shuang; LI YuTian; WANG HongBin; R S PRATHER; SONG Jun; WANG ZhenKun; TIAN JiangTian; KONG QingRan; ZHENG Zhong; YIN Zhi; GAO Li; MA HaiKun

    2008-01-01

    Transgenic somatic cell nuclear transfer is a very promising route for producing transgenic farm ani-mals. Research on GFP transgenic pigs can provide useful information for breeding transgenic pigs, human disease models and human organ xenotransplantation. In this study, a liposomal transfection system was screened and transgenic embryos were reconstructed by nuclear transfer of GFP positive cells into enucleated in vitro matured oocytes. The development of reconstructed embryos both in vitro and in vivo was observed, and GFP expression was determined. The results showed that porcine fe-tal-derived fibroblast cells cultured with 4.0 plJmL liposome and 1.6 pg/mL plasmid DNA for 6 h re-sulted in the highest transfection rate (3.6%). The percentage of GFP reconstructed embryos that de-veloped in vitro to the blastocyst stage was 10%. Of those the GFP positive percentage was 48%. Re-constructed transgenic embryos were transferred to 10 recipients. 5 of them were pregnant, and 3 de-livered 6 cloned piglets in which 4 piglets were transgenic for the GFP as verified by both GFP protein expression and GFP DNA sequence analysis. The percentage of reconstructed embryos that resulted in cloned piglets was 1.0%; while the percentage of piglets that were transgenic was 0.7%. This is the first group of transgenic cloned pigs born in China, marking a great progress in Chinese transgenic cloned pig research.

  7. Bacterial gastroenteritis

    Science.gov (United States)

    Infectious diarrhea - bacterial gastroenteritis; Acute gastroenteritis; Gastroenteritis - bacterial ... Bacterial gastroenteritis can affect 1 person or a group of people who all ate the same food. It is ...

  8. Protecting the environment through insect farming as a means to produce protein for use as livestock, poultry, and aquaculture feed

    OpenAIRE

    Tomberlin, J.K.; Huis, A.; Benbow, M.E.; Jordan, H; D.A. Astuti; Azzollini, D.; Banks, I.; Bava, V.; Borgemeister, C.; Cammack, J.A.; Chapkin, R.S.; Čičková, Helena; Crippen, T.L.; Day, A; Dicke, M.

    2015-01-01

    Securing protein for the approximate 10 billion humans expected to inhabit our planet by 2050 is a major priority for the global community. Evidence has accrued over the past 30 years that strongly supports and justifies the sustainable use of insects as a means to produce protein products as feed for pets, livestock, poultry, and aquacultured species. Researchers and entrepreneurs affiliated with universities and industries, respectively, from 18 nations distributed across North and South Am...

  9. Identification and Biochemical Characterization of Protein Phosphatase 5 from the Cantharidin-Producing Blister Beetle, Epicauta chinensis

    Directory of Open Access Journals (Sweden)

    Xi'en Chen

    2013-12-01

    Full Text Available Protein phosphatase 5 (PP5 is a unique member of serine/threonine phosphatases which has been recognized in regulation of diverse cellular processes. A cDNA fragment encoding PP5 (EcPP5 was cloned and characterized from the cantharidin-producing blister beetle, E. chinensis. EcPP5 contains an open reading frame of 1500 bp that encodes a protein of 56.89 kDa. The deduced amino acid sequence shares 88% and 68% identities to the PP5 of Tribolium castaneum and humans, respectively. Analysis of the primary sequence shows that EcPP5 has three TPR (tetratricopeptide repeat motifs at its N-terminal region and contains a highly conserved C-terminal catalytic domain. RT-PCR reveals that EcPP5 is expressed in all developmental stages and in different tissues. The recombinant EcPP5 (rEcPP5 was produced in Escherichia coli and purified to homogeneity. The purified protein exhibited phosphatase activity towards pNPP (p-nitrophenyl phosphate and phosphopeptides, and its activity can be enhanced by arachidonic acid. In vitro inhibition study revealed that protein phosphatase inhibitors, okadaic acid, cantharidin, norcantharidin and endothall, inhibited its activity. Further, protein phosphatase activity of total soluble protein extract from E. chinensis adults could be impeded by these inhibitors suggesting there might be some mechanism to protect this beetle from being damaged by its self-produced cantharidin.

  10. Phyloproteomic classification of unsequenced organisms by top-down identification of bacterial proteins using capLC-MS/MS on an Orbitrap.

    Science.gov (United States)

    Wynne, Colin; Edwards, Nathan J; Fenselau, Catherine

    2010-10-01

    Currently, most MS-based proteomic studies of bacteria and archea match experimental data to known amino acid sequences from the target organism. Top-down studies use a protein's molecular weight along with data gathered from MS/MS experiments to identify proteins by database matching. For Erwinia herbicola and Enterobacter cloacae, studied here, the necessary protein sequences are not available in protein sequence repositories. We apply top-down protein fragmentation, but match the experimental data with homologous proteins from related organisms with sequenced genomes, demonstrating considerable shared protein sequence between closely related bacteria. Using this homology-based approach, we are not only able to identify representative proteins, but are also able to place the two target bacteria in their correct phylogeny. Furthermore, we show that the unexpected mass delta between the experimental precursor and matched protein sequence can often be localized and characterized using accurate-mass precursor and fragment ion measurements. Finally, we demonstrate that proteins identified by top-down workflows provide strong experimental evidence for correct, missing, and misannotated bacterial protein sequences, not only in the analyzed organism, but also for homologous proteins in closely related species. PMID:20845332

  11. Characterization of pro-inflammatory flagellin proteins produced by Lactobacillus ruminis and related motile Lactobacilli.

    Directory of Open Access Journals (Sweden)

    B Anne Neville

    Full Text Available Lactobacillus ruminis is one of at least twelve motile but poorly characterized species found in the genus Lactobacillus. Of these, only L. ruminis has been isolated from mammals, and this species may be considered as an autochthonous member of the gastrointestinal microbiota of humans, pigs and cows. Nine L. ruminis strains were investigated here to elucidate the biochemistry and genetics of Lactobacillus motility. Six strains isolated from humans were non-motile while three bovine isolates were motile. A complete set of flagellum biogenesis genes was annotated in the sequenced genomes of two strains, ATCC25644 (human isolate and ATCC27782 (bovine isolate, but only the latter strain produced flagella. Comparison of the L. ruminis and L. mali DSM20444(T motility loci showed that their genetic content and gene-order were broadly similar, although the L. mali motility locus was interrupted by an 11.8 Kb region encoding rhamnose utilization genes that is absent from the L. ruminis motility locus. Phylogenetic analysis of 39 motile bacteria indicated that Lactobacillus motility genes were most closely related to those of motile carnobacteria and enterococci. Transcriptome analysis revealed that motility genes were transcribed at a significantly higher level in motile L. ruminis ATCC27782 than in non-motile ATCC25644. Flagellin proteins were isolated from L. ruminis ATCC27782 and from three other Lactobacillus species, while recombinant flagellin of aflagellate L. ruminis ATCC25644 was expressed and purified from E. coli. These native and recombinant Lactobacillus flagellins, and also flagellate L. ruminis cells, triggered interleukin-8 production in cultured human intestinal epithelial cells in a manner suppressed by short interfering RNA directed against Toll-Like Receptor 5. This study provides genetic, transcriptomic, phylogenetic and immunological insights into the trait of flagellum-mediated motility in the lactobacilli.

  12. Plant Ribosomal Proteins, RPL12 and RPL19, Play a Role in Nonhost Disease Resistance against Bacterial Pathogens.

    Science.gov (United States)

    Nagaraj, Satish; Senthil-Kumar, Muthappa; Ramu, Vemanna S; Wang, Keri; Mysore, Kirankumar S

    2015-01-01

    Characterizing the molecular mechanism involved in nonhost disease resistance is important to understand the adaptations of plant-pathogen interactions. In this study, virus-induced gene silencing (VIGS)-based forward genetics screen was utilized to identify genes involved in nonhost resistance in Nicotiana benthamiana. Genes encoding ribosomal proteins, RPL12 and RPL19, were identified in the screening. These genes when silenced in N. benthamiana caused a delay in nonhost bacteria induced hypersensitive response (HR) with concurrent increase in nonhost bacterial multiplication. Arabidopsis mutants of AtRPL12 and AtRPL19 also compromised nonhost resistance. The studies on NbRPL12 and NbRPL19 double silenced plants suggested that both RPL12 and RPL19 act in the same pathway to confer nonhost resistance. Our work suggests a role for RPL12 and RPL19 in nonhost disease resistance in N. benthamiana and Arabidopsis. In addition, we show that these genes also play a minor role in basal resistance against virulent pathogens. PMID:26779226

  13. Plant Ribosomal Proteins, RPL12 and RPL19, Play a Role in Nonhost Disease Resistance against Bacterial Pathogens

    Science.gov (United States)

    Nagaraj, Satish; Senthil-Kumar, Muthappa; Ramu, Vemanna S.; Wang, Keri; Mysore, Kirankumar S.

    2016-01-01

    Characterizing the molecular mechanism involved in nonhost disease resistance is important to understand the adaptations of plant-pathogen interactions. In this study, virus-induced gene silencing (VIGS)-based forward genetics screen was utilized to identify genes involved in nonhost resistance in Nicotiana benthamiana. Genes encoding ribosomal proteins, RPL12 and RPL19, were identified in the screening. These genes when silenced in N. benthamiana caused a delay in nonhost bacteria induced hypersensitive response (HR) with concurrent increase in nonhost bacterial multiplication. Arabidopsis mutants of AtRPL12 and AtRPL19 also compromised nonhost resistance. The studies on NbRPL12 and NbRPL19 double silenced plants suggested that both RPL12 and RPL19 act in the same pathway to confer nonhost resistance. Our work suggests a role for RPL12 and RPL19 in nonhost disease resistance in N. benthamiana and Arabidopsis. In addition, we show that these genes also play a minor role in basal resistance against virulent pathogens. PMID:26779226

  14. Bacterial mitosis: partitioning protein ParA oscillates in spiral-shaped structures and positions plasmids at mid-cell

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Gerdes, Kenn; Charbon, Gitte Ebersbach

    2004-01-01

    with a single plasmid focus, the focus located preferentially at mid-cell. In cells with two foci, these located at quarter-cell positions. In the absence of ParB and parC1/parC2, ParA-GFP formed stationary helices extending from one end of the nucleoid to the other. In the presence of ParB and parC1/parC2, ParA-GFP......The par2 locus of Escherichia coli plasmid pB171 encodes oscillating ATPase ParA, DNA binding protein ParB and two cis-acting DNA regions to which ParB binds (parC1 and parC2). Three independent techniques were used to investigate the subcellular localization of plasmids carrying par2. In cells...... oscillated in spiral-shaped structures. Amino acid substitutions in ParA simultaneously abolished ParA spiral formation, oscillation and either plasmid localization or plasmid separation at mid-cell. Therefore, our results suggest that ParA spirals position plasmids at the middle of the bacterial nucleoid...

  15. Identification of the Major ACE-Inhibitory Peptides Produced by Enzymatic Hydrolysis of a Protein Concentrate from Cuttlefish Wastewater

    OpenAIRE

    Isabel Rodríguez Amado; José Antonio Vázquez; Pilar González; Diego Esteban-Fernández; Mónica Carrera; Carmen Piñeiro

    2014-01-01

    The aim of this work was the purification and identification of the major angiotensin converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of a protein concentrate recovered from a cuttlefish industrial manufacturing effluent. This process consisted on the ultrafiltration of cuttlefish softening wastewater, with a 10 kDa cut-off membrane, followed by the hydrolysis with alcalase of the retained fraction. Alcalase produced ACE inhibitors reaching the highest activity (I...

  16. The effect of storage on the content of protein in RVNRL produced in different batches of latex production

    International Nuclear Information System (INIS)

    Life-threatening latex allergy caused by latex proteins has emerged as a serious problem for health care workers and others who use latex products. This study was aimed to determine the effect of storage on the protein content of radiation prevulcanized natural rubber latex (RVNRL) produced at pilot plant scale in RAYMINTEX plant, Malaysian Nuclear Agency. Rubber films were prepared from three batches of RVNRL by coagulant dipping method and their protein content was determine by extraction using phosphate buffer solution (PBS) and measurement using Micro BCA Protein Assay Kit. Within seven months of storage period employed in this study, it was found that protein content of RVNRL reduced with storage time. (Author)

  17. Serum level of C-reactive protein is not a parameter to determine the difference between viral and atypical bacterial infections.

    Science.gov (United States)

    Durán, Anyelo; González, Andrea; Delgado, Lineth; Mosquera, Jesús; Valero, Nereida

    2016-02-01

    C-reactive protein (CRP) is an acute-phase reactant that increases in the circulation in response to a variety of inflammatory stimuli. Elevated levels in serum during several infectious diseases have been reported. In this study, a highly sensitive CRP enzyme immunoassay was used to evaluate serum CRP values in patients with viral and atypical bacterial infections. Patients (n = 139) with different viral or atypical bacterial infections (systemic or respiratory) and healthy controls (n = 40) were tested for circulating CRP values. High levels of IgM antibodies against several viruses: Dengue virus (n = 36), Cytomegalovirus (n = 9), Epstein Barr virus (n = 17), Parvovirus B19 (n = 26), Herpes simplex 1 and 2 virus (n = 3) and Influenza A and B (n = 8) and against atypical bacteria: Legionella pneumophila (n = 15), Mycoplasma pneumoniae (n = 21) and Coxiella burnetii (n = 4) were found. High values of CRP in infected patients compared with controls (P < 0.001) were found; however, no significant differences between viral and atypical bacterial infections were found. Low levels of CRP in respiratory and Coxiella burnetii infections compared with exanthematic viral and other atypical bacterial infections were found. This study suggests that CRP values are useful to define viral and atypical bacterial infections compared with normal values, but, it is not useful to define type of infection. PMID:26241406

  18. Producing Recombinant mTEX101; a Murine Testis Specific Protein

    OpenAIRE

    Barzegar Yarmohammadi, Leila; Modarresi, Mohammad Hossein; Talebi, Saeed; Hadavi, Reza; Ostad Karampour, Mahyar; Mahmoudi, Ahmad Reza; Akhondi, Mohammad mehdi; Rabbani, Hodjattallah; Jeddi-Tehrani, Mahmood

    2009-01-01

    Introduction Production of antibodies against specific proteins of testis germ cells is of great significance for the investigation of processes involved in spermatogenesis, study of infertility problems and determination of the probable role of these proteins as cancer-testis antigens. Murine Testis Specific Recombinant Protein 101 (mTEX101) is a 38kDa, GPI-anchored protein which is expressed in testis germ cells of adult mice but it seems to be absent in other tissues. The structure and fun...

  19. Umami taste amino acids produced by hydrolyzing extracted protein from tomato seed meal

    Science.gov (United States)

    Enzymatic hydrolysis was performed for extracting protein to prepare umami taste amino acids from defatted tomato seed meal (DTSM) which is a by-product of tomato processing. Papain was used as an enzyme for the hydrolysis of DTSM. The particle size distribution of DTSM, protein concentration and fr...

  20. REDUCED CRUDE PROTEIN RATIONS FOR HIGH PRODUCING COWS: PRODUCTION AND ENVIRONMENTAL EFFECTS

    Science.gov (United States)

    Dairy cows utilize feed crude protein (CP) with greater efficiency than other ruminants, but still excrete about 2 to 3 times more N in manure than they secrete in milk. This increases milk production costs plus environmental N pollution. Optimizing microbial protein formation in the rumen is the mo...

  1. On the lipid-bacterial protein interaction studied by quartz crystal microbalance with dissipation, transmission electron microscopy and atomic force microscopy

    CERN Document Server

    Delcea, Mihaela; Pum, Dietmar; Sleytr, Uwe Bernd; Toca-Herrera, Jose Luis

    2009-01-01

    The interaction between the bacterial S-protein SbpA on different types of lipid membranes has been studied using atomic force microscopy, transmission electron microscopy, and quartz crystal microbalance with dissipation. On one hand, It has been found that the bacterial forms two dimensional nanocrystals on zwitterionic DOPC bilayers and negatively charged DMPG vesicles adsorbed on mica, on zwitterionic DPPC and charged DPPC/DMPG (1:1) monolayers adsorbed on carbon grids. On the other hand, SbpA protein adsorption took place on zwitterionic DOPC bilayers and DOPC/DOPS (4:1) bilayers, previously adsorbed on silicon supports. SbpA adsorption also took place on DPPC/DOPS (1:1) monolayers adsorbed on carbon grids. Finally, neither SbpA adsorption, nor recrystallization was observed on zwitterionic DMPC vesicles (previously adsorbed on polyelectrolyte multilayers), and on DPPC vesicles supported on silicon.

  2. Characterization of an immunomodulatory Der p 2-FIP-fve fusion protein produced in transformed rice suspension cell culture.

    Science.gov (United States)

    Su, Chin-Fen; Kuo, I-Chun; Chen, Peng-Wen; Huang, Chiung-Hui; Seow, See Voon; Chua, Kaw Yan; Yu, Su-May

    2012-02-01

    Der p 2, a major allergen of Dermatophagoides pteronyssinus mites, is one of the most clinically relevant allergens to allergic patients worldwide. FIP-fve protein (Fve) from the golden needle mushroom (Flammulina velutipes) is an immunomodulatory protein with potential Th1-skewed adjuvant properties. Here, we produced and immunologically evaluated a Der p 2-Fve fusion protein as a potential immunotherapeutic for allergic diseases. Using an inducible expression system in cultured rice suspension cells, the recombinant Der p 2-Fve fusion protein (designated as OsDp2Fve) was expressed in rice cells under the control of an α-amylase gene (αAmy8) promoter and secreted under sucrose starvation. OsDp2Fve was partially purified from the cultured medium. The conformation of Der p 2 in OsDp2Fve remains intact as reflected by its unaltered allergenicity, as assessed by human IgE ELISA and histamine release assays, compared to non-fusion Der p 2 protein. Furthermore, the Fve protein expressed in OsDp2Fve retains its in vitro lymphoproliferative activity but loses its hemagglutination and lymphoagglutination effects compared to the native protein. Notably, in vivo evaluation showed that mice administered with OsDp2Fve possessed an enhanced production of Der p 2-specific IgG antibodies without potentiating the production of Der p 2-specific IgE and Th2 effector cytokines in comparison with mice co-administered with native Fve and Der p 2 proteins. These results suggest that the recombinant Der p 2-Fve fusion protein produced in rice suspension cell cultures has a great potential for allergy immunotherapy. PMID:21556691

  3. Glucose enhances collectrin protein expression in insulin-producing MIN6 {beta} cells

    Energy Technology Data Exchange (ETDEWEB)

    Saisho, Kenji; Fukuhara, Atsunori [Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Osaka (Japan); Yasuda, Tomoko [Department of Medical Biochemistry, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto (Japan); Sato, Yoshifumi; Fukui, Kenji; Iwahashi, Hiromi; Imagawa, Akihisa [Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Osaka (Japan); Hatta, Mitsutoki [Department of Medical Biochemistry, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto (Japan); Shimomura, Iichiro [Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Osaka (Japan); Yamagata, Kazuya, E-mail: k-yamaga@kumamoto-u.ac.jp [Department of Medical Biochemistry, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto (Japan)

    2009-11-06

    Collectrin is a novel target gene of hepatocyte nuclear factor-1{alpha} in pancreatic {beta}-cells and controls insulin exocytosis. Although glucose is known to stimulate the expression of genes of the insulin secretory pathway, there is no information on how glucose regulates collectrin expression. We investigated the effects of glucose on the expression of collectrin in MIN6 {beta}-cell line. Glucose, in a dose-dependent manner, increased collectrin protein levels without changing collectrin mRNA levels and protein stability, indicating that glucose stimulation of collectrin protein expression is primarily mediated at a translational level. Although mannose and pyruvate also increased collectrin protein expression level, neither 2-deoxyglucose, mitochondrial fuels leucine and glutamate, sulphonylurea nor Ca{sup 2+} channel blockers, mimicked the effects of glucose. These data indicate the involvement of mitochondrial TCA cycle intermediates, distal to pyruvate, in the regulation of collectrin protein expression in {beta}-cells.

  4. Comparative proteome analysis of psychrophilic versus mesophilic bacterial species: Insights into the molecular basis of cold adaptation of proteins

    OpenAIRE

    Reddy Boojala; Metpally Raghu

    2009-01-01

    Abstract Background Cold adapted or psychrophilic organisms grow at low temperatures, where most of other organisms cannot grow. This adaptation requires a vast array of sequence, structural and physiological adjustments. To understand the molecular basis of cold adaptation of proteins, we analyzed proteomes of psychrophilic and mesophilic bacterial species and compared the differences in amino acid composition and substitution patterns to investigate their likely association with growth temp...

  5. Molecular cloning of the crr gene and evidence that it is the structural gene for IIIGlc, a phosphocarrier protein of the bacterial phosphotransferase system.

    OpenAIRE

    Meadow, N.D.; Saffen, D W; Dottin, R P; Roseman, S.

    1982-01-01

    Sugar substrates of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) normally prevent bacterial cells from utilizing sugars that are not substrates of this system (diauxic growth, "the glucose effect"). We have previously shown that this type of PTS-mediated repression can be completely reversed by a single mutation, designated crr. Two lines of evidence are presented in this report showing that crr is the structural gene for IIIGlc, one of the proteins of the PTS. First, homog...

  6. Ascorbic acid glycation of lens proteins produces UVA sensitizers similar to those in human lens

    International Nuclear Information System (INIS)

    Soluble calf lens proteins were extensively glycated during a 4 week incubation with ascorbic acid in the presence of oxygen. Amino acids analysis of the dialyzed proteins removed at weekly intervals showed an increasing loss of lysine, arginine and histidine, consistent with the extensive protein cross-linking observed. Irradiation of the dialyzed samples with UVA light (1.0 kJ/cm2 total illumination through a 338 nm cutoff filter) caused an increasing loss of tryptophan, an additional loss of histidine and the production of micromolar concentrations of hydrogen peroxide. No alteration in amino acid content and no photolytic effects were seen in proteins incubated without ascorbic acid in proteins incubated with glucose for 4 weeks. The rate of hydrogen peroxide formation was linear with each glycated sample with a maximum production of 25 nmol/mg protein illuminated. The possibility that the sensitizer activity was due to an ascorbate-induced oxidation of tryptophan was eliminated by the presence of a heavy metal ion chelator during the incubation and by showing equivalent effects with ascorbate-incubated ribonuclease A, which is devoid of tryptophan. The ascorbate-incubated samples displayed increasing absorbance at wavelengths above 300 nm and increasing fluorescence (340/430) as glycation proceeded. The spectra of the 4 week glycated proteins were identical to those obtained with a solubilized water-insoluble fraction from human lens, which is known to have UVA sensitizer activity. (Author)

  7. PCR-based gene synthesis to produce recombinant proteins for crystallization

    Directory of Open Access Journals (Sweden)

    Byrne-Steele Miranda L

    2008-04-01

    Full Text Available Abstract Background Gene synthesis technologies are an important tool for structural biology projects, allowing increased protein expression through codon optimization and facilitating sequence alterations. Existing methods, however, can be complex and not always reproducible, prompting researchers to use commercial suppliers rather than synthesize genes themselves. Results A PCR-based gene synthesis method, referred to as SeqTBIO, is described to efficiently assemble the coding regions of two novel hyperthermophilic proteins, PAZ (Piwi/Argonaute/Zwille domain, a siRNA-binding domain of an Argonaute protein homologue and a deletion mutant of a family A DNA polymerase (PolA. The gene synthesis procedure is based on sequential assembly such that homogeneous DNA products can be obtained after each synthesis step without extensive manipulation or purification requirements. Coupling the gene synthesis procedure to in vivo homologous recombination techniques allows efficient subcloning and site-directed mutagenesis for error correction. The recombinant proteins of PAZ and PolA were subsequently overexpressed in E. coli and used for protein crystallization. Crystals of both proteins were obtained and they were suitable for X-ray analysis. Conclusion We demonstrate, by using PAZ and PolA as examples, the feasibility of integrating the gene synthesis, error correction and subcloning techniques into a non-automated gene to crystal pipeline such that genes can be designed, synthesized and implemented for recombinant expression and protein crystallization.

  8. A minichaperone-based fusion system for producing insoluble proteins in soluble stable forms.

    Science.gov (United States)

    Sharapova, Olga A; Yurkova, Maria S; Fedorov, Alexey N

    2016-02-01

    We have developed a fusion system for reliable production of insoluble hydrophobic proteins in soluble stable forms. A carrier is thermophilic minichaperone, GroEL apical domain (GrAD), a 15 kDa monomer able to bind diverse protein substrates. The Met-less variant of GrAD has been made for further convenient use of Met-specific CNBr chemical cleavage, if desired. The Met-less GrAD retained stability and solubility of the original protein. Target polypeptides can be fused to either C-terminus or N-terminus of GrAD. The system has been tested with two unrelated insoluble proteins fused to the C-terminus of GrAD. One of the proteins was also fused to GrAD N-terminus. The fusions formed inclusion bodies at 25°C and above and were partly soluble only at lower expression temperatures. Most importantly, however, after denaturation in urea, all fusions without exception were completely renatured in soluble stable forms that safely survived freezing-thawing as well as lyophilization. All fusions for both tested target proteins retained solubility at high concentrations for days. Functional analysis revealed that a target protein may retain functionality in the fusion. Convenience features include potential thermostability of GrAD fusions, capacity for chemical and enzymatic cleavage of a target and His6 tag for purification. PMID:26612097

  9. Preparation of Core-Shell Hybrid Materials by Producing a Protein Corona Around Magnetic Nanoparticles

    Science.gov (United States)

    Weidner, A.; Gräfe, C.; von der Lühe, M.; Remmer, H.; Clement, J. H.; Eberbeck, D.; Ludwig, F.; Müller, R.; Schacher, F. H.; Dutz, S.

    2015-07-01

    Nanoparticles experience increasing interest for a variety of medical and pharmaceutical applications. When exposing nanomaterials, e.g., magnetic iron oxide nanoparticles (MNP), to human blood, a protein corona consisting of various components is formed immediately. The composition of the corona as well as its amount bound to the particle surface is dependent on different factors, e.g., particle size and surface charge. The actual composition of the formed protein corona might be of major importance for cellular uptake of magnetic nanoparticles. The aim of the present study was to analyze the formation of the protein corona during in vitro serum incubation in dependency of incubation time and temperature. For this, MNP with different shells were incubated in fetal calf serum (FCS, serving as protein source) within a water bath for a defined time and at a defined temperature. Before and after incubation the particles were characterized by a variety of methods. It was found that immediately (seconds) after contact of MNP and FCS, a protein corona is formed on the surface of MNP. This formation led to an increase of particle size and a slight agglomeration of the particles, which was relatively constant during the first minutes of incubation. A longer incubation (from hours to days) resulted in a stronger agglomeration of the FCS incubated MNP. Quantitative analysis (gel electrophoresis) of serum-incubated particles revealed a relatively constant amount of bound proteins during the first minutes of serum incubation. After a longer incubation (>20 min), a considerably higher amount of surface proteins was determined for incubation temperatures below 40 °C. For incubation temperatures above 50 °C, the influence of time was less significant which might be attributed to denaturation of proteins during incubation. Overall, analysis of the molecular weight distribution of proteins found in the corona revealed a clear influence of incubation time and temperature on corona

  10. Human Cytolytic Fusion Proteins: Modified Versions of Human Granzyme B and Angiogenin Have the Potential to Replace Bacterial Toxins in Targeted Therapies against CD64+ Diseases

    Directory of Open Access Journals (Sweden)

    Nina Berges

    2014-02-01

    Full Text Available Targeted therapies for the treatment of cancer, but also inflammation and autoimmune diseases will reduce major side effects accompanied with conventional treatment modalities. The immunotoxin concept uses bacterial or plant toxins, coupled to antibodies or natural ligands targeting cancer cells. Initially, immunotoxins suffered from drawbacks like nonspecific cytotoxicity. Even the third generation of immunotoxins comprised of truncated antibodies and modified effector molecules experienced clinical set-backs due to immune responses. Long-term treatment of cancer and non-life-threatening chronic inflammatory diseases requires their complete ‘humanization’. This lead to evaluating human cytolytic fusion proteins (hCFPs, based on human apoptosis-inducing proteins. Lacking an endogenous translocation domain dramatically reduces the cell-death inducing capacity of such proteins. Here, we report on optimizing hCFPs, based on the anti-CD64 single chain variable fragment H22(scFv, specifically eliminating CD64+ macrophages and malignant progenitor cells. We replaced the bacterial toxin in H22(scFv-ETA' with the pro-apoptotic human granzyme B or angiogenin. Translocation was promoted by a sophisticated adapter containing a membrane transfer peptide (MTD flanked by endosomal and cytosolic cleavable peptides, thus achieving in vitro cytotoxic activity comparable to bacterial immunotoxins. We demonstrate for the first time that optimized hCFPs, based on granzyme B or angiogenin, can compete with classical ETA-based immunotoxins.

  11. Structural and biochemical characterization of bacterial YpgQ protein reveals a metal-dependent nucleotide pyrophosphohydrolase.

    Science.gov (United States)

    Jeon, Ye Ji; Park, Sun Cheol; Song, Wan Seok; Kim, Ok-Hee; Oh, Byung-Chul; Yoon, Sung-Il

    2016-07-01

    The optimal balance of cellular nucleotides and the efficient elimination of non-canonical nucleotides are critical to avoiding erroneous mutation during DNA replication. One such mechanism involves the degradation of excessive or abnormal nucleotides by nucleotide-hydrolyzing enzymes. YpgQ contains the histidine-aspartate (HD) domain that is involved in the hydrolysis of nucleotides or nucleic acids, but the enzymatic activity and substrate specificity of YpgQ have never been characterized. Here, we unravel the catalytic activity and structural features of YpgQ to report the first Mn(2+)-dependent pyrophosphohydrolase that hydrolyzes (deoxy)ribonucleoside triphosphate [(d)NTP] to (deoxy)ribonucleoside monophosphate and pyrophosphate using the HD domain. YpgQ from Bacillus subtilis (bsYpgQ) displays a helical structure and assembles into a unique dimeric architecture that has not been observed in other HD domain-containing proteins. Each bsYpgQ monomer accommodates a metal ion and a nucleotide substrate in a cavity located between the N- and C-terminal lobes. The metal cofactor is coordinated by the canonical residues of the HD domain, namely, two histidine residues and two aspartate residues, and is positioned in close proximity to the β-phosphate group of the nucleotide, allowing us to propose a nucleophilic attack mechanism for the nucleotide hydrolysis reaction. YpgQ enzymes from other bacterial species also catalyze pyrophosphohydrolysis but exhibit different substrate specificity. Comparative structural and mutational studies demonstrated that residues outside the major substrate-binding site of bsYpgQ are responsible for the species-specific substrate preference. Taken together, our structural and biochemical analyses highlight the substrate-recognition mode and catalysis mechanism of YpgQ in pyrophosphohydrolysis. PMID:27062940

  12. Structural characterization of the major ampullate silk spidroin-2 protein produced by the spider Nephila clavipes.

    Science.gov (United States)

    Santos-Pinto, José Roberto Aparecido Dos; Arcuri, Helen Andrade; Lubec, Gert; Palma, Mario Sergio

    2016-10-01

    Major ampullate spidroin-2 (MaSp2) is one of the most important spider silk protein, but up to now no information is available regarding the post-translational modifications (PTMs) of this protein. A gel-based mass spectrometry strategy using collision-induced dissociation (CID) and electron-transfer dissociation (ETD) fragmentation methods was used to sequence Nephila clavipes MaSp2 (including the N- and C-terminal non-repetitive domains, and the great part of the central core), and to assign a series of post-translational modifications (PTMs) on to the MaSp2 sequence. Two forms of this protein were identified, with different levels of phosphorylation along their sequences. These findings provide a basis for understanding mechanoelastic properties and can support the future design of recombinant spider silk proteins for biotechnological applications. PMID:27208434

  13. Physicochemical and functional properties of protein isolate produced from Australian chia seeds.

    Science.gov (United States)

    Timilsena, Yakindra Prasad; Adhikari, Raju; Barrow, Colin J; Adhikari, Benu

    2016-12-01

    Protein was isolated from Australian chia seeds and converted to powders using spray, freeze and vacuum drying methods, to investigate the effect of drying methods on physicochemical and functional attributes of chia-seed protein isolate (CPI). It was found that there was no significant difference in the proximate composition; however vacuum dried CPI (VDCPI) had the highest bulk density and oil absorption capacity, whereas spray dried powder (SDCPI) demonstrated the highest solubility, water absorption capacity and lowest surface hydrophobicity. Solubility of all powders was higher at elevated temperature and alkaline pH. Foaming capacity and foam stability of CPI were found to increase with increasing pH and protein concentration. SDCPI was the least denatured and VDCPI the most denatured, demonstrating the poorest solubility and foaming properties of the latter. These findings are expected to be useful in selection of a drying process to yield chia seed protein powders with more desirable functionality. PMID:27374580

  14. Bio-inspired Silicification of Silica-binding Peptide-Silk Protein Chimeras: Comparison of Chemically and Genetically Produced Proteins

    OpenAIRE

    Canabady-Rochelle, Laetitia L.S.; Belton, David J.; Deschaume, Olivier; Currie, Heather A.; Kaplan, David L; Perry, Carole C.

    2012-01-01

    Novel protein chimeras constituted of ‘silk’ and a silica-binding peptide (KSLSRHDHIHHH) were synthesized by genetic or chemical approaches and their influence on silica-silk based chimera composite formation evaluated. Genetic chimeras were constructed from 6 or 15 repeats of the 32 amino acid consensus sequence of Nephila clavipes spider silk ([SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQG]n) to which one silica binding peptide was fused at the N terminus. For the chemical chimera, 25 equivalents of t...

  15. Use of γ-irradiation to produce films from whey, casein and soya proteins: structure and functionals characteristics

    International Nuclear Information System (INIS)

    γ-irradiation and thermal treatments have been used to produce sterilized cross-linked films. Formulations containing variable concentrations of calcium caseinate and whey proteins (whey protein isolate (WPI) and commercial whey protein concentrate) or mixture of soya protein isolate (SPI) with WPI was investigated on the physico-chemical properties of these films. Results showed that the mechanical properties of cross-linked films improved significantly the puncture strength for all types of films. Size-exclusion chromatography showed for no cross-linked proteins, a molecular mass of around 40 kDa. The soluble fractions of the cross-linked proteins molecular distributions were between 600 and 3800 kDa. γ-irradiation seems to modify to a certain extent the conformation of proteins which will adopt structures more ordered and more stable, as suggested by X-ray diffraction analysis. Microstructure observations showed that the mechanical characteristics of these films are closely related to their microscopic structure. Water vapor permeability of films based on SPI was also significantly decreased when irradiated. Microbial resistance was also evaluated for cross-linked films. Results showed that the level of biodegradation of cross-linked films was 36% after 60 d of fermentation in the presence of Pseudomonas aeruginosa

  16. Use of /γ-irradiation to produce films from whey, casein and soya proteins: structure and functionals characteristics

    Science.gov (United States)

    Lacroix, M.; Le, T. C.; Ouattara, B.; Yu, H.; Letendre, M.; Sabato, S. F.; Mateescu, M. A.; Patterson, G.

    2002-03-01

    γ-irradiation and thermal treatments have been used to produce sterilized cross-linked films. Formulations containing variable concentrations of calcium caseinate and whey proteins (whey protein isolate (WPI) and commercial whey protein concentrate) or mixture of soya protein isolate (SPI) with WPI was investigated on the physico-chemical properties of these films. Results showed that the mechanical properties of cross-linked films improved significantly the puncture strength for all types of films. Size-exclusion chromatography showed for no cross-linked proteins, a molecular mass of around 40 kDa. The soluble fractions of the cross-linked proteins molecular distributions were between 600 and 3800 kDa. γ-irradiation seems to modify to a certain extent the conformation of proteins which will adopt structures more ordered and more stable, as suggested by X-ray diffraction analysis. Microstructure observations showed that the mechanical characteristics of these films are closely related to their microscopic structure. Water vapor permeability of films based on SPI was also significantly decreased when irradiated. Microbial resistance was also evaluated for cross-linked films. Results showed that the level of biodegradation of cross-linked films was 36% after 60 d of fermentation in the presence of Pseudomonas aeruginosa.

  17. Levels of Serum Immunoglobulin G Specific to Bacterial Surface Protein A of Tannerella forsythia are Related to Periodontal Status

    Science.gov (United States)

    Hall, Lindsay M.; Dunford, Robert G.; Genco, Robert J.; Sharma, Ashu

    2015-01-01

    Background Tannerella forsythia (Tf) is a Gram-negative anaerobe implicated in the development of periodontal disease. Bacterial surface protein A (BspA) is a surface-expressed and -secreted protein that is recognized as an important virulence factor of Tf. This study was undertaken to determine whether Tf BspA induces an antibody response in periodontal disease. We hypothesized that serum immunoglobulin (Ig)G antibody levels against BspA correlate with the disease of patients. Methods Sera were obtained from 100 patients with cardiac disorders and periodontal disease and 73 patients who experienced myocardial infarction but were periodontally healthy. Sera samples were assayed for anti-BspA antibody (total IgG and IgG subtypes) by enzyme-linked immunosorbent assay (ELISA). Antibody levels were measured in ELISA units by using an arbitrary patient as a standard. Results A negative correlation was found with BspA-specific total IgG antibody titers and the severity of disease measured as the clinical attachment level (CAL) when healthy and diseased groups were analyzed separately (healthy group: [−0.23, correlation value] Student’s t value [73 degrees of freedom] = 1.99; P = 0.05; diseased group: [−0.21] t [100 degrees of freedom] = 2.12; P = 0.03]). However, there was a positive correlation ([0.18 correlation value] Student’s t value [173 degrees of freedom] = 2.39; P = 0.017) when healthy and diseased groups were combined. A strong positive correlation ([0.338 correlation value] Student’s t value [173 degrees of freedom] = 4.69; P <0.0001) between the BspA-specific IgG titers and periodontal probing depth was observed when healthy and disease groups were combined. Conclusions Data demonstrated that antibodies to Tf BspA were elicited in patients with periodontal disease, and antibody levels were associated with the disease severity. Furthermore, data suggested that anti-BspA IgG might have a protective function in periodontal disease by minimizing the

  18. Molecular insights into the enzymatic diversity of flavin-trafficking protein (Ftp; formerly ApbE) in flavoprotein biogenesis in the bacterial periplasm.

    Science.gov (United States)

    Deka, Ranjit K; Brautigam, Chad A; Liu, Wei Z; Tomchick, Diana R; Norgard, Michael V

    2016-02-01

    We recently reported a flavin-trafficking protein (Ftp) in the syphilis spirochete Treponema pallidum (Ftp_Tp) as the first bacterial metal-dependent FAD pyrophosphatase that hydrolyzes FAD into AMP and FMN in the periplasm. Orthologs of Ftp_Tp in other bacteria (formerly ApbE) appear to lack this hydrolytic activity; rather, they flavinylate the redox subunit, NqrC, via their metal-dependent FMN transferase activity. However, nothing has been known about the nature or mechanism of metal-dependent Ftp catalysis in either Nqr- or Rnf-redox-containing bacteria. In the current study, we identified a bimetal center in the crystal structure of Escherichia coli Ftp (Ftp_Ec) and show via mutagenesis that a single amino acid substitution converts it from an FAD-binding protein to a Mg(2+) -dependent FAD pyrophosphatase (Ftp_Tp-like). Furthermore, in the presence of protein substrates, both types of Ftps are capable of flavinylating periplasmic redox-carrying proteins (e.g., RnfG_Ec) via the metal-dependent covalent attachment of FMN. A high-resolution structure of the Ftp-mediated flavinylated protein of Shewanella oneidensis NqrC identified an essential lysine in phosphoester-threonyl-FMN bond formation in the posttranslationally modified flavoproteins. Together, these discoveries broaden our understanding of the physiological capabilities of the bacterial periplasm, and they also clarify a possible mechanism by which flavoproteins are generated. PMID:26626129

  19. Biostimulant action of a plant-derived protein hydrolysate produced through enzymatic hydrolysis

    Directory of Open Access Journals (Sweden)

    Giuseppe eColla

    2014-09-01

    Full Text Available The aim of this study was to evaluate the biostimulant action (hormone like activity, nitrogen uptake, and growth stimulation of a plant-derived protein hydrolysate by means of two laboratory bioassays: a corn (Zea mays L. coleoptile elongation rate test (experiment 1, a rooting test on tomato cuttings (experiment 2; and two greenhouse experiments: a dwarf pea (Pisum sativum L. growth test (experiment 3, and a tomato (Solanum lycopersicum L. nitrogen uptake trial (experiment 4. Protein hydrolysate treatments of corn caused an increase in coleoptile elongation rate when compared to the control, in a dose-dependent fashion, with no significant differences between the four concentrations tested (0.375, 0.75, 1.5, and 3.0 ml/L, and inodole-3-acetic acid (IAA treatment. The auxin-like effect of the protein hydrolysate on corn has been also observed in the rooting experiment of tomato cuttings. The shoot, root dry weight, root length, and root area were significantly higher by 21%, 35%, 24%, and 26%, respectively in tomato treated plants with the protein hydrolysate at 6 ml/L than untreated plants. In experiment 3, the application of the protein hydrolysate at all doses (0.375, 0.75, 1.5, and 3.0 ml/L significantly increased the shoot length of the giberellin (GA-deficient dwarf pea plants by an average value of 33% in comparison with the control treatment. Increasing the concentration of the protein hydrolysate from 0 to 10 ml/L increased the total dry biomass, SPAD index, and leaf nitrogen content by 20.5%, 15% and 21.5%, respectively. Thus the application of plant-derived protein hydrolysate containing amino acids and small peptides elicited a hormone-like activity, enhanced nitrogen uptake and consequently crop performances.

  20. Efficacy of locally produced papain enzyme for the production of protein bait for bactrocera invadens (diptera: tephritidae) control in Ghana

    International Nuclear Information System (INIS)

    Autolysed brewery yeast waste is currently being used as cost effective protein bait for Bactrocera invadens control the world over to replace commercial protein hydrolysate bait formulations. However, significant reduction in production cost can be achieved when all the production materials are from local sources. This experiment was aimed at assessing the efficacy of locally produced papain extracted from 'Red lady' pawpaw fruit latex and skin peel to be used for protein bait production. Aqueous two-phase extraction of papain from pawpaw fruit latex with 15 % (NH4)2SO4 - 8 % PEG recovered 64.72 ± 2.08 % papain into the supernatant with 7.33 % proteolytic activity yield and a fold purification of 58.11 ± 1.67. Proteolytic activity and protein concentration measured for the aqueous two-phase extracts of pawpaw skin peel were significantly higher (p= 0.00) than crude extracts of skin peel. However, the aqueous two phase extraction of papain from skin peel needs to be optimised further since SDS-PAGE showed no visible bands in the different phase extracts. Gamma irradiation at 10 KGy increased the proteolytic activity of crude papain by 21.69 % of the non-irradiated papain and subsequently increased the specific activity by 18.51 % but the protein concentration was not affected. Protein baits prepared with crude papain extracted from the pawpaw fruit latex and skin peels were evaluated in laboratory bioassays with wild flies reared from field collected infested mangoes. The source of papain did not affect the protein bait recovery, the pH and protein concentration though colour of bait differed for crude fruit latex papain bait (dark brown) and skin peel papain bait (light brown). The bait preparations had equal attractance to male and female B. invadens. Mean attractance to protein baits produced with fruit latex and skin peel papain baits were between 25.00 ± 7.56 % and 47.50 ± 11.09 % respectively for males, 25.00 ± 13.13 % and 32.86 ± 8.23 % for

  1. A recombinant West Nile virus envelope protein vaccine candidate produced in Spodoptera frugiperda expresSF+ cells

    OpenAIRE

    Bonafé, Nathalie; Rininger, Joseph A.; Chubet, Richard G.; Foellmer, Harald G.; Fader, Stacey; Anderson, John F.; Bushmich, Sandra L.; Anthony, Karen; Ledizet, Michel; Fikrig, Erol; Koski, Raymond A.; Kaplan, Paul

    2008-01-01

    In this study, a recombinant truncated West Nile virus envelope protein antigen (rWNV-E) was produced in serum-free cultures of the expresSF+ insect cell line via baculovirus infection. This production system was selected based on its use in the production of candidate human and animal vaccine antigens. A defined fermentation and purification process for the rWNV-E antigen was established to control for purity and immunogenicity of each protein batch. The material formulated with aluminum hyd...

  2. The Trichoderma-plant interaction is mediated by avirulence proteins produced by this fungus

    Institute of Scientific and Technical Information of China (English)

    Ruocco M; Kip N; P J G M de Wit; Lorito M; Lanzuise S; Woo S L; Ambrosino P; Marra R; Turrà D; Gigante S; Formisno E; Scala F

    2004-01-01

    @@ The molecular basis of Trichoderma -plant interaction is very complex and still not completely understood. The colonization of the root system by rhizosphere competent strains of Trichoderma results in increased development of root/aerial systems, in improved yields and in plant disease control.Other beneficial effects, such as the induction of plant systemic resistance, have also been described.To understand the mechanisms involved we are using different approaches, including the making of transformants expressing genes that encode for compounds able to affect plant response to pathogens.Trichoderma transformants carrying the avirulence gene Avr4 from Cladosporium fulvum under the control of constitutive and inducible promoters were obtained and tested on tomato plants having the Cf4 resistance gene. Necrosis and suberification zones, similar to the symptoms appearing during Cladosporium-tomato interaction, were found when the roots of the Cf4 plants were treated with Avr4-Trichoderma. This demonstrates that selected Trichoderma strains are able to transfer to the plant molecules that may deeply affect metabolism, disease resistance etc. Therefore, these beneficial fungi can be regarded as biotechnological tools to provide a variety of crops with useful compounds.Moreover, in in vitro competition assays the transformants were found to be more effective as antagonists against Alternaria alternata than the wild type. Trichoderma sends a variety of biochemical signals to the plants including avirulence molecules; therefore the presence of avr-like proteins in the fungus proteome was investigated. Proteome analysis has permitted us to isolate and sequence many proteins potentially having this function. From the extraeellular protein extracts, we have purified and sequenced a protein with structural characteristics similar to Avr4 of C. fulvum.The protein, Hytra1, was found to be a hydrophobin with chitin binding activity, the typical 8cysteine residues, and 4

  3. Combining a PagP fusion protein system with nickel ion-catalyzed cleavage to produce intrinsically disordered proteins in E. coli.

    Science.gov (United States)

    Zahran, Somaya; Pan, Jonathan S; Liu, Philip B; Hwang, Peter M

    2015-12-01

    Many proteins contain intrinsically disordered regions that are highly solvent-exposed and susceptible to post-translational modifications. Studying these protein segments is critical to understanding their physiologic regulation, but proteolytic degradation can make them difficult to express and purify. We have designed a new protein expression vector that fuses the target protein to the N-terminus of the integral membrane protein, PagP. The two proteins are connected by a short linker containing the sequence SRHW, previously shown to be optimal for nickel ion-catalyzed cleavage. The methodology is demonstrated for an intrinsically disordered segment of cardiac troponin I. cTnI[135-209]-SRHW-PagP-His6 fusion protein was overexpressed in Escherichia coli, accumulating in insoluble inclusion bodies. The protein was solubilized, purified using nickel affinity chromatography, and then cleaved with 0.5mM NiSO4 at pH 9.0 and 45 °C, all in 6M guanidine-HCl. Nickel ion-catalyzed peptide bond hydrolysis is an effective chemical cleavage technique under denaturing conditions that preclude the use of proteases. Moreover, nickel-catalyzed cleavage is more specific than the most commonly used agent, cyanogen bromide, which cleaves C-terminal to methionine residues. We were able to produce 15 mg of purified cTnI[135-209] from 1L of M9 minimal media using this protocol. The methodology is more generally applicable to the production of intrinsically disordered protein segments. PMID:26297994

  4. Biophysical and structural investigation of bacterially expressed and engineered CCR5, a G protein-coupled receptor

    International Nuclear Information System (INIS)

    The chemokine receptor CCR5 belongs to the class of G protein-coupled receptors. Besides its role in leukocyte trafficking, it is also the major HIV-1 coreceptor and hence a target for HIV-1 entry inhibitors. Here, we report Escherichia coli expression and a broad range of biophysical studies on E. coli-produced CCR5. After systematic screening and optimization, we obtained 10 mg of purified, detergent-solubilized, folded CCR5 from 1L culture in a triply isotope-labeled (2H/15N/13C) minimal medium. Thus the material is suitable for NMR spectroscopic studies. The expected α-helical secondary structure content is confirmed by circular dichroism spectroscopy. The solubilized CCR5 is monodisperse and homogeneous as judged by transmission electron microscopy. Interactions of CCR5 with its ligands, RANTES and MIP-1β were assessed by surface plasmon resonance yielding KD values in the nanomolar range. Using size exclusion chromatography, stable monomeric CCR5 could be isolated. We show that cysteine residues affect both the yield and oligomer distribution of CCR5. HSQC spectra suggest that the transmembrane domains of CCR5 are in equilibrium between several conformations. In addition we present a model of CCR5 based on the crystal structure of CXCR4 as a starting point for protein engineering.

  5. Biophysical and structural investigation of bacterially expressed and engineered CCR5, a G protein-coupled receptor

    Energy Technology Data Exchange (ETDEWEB)

    Wiktor, Maciej; Morin, Sebastien; Sass, Hans-Juergen [University of Basel, Focal Area Structural Biology and Biophysics, Biozentrum (Switzerland); Kebbel, Fabian [University of Basel, Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum (Switzerland); Grzesiek, Stephan, E-mail: stephan.grzesiek@unibas.ch [University of Basel, Focal Area Structural Biology and Biophysics, Biozentrum (Switzerland)

    2013-01-15

    The chemokine receptor CCR5 belongs to the class of G protein-coupled receptors. Besides its role in leukocyte trafficking, it is also the major HIV-1 coreceptor and hence a target for HIV-1 entry inhibitors. Here, we report Escherichia coli expression and a broad range of biophysical studies on E. coli-produced CCR5. After systematic screening and optimization, we obtained 10 mg of purified, detergent-solubilized, folded CCR5 from 1L culture in a triply isotope-labeled ({sup 2}H/{sup 15}N/{sup 13}C) minimal medium. Thus the material is suitable for NMR spectroscopic studies. The expected {alpha}-helical secondary structure content is confirmed by circular dichroism spectroscopy. The solubilized CCR5 is monodisperse and homogeneous as judged by transmission electron microscopy. Interactions of CCR5 with its ligands, RANTES and MIP-1{beta} were assessed by surface plasmon resonance yielding K{sub D} values in the nanomolar range. Using size exclusion chromatography, stable monomeric CCR5 could be isolated. We show that cysteine residues affect both the yield and oligomer distribution of CCR5. HSQC spectra suggest that the transmembrane domains of CCR5 are in equilibrium between several conformations. In addition we present a model of CCR5 based on the crystal structure of CXCR4 as a starting point for protein engineering.

  6. Molecular characterization of Indian isolate of peanut mottle virus and immunodiagnosis using bacterial expressed core capsid protein.

    Science.gov (United States)

    Soumya, K; Yogita, M; Prasanthi, Y; Anitha, K; Kishor, P B Kavi; Jain, R K; Mandal, Bikash

    2014-01-01

    Peanut mottle virus (PeMoV), a seed borne potyvirus was recorded in India in 1978, however the virus was not characterized at molecular level. In the present study, an isolate of PeMoV infecting peanut in southern India was characterized based on host reactions and coat protein (CP) gene sequence, which revealed that the Indian isolate was very close to a peanut isolate reported from Israel and distinct from pea isolate reported from USA. The core region of CP gene that contained majority of the predicted epitopes was successfully expressed (1.75 mg/l) in Escherichia coli as a 22 kDa protein. A high titer polyclonal antibody (PAb) to the expressed core CP was produced, which efficiently detected PeMoV. The antiserum was useful in specific detection of PeMoV as it showed negligible cross reactivity with the other potyviruses e.g., peanut stripe virus, potato virus Y, papaya ringspot virus and onion yellow dwarf virus. The PAb was validated in ELISA using 1,169 field and greenhouse samples of peanut which showed 1.85-26.3 % incidence of PeMoV in peanut seed multiplication field during 2011-2012. This is the first report of immunodiagnosis of PeMoV with a PAb to recombinant core CP of PeMoV. PMID:25674600

  7. Lipoxygenase activity of soybean and protein evaluation of soy milk produced from irradiated grains

    Energy Technology Data Exchange (ETDEWEB)

    Barros, Erica A., E-mail: ericabarros@fca.unesp.br [UNESP - Fazenda Experimental Lageado, Botucatu, SP (Brazil). Fac. de Ciencias Agronomicas; Broetto, Fernando, E-mail: broetto@ibb.unesp.br [UNESP - Universidade Estadual Paulista Julio de Mesquita Filho, Botucatu, SP (Brazil). Inst. de Biociencias. Dept. de Quimica e Bioquimica; Costa, Vladimir E., E-mail: vladimir@ibb.unesp.br [UNESP - Universidade Estadual Paulista Julio de Mesquita Filho, Botucatu, SP (Brazil). Inst. de Biociencias. Dept. de Fisica e Biofisica

    2011-07-01

    Soybean and its derivative are considered as a functional food because it has high quality protein and are used for the prevention of chronic degenerative diseases. The irradiation technique is used in soybeans to increase shelf life and avoid problems in plant products consumed raw or processed. However, the controversy in the literature that the irradiation dose up 10 kGy food can alter the functional properties and structures of macronutrients. With the prospect of more information on the use of radiation on soybeans, the objective of this study was to determine the activity of lipoxygenase in soybeans and to evaluate possible changes in the protein content of soymilk processed from grain-BRS 213, BRS 258 and Embrapa 48 subjected to dosages of 2.5 , 5.0 and 10.0 kGy of gamma radiation. The soybean cultivars were wrapped in plastic bags and subjected to gamma radiation source {sup 60}Co, Gammacell 220 (Atomic Energy of Canada Ltd.), except the control. The grains irradiated induced reduction of enzyme activity. The results for the protein content of soymilk were similar, appropriate to that required by ANVISA and showed little protein solubility for cultivars BRS-258 and Embrapa48. It was concluded that the technique of irradiation beyond to keep the nutritional value of soy can contribute to the organoleptic quality of soymilk. (author)

  8. PROPERTIES OF BIOPOLYMERS PRODUCED BY TRANSGLUTAMINASE TREATMENT OF WHEY PROTEIN ISOLATE AND GELATIN

    Science.gov (United States)

    Byproduct utilization is an important consideration in the development of sustainable processes. Whey protein isolate (WPI), a byproduct of the cheese industry, and gelatin, a byproduct of the leather industry, were reacted individually and in blends with microbial transglutaminase (mTGase) at pH 7...

  9. Lipoxygenase activity of soybean and protein evaluation of soy milk produced from irradiated grains

    International Nuclear Information System (INIS)

    Soybean and its derivative are considered as a functional food because it has high quality protein and are used for the prevention of chronic degenerative diseases. The irradiation technique is used in soybeans to increase shelf life and avoid problems in plant products consumed raw or processed. However, the controversy in the literature that the irradiation dose up 10 kGy food can alter the functional properties and structures of macronutrients. With the prospect of more information on the use of radiation on soybeans, the objective of this study was to determine the activity of lipoxygenase in soybeans and to evaluate possible changes in the protein content of soymilk processed from grain-BRS 213, BRS 258 and Embrapa 48 subjected to dosages of 2.5 , 5.0 and 10.0 kGy of gamma radiation. The soybean cultivars were wrapped in plastic bags and subjected to gamma radiation source 60Co, Gammacell 220 (Atomic Energy of Canada Ltd.), except the control. The grains irradiated induced reduction of enzyme activity. The results for the protein content of soymilk were similar, appropriate to that required by ANVISA and showed little protein solubility for cultivars BRS-258 and Embrapa48. It was concluded that the technique of irradiation beyond to keep the nutritional value of soy can contribute to the organoleptic quality of soymilk. (author)

  10. The bacterial signal transduction protein GlnB regulates the committed step in fatty acid biosynthesis by acting as a dissociable regulatory subunit of acetyl-CoA carboxylase.

    Science.gov (United States)

    Gerhardt, Edileusa C M; Rodrigues, Thiago E; Müller-Santos, Marcelo; Pedrosa, Fabio O; Souza, Emanuel M; Forchhammer, Karl; Huergo, Luciano F

    2015-03-01

    Biosynthesis of fatty acids is one of the most fundamental biochemical pathways in nature. In bacteria and plant chloroplasts, the committed and rate-limiting step in fatty acid biosynthesis is catalyzed by a multi-subunit form of the acetyl-CoA carboxylase enzyme (ACC). This enzyme carboxylates acetyl-CoA to produce malonyl-CoA, which in turn acts as the building block for fatty acid elongation. In Escherichia coli, ACC is comprised of three functional modules: the biotin carboxylase (BC), the biotin carboxyl carrier protein (BCCP) and the carboxyl transferase (CT). Previous data showed that both bacterial and plant BCCP interact with signal transduction proteins belonging to the PII family. Here we show that the GlnB paralogues of the PII proteins from E. coli and Azospirillum brasiliense, but not the GlnK paralogues, can specifically form a ternary complex with the BC-BCCP components of ACC. This interaction results in ACC inhibition by decreasing the enzyme turnover number. Both the BC-BCCP-GlnB interaction and ACC inhibition were relieved by 2-oxoglutarate and by GlnB uridylylation. We propose that the GlnB protein acts as a 2-oxoglutarate-sensitive dissociable regulatory subunit of ACC in Bacteria. PMID:25557370

  11. Context-dependent protein folding of a virulence peptide in the bacterial and host environments: structure of an SycH–YopH chaperone–effector complex

    International Nuclear Information System (INIS)

    The structure of a SycH–YopH chaperone–effector complex from Yersinia reveals the bacterial state of a protein that adopts different folds in the host and pathogen environments. Yersinia pestis injects numerous bacterial proteins into host cells through an organic nanomachine called the type 3 secretion system. One such substrate is the tyrosine phosphatase YopH, which requires an interaction with a cognate chaperone in order to be effectively injected. Here, the first crystal structure of a SycH–YopH complex is reported, determined to 1.9 Å resolution. The structure reveals the presence of (i) a nonglobular polypeptide in YopH, (ii) a so-called β-motif in YopH and (iii) a conserved hydrophobic patch in SycH that recognizes the β-motif. Biochemical studies establish that the β-motif is critical to the stability of this complex. Finally, since previous work has shown that the N-terminal portion of YopH adopts a globular fold that is functional in the host cell, aspects of how this polypeptide adopts radically different folds in the host and in the bacterial environments are analysed

  12. Bioinspired silicification of silica-binding peptide-silk protein chimeras: comparison of chemically and genetically produced proteins.

    Science.gov (United States)

    Canabady-Rochelle, Laetitia L S; Belton, David J; Deschaume, Olivier; Currie, Heather A; Kaplan, David L; Perry, Carole C

    2012-03-12

    Novel protein chimeras constituted of "silk" and a silica-binding peptide (KSLSRHDHIHHH) were synthesized by genetic or chemical approaches and their influence on silica-silk based chimera composite formation evaluated. Genetic chimeras were constructed from 6 or 15 repeats of the 32 amino acid consensus sequence of Nephila clavipes spider silk ([SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQG](n)) to which one silica binding peptide was fused at the N terminus. For the chemical chimera, 28 equiv of the silica binding peptide were chemically coupled to natural Bombyx mori silk after modification of tyrosine groups by diazonium coupling and EDC/NHS activation of all acid groups. After silica formation under mild, biomaterial-compatible conditions, the effect of peptide addition on the properties of the silk and chimeric silk-silica composite materials was explored. The composite biomaterial properties could be related to the extent of silica condensation and to the higher number of silica binding sites in the chemical chimera as compared with the genetically derived variants. In all cases, the structure of the protein/chimera in solution dictated the type of composite structure that formed with the silica deposition process having little effect on the secondary structural composition of the silk-based materials. Similarly to our study of genetic silk based chimeras containing the R5 peptide (SSKKSGSYSGSKGSKRRIL), the role of the chimeras (genetic and chemical) used in the present study resided more in aggregation and scaffolding than in the catalysis of condensation. The variables of peptide identity, silk construct (number of consensus repeats or silk source), and approach to synthesis (genetic or chemical) can be used to "tune" the properties of the composite materials formed and is a general approach that can be used to prepare a range of materials for biomedical and sensor-based applications. PMID:22229696

  13. Comparison study among advanced high protein lines of wheat produced by the IAEA under the local environmental conditions of Syria

    International Nuclear Information System (INIS)

    Ten high protein mutant lines of T. aestivum were, produced by the IAEA using gamma radiation and Ethyl Methane Sulphonite (EMS), studied under the Syrian environmental conditions for two years along with four local varieties. The experiments were sown in a complete randomized block design with four replicates. Yield was estimated by using Nearest Neighbour Analysis test and the effect of different locations on quality characters was carried out using Analysis Across Location test. Results showed that the effects of locations was higher than that of the lines. Under the dry conditions the two mutant lines E296 and 5074M were highest in protein percentage but lowest in yield. However, under irrigation they produced better yield than many others, hence, they were recommended to be grown under irrigation only. On the other hand 0827 mutant line was the highest stable line ranking first in yield among IAEA lines under both dry and irrigated conditions, whereas Buhuth4 and L92-6 surpassed 0827 in protein yield which was considered the best criterion for selecting high efficiency in uptake and transfer of nitrogenous compounds to the grain. The project showed the possibiligy of making use of some of these lines that could adapt with our environmental conditions either as introductions or through crosses with our Syrian material to produce new varieties. (author). 20 refs., 12 tabs., 2 figs

  14. Multiple growth hormone-binding proteins are expressed on insulin-producing cells

    DEFF Research Database (Denmark)

    Møldrup, A; Billestrup, N; Thorn, N A;

    1989-01-01

    disulfide-linked multimers of Mr 270-350K (300K). In addition, a minor Mr 180K GH-binding protein is identified which does not appear to be associated with other proteins by disulfide bridges. A plasma membrane-enriched fraction accounts for 86% of the RIN-cell GH-binding activity while cytosol and...... intracellular organelles are low in GH-binding activity. The plasma membrane-bound activity is soluble in Triton X-100 with intact hormone binding characteristics. The apparent KD in detergent solution is estimated to 18 ng/ml (8 x 10(-10) M). 125I-hGH-affinity cross-linking to intact and detergent...

  15. Influence of peptides and proteins produced by cyanobacterium Microcystis aeruginosa on the coagulation of turbid waters

    Czech Academy of Sciences Publication Activity Database

    Šafaříková, Jana; Barešová, Magdalena; Pivokonský, Martin; Kopecká, Ivana

    2013-01-01

    Roč. 18, October (2013), s. 49-57. ISSN 1383-5866 R&D Projects: GA ČR GAP105/11/0247 Institutional research plan: CEZ:AV0Z20600510 Institutional support: RVO:67985874 Keywords : Cellular organic matter (COM) * Coagulation * Microcystis aeruginosa * Peptides/proteins * Turbidity removal Subject RIV: BK - Fluid Dynamics Impact factor: 3.065, year: 2013 http://www.sciencedirect.com/science/article/pii/S1383586613004152

  16. Total Protein Extraction for Metaproteomics Analysis of Methane Producing Biofilm: The Effects of Detergents

    OpenAIRE

    Hung-Jen Huang; Wei-Yu Chen; Jer-Horng Wu

    2014-01-01

    Protein recovery is crucial for shotgun metaproteomics to study the in situ functionality of microbial populations from complex biofilms but still poorly addressed by far. To fill this knowledge gap, we systematically evaluated the sample preparation with extraction buffers comprising four detergents for the metaproteomics analysis of a terephthalate-degrading methanogenic biofilm using an on-line two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) system. Totally...

  17. Tapping-mode atomic force microscopy produces faithful high-resolution images of protein surfaces.

    OpenAIRE

    Möller, C; Allen, M.; Elings, V; Engel, A.; Müller, D J

    1999-01-01

    Compared to contact-mode atomic force microscopy (CMAFM), tapping-mode atomic force microscopy (TMAFM) has the advantage of allowing imaging surfaces of macromolecules, even when they are only weakly attached to the support. In this study, TMAFM is applied to two different regular protein layers whose structures are known to great detail, the purple membrane from Halobacterium salinarum and the hexagonally packed intermediate (HPI) layer from Deinococcus radiodurans, to assess the faithfulnes...

  18. Why Does Insect Antifreeze Protein from Tenebrio molitor Produce Pyramidal Ice Crystallites?

    OpenAIRE

    Strom, Christina S.; Liu, Xiang Yang; Jia, Zongchao

    2005-01-01

    The antifreeze protein (AFP) reduces the growth rates of the ice crystal facets. In that process the ice morphology undergoes a modification. An AFP-induced surface pinning mechanism, through matching of periodic bond chains in two dimensions, enables two-dimensional regular ice-binding surfaces (IBSs) of the insect AFPs to engage a certain class of ice surfaces, called primary surfaces. They are kinetically stable surfaces with unambiguous and predetermined orientations. In this work, the or...

  19. Application of Ionizing Radiations to Produce New Polysaccharides and Proteins with Enhanced Functionality

    International Nuclear Information System (INIS)

    Treatment of polysaccharides with ionizing radiation either in the solid state or in aqueous solution leads to degradation, whereas application of radiation to process synthetic polymers to introduce structural changes and special performance characteristics is now a thriving industry. Using a mediating gas associated during the radiation treatment prevents the degradation of natural polymers and enables the introduction of different molecular and functional characteristics, as previously achieved with synthetic polymers. For example, the molecular weight can be increased and standardised, protein distribution reorganised and modified to ensure better emulsification, viscosity and viscoelasticity enhanced, leading when required to hydrogel formation. More than one hydrocolloid can also be integrated into a single matrix using this process. Protein, within demineralised bone, too can be modified to give enhanced osteoinductive capacity. This experience has led to additional patented and proprietary processes, using standard food processing techniques, to promote changes in a wide range of hydrocolloids which emulates and extend those which occur naturally. The lecture will describe these structural changes and their functional role by reference to several hydrocolloids, including acacia gums, pectin, ispaghula and hyaluronan, bone morphogenic protein. Applications in food products, dietary fibre and medical products will be illustrated

  20. Identification of a novel calcium binding motif based on the detection of sequence insertions in the animal peroxidase domain of bacterial proteins.

    Directory of Open Access Journals (Sweden)

    Saray Santamaría-Hernando

    Full Text Available Proteins of the animal heme peroxidase (ANP superfamily differ greatly in size since they have either one or two catalytic domains that match profile PS50292. The orf PP_2561 of Pseudomonas putida KT2440 that we have called PepA encodes a two-domain ANP. The alignment of these domains with those of PepA homologues revealed a variable number of insertions with the consensus G-x-D-G-x-x-[GN]-[TN]-x-D-D. This motif has also been detected in the structure of pseudopilin (pdb 3G20, where it was found to be involved in Ca(2+ coordination although a sequence analysis did not reveal the presence of any known calcium binding motifs in this protein. Isothermal titration calorimetry revealed that a peptide containing this consensus motif bound specifically calcium ions with affinities ranging between 33-79 µM depending on the pH. Microcalorimetric titrations of the purified N-terminal ANP-like domain of PepA revealed Ca(2+ binding with a K(D of 12 µM and stoichiometry of 1.25 calcium ions per protein monomer. This domain exhibited peroxidase activity after its reconstitution with heme. These data led to the definition of a novel calcium binding motif that we have termed PERCAL and which was abundantly present in animal peroxidase-like domains of bacterial proteins. Bacterial heme peroxidases thus possess two different types of calcium binding motifs, namely PERCAL and the related hemolysin type calcium binding motif, with the latter being located outside the catalytic domains and in their C-terminal end. A phylogenetic tree of ANP-like catalytic domains of bacterial proteins with PERCAL motifs, including single domain peroxidases, was divided into two major clusters, representing domains with and without PERCAL motif containing insertions. We have verified that the recently reported classification of bacterial heme peroxidases in two families (cd09819 and cd09821 is unrelated to these insertions. Sequences matching PERCAL were detected in all kingdoms of

  1. 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.

  2. Channel-forming proteins in the cell wall of amino acid-producing Corynebacteria

    OpenAIRE

    Hünten, Peter

    2005-01-01

    Corynebacterium glutamicum is together with C. callunae and C. efficiens a member of the diverse group of mycolic-acid containing actinomycetes, the mycolata. These bacteria are potent producer of glutamate, lysine and other amino acids on industrial scale. The cell walls of most actinomycetes contain besides an arabinogalactan-peptidoglycan complex large amounts of mycolic acids. This three-layer envelope is called MAP (mycolyl-arabinogalactan-peptidoglycan) complex and it represents a secon...

  3. Isolation of a new Pseudomonas halophila strain possess bacteriorhodopsin-like protein by a novel method for screening of photoactive protein producing bacteria.

    Science.gov (United States)

    Fanaei, Maryam; Emtiazi, Giti

    2014-02-01

    Bacteriorhodopsin (bR) is a transmembrane protein deposited in the purple membrane of Halobacterium salinarum which absorbs energy from photons to create a photo-induced proton gradient across the membrane. A bR molecule can be considered as a natural solar device transforming light into other types of energy and therefore is of interest for a wide range of applications including two and three-dimensional memory storage, optical data processing, artificial cells, holographic media, the artificial retina and photo sensor devices. H. salinarum is a slow-growing, halophilic Archaea present in red salt waters. The present study introduces a novel bR-like pigment from a new strain of Pseudomonas halophila (with registered accession number KC959570 in the NCBI databank) which has a very significant degree of light-dependent activity. This is the first report on the presence of functional bR-like protein in the Pseudomonas family. The isolate is a fast-growing, halophilic bacterium and is comparable with other photoactive protein producer microorganisms. Also, in the present study a novel isolation method for screen light-stimulating protein producing microorganisms is introduced. For this purpose 2,3,5-triphenyltetrazolium chloride (TTC) was employed for the first time as an artificial hydrogen acceptor in the proton-transfer processes. The TTC test is an easy and susceptible method for estimating hydrogen production during the proton transport process. This is the first report of the use of TTC for photo activity measurement and selection of bacteria containing light dependent proteins. PMID:24002576

  4. Ice microsphere templating to produce highly porous nanocomposite PLA matrix scaffolds with pores selectively lined by bacterial cellulose nano-whiskers

    OpenAIRE

    Blaker, J. J.; Lee, K-Y; Mantalaris, A.; Bismarck, A.

    2010-01-01

    Abstract The production of 3D scaffolds for tissue engineering with provision of a controlled nano-topography remains a significant challenge. Here we have combined an ice microsphere templating technique with thermally induced phase separation, and by taking advantage of interactions between hydrophilic and hydrophobic phases, lined the pore walls with bacterial cellulose nano-whiskers. The cryogenic technique we have developed not only allows the decoration of the pore walls of 3...

  5. Crystallization and preliminary X-ray analysis of the FliH–FliI complex responsible for bacterial flagellar type III protein export

    International Nuclear Information System (INIS)

    The FliH–FliI complex from the bacterial flagellar type III export apparatus has been expressed, purified and crystallized, and the crystals have been characterized by X-ray diffraction. The bacterial flagellar proteins are translocated into the central channel of the flagellum by a specific protein-export apparatus for self-assembly at the distal growing end. FliH and FliI are soluble components of the export apparatus and form an FliH2–FliI heterotrimer in the cytoplasm. FliI is an ATPase and the FliH2–FliI complex delivers export substrates from the cytoplasm to an export gate made up of six integral membrane proteins of the export apparatus. In this study, an FliHC fragment consisting of residues 99–235 was co-purified with FliI and the FliHC2–FliI complex was crystallized. Crystals were obtained using the hanging-drop vapour-diffusion technique with PEG 400 as a precipitant. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 133.7, b = 147.3, c = 164.2 Å, and diffracted to 3.0 Å resolution

  6. Heme uptake in bacterial pathogens

    OpenAIRE

    Contreras, Heidi; Chim, Nicholas; Credali, Alfredo; Goulding, Celia W.

    2014-01-01

    Iron is an essential nutrient for the survival of organisms. Bacterial pathogens possess specialized pathways to acquire heme from their human hosts. In this review, we present recent structural and biochemical data that provide mechanistic insights into several bacterial heme uptake pathways, encompassing the sequestration of heme from human hemoproteins to secreted or membrane-associated bacterial proteins, the transport of heme across bacterial membranes, and the degradation of heme within...

  7. Co-expression of heat shock protein (HSP) 40 and HSP70 in Pinctada martensii response to thermal, low salinity and bacterial challenges.

    Science.gov (United States)

    Li, Jun; Zhang, Yuehuan; Liu, Ying; Zhang, Yang; Xiao, Shu; Yu, Ziniu

    2016-01-01

    Heat shock protein (HSP) 40 proteins are a family of molecular chaperones that bind to HSP70 through their J-domain and regulate the function of HSP70 by stimulating its adenosine triphosphatase activity. In the present study, a HSP40 homolog named PmHSP40 was cloned from the hemocytes of pearl oyster Pinctada martensii using EST and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of PmHSP40 was 1251 bp in length, which included a 5' untranslated region (UTR) of 75 bp, an open reading frame (ORF) of a 663 bp, and a 3' UTR of 513 bp. The deduced amino acid sequence of PmHSP40 contains a J domain in the N-terminus. In response to thermal and low salinity stress challenges, the expression of PmHSP40 in hemocytes and the gill were inducible in a time-dependent manner. After bacterial challenge, PmHSP40 transcripts in hemocytes increased and peaked at 6 h post injection. In the gill, PmHSP40 expression increased, similar to expression in hemocytes; however, transcript expression of PmHSP40 was significantly up-regulated at 12 h post injection. Furthermore, the transcripts of PmHSP70 showed similar kinetics as that of PmHSP40, with highest induction during thermal, low salinity stress and bacterial challenges. Altogether these results demonstrate that PmHSP40 is an inducible protein under thermal, low salinity and bacterial challenges, suggesting its involvement in both environmental and biological stresses, and in the innate immunity of the pearl oyster. PMID:26679110

  8. OppA of Listeria monocytogenes, an Oligopeptide-Binding Protein Required for Bacterial Growth at Low Temperature and Involved in Intracellular Survival

    OpenAIRE

    Borezee, Elise; Pellegrini, Elisabeth; Berche, Patrick

    2000-01-01

    We identified a new oligopeptide permease operon in the pathogen Listeria monocytogenes. This opp operon consists of five genes (oppA, oppB, oppC, oppD, and oppF) and displays the same genetic organization as those of several bacterial species. The first gene of this operon, oppA, encodes a 62-kDa protein sharing 33% identity with OppA of Bacillus subtilis and is expressed predominantly during exponential growth. The function of oppA was studied by constructing an oppA deletion mutant. The ph...

  9. A Burkholderia pseudomallei type III secreted protein, BopE, facilitates bacterial invasion of epithelial cells and exhibits guanine nucleotide exchange factor activity

    OpenAIRE

    M.P. Stevens; Friebel, A; Taylor, L A; Wood, M W; Brown, P. J.; Hardt, W D; Galyov, E E

    2003-01-01

    We report the characterization of BopE, a type III secreted protein that is encoded adjacent to the Burkholderia pseudomallei bsa locus and is homologous to Salmonella enterica SopE/SopE2. Inactivation of bopE impaired bacterial entry into HeLa cells, indicating that BopE facilitates invasion. Consistent with this notion, BopE expressed in eukaryotic cells induced rearrangements in the subcortical actin cytoskeleton, and purified BopE exhibited guanine nucleotide exchange factor activity for ...

  10. CCAAT/enhancer-binding protein δ facilitates bacterial dissemination during pneumococcal pneumonia in a platelet-activating factor receptor-dependent manner

    OpenAIRE

    Duitman, JanWillem; Schouten, Marcel; Groot, Angelique P.; Borensztajn, Keren S.; Daalhuisen, Joost B.; Florquin, Sandrine; van der Poll, Tom; Spek, C Arnold

    2012-01-01

    CCAAT/enhancer-binding protein δ (C/EBPδ) recently emerged as an essential player in the inflammatory response to bacterial infections. C/EBPδ levels increase rapidly after a proinflammatory stimulus, and increasing C/EBPδ levels seem to be indispensable for amplification of the inflammatory response. Here we aimed to elucidate the role of C/EBPδ in host defense in community-acquired pneumococcal pneumonia. We show that C/EBPδ−/− mice are relatively resistant to pneumococcal pneumonia, as ind...

  11. Effect of Carbon and Nitrogen Sources on the Production of Reducing Sugars, Extra-cellular Protein and Cellulolytic Enzymes by Two Cellulolytic Bacterial Isolates

    OpenAIRE

    Kashem, M. A.; M.A. Manchur; M.S. Rahman; M.N. Anwar

    2004-01-01

    Two thermophilic cellulolytic bacterial isolates were tested to determine the effect of carbon and nitrogen sources on the production of extra-cellular proteins, reducing sugars and cellulolytic enzymes. Lactose was found to be the most potential carbon source for Avicelase (342.52 U mL-1) and ß-glucosidase (256.89 U mL-1) activity where as NH4Cl was found to be the potential nitrogen source for CMCase (144.68 U mL-1) activity.

  12. Stearoyl CoA Desaturase Is Required to Produce Active, Lipid-Modified Wnt Proteins

    Directory of Open Access Journals (Sweden)

    Jessica Rios-Esteves

    2013-09-01

    Full Text Available Wnt proteins contain palmitoleic acid, an unusual lipid modification. Production of an active Wnt signal requires the acyltransferase Porcupine and depends on the attachment of palmitoleic acid to Wnt. The source of this monounsaturated fatty acid has not been identified, and it is not known how Porcupine recognizes its substrate and whether desaturation occurs before or after fatty acid transfer to Wnt. Here, we show that stearoyl desaturase (SCD generates a monounsaturated fatty acid substrate that is then transferred by Porcupine to Wnt. Treatment of cells with SCD inhibitors blocked incorporation of palmitate analogs into Wnt3a and Wnt5a and reduced Wnt secretion as well as autocrine and paracrine Wnt signaling. The SCD inhibitor effects were rescued by exogenous addition of monounsaturated fatty acids. We propose that SCD is a key molecular player responsible for Wnt biogenesis and processing and that SCD inhibition provides an alternative mechanism for blocking Wnt pathway activation.

  13. Cloning and molecular characterization of the murine macrophage "68-kDa" protein kinase C substrate and its regulation by bacterial lipopolysaccharide.

    OpenAIRE

    Seykora, J T; Ravetch, J V; Aderem, A

    1991-01-01

    We have isolated and characterized a cDNA clone encoding the murine macrophage 68-kDa protein kinase C substrate, which is homologous to the 80- to 87-kDa protein identified by the acronym MARCKS (myristoylated alanine-rich C kinase substrate). The murine MARCKS cDNA clone encodes an acidic protein of 309 amino acids with a calculated molecular weight of 29,661. Transfection of the murine MARCKS gene into TK-L fibroblasts produced a myristoylated protein kinase C substrate that migrated on SD...

  14. An Inner Membrane Protein (Imp) of Xanthomonas oryzae pv. oryzicola Functions in Carbon Acquisition, EPS Production, Bacterial Motility and Virulence in Rice

    Institute of Scientific and Technical Information of China (English)

    CHEN Gong-you

    2014-01-01

    Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak, a devastating disease in rice-growing regions worldwide. A Tn5-insertion mutant in Xoc_3248, encoding an inner membrane protein (Imp), showed reduced virulence in rice. To explore the potential function of this gene in virulence, a deletion mutant R∆imp was constructed in the wild-type RS105. The R∆imp mutant was signiifcantly impaired for bacterial virulence and growth in planta. The mutation in imp made the pathogen insufifciently utilize glucose, fructose, mannose or pyruvate as a sole carbon source, leading to less extracellular polysaccharide (EPS) production and reduced motility. The deifciencies noted for the mutant were restored to wild-type levels when imp was introduced in trans. Transcription of imp was signiifcantly declined when hrpG and hrpX was mutated and the expression of hrpG and hrpX was also signiifcantly declined when imp was deleted. Cell sublocalization in planta showed Imp membrane-binding feature. These results suggest that Imp is a virulence factor with roles in the catabolism of sugars, EPS production, and bacterial motility.

  15. A novel expression vector, designated as pHisJM, for producing recombinant His-fusion proteins.

    Science.gov (United States)

    Masuda, Junko; Takayama, Eiji; Satoh, Ayano; Kojima-Aikawa, Kyoko; Suzuki, Kimihiro; Matsumoto, Isamu

    2004-10-01

    Compared to glutathione S -transferase (GST), tagging with hexahistidine residues (His) has several merits: low levels of toxicity and immunogenicity, a smaller size and no electric charge. We have constructed a novel expression vector, designated as pHisJM (EMBL/GenBank/DDJB accession no. AB116367), for producing recombinant His-fusion proteins. This vector was constructed by replacing GST and multiple cloning site (MCS) cassettes in pGEX-5X-3 with those of hexahistidine and MCS derived from pRSET C vector. Human annexin IV (Anx IV) was used as target protein. His-Anx IV fusion protein was expressed using pHisJM and gave a 40 kDa band when immuno-stained with anti-His mAb or anti-Anx IV mAb as predicted. To compare expression efficiency, a Anx IV cDNA inserted-pHisJM or pGEX-5X-3 was transformed into Escherichia coli DH5alpha, JM109, BL21 and BL21(DE3). Using pHisJM, Anx IV protein was highly expressed in all cell strains. In addition to the merits of using His-tag, pHisJM has several advantages: 1) it has high expression efficiency; 2) it can be used in any Escherichia coli strain; and 3) it can be used in a single strain of Escherichia coli in all steps from plasmid construction to the expression of the target gene. PMID:15604794

  16. Selenite Reduction by Anaerobic Microbial Aggregates: Microbial Community Structure, and Proteins Associated to the Produced Selenium Spheres

    KAUST Repository

    Gonzalez-Gil, Graciela

    2016-04-26

    Certain types of anaerobic granular sludge, which consists of microbial aggregates, can reduce selenium oxyanions. To envisage strategies for removing those oxyanions from wastewater and recovering the produced elemental selenium (Se0), insights into the microbial community structure and synthesis of Se0 within these microbial aggregates are required. High-throughput sequencing showed that Veillonellaceae (c.a. 20%) and Pseudomonadaceae (c.a.10%) were the most abundant microbial phylotypes in selenite reducing microbial aggregates. The majority of the Pseudomonadaceae sequences were affiliated to the genus Pseudomonas. A distinct outer layer (∼200 μm) of selenium deposits indicated that bioreduction occurred in the outer zone of the microbial aggregates. In that outer layer, SEM analysis showed abundant intracellular and extracellular Se0 (nano)spheres, with some cells having high numbers of intracellular Se0 spheres. Electron tomography showed that microbial cells can harbor a single large intracellular sphere that stretches the cell body. The Se0 spheres produced by the microorganisms were capped with organic material. X-ray photoelectron spectroscopy (XPS) analysis of extracted Se0 spheres, combined with a mathematical approach to analyzing XPS spectra from biological origin, indicated that proteins and lipids were components of the capping material associated to the Se0 spheres. The most abundant proteins associated to the spheres were identified by proteomic analysis. Most of the proteins or peptide sequences capping the Se0 spheres were identified as periplasmic outer membrane porins and as the cytoplasmic elongation factor Tu protein, suggesting an intracellular formation of the Se0 spheres. In view of these and previous findings, a schematic model for the synthesis of Se0 spheres by the microorganisms inhabiting the granular sludge is proposed.

  17. Selenite Reduction by Anaerobic Microbial Aggregates: Microbial Community Structure, and Proteins Associated to the Produced Selenium Spheres

    Science.gov (United States)

    Gonzalez-Gil, Graciela; Lens, Piet N. L.; Saikaly, Pascal E.

    2016-01-01

    Certain types of anaerobic granular sludge, which consists of microbial aggregates, can reduce selenium oxyanions. To envisage strategies for removing those oxyanions from wastewater and recovering the produced elemental selenium (Se0), insights into the microbial community structure and synthesis of Se0 within these microbial aggregates are required. High-throughput sequencing showed that Veillonellaceae (c.a. 20%) and Pseudomonadaceae (c.a.10%) were the most abundant microbial phylotypes in selenite reducing microbial aggregates. The majority of the Pseudomonadaceae sequences were affiliated to the genus Pseudomonas. A distinct outer layer (∼200 μm) of selenium deposits indicated that bioreduction occurred in the outer zone of the microbial aggregates. In that outer layer, SEM analysis showed abundant intracellular and extracellular Se0 (nano)spheres, with some cells having high numbers of intracellular Se0 spheres. Electron tomography showed that microbial cells can harbor a single large intracellular sphere that stretches the cell body. The Se0 spheres produced by the microorganisms were capped with organic material. X-ray photoelectron spectroscopy (XPS) analysis of extracted Se0 spheres, combined with a mathematical approach to analyzing XPS spectra from biological origin, indicated that proteins and lipids were components of the capping material associated to the Se0 spheres. The most abundant proteins associated to the spheres were identified by proteomic analysis. Most of the proteins or peptide sequences capping the Se0 spheres were identified as periplasmic outer membrane porins and as the cytoplasmic elongation factor Tu protein, suggesting an intracellular formation of the Se0 spheres. In view of these and previous findings, a schematic model for the synthesis of Se0 spheres by the microorganisms inhabiting the granular sludge is proposed. PMID:27199909

  18. From the Cover: Specific chemical and structural damage to proteins produced by synchrotron radiation

    Science.gov (United States)

    Weik, Martin; Ravelli, Raimond B. G.; Kryger, Gitay; McSweeney, Sean; Raves, Maria L.; Harel, Michal; Gros, Piet; Silman, Israel; Kroon, Jan; Sussman, Joel L.

    2000-01-01

    Radiation damage is an inherent problem in x-ray crystallography. It usually is presumed to be nonspecific and manifested as a gradual decay in the overall quality of data obtained for a given crystal as data collection proceeds. Based on third-generation synchrotron x-ray data, collected at cryogenic temperatures, we show for the enzymes Torpedo californica acetylcholinesterase and hen egg white lysozyme that synchrotron radiation also can cause highly specific damage. Disulfide bridges break, and carboxyl groups of acidic residues lose their definition. Highly exposed carboxyls, and those in the active site of both enzymes, appear particularly susceptible. The catalytic triad residue, His-440, in acetylcholinesterase, also appears to be much more sensitive to radiation damage than other histidine residues. Our findings have direct practical implications for routine x-ray data collection at high-energy synchrotron sources. Furthermore, they provide a direct approach for studying the radiation chemistry of proteins and nucleic acids at a detailed, structural level and also may yield information concerning putative "weak links" in a given biological macromolecule, which may be of structural and functional significance.

  19. Engineering protein processing of the mammary gland to produce abundant hemophilia B therapy in milk.

    Science.gov (United States)

    Zhao, Jianguo; Xu, Weijie; Ross, Jason W; Walters, Eric M; Butler, Stephen P; Whyte, Jeff J; Kelso, Lindsey; Fatemi, Mostafa; Vanderslice, Nicholas C; Giroux, Keith; Spate, Lee D; Samuel, Melissa S; Murphy, Cliff N; Wells, Kevin D; Masiello, Nick C; Prather, Randall S; Velander, William H

    2015-01-01

    Both the low animal cell density of bioreactors and their ability to post-translationally process recombinant factor IX (rFIX) limit hemophilia B therapy to <20% of the world's population. We used transgenic pigs to make rFIX in milk at about 3,000-fold higher output than provided by industrial bioreactors. However, this resulted in incomplete γ-carboxylation and propeptide cleavage where both processes are transmembrane mediated. We then bioengineered the co-expression of truncated, soluble human furin (rFurin) with pro-rFIX at a favorable enzyme to substrate ratio. This resulted in the complete conversion of pro-rFIX to rFIX while yielding a normal lactation. Importantly, these high levels of propeptide processing by soluble rFurin did not preempt γ-carboxylation in the ER and therefore was compartmentalized to the Trans-Golgi Network (TGN) and also to milk. The Golgi specific engineering demonstrated here segues the ER targeted enhancement of γ-carboxylation needed to biomanufacture coagulation proteins like rFIX using transgenic livestock. PMID:26387706

  20. Contributions of adhesive proteins to the cellular and bacterial response to surfaces treated with bioactive polymers: case of poly(sodium styrene sulfonate) grafted titanium surfaces.

    Science.gov (United States)

    Felgueiras, Helena P; Aissa, Ines Ben; Evans, Margaret D M; Migonney, Véronique

    2015-11-01

    The research developed on functionalized model or prosthetic surfaces with bioactive polymers has raised the possibility to modulate and/or control the biological in vitro and in vivo responses to synthetic biomaterials. The mechanisms underlying the bioactivity exhibited by sulfonated groups on surfaces involves both selective adsorption and conformational changes of adsorbed proteins. Indeed, surfaces functionalized by grafting poly(sodium styrene sulfonate) [poly(NaSS)] modulate the cellular and bacterial response by inducing specific interactions with fibronectin (Fn). Once implanted, a biomaterial surface is exposed to a milieu of many proteins that compete for the surface which dictates the subsequent biological response. Once understood, this can be controlled by dictating exposure of active binding sites. In this in vitro study, we report the influence of binary mixtures of proteins [albumin (BSA), Fn and collagen type I (Col I)] adsorbed on poly(NaSS) grafted Ti6Al4V on the adhesion and differentiation of MC3T3-E1 osteoblast-like cells and the adhesion and proliferation of Staphylococcus aureus (S. aureus). Outcomes showed that poly(NaSS) stimulated cell spreading, attachment strength, differentiation and mineralization, whatever the nature of protein provided at the interface compared with ungrafted Ti6Al4V (control). While in competition, Fn and Col I were capable of prevailing over BSA. Fn played an important role in the early interactions of the cells with the surface, while Col I was responsible for increased alkaline phosphatase, calcium and phosphate productions associated with differentiation. Poly(NaSS) grafted surfaces decreased the adhesion of S. aureus and the presence of Fn on these chemically altered surfaces increased bacterial resistance ≈70% compared to the ungrafted Ti6Al4V. Overall, our study showed that poly(NaSS) grafted Ti6Al4V selectively adsorbed proteins (particularly Fn) promoting the adhesion and differentiation of osteoblast

  1. Nutritional Quality Assessment of Complementary Foods Produced from Fermented and Malted Quality Protein Maize Fortified with Soybean Flour

    Directory of Open Access Journals (Sweden)

    Abiose Sumbo H.

    2015-03-01

    Full Text Available Malnutrition of varying degrees has been associated with feeding infants with unwholesome and poor quality complementary foods. Therefore, the aim of this study was to produce complementary foods from quality protein maize (QPM using the processes of malting and fermentation. The resulting flour was blended with processed soy bean flour at a ratio of 70:30 (maize: soybean. The nutritional qualities of the complementary foods were assessed biologically using animal feeding experiment to determine the growth rate, feed intake, protein quality parameters, haematological properties and rehabilitation potentials. The results showed that the protein efficiency ratio (PER and food efficiency ratio of the malted QPM fortified with soybean were 2.44 and 0.24, respectively, which was the highest among the formulated diets and compared favourably with casein (2.5 and commercial diet (2.3. The QPM-based diets had a better biological value (<60% and true dig stibility (<60% than the products from normal maize. The packed cell volume of the samples ranged between 23.00 (basal and 46.00% (soy fermented normal maize. The QPM-based diets enhanced the quick recovery of protein starved/depleted animals better than the NM-based diets. Moreover, the addition of soybean further boosted the ability of the diet to rehabilitate the animals. The best result was seen in the group of rats fed with soy-malted QPM. The use of QPM in complementary food formulation gave better results and could alleviate the problem of protein and energy malnutrition, thereby reducing the mortality rate among infants.

  2. Use of in vivo-induced antigen technology (IVIAT for the identification of Streptococcus suis serotype 2 in vivo-induced bacterial protein antigens

    Directory of Open Access Journals (Sweden)

    Lu Chengping

    2009-09-01

    Full Text Available Abstract Background Streptococcus suis serotype 2 (SS2 is a zoonotic agent that causes death and disease in both humans and swine. A better understanding of SS2-host molecular interactions is crucial for understanding SS2 pathogenesis and immunology. Conventional genetic and biochemical approaches used to study SS2 virulence factors are unable to take into account the complex and dynamic environmental stimuli associated with the infection process. In this study, in vivo-induced antigen technology (IVIAT, an immunoscreening technique, was used to identify the immunogenic bacterial proteins that are induced or upregulated in vivo during SS2 infection. Results Convalescent-phase sera from pigs infected with SS2 were pooled, adsorbed against in vitro antigens, and used to screen SS2 genomic expression libraries. Upon analysis of the identified proteins, we were able to assign a putative function to 40 of the 48 proteins. These included proteins implicated in cell envelope structure, regulation, molecule synthesis, substance and energy metabolism, transport, translation, and those with unknown functions. The in vivo-induced changes in the expression of 10 of these 40 genes were measured using real-time reverse transcription (RT-PCR, revealing that the expression of 6 of the 10 genes was upregulated in the in vivo condition. The strain distribution of these 10 genes was analyzed by PCR, and they were found in the most virulent SS2 strains. In addition, protein sequence alignments of the newly identified proteins demonstrate that three are putative virulence-associated proteins. Conclusion Collectively, our results suggest that these in vivo-induced or upregulated genes may contribute to SS2 disease development. We hypothesize that the identification of factors specifically induced or upregulated during SS2 infection will aid in our understanding of SS2 pathogenesis and may contribute to the control SS2 outbreaks. In addition, the proteins identified

  3. Investigating the BSA protein adsorption and bacterial adhesion of Al-alloy surfaces after creating a hierarchical (micro/nano) superhydrophobic structure.

    Science.gov (United States)

    Moazzam, Parisa; Razmjou, Amir; Golabi, Mohsen; Shokri, Dariush; Landarani-Isfahani, Amir

    2016-09-01

    Bacterial adhesion and subsequent biofilm formation on metals such as aluminum (Al) alloys lead to serious issues in biomedical and industrial fields from both an economical and health perspective. Here, we showed that a careful manipulation of Al surface characteristics via a facile two-steps superhydrophobic modification can provide not only biocompatibility and an ability to control protein adsorption and bacterial adhesion, but also address the issue of apparent long-term toxicity of Al-alloys. To find out the roles of surface characteristics, surface modification and protein adsorption on microbial adhesion and biofilm formation, the surfaces were systematically characterized by SEM, EDX, XPS, AFM, FTIR, water contact angle (WCA) goniometry, surface free energy (SFE) measurement, MTT, Bradford, Lowry and microtiter plate assays and also flow-cytometry and potentiostat analyses. Results showed that WCA and SFE changed from 70° to 163° and 36.3 to 0.13 mN m(-1) , respectively. The stable and durable modification led to a substantial reduction in static/dynamic BSA adsorption. The effect of such a treatment on the biofilm formation was analyzed by using three different bacteria of Pseudomonas aeruginosa, Staphylococcus epidermidis, and Staphylococcus aureus. The microtiter plate assay and flow cytometry analysis showed that the modification not only could substantially reduce the bacterial adhesion but this biofouling resistance is independent of bacterium type. An excellent cell viability after exposure of HeLa cells to waters incubated with the modified samples was observed. Finally, the corrosion rate reduced sharply from 856.6 to 0.119 MPY after superhydrophobic modifications, which is an excellent stable corrosion inhibition property. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2220-2233, 2016. PMID:27104583

  4. Profiling of bacterial cells and cell-surface proteins of plant-associated bacteria by standard analytical techniques

    Czech Academy of Sciences Publication Activity Database

    Moravcová, Dana; Horká, Marie; Šalplachta, Jiří; Kubesová, Anna; Vykydalová, Marie; Kahle, Vladislav

    Latvia: Latvian Institute of Organic Synthesis, 2011 - (Kažoka, H.). s. 94 [Nordic Separation Science Society Conference /6./. 24.09.2011-27.09.2011, Riga] R&D Projects: GA AV ČR IAAX00310701; GA MV VG20112015021 Institutional research plan: CEZ:AV0Z40310501 Keywords : bacterial profiling * Rhizobium * MALDI-TOF MS Subject RIV: CB - Analytical Chemistry, Separation http://www.nosss.eu

  5. Inhibition of Bacterial Conjugation by Phage M13 and Its Protein g3p: Quantitative Analysis and Model

    OpenAIRE

    Lin, Abraham; Derr, Julien; Villanueva, Laura; Webber, Mark Alexander; Jimenez, Jose Ignacio; Vera, Pedro; Manapat, Michael; Esvelt, Kevin Michael; Liu, David Ruchien; Chen, Irene Ann

    2011-01-01

    Conjugation is the main mode of horizontal gene transfer that spreads antibiotic resistance among bacteria. Strategies for inhibiting conjugation may be useful for preserving the effectiveness of antibiotics and preventing the emergence of bacterial strains with multiple resistances. Filamentous bacteriophages were first observed to inhibit conjugation several decades ago. Here we investigate the mechanism of inhibition and find that the primary effect on conjugation is occlusion of the conju...

  6. The structure of BVU2987 from Bacteroides vulgatus reveals a superfamily of bacterial periplasmic proteins with possible inhibitory function

    International Nuclear Information System (INIS)

    The crystal structure of the BVU2987 gene product from B. vulgatus (UniProt A6L4L1) reveals that members of the new Pfam family PF11396 (domain of unknown function; DUF2874) are similar to β-lactamase inhibitor protein and YpmB. Proteins that contain the DUF2874 domain constitute a new Pfam family PF11396. Members of this family have predominantly been identified in microbes found in the human gut and oral cavity. The crystal structure of one member of this family, BVU2987 from Bacteroides vulgatus, has been determined, revealing a β-lactamase inhibitor protein-like structure with a tandem repeat of domains. Sequence analysis and structural comparisons reveal that BVU2987 and other DUF2874 proteins are related to β-lactamase inhibitor protein, PepSY and SmpA-OmlA proteins and hence are likely to function as inhibitory proteins

  7. Optimization of culture conditions of producing bacterial cellulose utilizing starch wastewater%淀粉废水发酵产细菌纤维素发酵条件的优化

    Institute of Scientific and Technical Information of China (English)

    徐伟; 张妍; 傅徐阳

    2012-01-01

    The culture conditions of Gluconacetobacter xylinus producing bacterial cellulose utilizing corn starch wastewater(adding glucose 20g/L,corn steep liquor 40g/L,ethanol 150mL/L) were investigated through singlefactor and orthogonal tests. The suitable culture conditions were as follows.liquid level was 80mL in 250mL triangle bottle,pH4.0,inoculation volume was 9% (VN),culture temperature was 28℃ ,the the yield of bacterial cellulose reached the peak(4.41g/L) at this time. The bacterial cellulose was verified by FTIR,SEM was used to observe the surface pattern of bacterial cellulose membrane.%以玉米淀粉废水添加葡萄糖20g/L,玉米浆40班,乙醇150mL/L为发酵基质,采用单因素和正交实验设计对葡糖醋杆菌(Gluconacetobacter xylinus)发酵产细菌纤维素条件进行优化。结果表明,最佳发酵条件为:装液量80mL/250mL,pH4.0,接种量9%(V/V),温度28℃;在此条件下得到细菌纤维素产量为4.41g/L。采用傅立叶转换红外光谱FTIR验证产物为细菌纤维素,并由SEM扫描电镜观察纤维素膜表面形貌。

  8. Vimentin in Bacterial Infections.

    Science.gov (United States)

    Mak, Tim N; Brüggemann, Holger

    2016-01-01

    Despite well-studied bacterial strategies to target actin to subvert the host cell cytoskeleton, thus promoting bacterial survival, replication, and dissemination, relatively little is known about the bacterial interaction with other components of the host cell cytoskeleton, including intermediate 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 virulence factors that target vimentin to subvert its function in order to change the host cell fate in the course of a bacterial infection. PMID:27096872

  9. Vimentin in Bacterial Infections

    Directory of Open Access Journals (Sweden)

    Tim N. Mak

    2016-04-01

    Full Text Available Despite well-studied bacterial strategies to target actin to subvert the host cell cytoskeleton, thus promoting bacterial survival, replication, and dissemination, relatively little is known about the bacterial interaction with other components of the host cell cytoskeleton, including intermediate 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 virulence factors that target vimentin to subvert its function in order to change the host cell fate in the course of a bacterial infection.

  10. Structural stability of Burkholderia cenocepacia biofilms is reliant on eDNA structure and presence of a bacterial nucleic acid binding protein.

    Directory of Open Access Journals (Sweden)

    Laura A Novotny

    Full Text Available Cystic fibrosis (CF is the most common lethal inherited genetic disorder affection Caucasians. Even with medical advances, CF is life-shortening with patients typically surviving only to age 38. Infection of the CF lung by Burkholderia cenocepacia presents exceptional challenges to medical management of these patients as clinically this microbe is resistant to virtually all antibiotics, is highly transmissible and infection of CF patients with this microbe renders them ineligible for lung transplant, often the last lifesaving option. Here we have targeted two abundant components of the B. cenocepacia biofilm for immune intervention: extracellular DNA and DNABII proteins, the latter of which are bacterial nucleic acid binding proteins. Treatment of B. cenocepacia biofilms with antiserum directed at one of these DNABII proteins (integration host factor or IHF resulted in significant disruption of the biofilm. Moreover, when anti-IHF mediated destabilization of a B. cenocepacia biofilm was combined with exposure to traditional antibiotics, B. cenocepacia resident within the biofilm and thereby typically highly resistant to the action of antibiotics, were now rendered susceptible to killing. Pre-incubation of B. cenocepacia with anti-IHF serum prior to exposure to murine CF macrophages, which are normally unable to effectively degrade ingested B. cenocepacia, resulted in a statistically significant increase in killing of phagocytized B. cenocepacia. Collectively, these findings support further development of strategies that target DNABII proteins as a novel approach for treatment of CF patients, particularly those whose lungs are infected with B. cenocepacia.

  11. Role of bacterial infection in the epigenetic regulation of Wnt antagonist WIF1 by PRC2 protein EZH2

    OpenAIRE

    Roy, Badal C.; Subramaniam, Dharmalingam; Ahmed, Ishfaq; Jala, Venkatakrishna R.; Hester, Christina; Greiner, K. Allen; Haribabu, Bodduluri; Anant, Shrikant; Umar, Shahid

    2014-01-01

    The Enhancer of Zeste Homolog-2 (EZH2) represses gene transcription through histone H3 lysine-27-trimethylation (H3K27me3). Citrobacter rodentium (CR) promotes crypt hyperplasia and tumorigenesis by aberrantly regulating Wnt/β-catenin signaling. We aimed at investigating EZH2’s role in epigenetically regulating Wnt/β-catenin signaling following bacterial infection. NIH:Swiss outbred and Apc Min/+ mice were infected with CR (108cfu); BLT1−/−ApcMin/+ mice, AOM/DSS-treated mice and de-identified...

  12. Ethanol extraction requirement for purification of protein labeled with [3H]leucine in aquatic bacterial production studies

    International Nuclear Information System (INIS)

    The trichloroacetic acid (TCA)-insoluble fraction of water column bacteria labeled with [3H]leucine contained an ethanol-soluble fraction accounting for up to 44% of the label. A component of the ethanol-soluble fraction is [3H]leucine. Labeled-protein purification requires an ethanol wash step. Cold TCA can replace hot TCA for precipitation of labeled proteins

  13. Xylo-oligosaccharides and inulin affect genotoxicity and bacterial populations differently in a human colonic simulator challenged with soy protein

    DEFF Research Database (Denmark)

    Christophersen, C. T.; Petersen, Anne; Licht, Tine Rask;

    2013-01-01

    High dietary intakes of some protein sources, including soy protein, can increase colonic DNA damage in animals, whereas some carbohydrates attenuate this. We investigated whether inulin and xylo-oligosaccharides (XOS) could be protective against DNA strand breaks by adding them to a human colonic...... simulator consisting of a proximal vessel (PV) (pH 5.5) and a distal vessel (DV) (pH 6.8) inoculated with human faeces and media containing soy protein. Genotoxicity of the liquid phase and microbial population changes in the vessels were measured. Soy protein (3%) was fermented with 1% low amylose...... cornstarch for 10 day followed by soy protein with 1% XOS or 1% inulin for 10 day. Inulin did not alter genotoxicity but XOS significantly reduced PV genotoxicity and increased DV genotoxicity. Inulin and XOS significantly increased butyrate concentration in the DV but not PV. Numbers of the key butyrate...

  14. Lipid transfer proteins (nsLTPs) from barley and maize leaves are potent inhibitors of bacterial and fungal plant pathogens

    OpenAIRE

    Molina Fernández, Antonio; Segura, Ana; García Olmedo, Francisco

    1993-01-01

    Four homogeneous proteins (Cw18, Cw20, Cw21, Cw22) were isolated from etiolated barley leaves by extraction of the insoluble pellet from a Tris-HCl (pH 7.5) homogenate with 1.5 M LiCl and fractionation by reverse-phase high-performance liquid chromatography. All 4 proteins inhibited growth of the pathogen Clavibacter michiganensis subsp. sepedonicus (EC50S = 1−3 × 10−7 M) and had closely related N-terminal amino acid sequences. The complete amino acid sequences of proteins Cw18 and Cw21 were ...

  15. Identification of Potential Plants Producing Tannin-protein Complex for a-amylase as Botanical Pesticide

    Directory of Open Access Journals (Sweden)

    Asriyah Firdausi

    2013-05-01

    Full Text Available Research  on  the  development  of  botanical  pesticides  should  be developed  through  new  methods,  such  as  by  inhibiting the  activity  of  digestive enzymes  by  secondary  metabolites.  The  aim  of  this  study  was  to  identify some  of  potential  plants  as  a  source  of  tannin-protein  complexes  to  inhibitthe  activity  of  - amylase.  The  study  of  identification  of  potential  plants producing  the  active  ingredient  tannin-protein  complex  was  divided  into  three stages,  1  identification  of  potential  plants  producing  tannin,  2  isolation  of tannin-protein  complexes,  and  3  in  vitro  test  of  tannin-protein  complexes effect  of  the  -amylase activity.  Some  of  the observed  plants  were  sidaguri  leaf (Sida rhombifolia, melinjo leaf (Gnetum gnemon, gamal leaf (Gliricidia sepium,lamtoro  leaf  (Leucaena  leucocephala ,  betel  nut  (Areca  catechu ,  and  crude gambier  (Uncaria  gambir a s  a  source of  tannins  and  melinjo  seed was  used  asprotein  source.  Betel  nut  and  melinjo  seed  were  the  best  source  of  tannin-protein  complex,  tannin  content  1.77  mg  TAE/mL  with  antioxidant  activity  of  90%,the  ability  to  inhibit  the  activity  of  -amylase by  95%  with  IC 50  values  of 10 mg/mL.Key words: Tannin, protein, -amylase, botanical pesticides,Areca catechu, Gnetum gnemon.

  16. Identification of Potential Plants Producing Tannin-protein Complex for a-amylase as Botanical Pesticide

    OpenAIRE

    Asriyah Firdausi; Tri Agus Siswoyo; Soekadar Wiryadiputra

    2013-01-01

    Research  on  the  development  of  botanical  pesticides  should  be developed  through  new  methods,  such  as  by  inhibiting the  activity  of  digestive enzymes  by  secondary  metabolites.  The  aim  of  this  study  was  to  identify some  of  potential  plants  as  a  source  of  tannin-protein  complexes  to  inhibitthe  activity  of  - amylase.  The  study  of  identification  of  potential  plants producing  the  active  ingredient  tannin-protein  complex  was  divided  into  th...

  17. Antioxidant and Chelating Activity of Nontoxic Jatropha curcas L. Protein Hydrolysates Produced by In Vitro Digestion Using Pepsin and Pancreatin

    Directory of Open Access Journals (Sweden)

    Santiago Gallegos Tintoré

    2015-01-01

    Full Text Available The antioxidant and metal chelating activities in J. curcas protein hydrolysates have been determined. The hydrolysates were produced by treatment of a nontoxic genotype with the digestive enzymes pepsin and pancreatin and then were characterized by fast protein liquid chromatography and reverse phase chromatography. Peptidic fractions with higher radical scavenging activity were analysed by matrix-assisted laser desorption/ionization mass spectrometry. The antioxidant activity was determined by measuring inhibition of the oxidative degradation of β-carotene and by measuring the reactive oxygen species (ROS in Caco-2 cell cultures. Cu2+ and Fe2+ chelating activities were also determined. The hydrolysates inhibited the degradation of β-carotene and the formation of ROS in Caco-2 cells. The lower molecular weight peptidic fractions from FPLC had stronger antioxidant activity in cell cultures compared with the hydrolysates, which correlated with a higher content in antioxidant and chelating amino acids. These fractions were characterized by a large presence of peptides with different molecular masses. The hydrolysates exhibited both Cu2+ and Fe2+ chelating activity. It was concluded that J. curcas is a good source of antioxidant and metal chelating peptides, which may have a positive impact on the economic value of this crop, as a potential source of food functional components.

  18. Characterization and expression analysis of a peptidoglycan recognition protein gene, SmPGRP2 in mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge.

    Science.gov (United States)

    Zhang, Linan; Gao, Chengbin; Liu, Fengqiao; Song, Lin; Su, Baofeng; Li, Chao

    2016-09-01

    Peptidoglycan recognition receptor proteins (PGRPs), a group of pattern recognition receptors (PRRs), can recognize peptidoglycan (PGN) of the bacteria cell wall and play an important role in host immune defense against pathogen infection. They are highly structurally conserved through evolution, but with different function in innate immunity between invertebrates and vertebrates. In teleost fish, several PGRPs have been characterized recently. They have both amidase activity and bactericidal activity and are involved in indirectly killing bacteria and regulating multiple signaling pathways. However, the knowledge of PGRPs in mucosal immunity of teleost fish is still limited. In this study, we identified a PGRPs gene (SmPGRP2) of turbot and investigated its expression patterns in mucosal tissues after challenge with Gram-positive bacteria Streptococcus iniae and Gram-negative bacteria Vibrio anguillarum. Phylogenetic analysis showed the strongest relationship of turbot PGRP to halibut, which was consistent with their phylogenetic relationships. In addition, SmPGRP2 was ubiquitously expressed in turbot tissues, and constitutive expression levels were higher in classical immune tissues (including liver, spleen, and head-kidney) than mucosal tissues (intestine, gill and skin). After bacterial challenge, the expression of SmPGRP2 was induced and showed a general trend of up-regulation in mucosal tissues, except in intestine following V. anguillarum infection. These different expression patterns varied depending on both pathogen and tissue type, suggesting its distinct roles in the host immune response to bacterial pathogen. PMID:27461422

  19. Surf1, Associated with Leigh Syndrome in Humans, Is a Heme-binding Protein in Bacterial Oxidase Biogenesis*

    OpenAIRE

    Bundschuh, Freya A.; Hannappel, Achim; Anderka, Oliver; Ludwig, Bernd

    2009-01-01

    Biogenesis of mitochondrial cytochrome c oxidase (COX) relies on a large number of assembly factors, among them the transmembrane protein Surf1. The loss of human Surf1 function is associated with Leigh syndrome, a fatal neurodegenerative disorder caused by severe COX deficiency. In the bacterium Paracoccus denitrificans, two homologous proteins, Surf1c and Surf1q, were identified, which we characterize in the present study. When coexpressed in Escherichia coli together with enzymes for heme ...

  20. Complete Genome Sequence of a Gluconacetobacter hansenii ATCC 23769 Isolate, AY201, Producer of Bacterial Cellulose and Important Model Organism for the Study of Cellulose Biosynthesis

    Science.gov (United States)

    Mehta, Kalpa

    2016-01-01

    The cellulose producer and model organism used for the study of cellulose biosynthesis, Gluconacetobacter hansenii AY201, is a variant of G. hansenii ATCC 23769. We report here the complete nucleotide sequence of G. hansenii AY201, information which may be utilized to further the research into understanding the genes necessary for cellulose biosynthesis. PMID:27516506