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Sample records for 16s rrna-targeted whole-cell

  1. Influence of DNA extraction method, 16S rRNA targeted hypervariable regions, and sample origin on microbial diversity detected by 454 pyrosequencing in marine chemosynthetic ecosystems.

    Science.gov (United States)

    Cruaud, Perrine; Vigneron, Adrien; Lucchetti-Miganeh, Céline; Ciron, Pierre Emmanuel; Godfroy, Anne; Cambon-Bonavita, Marie-Anne

    2014-08-01

    Next-generation sequencing (NGS) opens up exciting possibilities for improving our knowledge of environmental microbial diversity, allowing rapid and cost-effective identification of both cultivated and uncultivated microorganisms. However, library preparation, sequencing, and analysis of the results can provide inaccurate representations of the studied community compositions. Therefore, all these steps need to be taken into account carefully. Here we evaluated the effects of DNA extraction methods, targeted 16S rRNA hypervariable regions, and sample origins on the diverse microbes detected by 454 pyrosequencing in marine cold seep and hydrothermal vent sediments. To assign the reads with enough taxonomic precision, we built a database with about 2,500 sequences from Archaea and Bacteria from deep-sea marine sediments, affiliated according to reference publications in the field. Thanks to statistical and diversity analyses as well as inference of operational taxonomic unit (OTU) networks, we show that (i) while DNA extraction methods do not seem to affect the results for some samples, they can lead to dramatic changes for others; and (ii) the choice of amplification and sequencing primers also considerably affects the microbial community detected in the samples. Thereby, very different proportions of pyrosequencing reads were obtained for some microbial lineages, such as the archaeal ANME-1, ANME-2c, and MBG-D and deltaproteobacterial subgroups. This work clearly indicates that the results from sequencing-based analyses, such as pyrosequencing, should be interpreted very carefully. Therefore, the combination of NGS with complementary approaches, such as fluorescence in situ hybridization (FISH)/catalyzed reporter deposition (CARD)-FISH or quantitative PCR (Q-PCR), would be desirable to gain a more comprehensive picture of environmental microbial communities.

  2. Differentiation of Shewanella putrefaciens and Shewanella alga on the basis of whole-cell protein profiles, ribotyping, phenotypic characterization, and 16S rRNA gene sequence analysis

    DEFF Research Database (Denmark)

    Vogel, Birte Fonnesbech; Jørgensen, K.; Christensen, H.;

    1997-01-01

    by 16S rRNA gene sequence analysis. It is concluded that the isolates must be considered two different species, S. alga and S. putrefaciens, and that most mesophilic isolates formerly identified as S. putrefaciens belong to S. alga. The ecological role and potential pathogenicity of S. alga can...

  3. Differentiation of Shewanella putrefaciens and Shewanella alga on the basis of whole-cell protein profiles, ribotyping, phenotypic characterization, and 16S rRNA gene sequence analysis

    DEFF Research Database (Denmark)

    Vogel, Birte Fonnesbech; Jørgensen, K.; Christensen, H.

    1997-01-01

    Seventy-six presumed Shewanella putrefaciens isolates from fish, oil drillings, and clinical specimens, the type strain of Shewanella putrefaciens (ATCC 8071), the type strain of Shewanella alga (IAM 14159), and the type strain of Shewanella hanedai (ATCC 33224) were compared by several typing...... defined species S. alga (U. Simidu et at., Int. J. Syst. Bacteriol. 40:331-336, 1990). Fifty-two typically psychrotolerant strains formed the other, more heterogeneous major cluster, with 43 to 47 mol% G + C. The type strain of S. putrefaciens was included in this group. The two groups were confirmed...... by 16S rRNA gene sequence analysis. It is concluded that the isolates must be considered two different species, S. alga and S. putrefaciens, and that most mesophilic isolates formerly identified as S. putrefaciens belong to S. alga. The ecological role and potential pathogenicity of S. alga can...

  4. Whole cell entrapment techniques.

    Science.gov (United States)

    Trelles, Jorge A; Rivero, Cintia W

    2013-01-01

    Microbial whole cells are efficient, ecological, and low-cost catalysts that have been successfully applied in the pharmaceutical, environmental, and alimentary industries, among others. Microorganism immobilization is a good way to carry out the bioprocess under preparative conditions. The main advantages of this methodology lie in their high operational stability, easy upstream separation and bioprocess scale-up feasibility. Cell entrapment is the most widely used technique for whole cell immobilization. This technique-in which the cells are included within a rigid network-is porous enough to allow the diffusion of substrates and products, protects the selected microorganism from the reaction medium, and has high immobilization efficiency (100 % in most cases).

  5. Identification of sulfate reducers and Syntrophobacter sp. in anaerobic granular sludge by fatty-acid biomarkers and 16S rRNA probing

    NARCIS (Netherlands)

    Oude Elferink, S.J.W.H.; Boschker, H.T.S.; Stams, A.J.M.

    1998-01-01

    The sulfate-reducing bacterial sludge population in anaerobic bioreactors, treating different types of wastewater in the presence or absence of sulfate, was evaluated by polar-lipid fatty acid (PLFA) analyses, and by 16S rRNA dot-blot hybridizations using specific 16S rRNA- targeted oligonucleotide

  6. Identification of sulfate reducers and Syntrophobacter sp. in anaerobic granular sludge by fatty-acid biomarkers and 16S rRNA probing

    NARCIS (Netherlands)

    Oude Elferink, S.J.W.H.; Boschker, H.T.S.; Stams, A.J.M.

    1998-01-01

    The sulfate-reducing bacterial sludge population in anaerobic bioreactors, treating different types of wastewater in the presence or absence of sulfate, was evaluated by polar-lipid fatty acid (PLFA) analyses, and by 16S rRNA dot-blot hybridizations using specific 16S rRNA- targeted oligonucleotide

  7. 16S rRNA beacons for bacterial monitoring during human space missions.

    Science.gov (United States)

    Larios-Sanz, Maia; Kourentzi, Katerina D; Warmflash, David; Jones, Jeffrey; Pierson, Duane L; Willson, Richard C; Fox, George E

    2007-04-01

    Microorganisms are unavoidable in space environments and their presence has, at times, been a source of problems. Concerns about disease during human space missions are particularly important considering the significant changes the immune system incurs during spaceflight and the history of microbial contamination aboard the Mir space station. Additionally, these contaminants may have adverse effects on instrumentation and life-support systems. A sensitive, highly specific system to detect, characterize, and monitor these microbial populations is essential. Herein we describe a monitoring approach that uses 16S rRNA targeted molecular beacons to successfully detect several specific bacterial groupings. This methodology will greatly simplify in-flight monitoring by minimizing sample handling and processing. We also address and provide solutions to target accessibility problems encountered in hybridizations that target 16S rRNA.

  8. Whole cell biotransformation for reductive amination reactions

    OpenAIRE

    Klatte, Stephanie; Lorenz, Elisabeth; Wendisch, Volker F.

    2013-01-01

    Whole cell biotransformation systems with enzyme cascading increasingly find application in biocatalysis to complement or replace established chemical synthetic routes for production of, e.g., fine chemicals. Recently, we established an Escherichia coli whole cell biotransformation system for reductive amination by coupling a transaminase and an amino acid dehydrogenase with glucose catabolism for cofactor recycling. Transformation of 2-keto-3-methylvalerate to l-isoleucine by E. coli cells w...

  9. Whole cell biotransformation for reductive amination reactions

    Science.gov (United States)

    Klatte, Stephanie; Lorenz, Elisabeth; Wendisch, Volker F

    2014-01-01

    Whole cell biotransformation systems with enzyme cascading increasingly find application in biocatalysis to complement or replace established chemical synthetic routes for production of, e.g., fine chemicals. Recently, we established an Escherichia coli whole cell biotransformation system for reductive amination by coupling a transaminase and an amino acid dehydrogenase with glucose catabolism for cofactor recycling. Transformation of 2-keto-3-methylvalerate to l-isoleucine by E. coli cells was improved by genetic engineering of glucose metabolism for improved cofactor regeneration. Here, we compare this system with different strategies for cofactor regeneration such as cascading with alcohol dehydrogenases, with alternative production hosts such as Pseudomonas species or Corynebacterium glutamicum, and with improving whole cell biotransformation systems by metabolic engineering of NADPH regeneration. PMID:24406456

  10. Detection of Borrelia-specific 16S rRNA sequence in total RNA extracted from Ixodes ricinus ticks

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    Ž. Radulović

    2010-08-01

    Full Text Available A reverse transcriptase - polymerase chain reaction based assay for Borrelia species detection in ticks was developed. The method was based on amplification of 552 nucleotide bases long sequence of 16S rRNA, targeted by Borrelia specific primers. In the present study, total RNA extracted from Ixodes ricinus ticks was used as template. The results showed higher sensitivity for Borrelia detection as compared to standard dark-field microscopy. Method specificity was confirmed by cloning and sequencing of obtained 552 base pairs long amplicons. Phylogenetic analysis of obtained sequences showed that they belong to B. lusitaniae and B. afzelii genospecies. RT-PCR based method presented in this paper could be very useful as a screening test for detecting pathogen presence, especially when in investigations is required extraction of total RNA from ticks.

  11. Conversion of polyhydroxyalkanoates to methyl crotonate using whole cells

    NARCIS (Netherlands)

    Spekreijse, J.; Holgueras Ortega, J.; Sanders, J.P.M.; Bitter, J.H.; Scott, E.L.

    2016-01-01

    Isolated polyhydroxyalkanoates (PHA) can be used to produce biobased bulk chemicals. However, isolation is complex and costly. To circumvent this, whole cells containing PHA may be used. Here, PHA containing 3-hydroxybutyrate and small amounts of 3-hydroxyvalerate was produced from wastewater and

  12. Towards a whole-cell modeling approach for synthetic biology

    Science.gov (United States)

    Purcell, Oliver; Jain, Bonny; Karr, Jonathan R.; Covert, Markus W.; Lu, Timothy K.

    2013-06-01

    Despite rapid advances over the last decade, synthetic biology lacks the predictive tools needed to enable rational design. Unlike established engineering disciplines, the engineering of synthetic gene circuits still relies heavily on experimental trial-and-error, a time-consuming and inefficient process that slows down the biological design cycle. This reliance on experimental tuning is because current modeling approaches are unable to make reliable predictions about the in vivo behavior of synthetic circuits. A major reason for this lack of predictability is that current models view circuits in isolation, ignoring the vast number of complex cellular processes that impinge on the dynamics of the synthetic circuit and vice versa. To address this problem, we present a modeling approach for the design of synthetic circuits in the context of cellular networks. Using the recently published whole-cell model of Mycoplasma genitalium, we examined the effect of adding genes into the host genome. We also investigated how codon usage correlates with gene expression and find agreement with existing experimental results. Finally, we successfully implemented a synthetic Goodwin oscillator in the whole-cell model. We provide an updated software framework for the whole-cell model that lays the foundation for the integration of whole-cell models with synthetic gene circuit models. This software framework is made freely available to the community to enable future extensions. We envision that this approach will be critical to transforming the field of synthetic biology into a rational and predictive engineering discipline.

  13. Asymmetrische Totalsynthese von 16(S)-Iloprost und 3-Oxa-16(S)-Iloprost mittels der Azoen-Strategie

    OpenAIRE

    2005-01-01

    The present dissertation describes completely stereocontrolled asymmetric syntheses of 16S-iloprost and of 16S-3-oxa-iloprost by a azoene strategy. As Ilomedin and Ventavis registrated iloprost (1:1 mixture of diastereomers at C16) serves as a drug against ischemic heart disease, peripheral vascular disease and primary pulmonary hypertension. The 16S-diastereomer is 5 to 20 times more potent than the 16R-diastereomer in inhibiting collagen-induced platelet aggregation. 3-oxa-16S-iloprost shou...

  14. Synthesis of Gold Nanoparticles Using Whole Cells of Geotrichum candidum

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    Amit Kumar Mittal

    2013-01-01

    Full Text Available The synthesis of nanoparticles with desired size and shape is an important area of research in nanotechnology. Use of biological system is an alternative approach to chemical and physical procedures for the synthesis of metal nanoparticles. An efficient environment-friendly approach for the biosynthesis of rapid and stable Gold nanoparticles (AuNPs using whole cells of Geotrichum candidum is discussed in this paper. The enzymes/proteins present in the microorganism might be responsible for the reduction of metal salts to nanoparticles. Various reaction parameters such as culture age, temperature, pH, metal salt, and cell mass concentrations were optimized. The AuNPs were characterized by UV-visible spectroscopy, dynamic light scattering (DLS, energy dispersive spectroscopy (EDS, scanning electron microscope (SEM, and Fourier transform infrared spectroscopy (FTIR. Nanoparticles were isolated by sonicating the whole cells after treatment with Tween 80. The whole cell mediated process showed the simplistic, feasible, easy to scale up, and low-cost approach for the synthesis of AuNPs.

  15. Structural insights of microbial community of Deulajhari (India hot spring using 16s-rRNA based metagenomic sequencing

    Directory of Open Access Journals (Sweden)

    Archana Singh

    2016-03-01

    Full Text Available Insights about the distribution of the microbial community prove to be the major goal of understanding microbial ecology which remains to be fully deciphered. Hot springs being hub for the thermophilic microbiota attract the attention of the microbiologists. Deulajhari hot spring cluster is located in the Angul district of Odisha. Covered within a wooded area, Deulajhari hot spring is also fed by the plant litter resulting in a relatively high amount of total organic content (TOC. For the first time, Illumina sequencing based biodiversity analysis of microbial composition is studied through amplicon metagenome sequencing of 16s rRNA targeting V3‐V4 region using metagenomic DNA from the hot spring sediment. Over 28 phyla were detected through the amplicon metagenome sequencing of which the most dominating phyla at the existing physiochemical parameters like; temperature 69 °C, pH 8.09, electroconductivity 0.025 dSm−1 and total organic carbon 0.356%, were Proteobacteria (88.12%, Bacteriodetes (10.76%, Firmicutes (0.35%, Spirochetes (0.18% and chloroflexi (0.11%. Approximately 713 species were observed at the above physiochemical parameters. The analysis of the metagenome provides the quantitative insights into microbial populations based on the sequence data in Deulajhari hot spring. Metagenome sequence is deposited to SRA database which is available at NCBI with accession no. SRX1459736.

  16. Characterization of Aeromonas strains isolated from Indian foods using rpoD gene sequencing and whole cell protein analysis.

    Science.gov (United States)

    Nagar, Vandan; Shashidhar, Ravindranath; Bandekar, Jayant R

    2013-04-01

    Aeromonas are responsible for causing gastroenteritis and extra-intestinal infections in humans. Twenty-two Aeromonas strains isolated from different food sources were re-identified up to species level using rpoD gene sequence analysis. Biochemical tests and 16S rRNA gene sequencing were insufficient to identify Aeromonas till species level. However, incorporation of additional biochemical tests lead to correct identification of 95.5 % strains up to species level. The 16S rRNA gene sequencing was useful to identify Aeromonas isolates at the genus level only. Sequences of the rpoD gene showed greater discriminatory power than 16S rRNA gene and provided conclusive discrimination of the strains for which the phenotypic species identification was uncertain. All these 22 strains were accurately identified up to species level by rpoD gene as A. salmonicida (6), A. veronii bv. veronii (4), A. caviae (3), A. hydrophila (2), A. veronii bv. sobria (2), A. jandaei (1), A. trota (1), A. sobria (1), A. allosaccharophila (1) and A. bivalvium (1). All these strains were also characterized using whole cell protein (WCP) analysis by gradient SDS-PAGE and showed different whole cell protein (WCP) profile [22-28 polypeptide bands (~10 to >97 kDa)], indicating high genetic diversity. The present work emphasizes the use of molecular methods such as rpoD gene sequencing along with comprehensive biochemical tests for the rapid and accurate identification of Aeromonas isolates till species level. The WCP profile can be subsequently used to characterize Aeromonas isolates below species level.

  17. Whole-cell fungal transformation of precursors into dyes

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    Jarosz-Wilkołazka Anna

    2010-07-01

    Full Text Available Abstract Background Chemical methods of producing dyes involve extreme temperatures and unsafe toxic compounds. Application of oxidizing enzymes obtained from fungal species, for example laccase, is an alternative to chemical synthesis of dyes. Laccase can be replaced by fungal biomass acting as a whole-cell biocatalyst with properties comparable to the isolated form of the enzyme. The application of the whole-cell system simplifies the transformation process and reduces the time required for its completion. In the present work, four fungal strains with a well-known ability to produce laccase were tested for oxidation of 17 phenolic and non-phenolic precursors into stable and non-toxic dyes. Results An agar-plate screening test of the organic precursors was carried out using four fungal strains: Trametes versicolor, Fomes fomentarius, Abortiporus biennis, and Cerrena unicolor. Out of 17 precursors, nine were transformed into coloured substances in the presence of actively growing fungal mycelium. The immobilized fungal biomass catalyzed the transformation of 1 mM benzene and naphthalene derivatives in liquid cultures yielding stable and non-toxic products with good dyeing properties. The type of fungal strain had a large influence on the absorbance of the coloured products obtained after 48-hour transformation of the selected precursors, and the most effective was Fomes fomentarius (FF25. Whole-cell transformation of AHBS (3-amino-4-hydroxybenzenesulfonic acid into a phenoxazinone dye was carried out in four different systems: in aqueous media comprising low amounts of carbon and nitrogen source, in buffer, and in distilled water. Conclusions This study demonstrated the ability of four fungal strains belonging to the ecological type of white rot fungi to transform precursors into dyes. This paper highlights the potential of fungal biomass for replacing isolated enzymes as a cheaper industrial-grade biocatalyst for the synthesis of dyes and other

  18. Classification of methanogenic bacteria by 16S ribosomal RNA characterization

    Energy Technology Data Exchange (ETDEWEB)

    Fox, G.E.; Magrum, L.J.; Balch, W.E.; Wolfe, R.S.; Woese, C.R.

    1977-10-01

    The 16S ribosomal RNAs from 10 species of methanogenic bacteria have been characterized in terms of the oligonucleotides produced by T/sub 1/ RNase digestion. Comparative analysis of these data reveals the methanogens to constitute a distinct phylogenetic group containing two major divisions. These organisms appear to be only distantly related to typical bacteria.

  19. Whole-cell biocatalysts for biodiesel fuel production.

    Science.gov (United States)

    Fukuda, H; Hama, S; Tamalampudi, S; Noda, H

    2008-12-01

    Biodiesel fuel (BDF), which refers to fatty acid alkyl esters, has attracted considerable attention as an environmentally friendly alternative fuel for diesel engines. Alkali catalysis is widely applied for the commercial production of BDF. However, enzymatic transesterification offers considerable advantages, including reducing process operations in biodiesel fuel production and an easy separation of the glycerol byproduct. The high cost of the lipase enzyme is the main obstacle for a commercially feasible enzymatic production of biodiesel fuels. To reduce enzyme associated process costs, the immobilization of fungal mycelium within biomass support particles (BSPs) as well as expression of the lipase enzyme on the surface of yeast cells has been developed to generate whole-cell biocatalysts for industrial applications.

  20. Electrochemical As(III) whole-cell based biochip sensor.

    Science.gov (United States)

    Cortés-Salazar, Fernando; Beggah, Siham; van der Meer, Jan Roelof; Girault, Hubert H

    2013-09-15

    The development of a whole-cell based sensor for arsenite detection coupling biological engineering and electrochemical techniques is presented. This strategy takes advantage of the natural Escherichia coli resistance mechanism against toxic arsenic species, such as arsenite, which consists of the selective intracellular recognition of arsenite and its pumping out from the cell. A whole-cell based biosensor can be produced by coupling the intracellular recognition of arsenite to the generation of an electrochemical signal. Hereto, E. coli was equipped with a genetic circuit in which synthesis of beta-galactosidase is under control of the arsenite-derepressable arsR-promoter. The E. coli reporter strain was filled in a microchip containing 16 independent electrochemical cells (i.e. two-electrode cell), which was then employed for analysis of tap and groundwater samples. The developed arsenic-sensitive electrochemical biochip is easy to use and outperforms state-of-the-art bacterial bioreporters assays specifically in its simplicity and response time, while keeping a very good limit of detection in tap water, i.e. 0.8ppb. Additionally, a very good linear response in the ranges of concentration tested (0.94ppb to 3.75ppb, R(2)=0.9975 and 3.75 ppb to 30ppb, R(2)=0.9991) was obtained, complying perfectly with the acceptable arsenic concentration limits defined by the World Health Organization for drinking water samples (i.e. 10ppb). Therefore, the proposed assay provides a very good alternative for the portable quantification of As (III) in water as corroborated by the analysis of natural groundwater samples from Swiss mountains, which showed a very good agreement with the results obtained by atomic absorption spectroscopy.

  1. Romanian cyprinids phylogeny based on 16S ARN mitochondrial genes

    OpenAIRE

    Luca C.; Kevorkian S.; Elvira M.; Dinischiotu A.; Costache M.

    2007-01-01

    The vertebrate mitochondrial genome has been an important model system for studying molecular evolution, organism phylogeny, and genome structure. Phylogenetic relatioships were inferred from analysis of 570 base pairs (bp) of mithocondrial DNA (mtDNA), representing a conserved region of 16S rRNA. We sequenced 13 cyprinids species and one putative outgroup (Misgurnus fossilis) from Romania. Based upon nucleotide sequence comparisons of cyprinid mitochondrial 16SRNA genes, we established the p...

  2. Genetically modified whole-cell bioreporters for environmental assessment

    Science.gov (United States)

    Xu, Tingting; Close, Dan M.; Sayler, Gary S.; Ripp, Steven

    2015-01-01

    Living whole-cell bioreporters serve as environmental biosentinels that survey their ecosystems for harmful pollutants and chemical toxicants, and in the process act as human and other higher animal proxies to pre-alert for unfavorable, damaging, or toxic conditions. Endowed with bioluminescent, fluorescent, or colorimetric signaling elements, bioreporters can provide a fast, easily measured link to chemical contaminant presence, bioavailability, and toxicity relative to a living system. Though well tested in the confines of the laboratory, real-world applications of bioreporters are limited. In this review, we will consider bioreporter technologies that have evolved from the laboratory towards true environmental applications, and discuss their merits as well as crucial advancements that still require adoption for more widespread utilization. Although the vast majority of environmental monitoring strategies rely upon bioreporters constructed from bacteria, we will also examine environmental biosensing through the use of less conventional eukaryotic-based bioreporters, whose chemical signaling capacity facilitates a more human-relevant link to toxicity and health-related consequences. PMID:26594130

  3. Affinity of ribosomal protein S8 from mesophilic and (hyper)thermophilic archaea and bacteria for 16S rRNA correlates with the growth temperatures of the organisms.

    Science.gov (United States)

    Gruber, Thomas; Köhrer, Caroline; Lung, Birgit; Shcherbakov, Dmitri; Piendl, Wolfgang

    2003-08-14

    The ribosomal protein S8 plays a pivotal role in the assembly of the 30S ribosomal subunit. Using filter binding assays, S8 proteins from mesophilic, and (hyper)thermophilic species of the archaeal genus Methanococcus and from the bacteria Escherichia coli and Thermus thermophilus were tested for their affinity to their specific 16S rRNA target site. S8 proteins from hyperthermophiles exhibit a 100-fold and S8 from thermophiles exhibit a 10-fold higher affinity than their mesophilic counterparts. Thus, there is a striking correlation of affinity of S8 proteins for their specific RNA binding site and the optimal growth temperatures of the respective organisms. The stability of individual rRNA-protein complexes might modulate the stability of the ribosome, providing a maximum of thermostability and flexibility at the growth temperature of the organism.

  4. Accuracy of Conventional PCR Targeting the 16S rRNA Gene with the Ot-16sRF1 and Ot-16sRR1 Primers for Diagnosis of Scrub Typhus: a Case-Control Study.

    Science.gov (United States)

    Kim, Choon-Mee; Cho, Min Keun; Kim, Dong-Min; Yun, Na-Ra; Kim, Seok Won; Jang, Sook Jin; Ahn, Young-Joon; Lim, Donghoon

    2016-01-01

    We retrospectively evaluated the accuracy of conventional PCR targeting the 16S rRNA gene (16S C-PCR) using the Ot-16sRF1/Ot-16sRR1 primers for diagnosing scrub typhus. The diagnosis of Orientia tsutsugamushi infection by 16S C-PCR presented an increased sensitivity of 87.0% and specificity of 100% compared with those obtained with other targets and is thus a simple and clinically useful method with good diagnostic accuracy.

  5. High throughput 16S rRNA gene amplicon sequencing

    DEFF Research Database (Denmark)

    Nierychlo, Marta; Larsen, Poul; Jørgensen, Mads Koustrup

    S rRNA gene amplicon sequencing has been developed over the past few years and is now ready to use for more comprehensive studies related to plant operation and optimization thanks to short analysis time, low cost, high throughput, and high taxonomic resolution. In this study we show how 16S r...... to the presence of filamentous microorganisms was monitored weekly over 4 months. Microthrix was identified as a causative filament and suitable control measures were introduced. The level of Microthrix was reduced after 1-2 months but a number of other filamentous species were still present, with most of them...

  6. Whole-cell bioluminescent bioreporter sensing of foodborne toxicants

    Science.gov (United States)

    Ripp, Steve A.; Applegate, Bruce M.; Simpson, Michael L.; Sayler, Gary S.

    2001-03-01

    The presence of biologically derived toxins in foods is of utmost significance to food safety and human health concerns. Biologically active amines, referred to as biogenic amines, serve as a noteworthy example, having been implicated as the causative agent in numerous food poisoning episodes. Of the various biogenic amines encountered, histamine, putrescine, cadaverine, tyramine, tryptamine, beta-phenylethylamine, spermine, and spermidine are considered to be the most significant, and can be used as hygienic-quality indicators of food. Biogenic amines can be monitored using whole-cell bioluminescent bioreporters, which represent a family of genetically engineered microorganisms that generate visible light in response to specific chemical or physical agents in their environment. The light response occurs due to transcriptional activation of a genetically incorporated lux cassette, and can be measured using standard photomultiplier devices. We have successfully engineered a lux-based bioreporter capable of detecting and monitoring the biogenic amine beta-phenylethylamine. This research represents a biologically-based sensor technology that can be readily integrated into Hazard Analysis Critical Control Point programs to provide a rugged monitoring regime that can be uniformly applied for field-based and in-house laboratory quality control analyses. Since the bioreporter and biosensing elements are completely self-contained within the sensor design, this system provides ease of use, with operational capabilities realized by simply combining the food sample with the bioreporter and allowing the sensor to process the ensuing bioluminescent signal and communicate the results. The application of this technology to the critically important issue of food safety and hygienic quality represents a novel method for detecting, monitoring, and preventing biologically active toxins in food commodities.

  7. Full L1-regularized Traction Force Microscopy over whole cells.

    Science.gov (United States)

    Suñé-Auñón, Alejandro; Jorge-Peñas, Alvaro; Aguilar-Cuenca, Rocío; Vicente-Manzanares, Miguel; Van Oosterwyck, Hans; Muñoz-Barrutia, Arrate

    2017-08-10

    Traction Force Microscopy (TFM) is a widespread technique to estimate the tractions that cells exert on the surrounding substrate. To recover the tractions, it is necessary to solve an inverse problem, which is ill-posed and needs regularization to make the solution stable. The typical regularization scheme is given by the minimization of a cost functional, which is divided in two terms: the error present in the data or data fidelity term; and the regularization or penalty term. The classical approach is to use zero-order Tikhonov or L2-regularization, which uses the L2-norm for both terms in the cost function. Recently, some studies have demonstrated an improved performance using L1-regularization (L1-norm in the penalty term) related to an increase in the spatial resolution and sensitivity of the recovered traction field. In this manuscript, we present a comparison between the previous two regularization schemes (relying in the L2-norm for the data fidelity term) and the full L1-regularization (using the L1-norm for both terms in the cost function) for synthetic and real data. Our results reveal that L1-regularizations give an improved spatial resolution (more important for full L1-regularization) and a reduction in the background noise with respect to the classical zero-order Tikhonov regularization. In addition, we present an approximation, which makes feasible the recovery of cellular tractions over whole cells on typical full-size microscope images when working in the spatial domain. The proposed full L1-regularization improves the sensitivity to recover small stress footprints. Moreover, the proposed method has been validated to work on full-field microscopy images of real cells, what certainly demonstrates it is a promising tool for biological applications.

  8. Summary of the DREAM8 Parameter Estimation Challenge: Toward Parameter Identification for Whole-Cell Models.

    Directory of Open Access Journals (Sweden)

    Jonathan R Karr

    2015-05-01

    Full Text Available Whole-cell models that explicitly represent all cellular components at the molecular level have the potential to predict phenotype from genotype. However, even for simple bacteria, whole-cell models will contain thousands of parameters, many of which are poorly characterized or unknown. New algorithms are needed to estimate these parameters and enable researchers to build increasingly comprehensive models. We organized the Dialogue for Reverse Engineering Assessments and Methods (DREAM 8 Whole-Cell Parameter Estimation Challenge to develop new parameter estimation algorithms for whole-cell models. We asked participants to identify a subset of parameters of a whole-cell model given the model's structure and in silico "experimental" data. Here we describe the challenge, the best performing methods, and new insights into the identifiability of whole-cell models. We also describe several valuable lessons we learned toward improving future challenges. Going forward, we believe that collaborative efforts supported by inexpensive cloud computing have the potential to solve whole-cell model parameter estimation.

  9. Analysis of 16S rDNA Sequence and DNA朌NA Hybridization of Rhizobia Isolated from Indigofera sp.%木蓝根瘤菌的16S rDNA全序列分析及DNA朌NA杂交

    Institute of Scientific and Technical Information of China (English)

    韦革宏; 陈文新; 朱铭莪

    2000-01-01

    通过数值分类、SDS-全细胞蛋白电泳分析,对分离自西北黄土高原地区的木蓝根瘤菌进行了研究,获得了1个新类群。在此基础上,进行了中心菌株SHL042的16S rDNA全序列分析,得到系统发育树状图。SHL042与R.tropici A、R.tropici B、R. leguminosarum、R. etli、R. hananesis、R. mongolense和R. gallicum构成一个发育分支,其与这些种模式菌株16S rDNA全序列的相似性分别为95.4%、95.5%、96.3%、95.8%、96.3%、97.9%和97.7%,均大于95%,应属于同一个属。新类群群内DNA同源性大于80%,而中心菌株SHL042与分支内各已知种的DNA同源性小于50%,表明SHL042代表1个新的根瘤菌菌种。%Based on the previous studies on numerical taxonomy and SDS-PAGE of whole-cell protein, the rhizobia strains isolated from Indigofera sp. in loess plateau area of North-west China constituted a new cluster, the 16S rDNA sequence of representative strain SHL042 was tested, and the phylogenetic tree was produced. In this tree, the strain SHL042, R. tropici A, R. tropici B, R. leguminosarum, R. etli, R.hananesis, R. mongolense and R. gallicum constituted a branch of phylogenetic. Within this branch, the similarity values of 16S rDNA sequence were 95.4%, 95.5%,96.3%,95.8%,96.3%,97.9% and 97.7% respectively. The values were more than 95%. This indicated that these species should belong to the same genus. The values of DNA homology in the new cluster were all more than 80%, but the values between SHL042 and the strains of these species were less than 50%. Thus, the strain SH714 represented a new rhizobia species.

  10. Characteristic archaebacterial 16S rRNA oligonucleotides

    Science.gov (United States)

    McGill, T. J.; Jurka, J.; Sobieski, J. M.; Pickett, M. H.; Woese, C. R.; Fox, G. E.

    1986-01-01

    A method of analyzing 16S rRNA catalog data has been developed in which groupings at various taxonomic levels can be characterized in terms of specific "signature" oligonucleotides. This approach provides an alternative means for evaluating higher order branching possibilities and can be used to assess the phylogenetic position of isolates that are poorly placed by the usual clustering procedures. This signature approach has been applied to forty archaebacterial catalogs and every oligonucleotide with significant signature value has been identified. Sets of specific oligonucleotides were identified for every major group on a dendrogram produced by cluster analysis procedures. Signatures that would establish between group relationships were also sought and found. In the case of the Methanobacteriaceae the clustering methods suggest a specific relationship to the Methanococcaceae. This inclusion is in fact supported by six strong signature oligonucleotides. However there are also significant numbers of signature oligonucleotides supporting a specific relationship of the Methanobacteriaceae to either the Halobacteriaceae or the Methanomicrobiaceae. Thus the placement of the Methanobacteriaceae is less certain than the usual dendrograms imply. The signature approach also was used to assess the phylogenetic position of Thermoplasma acidophilum which is found to be more closely related to the methanogen/halophile Division than to the sulfur dependent Division of the archaebacteria. This does not imply however that Thermoplasma acidophilum is properly regarded as being in the methanogen/halophile Division.

  11. Incompatibility of lyophilized inactivated polio vaccine with liquid pentavalent whole-cell-pertussis-containing vaccine

    NARCIS (Netherlands)

    Kraan, H.; Have, Ten R.; Maas, van der L.; Kersten, G.F.A.; Amorij, J.P.

    2016-01-01

    A hexavalent vaccine containing diphtheria toxoid, tetanus toxoid, whole cell pertussis, Haemophilius influenza type B, hepatitis B and inactivated polio vaccine (IPV) may: (i) increase the efficiency of vaccination campaigns, (ii) reduce the number of injections thereby reducing needlestick injurie

  12. Risk of Brain Damage Following Pertussis Immunization with Whole-Cell cf Acellular Vaccines

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap

    2004-06-01

    Full Text Available Serious neurological disorders reported following whole-cell (WC in comparison to acellular (AC pertussis vaccines (PV were evaluated by the Genetic Centers of America, Silver Spring, MD.

  13. Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity

    Institute of Scientific and Technical Information of China (English)

    Mei-ling CHEN; Qin GUO; Rui-zhi WANG; Juan XU; Chen-wei ZHOU; Hui RUAN; Guo-qing HE

    2011-01-01

    Surface display is effectively utilized to construct a whole-cell biocatalyst.Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast.Here,the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae,and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor,recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed.Compared with the wild-type ROL-displaying yeast,the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate.To our knowledge,this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction.Consequently,the yeast whole-cell ROL biocatalyst was constructed with high activity.The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 ℃.Furthermore,this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.

  14. Experimental Conditions: SE16_S10_M03_D01 [Metabolonote[Archive

    Lifescience Database Archive (English)

    Full Text Available SE16_S10_M03_D01 SE16 Effect of agricultural films for spinach leaf metabolites 2 SE16_S10... Spinacia oleracea Kurohaminstarland Leaf SE16_S10_M03 6.7mg [MassBase ID] MDLC1_35977 SE16_MS1 LC-FT

  15. Signal amelioration of electrophoretically deposited whole-cell biosensors using external electric fields

    Energy Technology Data Exchange (ETDEWEB)

    Ben-Yoav, Hadar, E-mail: benyoav@post.tau.ac.il [Department of Physical Electronics, School of Electrical Engineering, Faculty of Engineering, Tel Aviv University, Tel-Aviv 69978 (Israel); Amzel, Tal [Department of Physical Electronics, School of Electrical Engineering, Faculty of Engineering, Tel Aviv University, Tel-Aviv 69978 (Israel); Sternheim, Marek [Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel-Aviv, 69978 (Israel); Belkin, Shimshon [Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, 91904 (Israel); Rubin, Adi [Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel Aviv University, Tel-Aviv, 69978 (Israel); Shacham-Diamand, Yosi [Department of Physical Electronics, School of Electrical Engineering, Faculty of Engineering, Tel Aviv University, Tel-Aviv 69978 (Israel); Freeman, Amihay [Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel-Aviv, 69978 (Israel)

    2011-11-01

    Highlights: > We present an electrochemical whole-cell biochip that can apply electric fields. > We examine the integration of cells on a biochip using electrophoretic deposition. > The effect of electric fields on the whole-cell biosensor has been demonstrated. > Relatively short DC electric pulse improves the performance of whole-cell biosensors. > Prolonged AC electric fields deteriorated the whole-cell biosensor performance. - Abstract: This paper presents an integrated whole-cell biochip system where functioning cells are deposited on the solid micro-machined surfaces while specially designed indium tin oxide electrodes that can be used to apply controllable electric fields during various stages; for example during cell deposition. The electrodes can be used also for sensing currents associated with the sensing mechanisms of electrochemical whole-cell biosensors. In this work a new approach integrating live bacterial cells on a biochip using electrophoretic deposition is presented. The biomaterial deposition technique was characterized under various driving potentials and chamber configurations. An analytical model of the electrophoretic deposition kinetics was developed and presented here. The deposited biomass included genetically engineered bacterial cells that may respond to toxic material exposure by expressing proteins that react with specific analytes generating electrochemically active byproducts. In this study the effect of external electric fields on the whole-cell biochips has been successfully developed and tested. The research hypothesis was that by applying electric fields on bacterial whole-cells, their permeability to the penetration of external analytes can be increased. This effect was tested and the results are shown here. The effect of prolonged and short external electric fields on the bioelectrochemical signal generated by sessile bacterial whole-cells in response to the presence of toxins was studied. It was demonstrated that relatively

  16. Contraception for the under 16s: better safe than sorry.

    Science.gov (United States)

    Cook, A

    1981-09-16

    acceptible if the couple was engaged, and 5.4% were totally against it, 9) 62% felt abortion was the right of every woman and 31.1% felt it was acceptible if the physical or mental well being of the mother was at risk, 10) 40.9% agreed with the British Medical Association policy on teenage contraception which advises doctors to encourage under 16's to tell their parents, but if they refuse, the doctor can still prescribe the pill, 11) 22.7% wanted contraception unconditionally available, 18.2% felt it should be dependent on parental knowledge, and 17% said it should not be available, 12) there was a trend for opinions to become less liberal as age increased, and 13) young girls appear to be less conscientious in using contraception than older women.

  17. Detection of the new cosmopolitan genus Thermoleptolyngbya (Cyanobacteria, Leptolyngbyaceae) using the 16S rRNA gene and 16S-23S ITS region.

    Science.gov (United States)

    Sciuto, Katia; Moro, Isabella

    2016-12-01

    Cyanobacteria are widespread prokaryotes that are able to live in extreme conditions such as thermal springs. Strains attributable to the genus Leptolyngbya are among the most common cyanobacteria sampled from thermal environments. Leptolyngbya is a character-poor taxon that was demonstrated to be polyphyletic based on molecular analyses. The recent joining of 16S rRNA gene phylogenies with 16S-23S ITS secondary structure analysis is a useful approach to detect new cryptic taxa and has led to the separation of new genera from Leptolyngbya and to the description of new species inside this genus and in other related groups. In this study, phylogenetic investigations based on both the 16S rRNA gene and the 16S-23S ITS region were performed alongside 16S rRNA and 16S-23S ITS secondary structure analyses on cyanobacteria of the family Leptolyngbyaceae. These analyses focused on filamentous strains sampled from thermal springs with a morphology ascribable to the genus Leptolyngbya. The phylogenetic reconstructions showed that the Leptolyngbya-like thermal strains grouped into a monophyletic lineage that was distinct from Leptolyngbya. The 16S-23S ITS secondary structure results supported the separation of this cluster. A new genus named Thermoleptolyngbya was erected to encompass these strains, and two species were described inside this new taxon: T. albertanoae and T. oregonensis.

  18. Environmental sensing of heavy metals through whole cell microbial biosensors: a synthetic biology approach.

    Science.gov (United States)

    Bereza-Malcolm, Lara Tess; Mann, Gülay; Franks, Ashley Edwin

    2015-05-15

    Whole cell microbial biosensors are offering an alternative means for rapid, on-site heavy metal detection. Based in microorganisms, biosensing constructs are designed and constructed to produce both qualitative and quantitative outputs in response to heavy metal ions. Previous microbial biosensors designs are focused on single-input constructs; however, development of multiplexed systems is resulting in more flexible designs. The movement of microbial biosensors from laboratory based designs toward on-site, functioning heavy metal detectors has been hindered by the toxic nature of heavy metals, along with the lack of specificity of heavy metals promoter elements. Applying a synthetic biology approach with alternative microbial chassis may increase the robustness of microbial biosensors and mitigate these issues. Before full applications are achieved, further consideration has to be made regarding the risk and regulations of whole cell microbial biosensor use in the environment. To this end, a standard framework for future whole cell microbial biosensor design and use is proposed.

  19. Whole-Cell Fluorescent Biosensors for Bioavailability and Biodegradation of Polychlorinated Biphenyls

    Directory of Open Access Journals (Sweden)

    David Ryan

    2010-02-01

    Full Text Available Whole-cell microbial biosensors are one of the newest molecular tools used in environmental monitoring. Such biosensors are constructed through fusing a reporter gene such as lux, gfp or lacZ,to a responsive promoter. There have been many reports of the applications of biosensors, particularly their use in assaying pollutant toxicity and bioavailability. This paper reviews the basic concepts behind the construction of whole-cell microbial biosensors for pollutant monitoring, and describes the applications of two such biosensors for detecting the bioavailability and biodegradation of Polychlorinated Biphenyls (PCBs.

  20. Whole-cell recordings of calcium and potassium currents in acutely isolated smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    Qing Cai; Zhong-Liang Zhu; Xiao-Li Fan

    2006-01-01

    AIM: To record calcium and potassium currents in acutely isolated smooth muscle cells of mesenteric arterial branches in rats.METHODS: Smooth muscle cells were freshly isolated by collagenase digest and mechanical trituration with polished pipettes. Patch clamp technique in whole-cell mode was employed to record calcium and potassium currents.RESULTS: The procedure dissociated smooth muscle cells without impairing the electrophysiological characteristics of the cells. The voltage-gated Ca2+ and potassium currents were successfully recorded using whole-cell patch clamp configuration.CONCLUSION: The method dissociates smooth muscle cells from rat mesenteric arterial branches. Voltage-gated channel currents can be recorded in this preparation.

  1. Genetic analysis on 16S rDNA of brucella%布鲁菌16S rDNA基因遗传分析

    Institute of Scientific and Technical Information of China (English)

    李克诚; 周邦谣; 夏菲

    2013-01-01

    目的 通过分析临床分离的布鲁菌、其他盐杆菌科细菌以及临床常见致病菌的16S rDNA基因片段的差异并构建16S rDNA系统发育树,为进一步研究布鲁菌打下基础.方法 PCR扩增临床分离株的16SrDNA并测序;从EMBL下载常见盐杆菌科细菌和临床上常见致病菌的16S rDNA序列.利用CLUSTALX和MEGA程序进行16S rDNA比对并构建系统发育树.结果 成功扩增了临床分离菌株的16S rDNA,得到测序结果,比对临床分离菌株和相关菌株后,发现了特异序列5’-ATCCCGGTCGCGGTTAGTGG-3';系统发育树表明不同种的布鲁菌间的距离非常接近;布鲁菌和醋菌属的进化距离较近,但是和其他的盐杆菌的进化距离较远.结论 在布鲁菌16S rDNA中存在特异序列5'-ATCCCGGTCGCGGTTAGTGG-3’,有可能作为探针来快速检测布鲁氏菌.%Objective To analyze and to compare the genetic characteristics of 16S rDNA gene of Brucella with other halobacteriaceae and pathogenic bacteria, and to construct phylogenetic tree to find the specific sequence. Methods 16s rDNA of Brucella isolated from clinical sample was amplified and sequenced, which was then compared with sequences of halobacteriaceae and other pathogenic bacteria downloaded from EM-BL. CLUSTALA and MEGA software were used for the comparison of the sequences and building of the phylogenetic tree. Results 16s rDNA was successfully amplified and sequenced. Meanwhile, a specific sequence of 5' -ATCCCGGTCGCGGTTAGTGG-3' was identified. Phylogenetic tree showed that the distance was very close among different species of Brucella and was also closer to acetobacter sp. . but was farther to other halobacteriaceae. Conclusions A specific sequence is present in 16S rDNA of brucella which could be used as a probe to detect Brucella.

  2. Visually guided whole cell patch clamp of mouse supraoptic nucleus neurons in cultured and acute conditions.

    Science.gov (United States)

    Stachniak, Tevye J E; Bourque, Charles W

    2006-07-01

    Recent advances in neuronal culturing techniques have supplied a new set of tools for studying neural tissue, providing effective means to study molecular aspects of regulatory elements in the supraoptic nucleus of the hypothalamus (SON). To combine molecular biology techniques with electrophysiological recording, we modified an organotypic culture protocol to permit transfection and whole cell patch-clamp recordings from SON cells. Neonatal mouse brain coronal sections containing the SON were dissected out, placed on a filter insert in culture medium, and incubated for at least 4 days to allow attachment to the insert. The SON was identifiable using gross anatomical landmarks, which remained intact throughout the culturing period. Immunohistochemical staining identified both vasopressinergic and oxytocinergic cells present in the cultures, typically appearing in well-defined clusters. Whole cell recordings from these cultures demonstrated that certain properties of the neonatal mouse SON were comparable to adult mouse magnocellular neurons. SON neurons in both neonatal cultures and acute adult slices showed similar sustained outward rectification above -60 mV and action potential broadening during evoked activity. Membrane potential, input resistance, and rapidly inactivating potassium current density (IA) were reduced in the cultures, whereas whole cell capacitance and spontaneous synaptic excitation were increased, perhaps reflecting developmental changes in cell physiology that warrant further study. The use of the outlined organotypic culturing procedures will allow the study of such electrophysiological properties of mouse SON using whole cell patch-clamp, in addition to various molecular, techniques that require longer incubation times.

  3. Whole cell fatty acid analysis as a tool for classification of phytopathogenic pseudomonas bacteria.

    NARCIS (Netherlands)

    Janse, J.D.

    1992-01-01

    In this thesis some members of the plant pathogenic bacterial genus Pseudomonas have been studied. Conventional morphological, biochemical, physiological and pathogenitcity tests as well as a 'finger-print' technique, viz. automated whole cell fatty acid analysis, were used. The taxonomy of the plan

  4. Lower risk of atopic disorders in whole cell pertussis-vaccinated children

    NARCIS (Netherlands)

    R.M.D. Bernsen (Roos); J.C. de Jongste (Johan); J.C. van der Wouden (Hans)

    2003-01-01

    textabstractThis study addressed whether whole cell pertussis-vaccinated children have a different risk of atopic disorders compared with children who did not receive this vaccination. Data on vaccination status, atopic disorders and child and family characteristics of the children

  5. A flow cytometer-based whole cell screening toolbox for directed hydrolase evolution through fluorescent hydrogels.

    Science.gov (United States)

    Lülsdorf, Nina; Pitzler, Christian; Biggel, Michael; Martinez, Ronny; Vojcic, Ljubica; Schwaneberg, Ulrich

    2015-05-21

    A high throughput whole cell flow cytometer screening toolbox was developed and validated by identifying improved variants (1.3-7-fold) for three hydrolases (esterase, lipase, cellulase). The screening principle is based on coupled enzymatic reaction using glucose derivatives which yield upon hydrolysis a fluorescent-hydrogel-layer on the surface of E. coli cells.

  6. Quantification of bioavailable chlortetracycline in pig feces using a bacterial whole-cell biosensor

    DEFF Research Database (Denmark)

    Hansen, Lars Hestbjerg; Aarestrup, Frank; Sørensen, Søren Johannes

    2002-01-01

    Bacterial whole-cell biosensors were used to measure the concentration of chlortetracycline (CTC) in the feces of pigs. In this study, the Escherichia coli biosensor used has a detection limit of 0.03 mg/kg CTC in pig feces. The tetracycline concentration was correlated with the appearance...

  7. Quantification of bioavailable chlortetracycline in pig feces using a bacterial whole-cell biosensor

    DEFF Research Database (Denmark)

    Hansen, L. H.; Aarestrup, Frank Møller; Sørensen, S. J.

    2002-01-01

    Bacterial whole-cell biosensors were used to measure the concentration of chlortetracycline (CTC) in the feces of pigs. In this study, the Escherichia coli biosensor used has a detection limit of 0.03 mg/kg CTC in pig feces. The tetracycline concentration was correlated with the appearance...

  8. Whole cell fatty acid analysis as a tool for classification of phytopathogenic pseudomonas bacteria

    NARCIS (Netherlands)

    Janse, J.D.

    1992-01-01

    In this thesis some members of the plant pathogenic bacterial genus Pseudomonas have been studied. Conventional morphological, biochemical, physiological and pathogenitcity tests as well as a 'finger-print' technique, viz. automated whole cell fatty acid analysis, were

  9. 蜡状芽胞杆菌群16S rDNA分析%Analysis of 16S rDNA in the Bacillus cereus Group

    Institute of Scientific and Technical Information of China (English)

    邹寰; 喻子牛; 孙明

    2008-01-01

    蜡状芽胞杆菌群主要包括炭疽芽胞杆菌(Bacillus anthracis)、蜡状芽胞杆菌(Bacillus cereus)、苏云金芽胞杆菌(Bacillus thuringiensis).GenBank已有这个群的8个菌株完成了全基因组序列测定.对这8株蜡状芽胞杆菌群菌株中98条16S rDNA的序列进行相互BLAST比较,发现在同一基因组内各个16S rDNA拷贝全局相似度最低为96.47%,在不同基因组间16S rDNA局部片段比对最小相似度达到99.72%,对应片段长度也有1417 bp.这点充分说明,该群的细菌完全共用同一种16S rDNA,根据16S rDNA给细菌分类的特点,它们应该属于同一个种.在亲缘关系上,枯草芽胞杆菌离蜡状芽胞杆菌群最近.

  10. The use of 16S and 16S-23S rDNA to easily detect and differentiate common Gram-negative orchard epiphytes.

    Science.gov (United States)

    Jeng, R S; Svircev, A M; Myers, A L; Beliaeva, L; Hunter, D M; Hubbes, M

    2001-02-01

    The identification of Gram-negative pathogenic and non-pathogenic bacteria commonly isolated from an orchard phylloplane may result in a time consuming and tedious process for the plant pathologist. The paper provides a simple "one-step" protocol that uses the polymerase chain reaction (PCR) to amplify intergenic spacer regions between 16S and 23S genes and a portion of 16S gene in the prokaryotic rRNA genetic loci. Amplified 16S rDNA, and restriction fragment length polymorphisms (RFLP) following EcoRI digestion produced band patterns that readily distinguished between the plant pathogen Erwinia amylovora (causal agent of fire blight in pear and apple) and the orchard epiphyte Pantoea agglomerans (formerly E. herbicola). The amplified DNA patterns of 16S-23S spacer regions may be used to differentiate E. amylovora at the intraspecies level. Isolates of E. amylovora obtained from raspberries exhibited two major fragments while those obtained from apples showed three distinct amplified DNA bands. In addition, the size of the 16S-23S spacer region differs between Pseudomonas syringae and Pseudomonas fluorescens. The RFLP pattern generated by HaeIII digestion may be used to provide a rapid and accurate identification of these two common orchard epiphytes.

  11. Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin

    Science.gov (United States)

    Chahine, Sarah; Okafor, Darius; Ong, Ana C.; Maybank, Rosslyn; Kwak, Yoon I.; Wilson, Kerry; Zapor, Michael; Lesho, Emil; Hinkle, Mary

    2015-01-01

    16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test. PMID:26537447

  12. Rules for biocatalyst and reaction engineering to implement effective, NAD(P)H-dependent, whole cell bioreductions

    DEFF Research Database (Denmark)

    Kratzer, Regina; Woodley, John; Nidetzky, Bernd

    2015-01-01

    recycling of catalytic quantities, which is mostly accomplished by enzymatic oxidation of a cheap co-substrate. The coupled oxidoreduction can be either performed by free enzymes in solution or by whole cells. Reductase selection, the decision between cell-free and whole cell reduction system, coenzyme...

  13. Adaptive immune response to whole cell pertussis vaccine reflects vaccine quality : A possible complementation to the Pertussis Serological Potency test

    NARCIS (Netherlands)

    Hoonakker, M E; Verhagen, L M; van der Maas, L; Metz, B; Uittenbogaard, J P; van de Waterbeemd, B; van Els, C A C M; van Eden, W; Hendriksen, C F M; Sloots, A; Han, W G H

    2016-01-01

    Whole cell Bordetella pertussis (wP) vaccines are still used in many countries to protect against the respiratory disease pertussis. The potency of whole-cell pertussis vaccine lots is determined by an intracerebral challenge test (the Kendrick test). This test is criticized due to lack of immunolog

  14. Binding of helix-threading peptides to E. coli 16S ribosomal RNA and inhibition of the S15-16S complex.

    Science.gov (United States)

    Gooch, Barry D; Krishnamurthy, Malathy; Shadid, Mohammad; Beal, Peter A

    2005-12-01

    Helix-threading peptides (HTPs) constitute a new class of small molecules that bind selectively to duplex RNA structures adjacent to helix defects and project peptide functionality into the dissimilar duplex grooves. To further explore and develop the capabilities of the HTP design for binding RNA selectively, we identified helix 22 of the prokaryotic ribosomal RNA 16S as a target. This helix is a component of the binding site for the ribosomal protein S15. In addition, the S15-16S RNA interaction is important for the ordered assembly of the bacterial ribosome. Here we present the synthesis and characterization of helix-threading peptides that bind selectively to helix 22 of E. coli 16S RNA. These compounds bind helix 22 by threading intercalation placing the N termini in the minor groove and the C termini in the major groove. Binding is dependent on the presence of a highly conserved purine-rich internal loop in the RNA, whereas removal of the loop minimally affects binding of the classical intercalators ethidium bromide and methidiumpropyl-EDTAFe (MPEFe). Moreover, binding selectivity translates into selective inhibition of formation of the S15-16S complex.

  15. 海南温泉嗜热菌的16S rDNA分析%Hainan Hot Spring Thermophiles' 16S rDNA Analysis

    Institute of Scientific and Technical Information of China (English)

    吴红萍; 孟甜; 张飞官; 陈永安; 李文芳; 王丙乾; 王锐萍

    2013-01-01

    目的:确定24株海南温泉嗜热菌菌株的分类地位.方法:Blastn分析菌株16S rDNA序列同源性;邻接法构建菌株16S rDNA序列系统发育进化树并分析菌株的进化位置;Clustax比对分析菌株的相似度和进化距离.结果:菌株LY5和LY4的16S rDNA序列与Geobacillus pallidus strain B1,partial sequence(GenBank:HM030740.1)的16SrDNA序列同源性分别为98%和97%,其他菌株的16S rDNA序列与Geobacillus subterraneus,strain R-35641(GenBank:FN428689.1)的16S rDNA序列的同源性均大于96%.Clustax比对分析表明26株菌16S rDNA序列前段(1~70bp)、中段(70bp~1420bp)、后段(1420~1484bp)的相似度分别为40%、100%和60%,进化距离分析表明菌株GT7、LY4和LY5与其他菌株进化距离较远,其余菌株之间进化距离差异不明显.综上所述,初步将24株温泉嗜热菌鉴定为土芽孢杆菌属(Geobacillus sp.).结论:16S rDNA序列分析可用于温泉嗜热菌的鉴定.

  16. Cancer preventive effects of whole cell type immunization against mice Ehrlich tumors.

    Science.gov (United States)

    Aysan, Erhan; Bayrak, Omer Faruk; Aydemir, Esra; Telci, Dilek; Sahin, Fikrettin; Yardimci, Cem; Muslumanoglu, Mahmut

    2013-01-01

    Effects of whole cell type immunization on mice Ehrlich tumours were evaluated. After preliminary study, mice were divided two major groups; 1 x 1000 and 100 x 1000 live Ehrlich cell transferred major groups, each divided into four subgroups (n: 10). Study groups were immunized with Ehrlich cell lysates in 0, 3, 7, 14th days and after 30 days of last immunization, live Ehrlich cells were transferred. Mice were observed for six months and evaluated for total and cancer free days. Out of 100 x 1000 cell transferred solid type study group, all study group mean and tumour free periods were statistically longer than control groups. All 1 x 1000 Ehrlich cell transferred study groups survived significantly longer than 100 x 1000 Ehrlich cell transferred groups. Ehrlich mice tumours were prevented and survival prolonged with whole cell type immunization. Effects are related to the number of transferred tumor cells.

  17. Application of genetically engineered microbial whole-cell biosensors for combined chemosensing.

    Science.gov (United States)

    He, Wei; Yuan, Sheng; Zhong, Wen-Hui; Siddikee, Md Ashaduzzaman; Dai, Chuan-Chao

    2016-02-01

    The progress of genetically engineered microbial whole-cell biosensors for chemosensing and monitoring has been developed in the last 20 years. Those biosensors respond to target chemicals and produce output signals, which offer a simple and alternative way of assessment approaches. As actual pollution caused by human activities usually contains a combination of different chemical substances, how to employ those biosensors to accurately detect real contaminant samples and evaluate biological effects of the combined chemicals has become a realistic object of environmental researches. In this review, we outlined different types of the recent method of genetically engineered microbial whole-cell biosensors for combined chemical evaluation, epitomized their detection performance, threshold, specificity, and application progress that have been achieved up to now. We also discussed the applicability and limitations of this biosensor technology and analyzed the optimum conditions for their environmental assessment in a combined way.

  18. A whole cell biocatalyst for cellulosic ethanol production from dilute acid-pretreated corn stover hydrolyzates.

    Science.gov (United States)

    Ryu, Seunghyun; Karim, Muhammad Nazmul

    2011-08-01

    In this research, a recombinant whole cell biocatalyst was developed by expressing three cellulases from Clostridium cellulolyticum--endoglucanase (Cel5A), exoglucanase (Cel9E), and β-glucosidase--on the surface of the Escherichia coli LY01. The modified strain is identified as LY01/pRE1H-AEB. The cellulases were displayed on the surface of the cell by fusing with an anchor protein, PgsA. The developed whole cell biocatalyst was used for single-step ethanol fermentation using the phosphoric acid-swollen cellulose (PASC) and the dilute acid-pretreated corn stover. Ethanol production was 3.59 ± 0.15 g/L using 10 g/L of PASC, which corresponds to a theoretical yield of 95.4 ± 0.15%. Ethanol production was 0.30 ± 0.02 g/L when 1 g/L equivalent of glucose in the cellulosic fraction of the dilute sulfuric acid-pretreated corn stover (PCS) was fermented for 84 h. A total of 0.71 ± 0.12 g/L ethanol was produced in 48 h when the PCS was fermented in the simultaneous saccharification and co-fermentation mode using the hemicellulosic (1 g/L of total soluble sugar) and as well as the cellulosic (1 g/L of glucose equivalent) parts of PCS. In a control experiment, 0.48 g/L ethanol was obtained from 1 g/L of hemicellulosic PCS. It was concluded that the whole cell biocatalyst could convert both cellulosic and hemicellulosic substrates into ethanol in a single reactor. The developed C. cellulolyticum-E. coli whole cell biocatalyst also overcame the incompatible temperature problem of the frequently reported fungal-yeast systems.

  19. A Bi-enzymatic Whole-Cell Algal Biosensor for Monitoring Waste Water Pollutants

    OpenAIRE

    2004-01-01

    International audience; Two algal whole cells biosensors are developed to measure specific toxicity of freshwater pollutants. Both optical and conductometric biosensors are based on inhibition of algal alkaline phosphatase (AP) and esterase activities. Chlorella vulgaris cells are immobilised on a membrane placed in front of an optical fiber bundle for optical sensing or deposited on the surface of an electrode for conductometric sensing. Phosphatase activity of the biosensor is strongly inhi...

  20. Differentiation of Bacillus anthracis from Bacillus cereus by gas chromatographic whole-cell fatty acid analysis.

    OpenAIRE

    Lawrence, D.; Heitefuss, S; Seifert, H S

    1991-01-01

    Three strains of Bacillus anthracis and seven strains of Bacillus cereus were grown on complex medium and on synthetic medium. Gas chromatographic analysis of whole-cell fatty acids of strains grown on complex medium gave nearly identical fatty acid patterns. Fatty acid patterns of strains grown on synthetic medium showed a high content of branched-chain fatty acids. Significant differences between the fatty acid patterns of the two species were found. Odd iso/anteiso fatty acid ratios were a...

  1. Bright field microscopy as an alternative to whole cell fluorescence in automated analysis of macrophage images.

    Directory of Open Access Journals (Sweden)

    Jyrki Selinummi

    Full Text Available BACKGROUND: Fluorescence microscopy is the standard tool for detection and analysis of cellular phenomena. This technique, however, has a number of drawbacks such as the limited number of available fluorescent channels in microscopes, overlapping excitation and emission spectra of the stains, and phototoxicity. METHODOLOGY: We here present and validate a method to automatically detect cell population outlines directly from bright field images. By imaging samples with several focus levels forming a bright field -stack, and by measuring the intensity variations of this stack over the -dimension, we construct a new two dimensional projection image of increased contrast. With additional information for locations of each cell, such as stained nuclei, this bright field projection image can be used instead of whole cell fluorescence to locate borders of individual cells, separating touching cells, and enabling single cell analysis. Using the popular CellProfiler freeware cell image analysis software mainly targeted for fluorescence microscopy, we validate our method by automatically segmenting low contrast and rather complex shaped murine macrophage cells. SIGNIFICANCE: The proposed approach frees up a fluorescence channel, which can be used for subcellular studies. It also facilitates cell shape measurement in experiments where whole cell fluorescent staining is either not available, or is dependent on a particular experimental condition. We show that whole cell area detection results using our projected bright field images match closely to the standard approach where cell areas are localized using fluorescence, and conclude that the high contrast bright field projection image can directly replace one fluorescent channel in whole cell quantification. Matlab code for calculating the projections can be downloaded from the supplementary site: http://sites.google.com/site/brightfieldorstaining.

  2. Development of a whole cell pneumococcal vaccine: BPL inactivation, cGMP production, and stability.

    Science.gov (United States)

    Gonçalves, Viviane M; Dias, Waldely O; Campos, Ivana B; Liberman, Celia; Sbrogio-Almeida, Maria E; Silva, Eliane P; Cardoso, Celso P; Alderson, Mark; Robertson, George; Maisonneuve, Jean-François; Tate, Andrea; Anderson, Porter; Malley, Richard; Fratelli, Fernando; Leite, Luciana C C

    2014-02-19

    Pneumococcal infections impose a large burden of disease on the human population, mainly in developing countries, and the current pneumococcal vaccines offer serotype-specific protection, but do not cover all pathogenic strains, leaving populations vulnerable to disease caused by non-vaccine serotypes. The pneumococcal whole cell vaccine is a low-cost strategy based on non-capsular antigens common to all strains, inducing serotype-independent immunity. Therefore, we developed the process for the cGMP production of this cellular vaccine. Initially, three engineering runs and two cGMP runs were performed in 60-L bioreactors, demonstrating the consistency of the production process, as evaluated by the growth curves, glucose consumption and metabolite formation (lactate and acetate). Cell recovery by tangential filtration was 92 ± 13 %. We optimized the conditions for beta-propiolactone (BPL) inactivation of the bacterial suspensions, establishing a maximum cell density of OD600 between 27 and 30, with a BPL concentration of 1:4000 (v/v) at 150 rpm and 4 °C for 30 h. BPL was hydrolyzed by heating for 2h at 37 °C. The criteria and methods for quality control were defined using the engineering runs and the cGMP Lots passed all specifications. cGMP vaccine Lots displayed high potency, inducing between 80 and 90% survival in immunized mice when challenged with virulent pneumococci. Sera from mice immunized with the cGMP Lots recognized several pneumococcal proteins in the extract of encapsulated strains by Western blot. The cGMP whole cell antigen bulk and whole cell vaccine product lots were shown to be stable for up to 12 and 18 months, respectively, based upon survival assays following i.p. challenge. Our results show the consistency and stability of the cGMP whole cell pneumococcal vaccine lots and demonstrate the feasibility of production in a developing country setting.

  3. Whole-Cell Chloride Currents in Rat Astrocytes Accompany Changes in Cell Morphology

    OpenAIRE

    Lascola, Christopher D.; Kraig, Richard P.

    1996-01-01

    Astrocytes can change shape dramatically in response to increased physiological and pathological demands, yet the functional consequences of morphological change are unknown. We report the expression of Cl− currents after manipulations that alter astrocyte morphology. Whole-cell Cl− currents were elicited after (1) rounding up cells by brief exposure to trypsin; (2) converting cells from a flat polygonal to a process-bearing (stellate) morphology by exposure to serum-free Ringer’s solution; a...

  4. Whole-cell chloride currents in rat astrocytes accompany changes in cell morphology.

    Science.gov (United States)

    Lascola, C D; Kraig, R P

    1996-04-15

    Astrocytes can change shape dramatically in response to increased physiological and pathological demands, yet the functional consequences of morphological change are unknown. We report the expression of Cl- currents after manipulations that alter astrocyte morphology. Whole-cell Cl- currents were elicited after (1) rounding up cells by brief exposure to trypsin; (2) converting cells from a flat polygonal to a process-bearing (stellate) morphology by exposure to serum-free Ringer's solution; and (3) swelling cells by exposure to hypo-osmotic solution. Zero-current potentials approximated the Nernst for Cl-, and rectification usually followed that predicted by the constant-field equation. We observed heterogeneity in the activation and inactivation kinetics, as well as in the relative degree of outward versus inward rectification. Cl- conductances were inhibited by 4, 4-diisothiocyanostilbene-2,2'-disulfonic acid (200 microM) and by Zn2+ (1 mM). Whole-cell Cl- currents were not expressed in cells without structural change. We investigated whether changes in cytoskeletal actin accompanying changes in astrocytic morphology play a role in the induction of shape-dependent Cl- currents. Cytochalasins, which disrupt actin polymers by enhancing actin-ATP hydrolysis, elicited whole-cell Cl- conductances in flat, polygonal astrocytes. In stellate cells, elevated intracellular Ca2+ (2 microM), which can depolymerize actin, enhanced Cl- currents, and high intracellular ATP (5 mM), required for repolymerization, reduced Cl- currents. Modulation of Cl- current by Ca2+ and ATP was blocked by concurrent whole-cell dialysis with phalloidin and DNase, respectively. Phalloidin stabilizes actin polymers and DNase inhibits actin polymerization. Dialysis with phalloidin also prevented hypo-osmotically activated Cl- currents. These results demonstrate how the expression of astrocyte Cl- currents can be dependent on cell morphology, the structure of actin, Ca2+ homeostasis, and metabolism.

  5. Distinguishing between whole cells and cell debris using surface plasmon coupled emission (Conference Presentation)

    Science.gov (United States)

    Talukder, Muhammad A.; Menyuk, Curtis R.; Kostov, Yordan

    2017-02-01

    Distinguishing between intact cells, dead but still whole cells, and cell debris is an important but difficult task in life sciences. The most common way to identify dead cells is using a cell-impermeant DNA binding dye, such as propidium iodide. A healthy living cell has an intact cell membrane and will act as a barrier to the dye so that it cannot enter the cell. A dead cell has a compromised cell membrane, and it will allow the dye into the cell to bind to the DNA and become fluorescent. The dead cells therefore will be positive and the live cells will be negative. The dead cells later deteriorate quickly into debris. Different pieces of debris from a single cell can be incorrectly identified as separate dead cells. Although a flow cytometer can quickly perform numerous quantitative, sensitive measurements on each individual cell to determine the viability of cells within a large, heterogeneous population, it is bulky, expensive, and only large hospitals and laboratories can afford them. In this work, we show that the distance-dependent coupling of fluorophore light to surface plasmon coupled emission (SPCE) from fluorescently-labeled cells can be used to distinguish whole cells from cell debris. Once the fluorescent labels are excited by a laser, the fluorescently-labeled whole cells create two distinct intensity rings in the far-field, in contrast to fluorescently-labeled cell debris, which only creates one ring. The distinct far-field patterns can be captured by camera and used to distinguish between whole cells and cell debris.

  6. Linking flickering to waves and whole-cell oscillations in a mitochondrial network model.

    Science.gov (United States)

    Nivala, Melissa; Korge, Paavo; Nivala, Michael; Weiss, James N; Qu, Zhilin

    2011-11-02

    It has been shown that transient single mitochondrial depolarizations, known as flickers, tend to occur randomly in space and time. On the other hand, many studies have shown that mitochondrial depolarization waves and whole-cell oscillations occur under oxidative stress. How single mitochondrial flickering events and whole-cell oscillations are mechanistically linked remains unclear. In this study, we developed a Markov model of the inner membrane anion channel in which reactive-oxidative-species (ROS)-induced opening of the inner membrane anion channel causes transient mitochondrial depolarizations in a single mitochondrion that occur in a nonperiodic manner, simulating flickering. We then coupled the individual mitochondria into a network, in which flickers occur randomly and sparsely when a small number of mitochondria are in the state of high superoxide production. As the number of mitochondria in the high-superoxide-production state increases, short-lived or abortive waves due to ROS-induced ROS release coexist with flickers. When the number of mitochondria in the high-superoxide-production state reaches a critical number, recurring propagating waves are observed. The origins of the waves occur randomly in space and are self-organized as a consequence of random flickering and local synchronization. We show that at this critical state, the depolarization clusters exhibit a power-law distribution, a signature of self-organized criticality. In addition, the whole-cell mitochondrial membrane potential changes from exhibiting small random fluctuations to more periodic oscillations as the superoxide production rate increases. These simulation results may provide mechanistic insight into the transition from random mitochondrial flickering to the waves and whole-cell oscillations observed in many experimental studies.

  7. Classifying compound mechanism of action for linking whole cell phenotypes to molecular targets

    OpenAIRE

    Bourne, Christina R.; Wakeham, Nancy; Richard A. Bunce; Berlin, K. Darrell; Barrow, William W.

    2012-01-01

    Drug development programs have proven successful when performed at a whole cell level, thus incorporating solubility and permeability into the primary screen. However, linking those results to the target within the cell has been a major set-back. The Phenotype Microarray system, marketed and sold by Biolog, seeks to address this need by assessing the phenotype in combination with a variety of chemicals with known mechanism of action (MOA). We have evaluated this system for usefulness in deduc...

  8. Defining an additivity framework for mixture research in inducible whole-cell biosensors

    Science.gov (United States)

    Martin-Betancor, K.; Ritz, C.; Fernández-Piñas, F.; Leganés, F.; Rodea-Palomares, I.

    2015-01-01

    A novel additivity framework for mixture effect modelling in the context of whole cell inducible biosensors has been mathematically developed and implemented in R. The proposed method is a multivariate extension of the effective dose (EDp) concept. Specifically, the extension accounts for differential maximal effects among analytes and response inhibition beyond the maximum permissive concentrations. This allows a multivariate extension of Loewe additivity, enabling direct application in a biphasic dose-response framework. The proposed additivity definition was validated, and its applicability illustrated by studying the response of the cyanobacterial biosensor Synechococcus elongatus PCC 7942 pBG2120 to binary mixtures of Zn, Cu, Cd, Ag, Co and Hg. The novel method allowed by the first time to model complete dose-response profiles of an inducible whole cell biosensor to mixtures. In addition, the approach also allowed identification and quantification of departures from additivity (interactions) among analytes. The biosensor was found to respond in a near additive way to heavy metal mixtures except when Hg, Co and Ag were present, in which case strong interactions occurred. The method is a useful contribution for the whole cell biosensors discipline and related areas allowing to perform appropriate assessment of mixture effects in non-monotonic dose-response frameworks PMID:26606975

  9. Yeast cell surface display for lipase whole cell catalyst and its applications

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yun; Zhang, Rui; Lian, Zhongshuai; Wang, Shihui; Wright, Aaron T.

    2014-08-01

    The cell surface display technique allows for the expression of target proteins or peptides on the microbial cell surface by fusing an appropriate protein as an anchoring motif. Yeast display systems, such as Pichia pastoris, Yarowia lipolytica and Saccharomyces cerevisiae, are ideal, alternative and extensive display systems with the advantage of simple genetic manipulation and post-translational modification of expressed heterologous proteins. Engineered yeasts show high performance characteristics and variant utilizations. Herein, we comprehensively summarize the variant factors affecting lipase whole cell catalyst activity and display efficiency, including the structure and size of target proteins, screening anchor proteins, type and chain length of linkers, and the appropriate matching rules among the above-mentioned display units. Furthermore, we also address novel approaches to enhance stability and activity of recombinant lipases, such as VHb gene co-expression, multi-enzyme co-display technique, and the micro-environmental interference and self-assembly techniques. Finally, we represent the variety of applications of whole cell surface displayed lipases on yeast cells in non-aqueous phases, including synthesis of esters, PUFA enrichment, resolution of chiral drugs, organic synthesis and biofuels. We demonstrate that the lipase surface display technique is a powerful tool for functionalizing yeasts to serve as whole cell catalysts, and increasing interest is providing an impetus for broad application of this technique.

  10. A novel whole-cell biocatalyst with NAD+ regeneration for production of chiral chemicals.

    Directory of Open Access Journals (Sweden)

    Zijun Xiao

    Full Text Available BACKGROUND: The high costs of pyridine nucleotide cofactors have limited the applications of NAD(P-dependent oxidoreductases on an industrial scale. Although NAD(PH regeneration systems have been widely studied, NAD(P(+ regeneration, which is required in reactions where the oxidized form of the cofactor is used, has been less well explored, particularly in whole-cell biocatalytic processes. METHODOLOGY/PRINCIPAL FINDINGS: Simultaneous overexpression of an NAD(+ dependent enzyme and an NAD(+ regenerating enzyme (H(2O producing NADH oxidase from Lactobacillus brevis in a whole-cell biocatalyst was studied for application in the NAD(+-dependent oxidation system. The whole-cell biocatalyst with (2R,3R-2,3-butanediol dehydrogenase as the catalyzing enzyme was used to produce (3R-acetoin, (3S-acetoin and (2S,3S-2,3-butanediol. CONCLUSIONS/SIGNIFICANCE: A recombinant strain, in which an NAD(+ regeneration enzyme was coexpressed, displayed significantly higher biocatalytic efficiency in terms of the production of chiral acetoin and (2S,3S-2,3-butanediol. The application of this coexpression system to the production of other chiral chemicals could be extended by using different NAD(P-dependent dehydrogenases that require NAD(P(+ for catalysis.

  11. Phosphoglycerate kinase enhanced immunity of the whole cell of Streptococcus agalactiae in tilapia, Oreochromis niloticus.

    Science.gov (United States)

    Wang, Yi-Ting; Huang, Hsing-Yen; Tsai, Ming-An; Wang, Pei-Chi; Jiang, Bo-Huang; Chen, Shih-Chu

    2014-12-01

    Streptococcus agalactiae is a Gram-positive bacterium and a severe aquaculture pathogen that can infect a wide range of warmwater fish species. The outer-surface proteins in bacterial pathogens play an important role in pathogenesis. We evaluated the immunogenicity of two of the identified surface proteins namely phosphoglycerate kinase (PGK) and ornithine carbamoyl-transferase (OCT). PGK and OCT were over-expressed and purified from Escherichia coli and used as the subunit vaccines in tilapia. Tilapia immunized with the S. agalactiae modified bacteria vaccine (whole cell preparations with recombinant PGK and OCT proteins) individually were tested for the efficacy. OCT and PGK combined with WC had a higher survival rate. A high-level protection and significant specific antibody responses against S. agalactiae challenge was observed upon the vaccinated tilapia with the purified PGK protein and S. agalactiae whole cells. The specific antibody titer against S. agalactiae antigen suggested that increased antibody titers were correlated with post-challenge survival rate. Il-1β expression profile was higher in PGK + WC-treated group. Tnf-α expression in the PGK + WC group was significantly increased. Taken together, our results suggested the combinations of recombinant protein and whole cell may elicit immune responses that reach greater protection than that of individual S. agalactiae components.

  12. Expression of a Dianthus flavonoid glucosyltransferase in Saccharomyces cerevisiae for whole-cell biocatalysis.

    Science.gov (United States)

    Werner, Sean R; Morgan, John A

    2009-07-15

    Glycosyltransferases are promising biocatalysts for the synthesis of small molecule glycosides. In this study, Saccharomyces cerevisiae expressing a flavonoid glucosyltransferase (GT) from Dianthus caryophyllus (carnation) was investigated as a whole-cell biocatalyst. Two yeast expression systems were compared using the flavonoid naringenin as a model substrate. Under in vitro conditions, naringenin-7-O-glucoside was formed and a higher specific glucosyl transfer activity was found using a galactose inducible expression system compared to a constitutive expression system. However, S. cerevisiae expressing the GT constitutively was significantly more productive than the galactose inducible system under in vivo conditions. Interestingly, the glycosides were recovered directly from the culture broth and did not accumulate intracellularly. A previously uncharacterized naringenin glycoside formed using the D. caryophyllus GT was identified as naringenin-4'-O-glucoside. It was found that S. cerevisiae cells hydrolyze naringenin-7-O-glucoside during whole-cell biocatalysis, resulting in a low final glycoside titer. When phloretin was added as a substrate to the yeast strain expressing the GT constitutively, the natural product phlorizin was formed. This study demonstrates S. cerevisiae is a promising whole-cell biocatalyst host for the production of valuable glycosides.

  13. Study in ovo immunisation with flagellin and whole cell protein antigens of Campylobacter jejuni in chickens

    Directory of Open Access Journals (Sweden)

    Susan Maphilindawati Noor

    2000-06-01

    Full Text Available In ovo immunisation of chickens with flagellin and whole cell protein antigens of Campylobacter jejuni was examined to determine Campylobacter infection. Four groups of embryonated chicken eggs (10 eggs per group were immunised in ovo at day 17 of incubation and booster was given at 7 days post-hatch. Group I was immunised in ovo and oral booster with whole cell protein of C. jejuni, group II was immunised in ovo and oral booster with C. jejuni flagellin protein, group III was immunised in ovo and intraperitoneal booster with whole cell, and group IV was treated as control. The humoral immune responses were determined by enzyme-linked immunosorbent assay (ELISA and the mucosal immune responses were examined by a direct fluorescent histology antibody technique. Immunised chickens of Group I, II, and III shown to have higher antibody titers than those of control chickens (group IV. The titres of anti-campylobacter antibodies of all isotypes in serum, bile, and intestinal scrapping after challenge were not significantly different in all groups. In addition, when immunised chickens were orally challenged with a homologous strain of viable C. jejuni organism, the chickens remained infected throughout the experiment based on cloacal swabs and caecal contents. These findings indicated that although in ovo immunisation resulted in increasing of the mucosal and humoral immune responses in chickens, it is not strong enough to protect the Campylobacter colonisation in the intestinal tract.

  14. Target-based identification of whole-cell active inhibitors of biotin biosynthesis in Mycobacterium tuberculosis.

    Science.gov (United States)

    Park, Sae Woong; Casalena, Dominick E; Wilson, Daniel J; Dai, Ran; Nag, Partha P; Liu, Feng; Boyce, Jim P; Bittker, Joshua A; Schreiber, Stuart L; Finzel, Barry C; Schnappinger, Dirk; Aldrich, Courtney C

    2015-01-22

    Biotin biosynthesis is essential for survival and persistence of Mycobacterium tuberculosis (Mtb) in vivo. The aminotransferase BioA, which catalyzes the antepenultimate step in the biotin pathway, has been established as a promising target due to its vulnerability to chemical inhibition. We performed high-throughput screening (HTS) employing a fluorescence displacement assay and identified a diverse set of potent inhibitors including many diversity-oriented synthesis (DOS) scaffolds. To efficiently select only hits targeting biotin biosynthesis, we then deployed a whole-cell counterscreen in biotin-free and biotin-containing medium against wild-type Mtb and in parallel with isogenic bioA Mtb strains that possess differential levels of BioA expression. This counterscreen proved crucial to filter out compounds whose whole-cell activity was off target as well as identify hits with weak, but measurable whole-cell activity in BioA-depleted strains. Several of the most promising hits were cocrystallized with BioA to provide a framework for future structure-based drug design efforts.

  15. The Preparation of Bioimprinted Whole-cell Biocatalyst and Its Application in Bioconversion of Biodiesel

    Directory of Open Access Journals (Sweden)

    Meiling Chen

    2015-08-01

    Full Text Available Biodiesel has attracted considerable attention as an environmentally friendly alternative fuel. Lipase is the most popular enzyme for biodiesel production and immobilization has been deployed to improve enzyme stability and reusability. Exploitation of high activity lipase is the key point for biodiesel production. Whole-cell biocatalysts have been applied in the biosynthesis of biodiesel and bioimprinting is a promising approach for enzyme performances improvement. In this study, based on the S. cerevisiae cell-surface display system with &alpha-agglutinin as anchor, a whole-cell biocatalyst of codon-optimized Rhizopus oryzae lipase was constructed and bioimprinted with oleic acid, gaining 5-fold increase on enzymatic activity in the alcoholysis of soybean oil to biodiesel. Moreover, the conversion of FAME was up to 95.45±2.73% after a 27-h reaction at 60°C. Our results indicated that combining bioimprinting with yeast display technique to prepare whole-cell biocatalyst could result in potential enzymes for bioconversion of biodiesel in organic solvents.

  16. Synthesis of vanillic acid using whole cell nitrilase of wild and mutant Gordonia terrae.

    Science.gov (United States)

    Bhalla, Tek Chand; Prashant; Kumari, Nisha; Kumar, Vijay; Kumar, Virender; Savitri

    2016-01-01

    The resting cells of Gordonia terrae mutant E9 having enhanced nitrilase activity were used for biotransformation of 4-hydroxy-3-methoxybenzonitrile into vanillic acid. The maximum conversion was observed in 0.1 M phosphate buffer (pH 8.0), using 60 mM substrate and 0.75 mgDCW resting cells in 1 mL reaction at 40 °C. Km of the whole cell nitrilase of wild and mutant strains of G. terrae for this substrate were 20 and 16.6 mM, and Vmax were 0.19 and 0.95 Umg(-1)(DCW), respectively. Fed batch reaction for transformation of 4-hydroxy-3-methoxybenzonitrile using whole cell nitrilase of wild G. terrae resulted in 2.36 g of vanillic acid in 5 h with a catalytic and volumetric productivity of 0.78 gg(-1)(DCW) h(-1) and 4.72 gL(-1)h(-1), respectively. The whole cell nitrilase of G. terrae mutant E9 resulted in higher catalytic and volumetric productivity, i.e., 1.68 gg(-1)DCW h(-1) and 10 gL(-1)h(-1). A total 5.04 g of vanillic acid with 99% purity were accumulated in 100 mL of reaction after 5 h.

  17. Defining an additivity framework for mixture research in inducible whole-cell biosensors

    DEFF Research Database (Denmark)

    Martin-Betancor, K; Ritz, Christian; Fernández-Piñas, F

    2015-01-01

    A novel additivity framework for mixture effect modelling in the context of whole cell inducible biosensors has been mathematically developed and implemented in R. The proposed method is a multivariate extension of the effective dose (EDp) concept. Specifically, the extension accounts for differe......A novel additivity framework for mixture effect modelling in the context of whole cell inducible biosensors has been mathematically developed and implemented in R. The proposed method is a multivariate extension of the effective dose (EDp) concept. Specifically, the extension accounts...... illustrated by studying the response of the cyanobacterial biosensor Synechococcus elongatus PCC 7942 pBG2120 to binary mixtures of Zn, Cu, Cd, Ag, Co and Hg. The novel method allowed by the first time to model complete dose-response profiles of an inducible whole cell biosensor to mixtures. In addition......, the approach also allowed identification and quantification of departures from additivity (interactions) among analytes. The biosensor was found to respond in a near additive way to heavy metal mixtures except when Hg, Co and Ag were present, in which case strong interactions occurred. The method is a useful...

  18. Defining an additivity framework for mixture research in inducible whole-cell biosensors

    DEFF Research Database (Denmark)

    Martin-Betancor, K; Ritz, Christian; Fernández-Piñas, F;

    2015-01-01

    A novel additivity framework for mixture effect modelling in the context of whole cell inducible biosensors has been mathematically developed and implemented in R. The proposed method is a multivariate extension of the effective dose (EDp) concept. Specifically, the extension accounts for differe......A novel additivity framework for mixture effect modelling in the context of whole cell inducible biosensors has been mathematically developed and implemented in R. The proposed method is a multivariate extension of the effective dose (EDp) concept. Specifically, the extension accounts...... for differential maximal effects among analytes and response inhibition beyond the maximum permissive concentrations. This allows a multivariate extension of Loewe additivity, enabling direct application in a biphasic dose-response framework. The proposed additivity definition was validated, and its applicability...... illustrated by studying the response of the cyanobacterial biosensor Synechococcus elongatus PCC 7942 pBG2120 to binary mixtures of Zn, Cu, Cd, Ag, Co and Hg. The novel method allowed by the first time to model complete dose-response profiles of an inducible whole cell biosensor to mixtures. In addition...

  19. Design and construction of a whole cell bacterial 4-hydroxyphenylacetic acid and 2-phenylacetic acid bioassay

    Directory of Open Access Journals (Sweden)

    Seppe eDierckx

    2015-06-01

    Full Text Available IntroductionAuxins are hormones that regulate plant growth and development. To accurately quantify the low levels of auxins present in plant and soil samples, sensitive detection methods are needed. In this study, the design and construction of two different whole cell auxin biosensors is illustrated. Both use the auxin responsive element HpaA as an input module and differ in output module. The first biosensor is combined with the gfp gene to produce a fluorescent biosensor. Whereas the second one is combined with the genes textit and phzS to produce a pyocyanin producing biosensor whose product can be measured electrochemically.section{Results} The fluorescent biosensor is able to detect 4-hydroxyphenylacetic acid (4-HPA and 2-phenylacetic acid (PAA concentrations from 15 µM to 3 mM in a dose-responsive manner. The pyocyanin producing biosensor is able to detect 4-HPA concentrations from 1.9 µM to 15.625 µM and PAA concentrations from 15.625 µM to 125 µM, both in a dose-responsive manner.ConclusionsA fluorescent whole cell auxin biosensor and an electrochemical whole cell auxin biosensor have been constructed and tested. Both are able to detect 4-HPA and PAA concentrations that are environmentally relevant in respect to plant growth.

  20. Scaling-up of complex whole-cell bioconversions in conventional and non-conventional media.

    Science.gov (United States)

    Marques, Marco P C; de Carvalho, Carla C C R; Cabral, Joaquim M S; Fernandes, Pedro

    2010-07-01

    The use of whole cells is becoming a more common approach in pharmaceutical and agrochemical industries in order to obtain pure compounds with fewer production steps, higher yields, and cleaner processes, as compared to those achieved with traditional strategies. Whole cells are often used as enzymes pools, in particular when multi-step reactions and/or co-factor regeneration are envisaged. Nonetheless, published information on the scale-up of such systems both in aqueous and in two-phase aqueous-organic systems is relatively scarce. The present work aims to evaluate suitable scale-up criteria in conventional and non-conventional medium for a whole-cell bioconversion that uses resting cells of Mycobacterium sp. NRRL B-3805 to cleave the side chain of beta-sitosterol, a poorly water-soluble substrate. The experiments were performed in 24-well microtiter plates and in 250 mL shaken flasks as orbital stirred systems, and in 300 mL stirred tanks as mechanically stirred systems. Results show that productivity yields were similar in all scales tested, when maintaining oxygen mass transfer coefficients constant in aqueous systems, or when maintaining constant volumetric power consumption in aqueous-organic two-phase systems.

  1. Oxyfunctionalization of pyridine derivatives using whole cells of Burkholderia sp. MAK1

    Science.gov (United States)

    Stankevičiūtė, Jonita; Vaitekūnas, Justas; Petkevičius, Vytautas; Gasparavičiūtė, Renata; Tauraitė, Daiva; Meškys, Rolandas

    2016-01-01

    Pyridinols and pyridinamines are important intermediates with many applications in chemical industry. The pyridine derivatives are in great demand as synthons for pharmaceutical products. Moreover, pyridines are used either as biologically active substances or as building blocks for polymers with unique physical properties. Application of enzymes or whole cells is an attractive strategy for preparation of hydroxylated pyridines since the methods for chemical synthesis of pyridinols, particularly aminopyridinols, are usually limited or inefficient. Burkholderia sp. MAK1 (DSM102049), capable of using pyridin-2-ol as the sole carbon and energy source, was isolated from soil. Whole cells of Burkholderia sp. MAK1 were confirmed to possess a good ability to convert different pyridin-2-amines and pyridin-2-ones into their 5-hydroxy derivatives. Moreover, several methylpyridines as well as methylated pyrazines were converted to appropriate N-oxides. In conclusion, regioselective oxyfunctionalization of pyridine derivatives using whole cells of Burkholderia sp. MAK1 is a promising method for the preparation of various pyridin-5-ols and pyridin-N-oxides. PMID:27982075

  2. Tsukamurella tyrosinosolvens intravascular catheter infection identified using 16S ribosomal DNA sequencing.

    Science.gov (United States)

    Sheridan, Elizabeth A S; Warwick, Simon; Chan, Anthony; Dall'Antonia, Martino; Koliou, Maria; Sefton, Armine

    2003-03-01

    Cultures of blood from a hemodialysis line repeatedly yielded a gram-positive rod. The organism was identified as Tsukamurella tyrosinosolvens by 16S ribosomal DNA sequencing, and the patient was treated successfully by removal of the line.

  3. Probing the structure of 16 S ribosomal RNA from Bacillus brevis.

    Science.gov (United States)

    Kop, J; Kopylov, A M; Magrum, L; Siegel, R; Gupta, R; Woese, C R; Noller, H F

    1984-12-25

    A majority (approximately 89%) of the nucleotide sequence of Bacillus brevis 16 S rRNA has been determined by a combination of RNA sequencing methods. Several experimental approaches have been used to probe its structure, including (a) partial RNase digestion of 30 S ribosomal subunits, followed by two-dimensional native/denatured gel electrophoresis, in which base-paired fragments were directly identified; (b) identification of positions susceptible to cleavage by RNase A and RNase T1 in 30 S subunits; (c) sites of attack by cobra venom RNase on naked 16 S rRNA; and (d) nucleotides susceptible to attack by bisulfite in 16 S rRNA. These data are discussed with respect to a secondary structure model for B. brevis 16 S rRNA derived by comparative sequence analysis.

  4. A Comparison between Transcriptome Sequencing and 16S Metagenomics for Detection of Bacterial Pathogens in Wildlife.

    Directory of Open Access Journals (Sweden)

    Maria Razzauti

    Full Text Available Rodents are major reservoirs of pathogens responsible for numerous zoonotic diseases in humans and livestock. Assessing their microbial diversity at both the individual and population level is crucial for monitoring endemic infections and revealing microbial association patterns within reservoirs. Recently, NGS approaches have been employed to characterize microbial communities of different ecosystems. Yet, their relative efficacy has not been assessed. Here, we compared two NGS approaches, RNA-Sequencing (RNA-Seq and 16S-metagenomics, assessing their ability to survey neglected zoonotic bacteria in rodent populations.We first extracted nucleic acids from the spleens of 190 voles collected in France. RNA extracts were pooled, randomly retro-transcribed, then RNA-Seq was performed using HiSeq. Assembled bacterial sequences were assigned to the closest taxon registered in GenBank. DNA extracts were analyzed via a 16S-metagenomics approach using two sequencers: the 454 GS-FLX and the MiSeq. The V4 region of the gene coding for 16S rRNA was amplified for each sample using barcoded universal primers. Amplicons were multiplexed and processed on the distinct sequencers. The resulting datasets were de-multiplexed, and each read was processed through a pipeline to be taxonomically classified using the Ribosomal Database Project. Altogether, 45 pathogenic bacterial genera were detected. The bacteria identified by RNA-Seq were comparable to those detected by 16S-metagenomics approach processed with MiSeq (16S-MiSeq. In contrast, 21 of these pathogens went unnoticed when the 16S-metagenomics approach was processed via 454-pyrosequencing (16S-454. In addition, the 16S-metagenomics approaches revealed a high level of coinfection in bank voles.We concluded that RNA-Seq and 16S-MiSeq are equally sensitive in detecting bacteria. Although only the 16S-MiSeq method enabled identification of bacteria in each individual reservoir, with subsequent derivation of

  5. Incorporating 16S Gene Copy Number Information Improves Estimates of Microbial Diversity and Abundance

    Science.gov (United States)

    Kembel, Steven W.; Wu, Martin; Eisen, Jonathan A.; Green, Jessica L.

    2012-01-01

    The abundance of different SSU rRNA (“16S”) gene sequences in environmental samples is widely used in studies of microbial ecology as a measure of microbial community structure and diversity. However, the genomic copy number of the 16S gene varies greatly – from one in many species to up to 15 in some bacteria and to hundreds in some microbial eukaryotes. As a result of this variation the relative abundance of 16S genes in environmental samples can be attributed both to variation in the relative abundance of different organisms, and to variation in genomic 16S copy number among those organisms. Despite this fact, many studies assume that the abundance of 16S gene sequences is a surrogate measure of the relative abundance of the organisms containing those sequences. Here we present a method that uses data on sequences and genomic copy number of 16S genes along with phylogenetic placement and ancestral state estimation to estimate organismal abundances from environmental DNA sequence data. We use theory and simulations to demonstrate that 16S genomic copy number can be accurately estimated from the short reads typically obtained from high-throughput environmental sequencing of the 16S gene, and that organismal abundances in microbial communities are more strongly correlated with estimated abundances obtained from our method than with gene abundances. We re-analyze several published empirical data sets and demonstrate that the use of gene abundance versus estimated organismal abundance can lead to different inferences about community diversity and structure and the identity of the dominant taxa in microbial communities. Our approach will allow microbial ecologists to make more accurate inferences about microbial diversity and abundance based on 16S sequence data. PMID:23133348

  6. Linkage disequilibrium mapping places the gene causing familial Mediterranean fever close to D16S246

    Energy Technology Data Exchange (ETDEWEB)

    Levy, E. N.; Aksentijevich, I.; Pras, E. [National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD (United States)] [and others

    1996-03-01

    This report presents refined genetic mapping data for the gene causing familial Mediterranean fever (FMF), a recessively inherited disorder of inflammation. We sampled 65 Jewish, Armenian, and Arab families and typed them for eight markers from chromosome 16p. Using a new algorithm that permits multipoint calculations for a dense map of markers in consanguineous families, we obtained a maximal LOD score of 49.2 at a location 1.6 cM centromeric to D16S246. A specific haplotype at D16S283-D16S94-D16S246 was found in 76% of Moroccan and 32% of non-Moroccan Jewish carrier chromosomes, but this haplotype was not overrepresented in Armenian or Arab FMF carriers. Moreover, the 2.5-kb allele at D16S246 was significantly associated with FMF in Moroccan and non-Moroccan Jews but not in Armenians or Arabs. Since the Moroccan Jewish community represents a relatively recently established and genetically isolated founder population, we analyzed the Moroccan linkage-disequilibrium data by using Luria-Delbruck formulas and simulations based on a Poisson branching process. These methods place the FMF susceptibility gene within 0.305 cM of D16S246 (2-LOD-unit range 0.02-0.64 cM). 41 refs., 3 figs., 5 tabs.

  7. Comparison of two approaches for the classification of 16S rRNA gene sequences.

    Science.gov (United States)

    Chatellier, Sonia; Mugnier, Nathalie; Allard, Françoise; Bonnaud, Bertrand; Collin, Valérie; van Belkum, Alex; Veyrieras, Jean-Baptiste; Emler, Stefan

    2014-10-01

    The use of 16S rRNA gene sequences for microbial identification in clinical microbiology is accepted widely, and requires databases and algorithms. We compared a new research database containing curated 16S rRNA gene sequences in combination with the lca (lowest common ancestor) algorithm (RDB-LCA) to a commercially available 16S rDNA Centroid approach. We used 1025 bacterial isolates characterized by biochemistry, matrix-assisted laser desorption/ionization time-of-flight MS and 16S rDNA sequencing. Nearly 80 % of isolates were identified unambiguously at the species level by both classification platforms used. The remaining isolates were mostly identified correctly at the genus level due to the limited resolution of 16S rDNA sequencing. Discrepancies between both 16S rDNA platforms were due to differences in database content and the algorithm used, and could amount to up to 10.5 %. Up to 1.4 % of the analyses were found to be inconclusive. It is important to realize that despite the overall good performance of the pipelines for analysis, some inconclusive results remain that require additional in-depth analysis performed using supplementary methods.

  8. Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians

    Directory of Open Access Journals (Sweden)

    Chiari Ylenia

    2005-03-01

    Full Text Available Abstract Background Identifying species of organisms by short sequences of DNA has been in the center of ongoing discussions under the terms DNA barcoding or DNA taxonomy. A C-terminal fragment of the mitochondrial gene for cytochrome oxidase subunit I (COI has been proposed as universal marker for this purpose among animals. Results Herein we present experimental evidence that the mitochondrial 16S rRNA gene fulfills the requirements for a universal DNA barcoding marker in amphibians. In terms of universality of priming sites and identification of major vertebrate clades the studied 16S fragment is superior to COI. Amplification success was 100% for 16S in a subset of fresh and well-preserved samples of Madagascan frogs, while various combination of COI primers had lower success rates.COI priming sites showed high variability among amphibians both at the level of groups and closely related species, whereas 16S priming sites were highly conserved among vertebrates. Interspecific pairwise 16S divergences in a test group of Madagascan frogs were at a level suitable for assignment of larval stages to species (1–17%, with low degrees of pairwise haplotype divergence within populations (0–1%. Conclusion We strongly advocate the use of 16S rRNA as standard DNA barcoding marker for vertebrates to complement COI, especially if samples a priori could belong to various phylogenetically distant taxa and false negatives would constitute a major problem.

  9. Efficiency Analysis and Mechanism Insight of that Whole-Cell Biocatalytic Production of Melibiose from Raffinose with Saccharomyces cerevisiae.

    Science.gov (United States)

    Zhou, Yingbiao; Zhu, Yueming; Dai, Longhai; Men, Yan; Wu, Jinhai; Zhang, Juankun; Sun, Yuanxia

    2017-01-01

    Melibiose is widely used as a functional carbohydrate. Whole-cell biocatalytic production of melibiose from raffinose could reduce its cost. However, characteristics of strains for whole-cell biocatalysis and mechanism of such process are unclear. We compared three different Saccharomyces cerevisiae strains (liquor, wine, and baker's yeasts) in terms of concentration variations of substrate (raffinose), target product (melibiose), and by-products (fructose and galactose) in whole-cell biocatalysis process. Distinct difference was observed in whole-cell catalytic efficiency among three strains. Furthermore, activities of key enzymes (invertase, α-galactosidase, and fructose transporter) involved in process and expression levels of their coding genes (suc2, mel1, and fsy1) were investigated. Conservation of key genes in S. cerevisiae strains was also evaluated. Results show that whole-cell catalytic efficiency of S. cerevisiae in the raffinose substrate was closely related to activity of key enzymes and expression of their coding genes. Finally, we summarized characteristics of producing strain that offered advantages, as well as contributions of key genes to excellent strains. Furthermore, we presented a dynamic mechanism model to achieve some mechanism insight for this whole-cell biocatalytic process. This pioneering study should contribute to improvement of whole-cell biocatalytic production of melibiose from raffinose.

  10. Progress in Clinical Application of 16S rRNA Gene%16S rRNA基因在临床上的应用进展

    Institute of Scientific and Technical Information of China (English)

    杨祖卿; 尚世强

    2005-01-01

    传统的细菌检测主要依靠血清学、生物化学、细菌形态学及细菌培养等方法进行分类鉴定,但前三者敏感性和特异性不高,后者费时且阳性率低.近10余年来分子生物学技术发展迅速,各种基因方法如DNA杂交、质粒图谱和16S rRNA序列分析等在临床上得到广泛应用.该文就近年来国外16S rRNA在细菌学研究及其应用的一些新进展作一综述.

  11. Activity profiles for marine sponge-associated bacteria obtained by 16S rRNA vs 16S rRNA gene comparisons.

    Science.gov (United States)

    Kamke, Janine; Taylor, Michael W; Schmitt, Susanne

    2010-04-01

    The phylogenetic diversity of microorganisms in marine sponges is becoming increasingly well described, yet relatively little is known about the activities of these symbionts. Given the seemingly favourable environment provided to microbes by their sponge hosts, as indicated by the extraordinarily high abundance of sponge symbionts, we hypothesized that the majority of sponge-associated bacteria are active in situ. To test this hypothesis we compared, for the first time in sponges, 16S rRNA gene- vs 16S rRNA-derived bacterial community profiles to gain insights into symbiont composition and activity, respectively. Clone libraries revealed a highly diverse bacterial community in Ancorina alata, and a much lower diversity in Polymastia sp., which were identified by electron microscopy as a high- and a low-microbial abundance sponge, respectively. Substantial overlap between DNA and RNA libraries was evident at both phylum and phylotype levels, indicating in situ activity for a large fraction of sponge-associated bacteria. This active fraction included uncultivated, sponge-specific lineages within, for example, Actinobacteria, Chloroflexi and Gemmatimonadetes. This study shows the potential of RNA vs DNA comparisons based on the 16S rRNA gene to provide insights into the activity of sponge-associated microorganisms.

  12. Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence.

    Science.gov (United States)

    Hao, Huijing; Liang, Junrong; Duan, Ran; Chen, Yuhuang; Liu, Chang; Xiao, Yuchun; Li, Xu; Su, Mingming; Jing, Huaiqi; Wang, Xin

    2016-01-01

    API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method.

  13. Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence.

    Directory of Open Access Journals (Sweden)

    Huijing Hao

    Full Text Available API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method.

  14. Differential activity profiles of translation inhibitors in whole-cell and cell-free approaches.

    Science.gov (United States)

    Weidlich, M; Klammt, C; Bernhard, F; Karas, M; Stein, T

    2008-02-01

    Evaluation of the activity profiles of standard prokaryotic translation inhibitors with different physicochemical properties under whole-cell and cell-free conditions. The minimal inhibitory concentration values (cell-free/whole-cell microg ml(-1)) for three aminoglycosides (neomycin, 0.01/6.92; paromomycin, 0.7/1.96; streptomycin 1.45/1.57), three macrolides (erythromycin, 1.53/56.9; josamycin, 1.61/87.7; oleandomycin, 5.12/565.9), chloramphenicol (11.9/3.04), and two tetracyclines (tetracycline hydrochloride, not determined/0.63; minocycline hydrochloride, 2.53/1.09), towards Escherichia coli A19 cells were determined with a microtitre plate-based broth dilution method and compared with values determined in a coupled transcription/translation system based on a S30 extract of the same E. coli strain (cell-free) for the production of the green fluorescent protein. The analysed prokaryotic translation inhibitors showed substance-specific activity profiles under cell-free vs whole-cell conditions that are explainable by the physicochemical properties of the molecules. This study shows the advantages and limits of cell- free transcription/translation (CFTT) experiments for the discovery of novel antimicrobials. The main advantage is the direct access of the target structures (ribosomes) for the inhibitors, and our results provide an estimation of the concentration necessary to detect new agents. The main limitations are that the inhibitory properties of different agents in CFTT experiments do not necessarily reflect their growth inhibition activity in cell cultures.

  15. Performance of a Cyanobacteria Whole Cell-Based Fluorescence Biosensor for Heavy Metal and Pesticide Detection

    Directory of Open Access Journals (Sweden)

    Salmijah Surif

    2013-05-01

    Full Text Available Whole cell biosensors always face the challenge of low stability of biological components and short storage life. This paper reports the effects of poly(2-hydroxyethyl methacrylate (pHEMA immobilization on a whole cell fluorescence biosensor for the detection of heavy metals (Cu, Pb, Cd, and pesticides (dichlorophenoxyacetic acid (2,4-D, and chlorpyrifos. The biosensor was produced by entrapping the cyanobacterium Anabaena torulosa on a cellulose membrane, followed by applying a layer of pHEMA, and attaching it to a well. The well was then fixed to an optical probe which was connected to a fluorescence spectrophotometer and an electronic reader. The optimization of the biosensor using several factors such as amount of HEMA and drying temperature were undertaken. The detection limits of biosensor without pHEMA for Cu, Cd, Pb, 2,4-D and chlorpyrifos were 1.195, 0.027, 0.0100, 0.025 and 0.025 µg/L respectively. The presence of pHEMA increased the limits of detection to 1.410, 0.250, 0.500, 0.235 and 0.117 µg/L respectively. pHEMA is known to enhance the reproducibility of the biosensor with average relative standard deviation (RSD of ±1.76% for all the pollutants tested, 48% better than the biosensor without pHEMA (RSD = ±3.73%. In storability test with Cu 5 µg/L, the biosensor with pHEMA performed 11.5% better than the test without pHEMA on day-10 and 5.2% better on day-25. pHEMA is therefore a good candidate to be used in whole cell biosensors as it increases reproducibility and enhances biosensor storability.

  16. A novel whole-cell mechanism for long-term memory enhancement.

    Directory of Open Access Journals (Sweden)

    Iris Reuveni

    Full Text Available Olfactory-discrimination learning was shown to induce a profound long-lasting enhancement in the strength of excitatory and inhibitory synapses of pyramidal neurons in the piriform cortex. Notably, such enhancement was mostly pronounced in a sub-group of neurons, entailing about a quarter of the cell population. Here we first show that the prominent enhancement in the subset of cells is due to a process in which all excitatory synapses doubled their strength and that this increase was mediated by a single process in which the AMPA channel conductance was doubled. Moreover, using a neuronal-network model, we show how such a multiplicative whole-cell synaptic strengthening in a sub-group of cells that form a memory pattern, sub-serves a profound selective enhancement of this memory. Network modeling further predicts that synaptic inhibition should be modified by complex learning in a manner that much resembles synaptic excitation. Indeed, in a subset of neurons all GABAA-receptors mediated inhibitory synapses also doubled their strength after learning. Like synaptic excitation, Synaptic inhibition is also enhanced by two-fold increase of the single channel conductance. These findings suggest that crucial learning induces a multiplicative increase in strength of all excitatory and inhibitory synapses in a subset of cells, and that such an increase can serve as a long-term whole-cell mechanism to profoundly enhance an existing Hebbian-type memory. This mechanism does not act as synaptic plasticity mechanism that underlies memory formation but rather enhances the response of already existing memory. This mechanism is cell-specific rather than synapse-specific; it modifies the channel conductance rather than the number of channels and thus has the potential to be readily induced and un-induced by whole-cell transduction mechanisms.

  17. Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst

    Directory of Open Access Journals (Sweden)

    Speight Robert

    2010-04-01

    Full Text Available Abstract Background Trigonopsis variabilis D-amino acid oxidase (TvDAO is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of TvDAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris TvDAO whole-cell biocatalyst. Results As compared to the native T. variabilis host, a more than seven-fold enhancement of the intracellular level of oxidase activity was achieved in P. pastoris through expression optimization by codon redesign as well as efficient subcellular targeting of the enzyme to peroxisomes. Multi copy integration further doubled expression and the specific activity of the whole cell catalyst. From a multicopy production strain, about 1.3 × 103 U/g wet cell weight (wcw were derived by standard induction conditions feeding pure methanol. A fed-batch cultivation protocol using a mixture of methanol and glycerol in the induction phase attenuated the apparent toxicity of the recombinant oxidase to yield final biomass concentrations in the bioreactor of ≥ 200 g/L compared to only 117 g/L using the standard methanol feed. Permeabilization of P. pastoris using 10% isopropanol yielded a whole-cell enzyme preparation that showed 49% of the total available intracellular oxidase activity and was notably stabilized (by three times compared to a widely used TvDAO expressing Escherichia coli strain under conditions of D-methionine conversion using vigorous

  18. Advances in in-situ product recovery (ISPR) in whole cell biotechnology during the last decade.

    Science.gov (United States)

    Van Hecke, Wouter; Kaur, Guneet; De Wever, Heleen

    2014-11-15

    The review presents the state-of-the-art in the applications of in-situ product recovery (ISPR) in whole-cell biotechnology over the last 10years. It summarizes various ISPR-integrated fermentation processes for the production of a wide spectrum of bio-based products. A critical assessment of the performance of various ISPR concepts with respect to the degree of product enrichment, improved productivity, reduced process flows and increased yields is provided. Requirements to allow a successful industrial implementation of ISPR are also discussed. Finally, supporting technologies such as online monitoring, mathematical modeling and use of recombinant microorganisms with ISPR are presented.

  19. Making bio-sense of toxicity: new developments in whole-cell biosencors

    DEFF Research Database (Denmark)

    Sørensen, Søren Johannes; Burmølle, Mette; Hansen, Lars Hestbjerg

    2006-01-01

    Bacterial whole-cell biosensors are very useful for toxicity measurements of various samples. Semi-specific biosensors, containing fusions of stress-regulated promoters and reporter genes, have several advantages over the traditional, general biosensors that are based on constitutively expressed ....... The application of in situ inoculation and single-cell detection, combined with the introduction of new reporter genes and refined detection equipment, could lead to the extensive use of semi-specific, stress-responsive biosensors for toxicity estimations in the future....

  20. Quantitation of recombinant protein in whole cells and cell extracts via solid-state NMR spectroscopy.

    Science.gov (United States)

    Vogel, Erica P; Weliky, David P

    2013-06-25

    Recombinant proteins (RPs) are commonly expressed in bacteria followed by solubilization and chromatography. Purified RP yield can be diminished by losses at any step with very different changes in methods that can improve the yield. Time and labor can therefore be saved by first identifying the specific reason for the low yield. This study describes a new solid-state nuclear magnetic resonance approach to RP quantitation in whole cells or cell extracts without solubilization or purification. The method is straightforward and inexpensive and requires only ∼50 mL culture and a low-field spectrometer.

  1. Reporter Proteins in Whole-Cell Optical Bioreporter Detection Systems, Biosensor Integrations, and Biosensing Applications

    Science.gov (United States)

    Close, Dan M.; Ripp, Steven; Sayler, Gary S.

    2009-01-01

    Whole-cell, genetically modified bioreporters are designed to emit detectable signals in response to a target analyte or related group of analytes. When integrated with a transducer capable of measuring those signals, a biosensor results that acts as a self-contained analytical system useful in basic and applied environmental, medical, pharmacological, and agricultural sciences. Historically, these devices have focused on signaling proteins such as green fluorescent protein, aequorin, firefly luciferase, and/or bacterial luciferase. The biochemistry and genetic development of these sensor systems as well as the advantages, challenges, and common applications of each one will be discussed. PMID:22291559

  2. Reporter Proteins in Whole-Cell Optical Bioreporter Detection Systems, Biosensor Integrations, and Biosensing Applications

    Directory of Open Access Journals (Sweden)

    Gary S. Sayler

    2009-11-01

    Full Text Available Whole-cell, genetically modified bioreporters are designed to emit detectable signals in response to a target analyte or related group of analytes. When integrated with a transducer capable of measuring those signals, a biosensor results that acts as a self-contained analytical system useful in basic and applied environmental, medical, pharmacological, and agricultural sciences. Historically, these devices have focused on signaling proteins such as green fluorescent protein, aequorin, firefly luciferase, and/or bacterial luciferase. The biochemistry and genetic development of these sensor systems as well as the advantages, challenges, and common applications of each one will be discussed.

  3. Non-Conventional Yeasts Whole Cells as Efficient Biocatalysts for the Production of Flavors and Fragrances

    Directory of Open Access Journals (Sweden)

    Luca Forti

    2015-06-01

    Full Text Available The rising consumer requests for natural flavors and fragrances have generated great interest in the aroma industry to seek new methods to obtain fragrance and flavor compounds naturally. An alternative and attractive route for these compounds is based on bio-transformations. In this review, the application of biocatalysis by Non Conventional Yeasts (NCYs whole cells for the production of flavor and fragrances is illustrated by a discussion of the production of different class of compounds, namely Aldehydes, Ketones and related compounds, Alcohols, Lactones, Terpenes and Terpenoids, Alkenes, and Phenols.

  4. Non-Conventional Yeasts Whole Cells as Efficient Biocatalysts for the Production of Flavors and Fragrances.

    Science.gov (United States)

    Forti, Luca; Di Mauro, Simone; Cramarossa, Maria Rita; Filippucci, Sara; Turchetti, Benedetta; Buzzini, Pietro

    2015-06-04

    The rising consumer requests for natural flavors and fragrances have generated great interest in the aroma industry to seek new methods to obtain fragrance and flavor compounds naturally. An alternative and attractive route for these compounds is based on bio-transformations. In this review, the application of biocatalysis by Non Conventional Yeasts (NCYs) whole cells for the production of flavor and fragrances is illustrated by a discussion of the production of different class of compounds, namely Aldehydes, Ketones and related compounds, Alcohols, Lactones, Terpenes and Terpenoids, Alkenes, and Phenols.

  5. Personalized Whole-Cell Kinetic Models of Metabolism for Discovery in Genomics and Pharmacodynamics

    DEFF Research Database (Denmark)

    Bordbar, Aarash; McCloskey, Douglas; Zielinski, Daniel C

    2015-01-01

    challenge. Here, we constructed multi-omic, data-driven, personalized whole-cell kinetic models of erythrocyte metabolism for 24 healthy individuals based on fasting-state plasma and erythrocyte metabolomics and whole-genome genotyping. We show that personalized kinetic rate constants, rather than......-induced anemia) and how genetic variation (inosine triphosphatase deficiency) may protect against this side effect. This study demonstrates the feasibility of personalized kinetic models, and we anticipate their use will accelerate discoveries in characterizing individual metabolic variation....

  6. Toxicity analysis of pesticides on cyanobacterial species by 16S rDNA molecular characterization

    Directory of Open Access Journals (Sweden)

    J. I. Nirmal Kumar

    2013-06-01

    Full Text Available Damaging effects of endosulfan on native structure of DNA, evident as a result of PCR based assay such as 16S rDNA amplification and sequencing, led to formation of gaps, mismatching of base pairs and dissimilarities in entire 16S rDNA sequences of treated cultures. Endosulfan was the most fatal to Westiellopsis prolifica of 16S rDNA region at 40ppm insecticide induced series of mispairing, and other lesions amounting up to 20% dissimilarity and 7% gaps. Whereas, 16S rDNA region of Anabaena fertilissima was comparatively less influenced with 18% dissimilarity and 7% gaps in response to 12ppm endosulfan, while 16S rDNA gene of Aulosira fertilissima was the least prone to changes with 17% dissimilarity, and 5% gaps under 60ppm endosulfan stress by the end of 16 days. On the other side, impact of fungicide tebuconazole after 16 days reflected identities up to 78% and 8% gaps for 30ppm treated A. fertilissima, while 60ppm treatment instilled 79% similarities with 10% gaps in W. prolifica and 83% identities with 5% gaps of Aulosira fertilissima after 16 days.

  7. 16s rRNA的保守字和进化树重建%Conserved Words in 16s Ribosomal RNA Deduced from Evolutionary Tree Reconstruction

    Institute of Scientific and Technical Information of China (English)

    罗辽复; 贾孟文

    2002-01-01

    Evolutionary distance is defined by oligonucleotide (n-bases) frequency difference of two sequences.Phylogenetic tree is reconstructed using a set of 16S (18S) rRNA sequences and the definition of distance.The quality of trees generally improves with increasing n and reaches a plateau of best fit at n=7 or 8.So,the 7-mer or 8-mer frequencies provides a basis to describe rRNA evolution.Then,a group of 7-mers are deduced which are correlate well with evolution.Evolution-related conservative words longer than 7 bases for Bacteria and Archaea in 16S rRNA sequences have been found.They are highly conserved in nearly all species of a kingdom (or a sub-kingdom) and are located on nearly same sites of sequences. The structural meaning of these conservative words is discussed briefly.%据寡核苷(n核苷)频数差定义进化距离,由此构成16s rRNA进化树,当n=7,8时和实验资料符合很好,在寻找出全部进化相关的7-核苷的基础上,本文进一步求得了长度大于7的保守字,它们在一个界别中的诸物种中高度保守,并出现于核糖体序列的基本相同的位置上,这些保守字对于核糖体的早期进化至关重要.

  8. Comparison of whole-cell SELEX methods for the identification of Staphylococcus aureus-specific DNA aptamers.

    Science.gov (United States)

    Moon, Jihea; Kim, Giyoung; Park, Saet Byeol; Lim, Jongguk; Mo, Changyeun

    2015-04-15

    Whole-cell Systemic Evolution of Ligands by Exponential enrichment (SELEX) is the process by which aptamers specific to target cells are developed. Aptamers selected by whole-cell SELEX have high affinity and specificity for bacterial surface molecules and live bacterial targets. To identify DNA aptamers specific to Staphylococcus aureus, we applied our rapid whole-cell SELEX method to a single-stranded ssDNA library. To improve the specificity and selectivity of the aptamers, we designed, selected, and developed two categories of aptamers that were selected by two kinds of whole-cell SELEX, by mixing and combining FACS analysis and a counter-SELEX process. Using this approach, we have developed a biosensor system that employs a high affinity aptamer for detection of target bacteria. FAM-labeled aptamer sequences with high binding to S. aureus, as determined by fluorescence spectroscopic analysis, were identified, and aptamer A14, selected by the basic whole-cell SELEX using a once-off FACS analysis, and which had a high binding affinity and specificity, was chosen. The binding assay was evaluated using FACS analysis. Our study demonstrated the development of a set of whole-cell SELEX derived aptamers specific to S. aureus; this approach can be used in the identification of other bacteria.

  9. Rare Events of Intragenus and Intraspecies Horizontal Transfer of the 16S rRNA Gene.

    Science.gov (United States)

    Tian, Ren-Mao; Cai, Lin; Zhang, Wei-Peng; Cao, Hui-Luo; Qian, Pei-Yuan

    2015-07-27

    Horizontal gene transfer (HGT) of operational genes has been widely reported in prokaryotic organisms. However, informational genes such as those involved in transcription and translation processes are very difficult to be horizontally transferred, as described by Woese's complexity hypothesis. Here, we analyzed all of the completed prokaryotic genome sequences (2,143 genomes) in the NCBI (National Center for Biotechnology Information) database, scanned for genomes with high intragenomic heterogeneity of 16S rRNA gene copies, and explored potential HGT events of ribosomal RNA genes based on the phylogeny, genomic organization, and secondary structures of the ribosomal RNA genes. Our results revealed 28 genomes with relatively high intragenomic heterogeneity of multiple 16S rRNA gene copies (lowest pairwise identity 16S rRNA gene only occurred at intragenus or intraspecies levels, which is quite different from the HGT of operational genes. Our results improve our understanding regarding the exchange of informational genes.

  10. Sequence of the 16S Ribosomal RNA from Halobacterium volcanii, an Archaebacterium.

    Science.gov (United States)

    Gupta, R; Lanter, J M; Woese, C R

    1983-08-12

    The sequence of the 16S ribosomal RNA (rRNA) from the archaebacterium Halobacterium volcanii has been determined by DNA sequencing methods. The archaebacterial rRNA is similar to its eubacterial counterpart in secondary structure. Although it is closer in sequence to the eubacterial 16S rRNA than to the eukaryotic 16S-like rRNA, the H. volcanii sequence also shows certain points of specific similarity to its eukaryotic counterpart. Since the H. volcanii sequence is closer to both the eubacterial and the eukaryotic sequences than these two are to one another, it follows that the archaebacterial sequence resembles their common ancestral sequence more closely than does either of the other two versions.

  11. Application of Whole-Cell Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Rapid Identification and Clustering Analysis of Pantoea Species ▿ †

    Science.gov (United States)

    Rezzonico, Fabio; Vogel, Guido; Duffy, Brion; Tonolla, Mauro

    2010-01-01

    Pantoea agglomerans is an ecologically diverse taxon that includes commercially important plant-beneficial strains and opportunistic clinical isolates. Standard biochemical identification methods in diagnostic laboratories were repeatedly shown to run into false-positive identifications of P. agglomerans, a fact which is also reflected by the high number of 16S rRNA gene sequences in public databases that are incorrectly assigned to this species. More reliable methods for rapid identification are required to ascertain the prevalence of this species in clinical samples and to evaluate the biosafety of beneficial isolates. Whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) methods and reference spectra (SuperSpectrum) were developed for accurate identification of P. agglomerans and related bacteria and used to detect differences in the protein profile within variants of the same strain, including a ribosomal point mutation conferring streptomycin resistance. MALDI-TOF MS-based clustering was shown to generally agree with classification based on gyrB sequencing, allowing rapid and reliable identification at the species level. PMID:20453125

  12. Streptomycin binds to the decoding center of 16 S ribosomal RNA.

    Science.gov (United States)

    Spickler, C; Brunelle, M N; Brakier-Gingras, L

    1997-10-31

    Streptomycin, an error-inducing aminoglycoside antibiotic, binds to a single site on the small ribosomal subunit of bacteria, but this site has not yet been defined precisely. Here, we demonstrate that streptomycin binds to E. coli 16 S rRNA in the absence of ribosomal proteins, and protects a set of bases in the decoding region against dimethyl sulfate attack. The binding studies were performed in a high ionic strength buffer containing 20 mM Mg2+. The pattern of protection in the decoding region was similar to that observed when streptomycin binds to the 30 S subunit. However, streptomycin also protects the 915 region of 16 S rRNA within the 30 S subunit, whereas it did not protect the 915 region of the naked 16 S rRNA. The interaction of streptomycin with 16 S rRNA was further defined by using two fragments that correspond to the 3' minor domain of 16 S rRNA and to the decoding analog, a portion of this domain encompassing the decoding center. In the presence of streptomycin, the pattern of protection against dimethyl sulfate attack for the two fragments was similar to that seen with the full-length 16 S rRNA. This indicates that the 3' minor domain as well as the decoding analog contain the recognition signals for the binding of streptomycin. However, streptomycin could not bind to the decoding analog in the absence of Mg2+. This contrasts with neomycin, another error-inducing aminoglycoside antibiotic, that binds to the decoding analog in the absence of Mg2+, but not at 20 mM Mg2+. Our results suggest that both neomycin and streptomycin interact with the decoding center, but recognize alternative conformations of this region.

  13. Genomic Insights into Geothermal Spring Community Members using a 16S Agnostic Single-Cell Approach

    Science.gov (United States)

    Bowers, R. M.

    2016-12-01

    INSTUTIONS (ALL): DOE Joint Genome Institute, Walnut Creek, CA USA. Bigelow Laboratory for Ocean Sciences, East Boothbay, ME USA. Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada. ABSTRACT BODY: With recent advances in DNA sequencing, rapid and affordable screening of single-cell genomes has become a reality. Single-cell sequencing is a multi-step process that takes advantage of any number of single-cell sorting techniques, whole genome amplification (WGA), and 16S rRNA gene based PCR screening to identify the microbes of interest prior to shotgun sequencing. However, the 16S PCR based screening step is costly and may lead to unanticipated losses of microbial diversity, as cells that do not produce a clean 16S amplicon are typically omitted from downstream shotgun sequencing. While many of the sorted cells that fail the 16S PCR step likely originate from poor quality amplified DNA, some of the cells with good WGA kinetics may instead represent bacteria or archaea with 16S genes that fail to amplify due to primer mis-matches or the presence of intervening sequences. Using cell material from Dewar Creek, a hot spring in British Columbia, we sequenced all sorted cells with good WGA kinetics irrespective of their 16S amplification success. We show that this high-throughput approach to single-cell sequencing (i) can reduce the overall cost of single-cell genome production, and (ii). may lead to the discovery of previously unknown branches on the microbial tree of life.

  14. Engineering Pichia pastoris for improved NADH regeneration: A novel chassis strain for whole-cell catalysis

    Directory of Open Access Journals (Sweden)

    Martina Geier

    2015-09-01

    Full Text Available Many synthetically useful reactions are catalyzed by cofactor-dependent enzymes. As cofactors represent a major cost factor, methods for efficient cofactor regeneration are required especially for large-scale synthetic applications. In order to generate a novel and efficient host chassis for bioreductions, we engineered the methanol utilization pathway of Pichia pastoris for improved NADH regeneration. By deleting the genes coding for dihydroxyacetone synthase isoform 1 and 2 (DAS1 and DAS2, NADH regeneration via methanol oxidation (dissimilation was increased significantly. The resulting Δdas1 Δdas2 strain performed better in butanediol dehydrogenase (BDH1 based whole-cell conversions. While the BDH1 catalyzed acetoin reduction stopped after 2 h reaching ~50% substrate conversion when performed in the wild type strain, full conversion after 6 h was obtained by employing the knock-out strain. These results suggest that the P. pastoris Δdas1 Δdas2 strain is capable of supplying the actual biocatalyst with the cofactor over a longer reaction period without the over-expression of an additional cofactor regeneration system. Thus, focusing the intrinsic carbon flux of this methylotrophic yeast on methanol oxidation to CO2 represents an efficient and easy-to-use strategy for NADH-dependent whole-cell conversions. At the same time methanol serves as co-solvent, inductor for catalyst and cofactor regeneration pathway expression and source of energy.

  15. Evaluation of enteric-coated tablets as a whole cell inactivated vaccine candidate against Vibrio cholerae.

    Science.gov (United States)

    Fernández, Sonsire; Año, Gemma; Castaño, Jorge; Pino, Yadira; Uribarri, Evangelina; Riverón, Luis A; Cedré, Bárbara; Valmaseda, Tania; Falero, Gustavo; Pérez, José L; Infante, Juan F; García, Luis G; Solís, Rosa L; Sierra, Gustavo; Talavera, Arturo

    2013-01-01

    A vaccine candidate against cholera was developed in the form of oral tablets to avoid difficulties during application exhibited by current whole cell inactivated cholera vaccines. In this study, enteric-coated tablets were used to improve the protection of the active compound from gastric acidity. Tablets containing heat-killed whole cells of Vibrio cholerae strain C7258 as the active pharmaceutical compound was enteric-coated with the polymer Kollicoat(®) MAE-100P, which protected them efficiently from acidity when a disintegration test was carried out. Enzyme-linked immunosorbent assay (ELISA) anti-lipopolysaccharide (LPS) inhibition test and Western blot assay revealed the presence of V. cholerae antigens as LPS, mannose-sensitive haemagglutinin (MSHA) and outer membrane protein U (Omp U) in enteric-coated tablets. Immunogenicity studies (ELISA and vibriocidal test) carried out by intraduodenal administration in rabbits showed that the coating process of tablets did not affect the immunogenicity of V. cholerae-inactivated cells. In addition, no differences were observed in the immune response elicited by enteric-coated or uncoated tablets, particularly because the animal model and immunization route used did not allow discriminating between acid resistances of both tablets formulations in vivo. Clinical studies with volunteers will be required to elucidate this aspect, but the results suggest the possibility of using enteric-coated tablets as a final pharmaceutical product for a cholera vaccine.

  16. Whole-Cell Biocatalysis for Producing Ginsenoside Rd from Rb1 Using Lactobacillus rhamnosus GG.

    Science.gov (United States)

    Ku, Seockmo; You, Hyun Ju; Park, Myeong Soo; Ji, Geun Eog

    2016-07-28

    Ginsenosides are the major active ingredients in ginseng used for human therapeutic plant medicines. One of the most well-known probiotic bacteria among the various strains on the functional food market is Lactobacillus rhamnosus GG. Biocatalytic methods using probiotic enzymes for producing deglycosylated ginsenosides such as Rd have a growing significance in the functional food industry. The addition of 2% cellobiose (w/v) to glucose-free de Man-Rogosa-Sharpe broths notably induced β-glucosidase production from L. rhamnosus GG. Enzyme production and activity were optimized at a pH, temperature, and cellobiose concentration of 6.0, 40°C, and 2% (w/v), respectively. Under these controlled conditions, β-glucosidase production in L. rhamnosus GG was enhanced by 25-fold. Additionally, whole-cell homogenates showed the highest β-glucosidase activity when compared with disrupted cell suspensions; the cell disruption step significantly decreased the β-glucosidase activity. Based on the optimized enzyme conditions, whole-cell L. rhamnosus GG was successfully used to convert ginsenoside Rb1 into Rd.

  17. Optimization of a whole-cell biocatalyst by employing genetically encoded product sensors inside nanolitre reactors

    Science.gov (United States)

    Meyer, Andreas; Pellaux, René; Potot, Sébastien; Becker, Katja; Hohmann, Hans-Peter; Panke, Sven; Held, Martin

    2015-08-01

    Microcompartmentalization offers a high-throughput method for screening large numbers of biocatalysts generated from genetic libraries. Here we present a microcompartmentalization protocol for benchmarking the performance of whole-cell biocatalysts. Gel capsules served as nanolitre reactors (nLRs) for the cultivation and analysis of a library of Bacillus subtilis biocatalysts. The B. subtilis cells, which were co-confined with E. coli sensor cells inside the nLRs, converted the starting material cellobiose into the industrial product vitamin B2. Product formation triggered a sequence of reactions in the sensor cells: (1) conversion of B2 into flavin mononucleotide (FMN), (2) binding of FMN by a RNA riboswitch and (3) self-cleavage of RNA, which resulted in (4) the synthesis of a green fluorescent protein (GFP). The intensity of GFP fluorescence was then used to isolate B. subtilis variants that convert cellobiose into vitamin B2 with elevated efficiency. The underlying design principles of the assay are general and enable the development of similar protocols, which ultimately will speed up the optimization of whole-cell biocatalysts.

  18. Simulation step size analysis of a whole-cell computational model of bacteria

    Science.gov (United States)

    Abreu, Raphael; Castro, Maria Clicia S.; Silva, Fabrício Alves B.

    2016-12-01

    Understanding how complex phenotypes arise from individual molecules and their interactions is a major challenge in biology and, to meet this challenge, computational approaches are increasingly employed. As an example, a recent paper [1] proposed a whole-cell model Mycoplasma genitalium including all cell components and their interactions. 28 modules representing several cell functions were modeled independently, and then integrated into a single computational model. One assumption considered in the whole-cell model of M.Genitalium is that all 28 modules can be modeled independently given the 1 second step size used in simulations. This is a major assumption, since it simplifies the modeling of several cell functions and makes the modeling of the system as a whole feasible. In this paper we investigate the dependency of experimental results on that assumption. We have simulated the M.Genitalium cell cycle using several simulation time step sizes and compared the results to the ones obtained with the system using 1 second simulation time step.

  19. Qualification status of hybrid crystal oscillators style OTO 16S for space application

    Science.gov (United States)

    Gerard, E.; Deviller, J. L.

    1991-03-01

    The qualification status of a crystal clock oscillator, OTO 16S, is described. Specifically designed for the Telecom 2 and Intelsat 7 programs, the oscillator is available in frequencies between 3 and 25 MHz with Transistor Transistor Logic (TTL) compatible outputs. Qualification tests results are presented to demonstrate that all the OTO 16S performances are in compliance with space requirements. From a mechanical viewpoint, no degradation is seen from a vibration level of 50 g sinus 10 to 2000 Hz. From a life test viewpoint, no significant variations are observed after 2000 hours of testing.

  20. Binding of 16S rRNA to chloroplast 30S ribosomal proteins blotted on nitrocellulose

    OpenAIRE

    Rozier, Claude; Mache, Régis

    1984-01-01

    Protein-RNA associations were studied by a method using proteins blotted on a nitrocellulose sheet. This method was assayed with Escherichia Coli 30S ribosomal components. In stringent conditions (300 mM NaCl or 20° C) only 9 E. coli ribosomal proteins strongly bound to the 16S rRNA: S4, S5, S7, S9, S12, S13, S14, S19, S20. 8 of these proteins have been previously found to bind independently to the 16S rRNA. The same method was applied to determine protein-RNA interactions in spinach chloropl...

  1. Analysis of Rhizobia Isolated from Melilotus by 16S rDNA-RFLP%草木樨属根瘤菌的16S rDNA-RFLP分析

    Institute of Scientific and Technical Information of China (English)

    李香香; 张美玲; 付芸芸; 赵美玲; 韦革宏

    2008-01-01

    采用16S rDNA-RFLP分析方法对草木樨属根瘤菌进行了遗传多样性及系统分类研究.结果表明,3种限制性内切酶(HaeⅢ、Hinf I、Msp I)对所有供试菌株的酶切图谱类型组合只有2种.类型I的代表菌株CCNWSX0003-1与豌豆根瘤菌(R.leguminosarum)的模式菌株USDA2370的序列相似性达到99.8%,在分类地位上属于根瘤菌属(Rhizobium)分支;类型Ⅱ的代表菌株CCNWGS0006与草木樨中华根瘤菌(Sinorhizobium meliloti)的16S rDNA相似性为100%,属于中华根瘤菌属(Sinorhizobium)分支.

  2. Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification

    NARCIS (Netherlands)

    Ziesemer, K.A.; Mann, A.E.; Sankaranarayanan, K.; Schroeder, H.; Ozga, A.T.; Brandt, B.W.; Zaura, E.; Waters-Rist, A.; Hoogland, M.; Salazar-García, D.C.; Aldenderfer, M.; Speller, C.; Hendy, J.; Weston, D.A.; MacDonald, S.J.; Thomas, G.H.; Collins, M.J.; Lewis, C.M.; Hofman, C.; Warinner, C.

    2015-01-01

    To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341-534) of this gen

  3. Problem-Based Test: Functional Analysis of Mutant 16S rRNAs

    Science.gov (United States)

    Szeberenyi, Jozsef

    2010-01-01

    Terms to be familiar with before you start to solve the test: ribosome, ribosomal subunits, antibiotics, point mutation, 16S, 5S, and 23S rRNA, Shine-Dalgarno sequence, mRNA, tRNA, palindrome, hairpin, restriction endonuclease, fMet-tRNA, peptidyl transferase, initiation, elongation, termination of translation, expression plasmid, transformation,…

  4. The Characterization of Novel Tissue Microbiota Using an Optimized 16S Metagenomic Sequencing Pipeline.

    Directory of Open Access Journals (Sweden)

    Jérôme Lluch

    Full Text Available Substantial progress in high-throughput metagenomic sequencing methodologies has enabled the characterisation of bacteria from various origins (for example gut and skin. However, the recently-discovered bacterial microbiota present within animal internal tissues has remained unexplored due to technical difficulties associated with these challenging samples.We have optimized a specific 16S rDNA-targeted metagenomics sequencing (16S metabarcoding pipeline based on the Illumina MiSeq technology for the analysis of bacterial DNA in human and animal tissues. This was successfully achieved in various mouse tissues despite the high abundance of eukaryotic DNA and PCR inhibitors in these samples. We extensively tested this pipeline on mock communities, negative controls, positive controls and tissues and demonstrated the presence of novel tissue specific bacterial DNA profiles in a variety of organs (including brain, muscle, adipose tissue, liver and heart.The high throughput and excellent reproducibility of the method ensured exhaustive and precise coverage of the 16S rDNA bacterial variants present in mouse tissues. This optimized 16S metagenomic sequencing pipeline will allow the scientific community to catalogue the bacterial DNA profiles of different tissues and will provide a database to analyse host/bacterial interactions in relation to homeostasis and disease.

  5. Novel essential gene Involved in 16S rRNA processing in Escherichia coli.

    Science.gov (United States)

    Kurata, Tatsuaki; Nakanishi, Shinobu; Hashimoto, Masayuki; Taoka, Masato; Yamazaki, Yukiko; Isobe, Toshiaki; Kato, Jun-ichi

    2015-02-27

    Biogenesis of ribosomes is a complex process mediated by many factors. While its transcription proceeds, ribosomal RNA (rRNA) folds itself into a characteristic three-dimensional structure through interaction with ribosomal proteins, during which its ends are processed. Here, we show that the essential protein YqgF, a RuvC family protein with an RNase-H-like motif, is involved in the processing of pre-16S rRNA during ribosome maturation. Indeed, pre-16S rRNA accumulated in cells of a temperature-sensitive yqgF mutant (yqgF(ts)) cultured at a non-permissive temperature. In addition, purified YqgF was shown to process the 5' end of pre-16S rRNA within 70S ribosomes in vitro. Mass spectrometry analysis of the total proteins in the yqgF(ts) mutant cells showed that the expression of genes containing multiple Shine-Dalgarno-like sequences was observed to be lower than in wild type. These results are interpreted to indicate that YqgF is involved in a novel enzymic activity necessary for the processing of pre-16S rRNA, thereby affecting elongation of translation.

  6. Effects of 16S rRNA gene mutations on tetracycline resistance in Helicobacter pylori

    NARCIS (Netherlands)

    M.M. Gerrits (Monique); M. Berning; A.H.M. van Vliet (Arnoud); E.J. Kuipers (Ernst); J.G. Kusters (Johannes)

    2003-01-01

    textabstractThe triple-base-pair 16S rDNA mutation AGA(926-928)-->TTC mediates high-level tetracycline resistance in Helicobacter pylori. In contrast, single- and double-base-pair mutations mediated only low-level tetracycline resistance and decreased growth rates in the presence o

  7. Prosthetic joint infection due to Lysobacter thermophilus diagnosed by 16S rRNA gene sequencing.

    Science.gov (United States)

    Dhawan, B; Sebastian, S; Malhotra, R; Kapil, A; Gautam, D

    2016-01-01

    We report the first case of prosthetic joint infection caused by Lysobacter thermophilus which was identified by 16S rRNA gene sequencing. Removal of prosthesis followed by antibiotic treatment resulted in good clinical outcome. This case illustrates the use of molecular diagnostics to detect uncommon organisms in suspected prosthetic infections.

  8. Isolation of temperature-sensitive mutants of 16 S rRNA in Escherichia coli

    DEFF Research Database (Denmark)

    Triman, K; Becker, E; Dammel, C;

    1989-01-01

    Temperature-sensitive mutants have been isolated following hydroxylamine mutagenesis of a plasmid containing Escherichia coli rRNA genes carrying selectable markers for spectinomycin resistance (U1192 in 16 S rRNA) and erythromycin resistance (G2058 in 23 S rRNA). These antibiotic resistance alle...

  9. Globicatella sanguinis bacteraemia identified by partial 16S rRNA gene sequencing

    DEFF Research Database (Denmark)

    Abdul-Redha, Rawaa Jalil; Balslew, Ulla; Christensen, Jens Jørgen;

    2007-01-01

    Globicatella sanguinis is a gram-positive coccus, resembling non-haemolytic streptococci. The organism has been isolated infrequently from normally sterile sites of humans. Three isolates obtained by blood culture could not be identified by Rapid 32 ID Strep, but partial sequencing of the 16S r...

  10. Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects

    Directory of Open Access Journals (Sweden)

    Vasco Elbrecht

    2016-04-01

    Full Text Available Cytochrome c oxidase I (COI is a powerful marker for DNA barcoding of animals, with good taxonomic resolution and a large reference database. However, when used for DNA metabarcoding, estimation of taxa abundances and species detection are limited due to primer bias caused by highly variable primer binding sites across the COI gene. Therefore, we explored the ability of the 16S ribosomal DNA gene as an alternative metabarcoding marker for species level assessments. Ten bulk samples, each containing equal amounts of tissue from 52 freshwater invertebrate taxa, were sequenced with the Illumina NextSeq 500 system. The 16S primers amplified three more insect species than the Folmer COI primers and amplified more equally, probably due to decreased primer bias. Estimation of biomass might be less biased with 16S than with COI, although variation in read abundances of two orders of magnitudes is still observed. According to these results, the marker choice depends on the scientific question. If the goal is to obtain a taxonomic identification at the species level, then COI is more appropriate due to established reference databases and known taxonomic resolution of this marker, knowing that a greater proportion of insects will be missed using COI Folmer primers. If the goal is to obtain a more comprehensive survey the 16S marker, which requires building a local reference database, or optimised degenerated COI primers could be more appropriate.

  11. 16S rRNA、16S-23S rRNA基因测序分析检测主要血流感染病原菌比较%Comparison of the role of 16S rRNA and 16S-23S rRNA gene sequence-based identification of bacteria in bloodsteam infection

    Institute of Scientific and Technical Information of China (English)

    金中淦; 葛平; 徐蓉; 陈蓉; 宣瑛; 刘学杰; 王庆忠

    2012-01-01

    目的 比较细菌16S rRNA、16S-23S rRNA基因测序分析在血流感染病原菌检测中的作用.方法 提取临床上血流感染常见的金黄色葡萄菌、表皮葡萄球菌、大肠埃希菌、粪肠球菌、肺炎链球菌、铜绿假单胞菌、阴沟肠杆菌、鲍曼不动杆菌、洛菲不动杆菌、肺炎克雷伯杆菌、化脓性链球菌、奇异变形杆菌、潘尼变形杆菌、屎肠球菌、粘质沙雷菌、宋内志贺菌、产气肠杆菌、小肠结肠炎耶尔森菌、腐生葡萄球菌基因组DNA,运用16S rRNA、16S-23S rRNA基因进行PCR扩增.扩增产物经测序后在美国国家生物技术中心( NCBI)上进行比对分析,确定菌种.结果 在所分析的19种临床血流感染常见细菌中,16S rRNA基因测序分析可将除粘质沙雷菌外的细菌鉴定到种的水平,但无法完全区分近缘种属;16S-23SrRNA成功鉴定17种细菌,除大肠埃希菌、宋内志贺菌外所有细菌均成功鉴定到单一种的水平.结论 16S-23S rRNA基因可作为血流感染细菌检测较好的分子靶标.

  12. Oral microbiome profiles: 16S rRNA pyrosequencing and microarray assay comparison.

    Directory of Open Access Journals (Sweden)

    Jiyoung Ahn

    Full Text Available OBJECTIVES: The human oral microbiome is potentially related to diverse health conditions and high-throughput technology provides the possibility of surveying microbial community structure at high resolution. We compared two oral microbiome survey methods: broad-based microbiome identification by 16S rRNA gene sequencing and targeted characterization of microbes by custom DNA microarray. METHODS: Oral wash samples were collected from 20 individuals at Memorial Sloan-Kettering Cancer Center. 16S rRNA gene survey was performed by 454 pyrosequencing of the V3-V5 region (450 bp. Targeted identification by DNA microarray was carried out with the Human Oral Microbe Identification Microarray (HOMIM. Correlations and relative abundance were compared at phylum and genus level, between 16S rRNA sequence read ratio and HOMIM hybridization intensity. RESULTS: The major phyla, Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria were identified with high correlation by the two methods (r = 0.70∼0.86. 16S rRNA gene pyrosequencing identified 77 genera and HOMIM identified 49, with 37 genera detected by both methods; more than 98% of classified bacteria were assigned in these 37 genera. Concordance by the two assays (presence/absence and correlations were high for common genera (Streptococcus, Veillonella, Leptotrichia, Prevotella, and Haemophilus; Correlation = 0.70-0.84. CONCLUSION: Microbiome community profiles assessed by 16S rRNA pyrosequencing and HOMIM were highly correlated at the phylum level and, when comparing the more commonly detected taxa, also at the genus level. Both methods are currently suitable for high-throughput epidemiologic investigations relating identified and more common oral microbial taxa to disease risk; yet, pyrosequencing may provide a broader spectrum of taxa identification, a distinct sequence-read record, and greater detection sensitivity.

  13. A new insight to adsorption and accumulation of high lead concentration by exopolymer and whole cells of lead-resistant bacterium Acinetobacter junii L. Pb1 isolated from coal mine dump.

    Science.gov (United States)

    Kushwaha, Anamika; Rani, Radha; Kumar, Sanjay; Thomas, Tarence; David, Arun Alfred; Ahmed, Meraz

    2017-03-11

    A lead-resistant bacterial strain was isolated from coal mine dump and identified as Acinetobacter junii Pb1 on basis of 16S rRNA (ribosomal ribonucleic acid) gene sequencing. The minimum inhibitory concentration of lead for the strain was 16,000 mg l(-1) and it showed antibiotic and multi metal resistance. In aqueous culture, at an initial lead (Pb(II)) concentration of 100 and 500 mg l(-1), lead adsorption and accumulation by the isolate was 100 and 60%, at pH 7 at 30 °C after 48 and 120 h, respectively. The two fractions of exopolysaccharide (EPS), loosely associated EPS (laEPS) and bound EPS (bEPS), and whole cells (devoid of EPS) showed high binding affinity towards Pb(II). The binding affinity of laEPS towards Pb(II) (1071 mg Pb g(-1)) was three times higher than that of bEPS (321.5 mg Pb g(-1)) and 6.5 times higher than that of whole cells (165 mg Pb g(-1)). The binding affinity of EPS and whole cells with Pb(II), reported in the current study, is considerably higher as compared to that reported in the literature, till date. SEM analysis, showed an increase in thickness of cells on exposure to Pb(II) and TEM analysis, revealed its accumulation (interior of cell) and its adsorption (with the external cell surface). The isolate was also found to be positive for indole acetic acid (IAA) and 1-aminocyclopropane-1-carboxylate (ACC) deaminase production which helps in promoting plant growth. Thus, this study provides a new understanding towards Pb(II) uptake by A. junii Pb1, highlighting its potential on the restoration of Pb(II) contaminated repositories.

  14. Formate production through carbon dioxide hydrogenation with recombinant whole cell biocatalysts.

    Science.gov (United States)

    Alissandratos, Apostolos; Kim, Hye-Kyung; Easton, Christopher J

    2014-07-01

    The biological conversion of CO2 and H2 into formate offers a sustainable route to a valuable commodity chemical through CO2 fixation, and a chemical form of hydrogen fuel storage. Here we report the first example of CO2 hydrogenation utilising engineered whole-cell biocatalysts. Escherichia coli JM109(DE3) cells transformed for overexpression of either native formate dehydrogenase (FDH), the FDH from Clostridium carboxidivorans, or genes from Pyrococcus furiosus and Methanobacterium thermoformicicum predicted to express FDH based on their similarity to known FDH genes were all able to produce levels of formate well above the background, when presented with H2 and CO2, the latter in the form of bicarbonate. In the case of the FDH from P. furiosus the yield was highest, reaching more than 1 g L(-1)h(-1) when a hydrogen-sparging reactor design was used.

  15. Immobilization of Bacillus acidocaldarius whole-cell rhodanese in polysaccharide and insolubilized gelatin gels

    Energy Technology Data Exchange (ETDEWEB)

    De Riso, L.; Alteriis, E. de; Parascandola, P. [Universita `Federico II` Napoli (Italy)]|[Istituto de Biochimica delle Proteine ed Enzimologia, Napoli (Italy); La Cara, F.; Sada, A. [Istituto di Biochimica delle Proteine ed Enzimologia, Napoli (Italy)

    1996-04-01

    The presence of rhodanese activity has been investigated in two strains of thermophilic eubacteria and two strains of extremophiles. Bacillus acidocaldarius, a thermoacidophilic eubacterium, showed the highest levels of enzyme activity. Whole cells, previously subjected to one cycle of freeze-thawing, were immobilized by entrapment in the polysaccharide matrices Ca-alginate, {kappa}-carrageenan and chitosan, and in an insolubilized gelatin gel. The results obtained with the different immobilizates in terms of activity yield, possibility of regeneration and operative stability were evaluated with the aim of setting up a continuous system. This was achieved with a system consisting of B. acidocaldarius cells entrapped in an insolubilized gelatin matrix. The latter, in the form of a thin membrane, was employed in a custom-conceived reactor operating as a plug flow reactor. 21 refs., 3 figs., 2 tabs.

  16. Whole-cell biotransformation with recombinant cytochrome P450 for the selective oxidation of Grundmann's ketone.

    Science.gov (United States)

    Hernández-Martín, Alba; von Bühler, Clemens J; Tieves, Florian; Fernández, Susana; Ferrero, Miguel; Urlacher, Vlada B

    2014-10-15

    25-Hydroxy-Grundmann's ketone is a key building block in the chemical synthesis of vitamin D3 and its derivatives through convergent routes. Generally, the chemical synthesis of this compound involves tedious procedures and results in a mixture of several products. Recently, the selective hydroxylation of Grundmann's ketone at position C25 by cytochrome P450 (CYP) 154E1 from Thermobifida fusca YX was described. In this study a recombinant whole-cell biocatalyst was developed and applied for hydroxylation of Grundmann's ketone. Biotransformation was performed by Escherichia coli cells expressing CYP154E1 along with two redox partner systems, Pdx/PdR and YkuN/FdR. The system comprising CYP154E1/Pdx/PdR showed the highest production of 25-hydroxy-Grundmann's ketone and resulted in 1.1mM (300mgL(-1)) product concentration. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Chemical nature of algal (chlorella) starch and its estimation in whole cells

    Energy Technology Data Exchange (ETDEWEB)

    Watts, P.M.; Pirt, S.J.

    1981-01-01

    The estimation of the starch content of chlorella cells was based on the sonication of whole cells in 2M NaOH followed by heating to solubilize the starch. The starch content of the solution was estimated by measurement of the glucose equivalents formed by acid hydrolysis. The starch content of the neutral solution was estimated by measuring light absorbance of its blue complex with iodine. The chlorella starch was purified from cells which had been disrupted in a French press, and the starch was solubilized by boiling with water. The starch was deproteinized, desalted and finally precipitated with ethanol before drying. This purification procedure avoided alkali extraction which degrades the starch. Beta-amylase digestion of the purified starch was found to convert 67% of it to maltose. The purified starch gave a bright blue complex with iodine with peak absorbance at 590 nm.

  18. Copper sulfate improves pullulan production by bioconversion using whole cells of Aureobasidium pullulans as the catalyst.

    Science.gov (United States)

    Wang, Dahui; Ju, Xiaomin; Zhang, Gaochuan; Wang, Donghua; Wei, Gongyuan

    2016-10-01

    The effects of mineral salts on pullulan production by bioconversion using whole cells of Aureobasidium pullulans CCTCC M 2012259 as the catalyst were investigated. Copper sulfate (CuSO4) improved pullulan production by 36.2% and 42.3% when added at the optimum concentration of 0.2mg/L to the bioconversion broth or seed medium, respectively, as compared with controls without CuSO4 addition. Pullulan production was further enhanced when CuSO4 was added to both seed medium and bioconversion broth simultaneously. In order to probe the mechanism of CuSO4 improvement, cell viability, membrane integrity, intracellular adenosine triphosphate (ATP) levels and the activities of key enzymes involved in pullulan biosynthesis were determined. As a result, CuSO4 increased the activities of key biosynthetic enzymes, maintained intracellular ATP at a higher level, and accelerated the rate of pullulan secretion, all of which contributed to improved pullulan production by bioconversion.

  19. 16S rRNA荧光定量PCR法检测双歧杆菌%DETECTING BIFIDOBACTERIA BY 16S RRNA FLUORESECENT QUANTIFICATIVE PCR

    Institute of Scientific and Technical Information of China (English)

    沈永才; 袁佩娜

    2001-01-01

    目的应用16S rRNA序列设计引物定量测定双歧杆菌。方法应用青春型双歧杆菌2627号作常规PCR扩增出的模板经系列稀释做荧光定量PCR,制作标准曲线;对青春型、长型、短型、婴儿型、两歧型、链型等6个种共15株双歧杆菌和大肠杆菌等10株其他细菌做荧光定量PCR,计算机通过与标准曲线比较给出定量结果;对青春型双歧杆菌2627号进行敏感性测定。结果 15株双歧杆菌(106个菌)荧光定量PCR法得到拷贝数(1.0×106~1.4×106拷贝)基本一致。结论通过荧光定量PCR可正确定量样品中双歧杆菌数量。%Objective:Quantificative detecting Bifidobacteria by 16S rRNA-targeted PCR.Methods:The standard curve of fluorescent quantificative PCR was deveoped by using a series of dilution of PCR products from Bifidobacterium 2627.Compared with the standard curve,15 strains of Bifidobacteria and 10 strains other bacteria were measured by fluorescent quantificative PCR.The sensitivity was detected by fluorescent quantificative PCR of B.adolescentis 2627.Results:The range of fluorescent quantificative PCR of 15 strains of Bifidobacteria(106bacteria)was nearly equal(1.0×106~1.4×106copies).Conclusion:fluorescent quantificative PCR can be applicable for the quantification fo Bifidobadteria.

  20. Fragment-based whole cell screen delivers hits against M. tuberculosis and non-tuberculous mycobacteria

    Directory of Open Access Journals (Sweden)

    Wilfried Moreira

    2016-09-01

    Full Text Available Reactive multi-target ‘fragment drugs’ represent critical components of current tuberculosis regimens. These compounds, such as pyrazinamide, are old synthetic antimycobacterials that are activated inside Mycobacterium tuberculosis bacilli and are smaller than the usual drug-like, single-target molecules. Based on the success of small ‘dirty’ drugs in the chemotherapy of tuberculosis, we suggested previously that fragment-based whole cell screens should be introduced in our current antimycobacterial drug discovery efforts. Here, we carried out such a screen and characterized bactericidal activity, selectivity and spectrum of hits we obtained. A library of 1725 fragments was tested at a single concentration for growth inhibitory activity against M. bovis BCG as screening strain and 38 of 116 primary hits were confirmed in dose response analyses to be active against virulent M. tuberculosis. Bacterial kill experiments showed that most hits displayed bactericidal activity at their minimal inhibitory concentration. Cytotoxicity assays established that a large proportion of hits displayed a favorable selectivity index for mammalian cells. Importantly, one third of M. tuberculosis active fragments were also active against M. abscessus and M. avium, two emerging non-tuberculous mycobacterial pathogens, opening the opportunity to develop broad spectrum antimycobacterials. Activity determination against Gram positive (Staphylococcus aureus and Gram negative (Escherichia coli, Klebsiella pneumonia, Acinetobacter baumanii, Pseudomonas aeruginosa bacteria, as well as fungi (Candida albicans, Cryptococcus neoformans showed only a small overlap indicating a generally narrow spectrum of these novel antimicrobial hits for mycobacteria. In conclusion, we carried out the first fragment-based whole cell screen against bacteria and identified a substantial number of hits with excellent physicochemical properties and dual activity against M. tuberculosis and

  1. A computational framework for particle and whole cell tracking applied to a real biological dataset.

    Science.gov (United States)

    Yang, Feng Wei; Venkataraman, Chandrasekhar; Styles, Vanessa; Kuttenberger, Verena; Horn, Elias; von Guttenberg, Zeno; Madzvamuse, Anotida

    2016-05-24

    Cell tracking is becoming increasingly important in cell biology as it provides a valuable tool for analysing experimental data and hence furthering our understanding of dynamic cellular phenomena. The advent of high-throughput, high-resolution microscopy and imaging techniques means that a wealth of large data is routinely generated in many laboratories. Due to the sheer magnitude of the data involved manual tracking is often cumbersome and the development of computer algorithms for automated cell tracking is thus highly desirable. In this work, we describe two approaches for automated cell tracking. Firstly, we consider particle tracking. We propose a few segmentation techniques for the detection of cells migrating in a non-uniform background, centroids of the segmented cells are then calculated and linked from frame to frame via a nearest-neighbour approach. Secondly, we consider the problem of whole cell tracking in which one wishes to reconstruct in time whole cell morphologies. Our approach is based on fitting a mathematical model to the experimental imaging data with the goal being that the physics encoded in the model is reflected in the reconstructed data. The resulting mathematical problem involves the optimal control of a phase-field formulation of a geometric evolution law. Efficient approximation of this challenging optimal control problem is achieved via advanced numerical methods for the solution of semilinear parabolic partial differential equations (PDEs) coupled with parallelisation and adaptive resolution techniques. Along with a detailed description of our algorithms, a number of simulation results are reported on. We focus on illustrating the effectivity of our approaches by applying the algorithms to the tracking of migrating cells in a dataset which reflects many of the challenges typically encountered in microscopy data.

  2. Safety and immunogenicity of oral killed whole cell recombinant B subunit cholera vaccine in Barranquilla, Colombia.

    Science.gov (United States)

    Concha, A; Giraldo, A; Castañeda, E; Martínez, M; de la Hoz, F; Rivas, F; Depetris, A; Svennerholm, A M; Sack, D A

    1995-12-01

    In January and February 1992, an assessment was conducted of the safety and immunogenicity of two doses of a new oral cholera vaccine prepared from the recombinant B subunit of the toxin and from killed whole cells (rBS/WC) in 1,165 individuals between the ages of 12 months and 64 years in Barranquilla, Colombia. This was a randomized, double-blind placebo-controlled study. Participants received two doses of either the vaccine or a placebo (killed Escherichia coli K12) over a two-week interval. Few symptoms were detected during the three days following administration of the initial dose and even fewer following the second. Sera obtained upon administration of the first dose and two weeks after administration of the second were tested for Vibrio cholerae 01 Inaba vibriocidal antibodies and antitoxins. Geometric mean titers (GMT) of vibriocidal antibodies were found to increase two-fold in subjects receiving the vaccine. In the paired samples taken from vaccinated subjects, two-fold or greater increases were observed in 44% and four-fold or greater increases were observed in 34%, as compared to similar increases in 9.2% and 2.2% of the sera taken from those receiving the placebo (P or = 1.5, as compared to only about 20% of those in the placebo group (P < 0.000001). Belonging to the O blood group did not significantly affect the immune response. Children under age four tended to show a weaker vibriocidal antibody response and a stronger antitoxin response than older subjects. The two doses of oral vaccine were found to be safe and without attributable side-effects. The vibriocidal antibody and antitoxin responses were similar to those obtained previously with the conventional oral killed whole cell B subunit cholera vaccine.

  3. Malaria chemoprophylaxis and the serologic response to measles and diphtheria-tetanus-whole-cell pertussis vaccines

    Directory of Open Access Journals (Sweden)

    Saliou Pierre

    2005-11-01

    Full Text Available Abstract Background Acute malaria has been associated with a decreased antibody response to tetanus and diphtheria toxoids, meningococcal, salmonella, and Hib vaccines. Interest in giving malaria drug therapy and prevention at the time of childhood immunizations has increased greatly following recent trials of intermittent preventive therapy during infancy (IPTi, stimulating this re-analysis of unpublished data. The effect of malaria chemoprophylaxis on vaccine response was studied following administration of measles vaccines and diphtheria-tetanus-whole cell pertussis (DTP vaccines. Methods In 1975, six villages divided into two groups of children ≤74 months of age from Burkina Faso, were assigned to receive amodiaquine hydrochloride chemoprophylaxis (CH+ every two weeks for seven months or no chemoprophylaxis (CH-. After five months, children in each group received either one dose of measles or two doses of DTP vaccines. Results For recipients of the measles vaccine, the seroconversion rates in CH+ and CH- children, respectively, were 93% and 96% (P > 0.05. The seroresponse rates in CH+ and CH- children respectively, were 73% and 86% for diphtheria (P > 0.05 and 77% and 91% for tetanus toxoid (P > 0.05. In a subset analysis, in which only children who strictly adhered to chemoprophylaxis criteria were included, there were, likewise, no significant differences in seroconversion or seroresponse for measles, diphtheria, or tetanus vaccines (P > 0.05. While analysis for pertussis showed a 43% (CH+ and 67% (CH- response (P Conclusion Malaria chemoprophylaxis prior to vaccination in malaria endemic settings did not improve or impair immunogenicity of DTP and measles vaccines. This is the first human study to look at the association between malaria chemoprophylaxis and the serologic response to whole-cell pertussis vaccine.

  4. Killed but metabolically active Leishmania infantum as a novel whole-cell vaccine for visceral leishmaniasis.

    Science.gov (United States)

    Bruhn, Kevin W; Birnbaum, Ron; Haskell, Jacquelyn; Vanchinathan, Veena; Greger, Stephanie; Narayan, Rupa; Chang, Pei-Lin; Tran, Thu Anh; Hickerson, Suzanne M; Beverley, Stephen M; Wilson, Mary E; Craft, Noah

    2012-04-01

    There are currently no effective vaccines for visceral leishmaniasis, the second most deadly parasitic infection in the world. Here, we describe a novel whole-cell vaccine approach using Leishmania infantum chagasi promastigotes treated with the psoralen compound amotosalen (S-59) and low doses of UV A radiation. This treatment generates permanent, covalent DNA cross-links within parasites and results in Leishmania organisms termed killed but metabolically active (KBMA). In this report, we characterize the in vitro growth characteristics of both KBMA L. major and KBMA L. infantum chagasi. Concentrations of S-59 that generate optimally attenuated parasites were identified. Like live L. infantum chagasi, KBMA L. infantum chagasi parasites were able to initially enter liver cells in vivo after intravenous infection. However, whereas live L. infantum chagasi infection leads to hepatosplenomegaly in mice after 6 months, KBMA L. infantum chagasi parasites were undetectable in the organs of mice at this time point. In vitro, KBMA L. infantum chagasi retained the ability to enter macrophages and induce nitric oxide production. These characteristics of KBMA L. infantum chagasi correlated with the ability to prophylactically protect mice via subcutaneous vaccination at levels similar to vaccination with live, virulent organisms. Splenocytes from mice vaccinated with either live L. infantum chagasi or KBMA L. infantum chagasi displayed similar cytokine patterns in vitro. These results suggest that KBMA technology is a potentially safe and effective novel vaccine strategy against the intracellular protozoan L. infantum chagasi. This approach may represent a new method for whole-cell vaccination against other complex intracellular pathogens.

  5. Whole cell-SELEX aptamers for highly specific fluorescence molecular imaging of carcinomas in vivo.

    Directory of Open Access Journals (Sweden)

    Hui Shi

    Full Text Available BACKGROUND: Carcinomas make up the majority of cancers. Their accurate and specific diagnoses are of great significance for the improvement of patients' curability. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we report an effectual example of the in vivo fluorescence molecular imaging of carcinomas with extremely high specificity based on whole cell-SELEX aptamers. Firstly, S6, an aptamer against A549 lung carcinoma cells, was adopted and labeled with Cy5 to serve as a molecular imaging probe. Flow cytometry assays revealed that Cy5-S6 could not only specifically label in vitro cultured A549 cells in buffer, but also successfully achieve the detection of ex vivo cultured target cells in serum. When applied to in vivo imaging, Cy5-S6 was demonstrated to possess high specificity in identifying A549 carcinoma through a systematic comparison investigation. Particularly, after Cy5-S6 was intravenously injected into nude mice which were simultaneously grafted with A549 lung carcinoma and Tca8113 tongue carcinoma, a much longer retention time of Cy5-S6 in A549 tumor was observed and a clear targeted cancer imaging result was presented. On this basis, to further promote the application to imaging other carcinomas, LS2 and ZY8, which are two aptamers selected by our group against Bel-7404 and SMMC-7721 liver carcinoma cells respectively, were tested in a similar way, both in vitro and in vivo. Results showed that these aptamers were even effective in differentiating liver carcinomas of different subtypes in the same body. CONCLUSIONS/SIGNIFICANCE: This work might greatly advance the application of whole cell-SELEX aptamers to carcinomas-related in vivo researches.

  6. Development of a dual-internal-reference technique to improve accuracy when determining bacterial 16S rRNA:16S rRNA gene ratio with application to Escherichia coli liquid and aerosol samples.

    Science.gov (United States)

    Zhen, Huajun; Krumins, Valdis; Fennell, Donna E; Mainelis, Gediminas

    2015-10-01

    Accurate enumeration of rRNA content in microbial cells, e.g. by using the 16S rRNA:16S rRNA gene ratio, is critical to properly understand its relationship to microbial activities. However, few studies have considered possible methodological artifacts that may contribute to the variability of rRNA analysis results. In this study, a technique utilizing genomic DNA and 16S rRNA from an exogenous species (Pseudomonas fluorescens) as dual internal references was developed to improve accuracy when determining the 16S rRNA:16S rRNA gene ratio of a target organism, Escherichia coli. This technique was able to adequately control the variability in sample processing and analysis procedures due to nucleic acid (DNA and RNA) losses, inefficient reverse transcription of RNA, and inefficient PCR amplification. The measured 16S rRNA:16S rRNA gene ratio of E. coli increased by 2-3 fold when E. coli 16S rRNA gene and 16S rRNA quantities were normalized to the sample-specific fractional recoveries of reference (P. fluorescens) 16S rRNA gene and 16S rRNA, respectively. In addition, the intra-sample variation of this ratio, represented by coefficients of variation from replicate samples, decreased significantly after normalization. This technique was applied to investigate the temporal variation of 16S rRNA:16S rRNA gene ratio of E. coli during its non-steady-state growth in a complex liquid medium, and to E. coli aerosols when exposed to particle-free air after their collection on a filter. The 16S rRNA:16S rRNA gene ratio of E. coli increased significantly during its early exponential phase of growth; when E. coli aerosols were exposed to extended filtration stress after sample collection, the ratio also increased. In contrast, no significant temporal trend in E. coli 16S rRNA:16S rRNA gene ratio was observed when the determined ratios were not normalized based on the recoveries of dual references. The developed technique could be widely applied in studies of relationship between

  7. A renaissance for the pioneering 16S rRNA gene

    Energy Technology Data Exchange (ETDEWEB)

    Tringe, Susannah; Hugenholtz, Philip

    2008-09-07

    Culture-independent molecular surveys using the 16S rRNA gene have become a mainstay for characterizing microbial community structure over the last quarter century. More recently this approach has been overshadowed by metagenomics, which provides a global overview of a community's functional potential rather than just an inventory of its inhabitants. However, the pioneering 16S rRNA gene is making a comeback in its own right thanks to a number of methodological advancements including higher resolution (more sequences), analysis of multiple related samples (e.g. spatial and temporal series) and improved metadata and use of metadata. The standard conclusion that microbial ecosystems are remarkably complex and diverse is now being replaced by detailed insights into microbial ecology and evolution based only on this one historically important marker gene.

  8. Sequence and secondary structure of the mitochondrial 16S ribosomal RNA gene of Ixodes scapularis.

    Science.gov (United States)

    Krakowetz, Chantel N; Chilton, Neil B

    2015-02-01

    The complete DNA sequences and secondary structure of the mitochondrial (mt) 16S ribosomal (r) RNA gene were determined for six Ixodes scapularis adults. There were 44 variable nucleotide positions in the 1252 bp sequence alignment. Most (95%) nucleotide alterations did not affect the integrity of the secondary structure of the gene because they either occurred at unpaired positions or represented compensatory changes that maintained the base pairing in helices. A large proportion (75%) of the intraspecific variation in DNA sequence occurred within Domains I, II and VI of the 16S gene. Therefore, several regions within this gene may be highly informative for studies of the population genetics and phylogeography of I. scapularis, a major vector of pathogens of humans and domestic animals in North America.

  9. An intron within the 16S ribosomal RNA gene of the archaeon Pyrobaculum aerophilum

    Science.gov (United States)

    Burggraf, S.; Larsen, N.; Woese, C. R.; Stetter, K. O.

    1993-01-01

    The 16S rRNA genes of Pyrobaculum aerophilum and Pyrobaculum islandicum were amplified by the polymerase chain reaction, and the resulting products were sequenced directly. The two organisms are closely related by this measure (over 98% similar). However, they differ in that the (lone) 16S rRNA gene of Pyrobaculum aerophilum contains a 713-bp intron not seen in the corresponding gene of Pyrobaculum islandicum. To our knowledge, this is the only intron so far reported in the small subunit rRNA gene of a prokaryote. Upon excision the intron is circularized. A secondary structure model of the intron-containing rRNA suggests a splicing mechanism of the same type as that invoked for the tRNA introns of the Archaea and Eucarya and 23S rRNAs of the Archaea. The intron contains an open reading frame whose protein translation shows no certain homology with any known protein sequence.

  10. An intron within the 16S ribosomal RNA gene of the archaeon Pyrobaculum aerophilum

    Science.gov (United States)

    Burggraf, S.; Larsen, N.; Woese, C. R.; Stetter, K. O.

    1993-01-01

    The 16S rRNA genes of Pyrobaculum aerophilum and Pyrobaculum islandicum were amplified by the polymerase chain reaction, and the resulting products were sequenced directly. The two organisms are closely related by this measure (over 98% similar). However, they differ in that the (lone) 16S rRNA gene of Pyrobaculum aerophilum contains a 713-bp intron not seen in the corresponding gene of Pyrobaculum islandicum. To our knowledge, this is the only intron so far reported in the small subunit rRNA gene of a prokaryote. Upon excision the intron is circularized. A secondary structure model of the intron-containing rRNA suggests a splicing mechanism of the same type as that invoked for the tRNA introns of the Archaea and Eucarya and 23S rRNAs of the Archaea. The intron contains an open reading frame whose protein translation shows no certain homology with any known protein sequence.

  11. Inhibition of Escherichia coli precursor-16S rRNA processing by mouse intestinal contents

    DEFF Research Database (Denmark)

    Licht, Tine Rask; Tolker-Nielsen, Tim; Holmstrøm, Kim;

    1999-01-01

    . We have applied fluorescence in situ hybridization of pre-16S rRNA to Escherichia coli cells growing in vitro in extracts from two different compartments of the mouse intestine: the caecal mucus layer, where E. coli grew rapidly, and the contents of the caecum, which supported much slower bacterial......The correlation between ribosome content and growth rate found in many bacterial species has proved useful for estimating the growth activity of individual cells by quantitative in situ rRNA hybridization. However, in dynamic environments, the stability of mature ribosomal RNA causes problems...... growth. The amounts of 23S rRNA and pre-16S rRNA measured for E. coli growing in intestinal mucus corresponded to that expected for bacteria with the observed growth rate. In contrast, the slow-growing E. coli cells present in intestinal contents turned out to have an approximately ninefold higher...

  12. Greengenes: Chimera-checked 16S rRNA gene database and workbenchcompatible in ARB

    Energy Technology Data Exchange (ETDEWEB)

    DeSantis, T.Z.; Hugenholtz, P.; Larsen, N.; Rojas, M.; Brodie,E.L; Keller, K.; Huber, T.; Dalevi, D.; Hu, P.; Andersen, G.L.

    2006-02-01

    A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera-screening, standard alignments and taxonomic classification using multiple published taxonomies. It was revealed that incongruent taxonomic nomenclature exists among curators even at the phylum-level. Putative chimeras were identified in 3% of environmental sequences and 0.2% of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages within the Archaea and Bacteria.

  13. [Characterization of Black and Dichothrix Cyanobacteria Based on the 16S Ribosomal RNA Gene Sequence

    Science.gov (United States)

    Ortega, Maya

    2010-01-01

    My project focuses on characterizing different cyanobacteria in thrombolitic mats found on the island of Highborn Cay, Bahamas. Thrombolites are interesting ecosystems because of the ability of bacteria in these mats to remove carbon dioxide from the atmosphere and mineralize it as calcium carbonate. In the future they may be used as models to develop carbon sequestration technologies, which could be used as part of regenerative life systems in space. These thrombolitic communities are also significant because of their similarities to early communities of life on Earth. I targeted two cyanobacteria in my research, Dichothrix spp. and whatever black is, since they are believed to be important to carbon sequestration in these thrombolitic mats. The goal of my summer research project was to molecularly identify these two cyanobacteria. DNA was isolated from each organism through mat dissections and DNA extractions. I ran Polymerase Chain Reactions (PCR) to amplify the 16S ribosomal RNA (rRNA) gene in each cyanobacteria. This specific gene is found in almost all bacteria and is highly conserved, meaning any changes in the sequence are most likely due to evolution. As a result, the 16S rRNA gene can be used for bacterial identification of different species based on the sequence of their 16S rRNA gene. Since the exact sequence of the Dichothrix gene was unknown, I designed different primers that flanked the gene based on the known sequences from other taxonomically similar cyanobacteria. Once the 16S rRNA gene was amplified, I cloned the gene into specialized Escherichia coli cells and sent the gene products for sequencing. Once the sequence is obtained, it will be added to a genetic database for future reference to and classification of other Dichothrix sp.

  14. Distinct genetic lineages of Bactrocera caudata (Insecta: Tephritidae revealed by COI and 16S DNA sequences.

    Directory of Open Access Journals (Sweden)

    Phaik-Eem Lim

    Full Text Available The fruit fly Bactrocera caudata is a pest species of economic importance in Asia. Its larvae feed on the flowers of Cucurbitaceae such as Cucurbita moschata. To-date it is distinguished from related species based on morphological characters. Specimens of B. caudata from Peninsular Malaysia and Indonesia (Bali and Lombok were analysed using the partial DNA sequences of cytochrome c oxidase subunit I (COI and 16S rRNA genes. Both gene sequences revealed that B. caudata from Peninsular Malaysia was distinctly different from B. caudata of Bali and Lombok, without common haplotype between them. Phylogenetic analysis revealed two distinct clades, indicating distinct genetic lineage. The uncorrected 'p' distance for COI sequences between B. caudata of Malaysia-Thailand-China and B. caudata of Bali-Lombok was 5.65%, for 16S sequences from 2.76 to 2.99%, and for combined COI and 16S sequences 4.45 to 4.46%. The 'p' values are distinctly different from intraspecific 'p' distance (0-0.23%. Both the B. caudata lineages are distinctly separated from related species in the subgenus Zeugodacus - B. ascita, B. scutellata, B. ishigakiensis, B. diaphora, B. tau, B. cucurbitae, and B. depressa. Molecular phylogenetic analysis indicates that the B. caudata lineages are closely related to B. ascita sp. B, and form a clade with B. scutellata, B. ishigakiensis, B. diaphora and B. ascita sp. A. This study provides additional baseline for the phylogenetic relationships of Bactrocera fruit flies of the subgenus Zeugodacus. Both the COI and 16S genes could be useful markers for the molecular differentiation and phylogenetic analysis of tephritid fruit flies.

  15. Distinct genetic lineages of Bactrocera caudata (Insecta: Tephritidae) revealed by COI and 16S DNA sequences.

    Science.gov (United States)

    Lim, Phaik-Eem; Tan, Ji; Suana, I Wayan; Eamsobhana, Praphathip; Yong, Hoi Sen

    2012-01-01

    The fruit fly Bactrocera caudata is a pest species of economic importance in Asia. Its larvae feed on the flowers of Cucurbitaceae such as Cucurbita moschata. To-date it is distinguished from related species based on morphological characters. Specimens of B. caudata from Peninsular Malaysia and Indonesia (Bali and Lombok) were analysed using the partial DNA sequences of cytochrome c oxidase subunit I (COI) and 16S rRNA genes. Both gene sequences revealed that B. caudata from Peninsular Malaysia was distinctly different from B. caudata of Bali and Lombok, without common haplotype between them. Phylogenetic analysis revealed two distinct clades, indicating distinct genetic lineage. The uncorrected 'p' distance for COI sequences between B. caudata of Malaysia-Thailand-China and B. caudata of Bali-Lombok was 5.65%, for 16S sequences from 2.76 to 2.99%, and for combined COI and 16S sequences 4.45 to 4.46%. The 'p' values are distinctly different from intraspecific 'p' distance (0-0.23%). Both the B. caudata lineages are distinctly separated from related species in the subgenus Zeugodacus - B. ascita, B. scutellata, B. ishigakiensis, B. diaphora, B. tau, B. cucurbitae, and B. depressa. Molecular phylogenetic analysis indicates that the B. caudata lineages are closely related to B. ascita sp. B, and form a clade with B. scutellata, B. ishigakiensis, B. diaphora and B. ascita sp. A. This study provides additional baseline for the phylogenetic relationships of Bactrocera fruit flies of the subgenus Zeugodacus. Both the COI and 16S genes could be useful markers for the molecular differentiation and phylogenetic analysis of tephritid fruit flies.

  16. Binding of 16S rRNA to chloroplast 30S ribosomal proteins blotted on nitrocellulose.

    Science.gov (United States)

    Rozier, C; Mache, R

    1984-10-11

    Protein-RNA associations were studied by a method using proteins blotted on a nitrocellulose sheet. This method was assayed with Escherichia Coli 30S ribosomal components. In stringent conditions (300 mM NaCl or 20 degrees C) only 9 E. coli ribosomal proteins strongly bound to the 16S rRNA: S4, S5, S7, S9, S12, S13, S14, S19, S20. 8 of these proteins have been previously found to bind independently to the 16S rRNA. The same method was applied to determine protein-RNA interactions in spinach chloroplast 30S ribosomal subunits. A set of only 7 proteins was bound to chloroplast rRNA in stringent conditions: chloroplast S6, S10, S11, S14, S15, S17 and S22. They also bound to E. coli 16S rRNA. This set includes 4 chloroplast-synthesized proteins: S6, S11, S15 and S22. The core particles obtained after treatment by LiCl of chloroplast 30S ribosomal subunit contained 3 proteins (S6, S10 and S14) which are included in the set of 7 binding proteins. This set of proteins probably play a part in the early steps of the assembly of the chloroplast 30S ribosomal subunit.

  17. Detection of bacterial 16S ribosomal RNA genes for forensic identification of vaginal fluid.

    Science.gov (United States)

    Akutsu, Tomoko; Motani, Hisako; Watanabe, Ken; Iwase, Hirotaro; Sakurada, Koichi

    2012-05-01

    To preliminarily evaluate the applicability of bacterial DNA as a marker for the forensic identification of vaginal fluid, we developed and performed PCR-based detection of 16S ribosomal RNA genes of Lactobacillus spp. dominating the vagina and of bacterial vaginosis-related bacteria from DNA extracted from body fluids and stains. As a result, 16S ribosomal RNA genes of Lactobacillus crispatus, Lactobacillus jensenii and Atopobium vaginae were specifically detected in vaginal fluid and female urine samples. Bacterial genes detected in female urine might have originated from contaminated vaginal fluid. In addition, those of Lactobacillus iners, Lactobacillus gasseri and Gardnerella vaginalis were also detected in non-vaginal body fluids such as semen. Because bacterial genes were successfully amplified in DNA samples extracted by using the general procedure for animal tissues without any optional treatments, DNA samples prepared for the identification of vaginal fluid can also be used for personal identification. In conclusion, 16S ribosomal RNA genes of L. crispatus, L. jensenii and A. vaginae could be effective markers for forensic identification of vaginal fluid.

  18. Comparison of 16S ribosomal RNA genes in Clavibacter michiganensis subspecies with other coryneform bacteria.

    Science.gov (United States)

    Li, X; De Boer, S H

    1995-10-01

    Nearly complete sequences (97-99%) of the 16S rRNA genes were determined for type strains of Clavibacter michiganensis subsp. michiganensis, Clavibacter michiganensis subsp. insidiosus, Clavibacter michiganensis subsp. sepedonicus, and Clavibacter michiganensis subsp. nebraskensis. The four subspecies had less than 1% dissimilarity in their 16S rRNA genes. Comparative studies indicated that the C. michiganensis subsp. shared relatively high homology with the 16S rRNA gene of Clavibacter xyli. Further comparison with representatives of other Gram-positive coryneform and related bacteria with high G+C% values showed that this group of bacteria was subdivided into three clusters. One cluster consisted of the Clavibacter michiganensis subsp., Clavibacter xyli, Arthrobacter globiformis, Arthrobacter simplex, and Frankia sp.; another cluster consisted of members of the corynebacteria-mycobacteria-nocardia (CMN) group of Mycobacteriaceae including Tsukamurella paurometabolum; and Propionibacterium freudenreichii alone formed a unique cluster, which was remote from other coryneform bacteria analyzed. The three clusters may reflect a systematic rank higher than the genus level among these bacteria.

  19. Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing

    Science.gov (United States)

    Ranjan, Ravi; Rani, Asha; Metwally, Ahmed; McGee, Halvor S.; Perkins, David L.

    2016-01-01

    The human microbiome has emerged as a major player in regulating human health and disease. Translation studies of the microbiome have the potential to indicate clinical applications such as fecal transplants and probiotics. However, one major issue is accurate identification of microbes constituting the microbiota. Studies of the microbiome have frequently utilized sequencing of the conserved 16S ribosomal RNA (rRNA) gene. We present a comparative study of an alternative approach using shotgun whole genome sequencing (WGS). In the present study, we analyzed the human fecal microbiome compiling a total of 194.1×106 reads from a single sample using multiple sequencing methods and platforms. Specifically, after establishing the reproducibility of our methods with extensive multiplexing, we compared: 1) The 16S rRNA amplicon versus the WGS method, 2) the Illumina HiSeq versus MiSeq platforms, 3) the analysis of reads versus de novo assembled contigs, and 4) the effect of shorter versus longer reads. Our study demonstrates that shotgun whole genome sequencing has multiple advantages compared with the 16S amplicon method including enhanced detection of bacterial species, increased detection of diversity and increased prediction of genes. In addition, increased length, either due to longer reads or the assembly of contigs, improved the accuracy of species detection. PMID:26718401

  20. Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification.

    Science.gov (United States)

    Ziesemer, Kirsten A; Mann, Allison E; Sankaranarayanan, Krithivasan; Schroeder, Hannes; Ozga, Andrew T; Brandt, Bernd W; Zaura, Egija; Waters-Rist, Andrea; Hoogland, Menno; Salazar-García, Domingo C; Aldenderfer, Mark; Speller, Camilla; Hendy, Jessica; Weston, Darlene A; MacDonald, Sandy J; Thomas, Gavin H; Collins, Matthew J; Lewis, Cecil M; Hofman, Corinne; Warinner, Christina

    2015-11-13

    To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341-534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.

  1. Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing.

    Science.gov (United States)

    Ranjan, Ravi; Rani, Asha; Metwally, Ahmed; McGee, Halvor S; Perkins, David L

    2016-01-22

    The human microbiome has emerged as a major player in regulating human health and disease. Translational studies of the microbiome have the potential to indicate clinical applications such as fecal transplants and probiotics. However, one major issue is accurate identification of microbes constituting the microbiota. Studies of the microbiome have frequently utilized sequencing of the conserved 16S ribosomal RNA (rRNA) gene. We present a comparative study of an alternative approach using whole genome shotgun sequencing (WGS). In the present study, we analyzed the human fecal microbiome compiling a total of 194.1 × 10(6) reads from a single sample using multiple sequencing methods and platforms. Specifically, after establishing the reproducibility of our methods with extensive multiplexing, we compared: 1) The 16S rRNA amplicon versus the WGS method, 2) the Illumina HiSeq versus MiSeq platforms, 3) the analysis of reads versus de novo assembled contigs, and 4) the effect of shorter versus longer reads. Our study demonstrates that whole genome shotgun sequencing has multiple advantages compared with the 16S amplicon method including enhanced detection of bacterial species, increased detection of diversity and increased prediction of genes. In addition, increased length, either due to longer reads or the assembly of contigs, improved the accuracy of species detection.

  2. PCR Conditions for 16S Primers for Analysis of Microbes in the Colon of Rats.

    Science.gov (United States)

    Guillen, I A; Camacho, H; Tuero, A D; Bacardí, D; Palenzuela, D O; Aguilera, A; Silva, J A; Estrada, R; Gell, O; Suárez, J; Ancizar, J; Brown, E; Colarte, A B; Castro, J; Novoa, L I

    2016-09-01

    The study of the composition of the intestinal flora is important to the health of the host, playing a key role in maintaining intestinal homeostasis and the evolution of the immune system. For these studies, various universal primers of the 16S rDNA gene are used in microbial taxonomy. Here, we report an evaluation of 5 universal primers to explore the presence of microbial DNA in colon biopsies preserved in RNAlater solution. The DNA extracted was used for the amplification of PCR products containing the variable (V) regions of the microbial 16S rDNA gene. The PCR products were studied by restriction fragment length polymorphism (RFLP) analysis and DNA sequence, whose percent of homology with microbial sequences reported in GenBank was verified using bioinformatics tools. The presence of microbes in the colon of rats was quantified by the quantitative PCR (qPCR) technique. We obtained microbial DNA from rat, useful for PCR analysis with the universal primers for the bacteria 16S rDNA. The sequences of PCR products obtained from a colon biopsy of the animal showed homology with the classes bacilli (Lactobacillus spp) and proteobacteria, normally represented in the colon of rats. The proposed methodology allowed the attainment of DNA of bacteria with the quality and integrity for use in qPCR, sequencing, and PCR-RFLP analysis. The selected universal primers provided knowledge of the abundance of microorganisms and the formation of a preliminary test of bacterial diversity in rat colon biopsies.

  3. Nested polymerase chain reaction (PCR) targeting 16S rDNA for bacterial identification in empyema.

    Science.gov (United States)

    Prasad, Rajniti; Kumari, Chhaya; Das, B K; Nath, Gopal

    2014-05-01

    Empyema in children causes significant morbidity and mortality. However, identification of organisms is a major concern. To detect bacterial pathogens in pus specimens of children with empyema by 16S rDNA nested polymerase chain reaction (PCR) and correlate it with culture and sensitivity. Sixty-six children admitted to the paediatric ward with a diagnosis of empyema were enrolled prospectively. Aspirated pus was subjected to cytochemical examination, culture and sensitivity, and nested PCR targeting 16S rDNA using a universal eubacterial primer. Mean (SD) age was 5·8 (1·8) years (range 1-13). Analysis of aspirated pus demonstrated total leucocyte count >1000×10(6)/L, elevated protein (≧20 g/L) and decreased glucose (≤2·2 mmol/L) in 80·3%, 98·5% and 100%, respectively. Gram-positive cocci were detected in 29 (43·9%) and Gram-negative bacilli in two patients. Nested PCR for the presence of bacterial pathogens was positive in 50·0%, compared with 36·3% for culture. 16S rDNA PCR improves rates of detection of bacteria in pleural fluid, and can detect bacterial species in a single assay as well as identifying unusual and unexpected causal agents.

  4. Culture-Negative Endocarditis Diagnosed Using 16S DNA Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Stephen Duffett

    2012-01-01

    Full Text Available 16S DNA polymerase chain reaction (PCR is a molecular amplification technique that can be used to identify bacterial pathogens in culture-negative endocarditis. Bacterial DNA can be isolated from surgically excised valve tissue or from blood collected in EDTA vials. Use of this technique is particularly helpful in identifying the bacterial pathogen in cases of culture-negative endocarditis. A case involving a 48-year-old man who presented with severe aortic regurgitation and a four-month prodrome of low-grade fever is reported. Blood and valve tissue cultures following valve replacement were negative. A valve tissue sample was sent for investigation with 16S DNA PCR, which successfully identified Streptococcus salivarius and was interpreted as the true diagnosis. A review of the literature suggests that 16S DNA PCR from valve tissue is a more sensitive diagnostic test than culture. It is also extremely specific, based on a sequence match of at least 500 base pairs.

  5. Detection of Enterobacter sakazakii in neonatal sepsis by PCR on 16S ribosomal RNA

    Directory of Open Access Journals (Sweden)

    Khadijeh Ahmadi

    2014-08-01

    Full Text Available Background: Enterobacter sakazakii is a gram negative, facultative anaerobic, straight rod-shaped bacterium that belongs to the family Enterobacteriaceae. It is also considered an emerging opportunistic pathogen, responsible of cases of neonatal infections including sepsis, meningitis, necrotizing enterocolitis ad bacteremia. The goal of this study was detection of Enterobacter salazakii in neonates with sepsis by PCR on 16S ribosomal RNA gene. Material and Methods: This cross-sectional study was conducted on 405 blood specimens that were taken from hospitalized neonates suspected to sepsis in Ahvaz Abuzar Hospital in 2011. From each neonate 0.5 ml blood sample was taken and placed in CBC tubes containing EDTA at -200C for polymerase chain reaction. For detection of Enterobacter sakazakii, PCR was performed on DNA for amplification of 16S ribosomal RNA gene. Results: In all 405 neonates blood samples’ PCR reactions for Enterobacter sakazakii 16S ribosomal RNA gene were negative. Blood cultures were positive for Streptococcus agalactiae in 8 (1.4 % patients. Conclusion: Because Enterobacter sakazakii is an opportunistic pathogen with high pathogenicity power, more investigation on high risk groups is required. For detection of infection caused by this organism using of different diagnostic methods with high specificity and sensitivity is necessary.

  6. The Role of 16S rRNA Gene Sequencing in Confirmation of Suspected Neonatal Sepsis.

    Science.gov (United States)

    El Gawhary, Somaia; El-Anany, Mervat; Hassan, Reem; Ali, Doaa; El Gameel, El Qassem

    2016-02-01

    Different molecular assays for the detection of bacterial DNA in the peripheral blood represented a diagnostic tool for neonatal sepsis. We targeted to evaluate the role of 16S rRNA gene sequencing to screen for bacteremia to confirm suspected neonatal sepsis (NS) and compare with risk factors and septic screen testing. Sixty-two neonates with suspected NS were enrolled. White blood cells count, I/T ratio, C-reactive protein, blood culture and 16S rRNA sequencing were performed. Blood culture was positive in 26% of cases, and PCR was positive in 26% of cases. Evaluation of PCR for the diagnosis of NS showed sensitivity 62.5%, specificity 86.9%, PPV 62.5%, NPV 86.9% and accuracy of 79.7%. 16S rRNA PCR increased the sensitivity of detecting bacterial DNA in newborns with signs of sepsis from 26 to 35.4%, and its use can be limited to cases with the most significant risk factors and positive septic screen. © The Author [2015]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  7. Microarray analysis of cytoplasmic versus whole cell RNA reveals a considerable number of missed and false positive mRNAs

    OpenAIRE

    Trask, Heidi W.; Cowper-Sal-lari, Richard; Sartor, Maureen A.; Gui, Jiang; Heath, Catherine V.; Renuka, Janhavi; Higgins, Azara-Jane; Andrews,Peter; Korc, Murray; Moore, Jason H; Craig R Tomlinson

    2009-01-01

    With no known exceptions, every published microarray study to determine differential mRNA levels in eukaryotes used RNA extracted from whole cells. It is assumed that the use of whole cell RNA in microarray gene expression analysis provides a legitimate profile of steady-state mRNA. Standard labeling methods and the prevailing dogma that mRNA resides almost exclusively in the cytoplasm has led to the long-standing belief that the nuclear RNA contribution is negligible. We report that unadulte...

  8. Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies

    KAUST Repository

    Wang, Yong

    2009-10-09

    Bacterial 16S ribosomal DNA (rDNA) amplicons have been widely used in the classification of uncultured bacteria inhabiting environmental niches. Primers targeting conservative regions of the rDNAs are used to generate amplicons of variant regions that are informative in taxonomic assignment. One problem is that the percentage coverage and application scope of the primers used in previous studies are largely unknown. In this study, conservative fragments of available rDNA sequences were first mined and then used to search for candidate primers within the fragments by measuring the coverage rate defined as the percentage of bacterial sequences containing the target. Thirty predicted primers with a high coverage rate (>90%) were identified, which were basically located in the same conservative regions as known primers in previous reports, whereas 30% of the known primers were associated with a coverage rate of <90%. The application scope of the primers was also examined by calculating the percentages of failed detections in bacterial phyla. Primers A519-539, E969- 983, E1063-1081, U515 and E517, are highly recommended because of their high coverage in almost all phyla. As expected, the three predominant phyla, Firmicutes, Gemmatimonadetes and Proteobacteria, are best covered by the predicted primers. The primers recommended in this report shall facilitate a comprehensive and reliable survey of bacterial diversity in metagenomic studies. © 2009 Wang, Qian.

  9. Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies.

    Science.gov (United States)

    Wang, Yong; Qian, Pei-Yuan

    2009-10-09

    Bacterial 16S ribosomal DNA (rDNA) amplicons have been widely used in the classification of uncultured bacteria inhabiting environmental niches. Primers targeting conservative regions of the rDNAs are used to generate amplicons of variant regions that are informative in taxonomic assignment. One problem is that the percentage coverage and application scope of the primers used in previous studies are largely unknown. In this study, conservative fragments of available rDNA sequences were first mined and then used to search for candidate primers within the fragments by measuring the coverage rate defined as the percentage of bacterial sequences containing the target. Thirty predicted primers with a high coverage rate (>90%) were identified, which were basically located in the same conservative regions as known primers in previous reports, whereas 30% of the known primers were associated with a coverage rate of primers was also examined by calculating the percentages of failed detections in bacterial phyla. Primers A519-539, E969-983, E1063-1081, U515 and E517, are highly recommended because of their high coverage in almost all phyla. As expected, the three predominant phyla, Firmicutes, Gemmatimonadetes and Proteobacteria, are best covered by the predicted primers. The primers recommended in this report shall facilitate a comprehensive and reliable survey of bacterial diversity in metagenomic studies.

  10. Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies.

    Directory of Open Access Journals (Sweden)

    Yong Wang

    Full Text Available Bacterial 16S ribosomal DNA (rDNA amplicons have been widely used in the classification of uncultured bacteria inhabiting environmental niches. Primers targeting conservative regions of the rDNAs are used to generate amplicons of variant regions that are informative in taxonomic assignment. One problem is that the percentage coverage and application scope of the primers used in previous studies are largely unknown. In this study, conservative fragments of available rDNA sequences were first mined and then used to search for candidate primers within the fragments by measuring the coverage rate defined as the percentage of bacterial sequences containing the target. Thirty predicted primers with a high coverage rate (>90% were identified, which were basically located in the same conservative regions as known primers in previous reports, whereas 30% of the known primers were associated with a coverage rate of <90%. The application scope of the primers was also examined by calculating the percentages of failed detections in bacterial phyla. Primers A519-539, E969-983, E1063-1081, U515 and E517, are highly recommended because of their high coverage in almost all phyla. As expected, the three predominant phyla, Firmicutes, Gemmatimonadetes and Proteobacteria, are best covered by the predicted primers. The primers recommended in this report shall facilitate a comprehensive and reliable survey of bacterial diversity in metagenomic studies.

  11. Short Term Reactogenicity of a Triple Diphtheria-Tetanus-Whole Cell Pertussis Vaccine in Iranian Infants

    Directory of Open Access Journals (Sweden)

    S Zarei

    2009-03-01

    Full Text Available "nBackground: Immunization against diphtheria, tetanus and pertussis (DTP has long been applied in Iran using whole cell vac­cine. Despite the role of whole cell DTP (DTwP vaccine in reduction of mortality as a result of disastrous diseases such as diphtheria, tetanus, and pertussis, serious local and systemic complications have been attributed to these vaccines. This study was performed to determine the complications of DTwP vaccine in infants attending some of the health centers of Te­hran in 2006-2007."nMethods:  In this prospective study, 330 infants were injected with DTwP vaccine manufactured by Razi Institute of Iran. All subjects received DTwP vaccine at 2, 4, and 6 months of age following the national vaccination schedule of Iran. Re­actogenicity was assessed by the parents for 7 days post-vaccination using diary cards."nResults: Of the 279 infants who completed the vaccination study, pain was the most frequent local reaction after the pri­mary vaccination (68.1-75.3%. The mean diameters of the redness and swelling at first day post-vaccination were 2.81±6.91 and 2.60±7.93 mm in the first dose, 2.40±6.25 and 1.94±5.74 mm in the second dose and 2.24±5.66 and 2.16±6.03 in the third dose, respectively. Fever (axillary temperature >37.5° C was the most frequently reported systemic re­action during the pri­mary vaccination (53.8-58.8%. All systemic reactions observed after each dose were either reduced or completely disap­peared during a week."nConclusion: The high incident of complications observed following vaccination with this cellular triple vaccine may be re­lated to the formulation or the bacterial cell fragments used in vaccine production.

  12. Surface complexation of neptunium (V) onto whole cells and cell componets of Shewanella alga

    Energy Technology Data Exchange (ETDEWEB)

    Reed, Donald Timothy [Los Alamos National Laboratory; Deo, Randhir P [ASU; Rittmann, Bruce E [ASU; Songkasiri, Warinthorn [UNAFFILIATED

    2008-01-01

    We systematically quantified surface complexation of neptunium(V) onto whole cells of Shewanella alga strain BrY and onto cell wall and extracellular polymeric substances (EPS) of S. alga. We first performed acid and base titrations and used the mathematical model FITEQL with constant-capacitance surface-complexation to determine the concentrations and deprotonation constants of specific surface functional groups. Deprotonation constants most likely corresponded to a carboxyl site associated with amino acids (pK{sub a} {approx} 2.4), a carboxyl group not associated with amino acids (pK{sub a} {approx} 5), a phosphoryl site (pK{sub a} {approx} 7.2), and an amine site (pK{sub a} > 10). We then carried out batch sorption experiments with Np(V) and each of the S. alga components at different pHs. Results show that solution pH influenced the speciation of Np(V) and each of the surface functional groups. We used the speciation sub-model of the biogeochemical model CCBATCH to compute the stability constants for Np(V) complexation to each surface functional group. The stability constants were similar for each functional group on S. alga bacterial whole cells, cell walls, and EPS, and they explain the complicated sorption patterns when they are combined with the aqueous-phase speciation of Np(V). For pH < 8, NpO{sub 2}{sup +} was the dominant form of Np(V), and its log K values for the low-pK{sub a} carboxyl, other carboxyl, and phosphoryl groups were 1.75, 1.75, and 2.5 to 3.1, respectively. For pH greater than 8, the key surface ligand was amine >XNH3+, which complexed with NpO{sub 2}(CO{sub 3}){sub 3}{sup 5-}. The log K for NpO{sub 2}(CO{sub 3}){sub 3}{sup 5-} complexed onto the amine groups was 3.1 to 3.6. All of the log K values are similar to those of Np(V) complexes with aqueous carboxyl and N-containing carboxyl ligands. These results point towards the important role of surface complexation in defining key actinide-microbiological interactions in the subsurface.

  13. Characterization of copy numbers of 16S rDNA and 16S rRNA of Candidatus Liberibacter asiaticus and the implication in detection in planta using quantitative PCR

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    Wang Nian

    2009-03-01

    Full Text Available Abstract Background Citrus Huanglongbing (HLB is one of the most devastating diseases on citrus and is associated with Candidatus Liberibacter spp.. The pathogens are phloem limited and have not been cultured in vitro. The current management strategy of HLB is to remove infected citrus trees and reduce psyllid populations with insecticides to prevent the spreading. This strategy requires sensitive and reliable diagnostic methods for early detection. Results We investigated the copy numbers of the 16S rDNA and 16S rRNA of the HLB pathogen and the implication of improving the diagnosis of HLB for early detection using Quantitative PCR. We compared the detection of HLB with different Quantitative PCR based methods with primers/probe targeting either 16S rDNA, beta-operon DNA, 16S rRNA, or beta-operon RNA. The 16S rDNA copy number of Ca. Liberibacter asiaticus was estimated to be three times of that of the beta-operon region, thus allowing detection of lower titer of Ca. L. asiaticus. Quantitative reverse transcriptional PCR (QRT-PCR indicated that the 16S rRNA averaged 7.83 times more than that of 16S rDNA for the same samples. Dilution analysis also indicates that QRT-PCR targeting 16S rRNA is 10 time more sensitive than QPCR targeting 16S rDNA. Thus QRT-PCR was able to increase the sensitivity of detection by targeting 16S rRNA. Conclusion Our result indicates that Candidatus Liberibacter asiaticus contains three copies of 16S rDNA. The copy number of 16S rRNA of Ca. L. asiaticus in planta averaged about 7.8 times of 16S rDNA for the same set of samples tested in this study. Detection sensitivity of HLB could be improved through the following approaches: using 16S rDNA based primers/probe in the QPCR assays; and using QRT-PCR assays targeting 16S rRNA.

  14. Biocatalytic Production of Trehalose from Maltose by Using Whole Cells of Permeabilized Recombinant Escherichia coli.

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    Zhaojuan Zheng

    Full Text Available Trehalose is a non-reducing disaccharide, which can protect proteins, lipid membranes, and cells from desiccation, refrigeration, dehydration, and other harsh environments. Trehalose can be produced by different pathways and trehalose synthase pathway is a convenient, practical, and low-cost pathway for the industrial production of trehalose. In this study, 3 candidate treS genes were screened from genomic databases of Pseudomonas and expressed in Escherichia coli. One of them from P. stutzeri A1501 exhibited the best transformation ability from maltose into trehalose and the least byproduct. Thus, whole cells of this recombinant E. coli were used as biocatalyst for trehalose production. In order to improve the conversion rate of maltose to trehalose, optimization of the permeabilization and biotransformation were carried out. Under optimal conditions, 92.2 g/l trehalose was produced with a high productivity of 23.1 g/(l h. No increase of glucose was detected during the whole course. The biocatalytic process developed in this study might serve as a candidate for the large scale production of trehalose.

  15. A UAV-Mounted Whole Cell Biosensor System for Environmental Monitoring Applications.

    Science.gov (United States)

    Lu, Yi; Macias, Dominique; Dean, Zachary S; Kreger, Nicole R; Wong, Pak Kin

    2015-12-01

    This study reports the development of a portable whole cell biosensor system for environmental monitoring applications, such as air quality control, water pollution monitoring, and radiation leakage detection. The system consists of a lightweight mechanical housing, a temperature regulating system, and a microfluidic bacterial inoculation channel. The overall system, which is less than 200 g, serves as a portable incubator for cell inoculation and can be mounted on an unmanned aerial vehicle for monitoring remote and unreachable locations. The feedback control system maintains the inoculation temperature within 0.05 °C. The large surface-to-volume ratio of the polydimethylsiloxane microchannel facilitates effective gas exchange for rapid bacterial growth. Molecular dynamic simulation shows effective diffusion of major gas pollutants in PDMS toward gas sensing applications. By optimizing the design, we demonstrate the operation of the system in ambient temperatures from 5 °C to 32 °C and rapid bacterial growth in microchannels compared to standard bacterial culture techniques.

  16. Bacterial whole-cell biocatalysts by surface display of enzymes: toward industrial application.

    Science.gov (United States)

    Schüürmann, Jan; Quehl, Paul; Festel, Gunter; Jose, Joachim

    2014-10-01

    Despite the first report on the bacterial display of a recombinant peptide appeared almost 30 years ago, industrial application of cells with surface-displayed enzymes is still limited. To display an enzyme on the surface of a living cell bears several advantages. First of all, neither the substrate nor the product of the enzymatic reaction needs to cross a membrane barrier. Second, the enzyme being linked to the cell can be separated from the reaction mixture and hence the product by simple centrifugation. Transfer to a new substrate preparation results in multiple cycles of enzymatic conversion. Finally, the anchoring in a matrix, in this case, the cell envelope stabilizes the enzyme and makes it less accessible to proteolytic degradation and material adsorption resulting in continuous higher activities. These advantages in common need to balance some disadvantages before this application can be taken into account for industrial processes, e.g., the exclusion of the enzyme from the cellular metabolome and hence from redox factors or other co-factors that need to be supplied. Therefore, this digest describes the different systems in Gram-positive and Gram-negative bacteria that have been used for the surface display of enzymes so far and focuses on examples among these which are suitable for industrial purposes or for the production of valuable resources, not least in order to encourage a broader application of whole-cell biocatalysts with surface-displayed enzymes.

  17. Efficient conversion of phenylpyruvic acid to phenyllactic acid by using whole cells of Bacillus coagulans SDM.

    Directory of Open Access Journals (Sweden)

    Zhaojuan Zheng

    Full Text Available BACKGROUND: Phenyllactic acid (PLA, a novel antimicrobial compound with broad and effective antimicrobial activity against both bacteria and fungi, can be produced by many microorganisms, especially lactic acid bacteria. However, the concentration and productivity of PLA have been low in previous studies. The enzymes responsible for conversion of phenylpyruvic acid (PPA into PLA are equivocal. METHODOLOGY/PRINCIPAL FINDINGS: A novel thermophilic strain, Bacillus coagulans SDM, was isolated for production of PLA. When the solubility and dissolution rate of PPA were enhanced at a high temperature, whole cells of B. coagulans SDM could effectively convert PPA into PLA at a high concentration (37.3 g l(-1 and high productivity (2.3 g l(-1 h(-1 under optimal conditions. Enzyme activity staining and kinetic studies identified NAD-dependent lactate dehydrogenases as the key enzymes that reduced PPA to PLA. CONCLUSIONS/SIGNIFICANCE: Taking advantage of the thermophilic character of B. coagulans SDM, a high yield and productivity of PLA were obtained. The enzymes involved in PLA production were identified and characterized, which makes possible the rational design and construction of microorganisms suitable for PLA production with metabolic engineering.

  18. A microbial method using whole cells of Thiobacillus thiooxidans for measuring sulphate in waters

    Energy Technology Data Exchange (ETDEWEB)

    Nakamura, K.; Yudiarto, M.A.; Kaneko, N.; Kurosawa, H.; Amano, Y. [Dept. of Applied Chemistry and Biotechnology, Faculty of Engineering, Yamanashi Univ. (Japan)

    1997-12-31

    A microbial method to determine sulphate concentration in water was developed on the basis of sulphate-dependent acid phosphatase (APase) in whole cells of Thiobacillus thiooxidans. The activity of the APase was determined colorimetrically by using p-nitrophenylphosphate as substrate. The APase was activated by sulphate. A linear relationship was obtained between the activity of the APase and the concentration of sulphate in the range 0-0.06 mM. Therefore, the sulphate concentration was estimated from the APase activity, represented by the absorbance (A{sub 400}). The microbial method was applied to the determination sulphate in water. The lower limit of detection was 0.02 mM, the relative standard deviation being 2% for 10 measurements on a standard sample. As for practical samples, which were taken from rain, river and tap water, good agreement was obtained between the values measured by the microbial method and those given by a conventional barium chloranilate method. The relative standard deviation was 2.1% for 12 measurements of tap water. The activity of the APase was stable over a period of more than 100 days when the cells were stored in 0.1 M sodium acetate/acetic acid buffer (pH 5.0) at 4 C. (orig.)

  19. Development of novel conductometric biosensors based on immobilised whole cell Chlorella vulgaris microalgae.

    Science.gov (United States)

    Chouteau, Celine; Dzyadevych, Sergei; Chovelon, Jean-Marc; Durrieu, Claude

    2004-04-15

    A novel biosensor based on immobilised whole cell Chlorella vulgaris microalgae as a bioreceptor and interdigitated conductometric electrodes as a transducer has been developed and tested for alkaline phosphatase activity (APA) analysis. These sensors were also used for the detection of toxic compounds, namely cadmium ions, in aquatic habitats. Algae were immobilised inside bovine serum albumin (BSA) membranes cross-linked with glutaraldehyde vapours. The detection of the local conductivity variations caused by algae enzymatic reactions could be achieved. The inhibition of C. vulgaris microalgae Alkaline phosphatase activities in presence of cadmium ions was measured. These results were compared with measurements in bioassays. It finally appeared that conductometric biosensors using algae seemed more sensitive than bioassays to detect low levels of cadmium ions (the detection limit for the first experiments was 1 ppb of Cd2+). The main advantages of these alkaline phosphatase biosensors consist of their high specificity in regard to the toxic compounds they enable to detect, but also on their high stability since contrary to enzymatic biosensors, they use whole algae cells with APs on their walls.

  20. Development of microbial engineered whole-cell systems for environmental benzene determination.

    Science.gov (United States)

    Di Gennaro, P; Bruzzese, N; Anderlini, D; Aiossa, M; Papacchini, M; Campanella, L; Bestetti, G

    2011-03-01

    This paper reports the development of two recombinant bacterial systems that can be used to monitor environmental benzene contamination based on Escherichia coli, which carry genes coding for benzene dioxygenase and benzene dihydrodiol dehydrogenase from Pseudomonas putida MST. E. coli strains express these two enzymes under the control of the Ptac promoter or without any induction. These activities can be detected electrochemically or colorimetrically and used to monitor benzene pollution in environmental air samples collected from an oil refinery assessing benzene by different laboratory experimental procedures. The procedures involving whole-cell bioassays determine the concentration of benzene through benzene dioxygenase activity, which allows for direct correlation of oxygen consumption, and through the benzene dihydrodiol dehydrogenase that causes catechol accumulation and restores NADH necessary for the activity of the first enzyme. Oxygen consumption and catechol production deriving from both enzymatic activities are related to benzene concentration and their measurements determined the sensitivity of the system. The results indicated that the sensitivity was enough to detect the benzene vapor at a lower concentration level of 0.01 mM in about 30 min. The possibility for on-line monitoring of benzene concentration by our new recombinant cells results from the fact that no particular treatment of environmental samples is required. This is a major advantage over other biosensors or assays. Moreover, the development of microbial cells that did not require any addition or effectors for the transcription of the specific enzymes, allowed these systems to be more versatile in automated environmental benzene monitoring.

  1. Pharmacology and toxicology of an oral tablet whole cells inactivated cholera vaccine in Sprague Dawley rats.

    Science.gov (United States)

    López, Yulieé; Infante, Juan Francisco; Sifontes, Sergio; Díaz, Daiyana; Pérez, Viviana; Año, Gemma; Hernández, Tamara; Fernández, Sonsire; Castaño, Jorge Luis; Cedré, Bárbara; Oliva, Reynaldo; García, Luis; Solís, Rosa L; Talavera, Arturo

    2011-04-27

    Here we further investigate the pharmacological and toxicological properties of a cholera vaccine based on inactivated whole cells presented in either enteric coated (COA) or uncoated (U/C) tablet formulation from Vibrio cholerae C7258 strain. Tablets were dispersed in 2mL drinking water and administered orally to Sprague Dawley rats distributed in five groups (I COA7, II U/C7 immunized at 0, 7, 69days and III COA14, IV U/C14 immunized at 0, 14, 69days and V control group). Serum vibriocidal antibody response was measured after the administration of two doses with an interval of 7-14days. To further investigate the toxicological aspects a third dose was applied 10 weeks after the initial one. Animals were observed daily and water and food consumption was measured every other day. Periodic blood extractions were performed for hematology, biochemistry, and the titer of serum vibriocidal antibodies was determined. Anatomopathological analysis was performed at days 3 or 14 after the third dose. Results from clinical observations, as well as from water and food consumption and body weigh indicated no toxicity of the vaccine product. Meanwhile, no biological differences were found among different groups in hematological, hemo-chemistry, and anatomopathological studies. Moreover, enteric coated and uncoated tablets against human cholera were found to induce an immune response in rats.

  2. Whole-cell Gluconobacter oxydans biosensor for 2-phenylethanol biooxidation monitoring.

    Science.gov (United States)

    Schenkmayerová, Andrea; Bertóková, Anikó; Sefčovičová, Jana; Stefuca, Vladimír; Bučko, Marek; Vikartovská, Alica; Gemeiner, Peter; Tkáč, Ján; Katrlík, Jaroslav

    2015-01-07

    A microbial biosensor for 2-phenylethanol (2-PE) based on the bacteria Gluconobacter oxydans was developed and applied in monitoring of a biotechnological process. The cells of G. oxydans were immobilized within a disposable polyelectrolyte complex gel membrane consisting of sodium alginate, cellulose sulphate and poly(methylene-co-guanidine) attached onto a miniaturized Clark oxygen electrode, forming whole cell amperometric biosensor. Measured changes in oxygen concentration were proportional to changes in 2-PE concentration. The biosensor sensitivity was 864 nA mM(-1) (RSD=6%), a detection limit of 1 μM, and the biosensor response towards 2-PE was linear in the range 0.02-0.70 mM. The biosensor preserved 93% of its initial sensitivity after 7h of continuous operation and exhibited excellent storage stability with loss of only 6% of initial sensitivity within two months, when stored at 4°C. The developed system was designed and successfully used for an off-line monitoring of whole course of 2-PE biooxidation process producing phenylacetic acid (PA) as industrially valuable aromatic compound. The biosensor measurement did not require the use of hazardous organic solvent. The biosensor response to 2-PE was not affected by interferences from PA and phenylacetaldehyde at concentrations present in real samples during the biotransformation and the results were in a very good agreement with those obtained via gas chromatography.

  3. Identification of novel bacterial histidine biosynthesis inhibitors using docking, ensemble rescoring, and whole-cell assays.

    Science.gov (United States)

    Henriksen, S T; Liu, J; Estiu, G; Oltvai, Z N; Wiest, O

    2010-07-15

    The rapid spread on multidrug-resistant strains of Staphylococcus aureus requires not just novel treatment options, but the development of faster methods for the identification of new hits for drug development. The exponentially increasing speed of computational methods makes a more extensive use in the early stages of drug discovery attractive if sufficient accuracy can be achieved. Computational target identification using systems-level methods suggested the histidine biosynthesis pathway as an attractive target against S. aureus. Potential inhibitors for the pathway were identified through docking, followed by ensemble rescoring, that is sufficiently accurate to justify immediate testing of the identified compounds by whole-cell assays, avoiding the need for time-consuming and often difficult intermediary enzyme assays. This novel strategy is demonstrated for three key enzymes of the S. aureus histidine biosynthesis pathway, which is predicted to be essential for bacterial biomass productions. Virtual screening of a library of approximately 10(6) compounds identified 49 potential inhibitors of three enzymes of this pathway. Eighteen representative compounds were directly tested on three S. aureus- and two Escherichia coli strains in standard disk inhibition assays. Thirteen compounds are inhibitors of some or all of the S. aureus strains, while 14 compounds weakly inhibit growth in one or both E. coli strains. The high hit rate obtained from a fast virtual screen demonstrates the applicability of this novel strategy to the histidine biosynthesis pathway.

  4. Whole cell structural imaging at 20 nanometre resolutions using MeV ions

    Science.gov (United States)

    Watt, F.; Chen, X.; Chen, C.-B.; Udalagama, CNB; van Kan, J. A.; Bettiol, A. A.

    2013-07-01

    MeV proton and alpha (helium ion) particle beams can now be focused to 20 nm spot sizes, and ion/matter simulations using the DEEP computer code show that these resolutions are maintained through the top micrometre or so of organic material. In addition, the energy deposition profiles of the transmitted ions are laterally constrained to a few nanometers from the initial ion path. This paves the way for high resolution structural imaging of relatively thick biological material, e.g. biological cells. Examples are shown of high resolution structural imaging of whole biological cells (MRC5) using on-axis scanning transmission ion microscopy (STIM). Nanoparticles have the ability to cross the cell membrane, and may therefore prove useful as drug delivery probes. We show that the combination of on-axis STIM for imaging the cell interior, and off-axis STIM for imaging gold nanoparticles with enhanced contrast within the cell, represents a powerful set of ion beam techniques for tracking gold nanoparticles in biological cells. Whole cell imaging at high spatial resolutions represents a new area for nuclear microprobes.

  5. Whole cell strategies based on lux genes for high throughput applications toward new antimicrobials.

    Science.gov (United States)

    Galluzzi, Lorenzo; Karp, Matti

    2006-08-01

    The discovery/development of novel drug candidates has witnessed dramatic changes over the last two decades. Old methods to identify lead compounds are not suitable to screen wide libraries generated by combinatorial chemistry techniques. High throughput screening (HTS) has become irreplaceable and hundreds of different approaches have been described. Assays based on purified components are flanked by whole cell-based assays, in which reporter genes are used to monitor, directly or indirectly, the influence of a chemical over the metabolism of living cells. The most convenient and widely used reporters for real-time measurements are luciferases, light emitting enzymes from evolutionarily distant organisms. Autofluorescent proteins have been also extensively employed, but proved to be more suitable for end-point measurements, in situ applications - such as the localization of fusion proteins in specific subcellular compartments - or environmental studies on microbial populations. The trend toward miniaturization and the technical advances in detection and liquid handling systems will allow to reach an ultra high throughput screening (uHTS), with 100,000 of compounds routinely screened each day. Here we show how similar approaches may be applied also to the search for new and potent antimicrobial agents.

  6. Nanoscale imaging of whole cells using a liquid enclosure and a scanning transmission electron microscope.

    Directory of Open Access Journals (Sweden)

    Diana B Peckys

    Full Text Available Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7 were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM laboratory.

  7. Proteomic analysis using 2-D liquid separations of intact proteins from whole-cell lysates.

    Science.gov (United States)

    Zhu, Kan; Yan, Fang; O'Neil, Kimberly A; Hamler, Rick; Lubman, David M; Lin, Linda; Barder, Timothy J

    2004-02-01

    This unit describes procedures for 2-D liquid separations of proteins from whole-cell lysates. Protocols for protein isoelectric point (pI) fractionation in the first dimension include the use of liquid isoelectric focusing (IEF) and chromatofocusing. The liquid IEF provides a pI-based fractionation using a batch-phase electrophoretic method, while chromatofocusing uses a column-based chromatographic method to generate the pH gradient. Using either method, a second-dimension fractionation is provided in the liquid phase using nonporous silica-based reversed-phase HPLC (NPS-RP-HPLC) to generate a 2-D liquid map of the protein content of the cell. The eluate of the 2-D liquid fractionation is directly coupled to a mass spectrometer for on-line detection of the intact molecular weights of proteins. As a result, a multidimensional map of protein expression is obtained that characterizes cellular proteins by pI, hydrophobicity, and intact molecular weight. Such expression maps are useful for differential proteomic comparison between different cell samples.

  8. Electrical field manipulation of cancer cell behavior monitored by whole cell biosensing device.

    Science.gov (United States)

    Hondroulis, Evangelia; Melnick, Steven J; Zhang, Xueji; Wu, Ze-Zhi; Li, Chen-Zhong

    2013-08-01

    All living cells possess electrical characteristics and are thus responsive to, and even generate electric fields and currents. It has been shown that the electrical properties of cancer cells differ from normal proliferating cells, thus electric fields may induce differential effects in normal and cancer cells. Manipulation of these electrical properties may provide a powerful direct and/or adjuvant therapeutic option for cancer. A whole cell impedance-based biosensor to monitor the effects of a range of different frequencies (50 kHz-2 MHz) at low-intensity (electric fields were applied and the impedimetric response of the cells was recorded. The cells were also labeled with propidium iodide to examine morphological changes and cell viability with fluorescence microscopy with trypan blue for comparison. A noticeable decrease in the growth profile of the SKOV3 was observed with the application of 200 kHz alternating electric fields indicating specific inhibitory effects on dividing cells in culture in contrast to the HUVECs. The outcome of this research will improve our fundamental understanding of the behavior of cancer cells when exposed to alternating electric fields at specific frequencies and foster the development strategies and optimal parameters for alternating electric field therapies for clinical and drug delivery applications.

  9. Immunogenicity of Coxiella burnetii whole cells and their outer membrane components.

    Science.gov (United States)

    Gajdosová, E; Kovácová, E; Toman, R; Skultéty, L; Lukácová, M; Kazár, J

    1994-12-01

    The immunogenicity and protective efficacy of the phase I and phase II Coxiella burnetii whole cells (Cb I and Cb II) and their outer membrane components (OMC), i.e. phase I trichloroacetic acid extract (TCAE), phase I 29 K protein (PRO), phase I and II lipopolysaccharides (LPS I, LPS II), polysaccharides (PS I, PS II), and lipid A (LA I, LA II), were compared. The highest immune response was observed in BALB/c mice by Cb I in both humoral immunity and lymphocyte transformation assays, and in the protective effect as well. The immune response was also significant by Cb II, but their protective capacity was low. The OMC reacted variously. Only TCAE and PRO gave a high value of humoral immunity evaluated by the serological methods. All OMC reacted in the haemolytic plaque assay giving different responses. Lymphoproliferation of splenocytes was positive with all OMC using both Cb I and Cb II antigens with the exception of PS I and PS II in the case of Cb II antigen. The induction of protection against infectious Cb I was demonstrated after immunization with TCAE, PRO, and LPS I. Other OMC did not induce protection against this agent.

  10. Toxicity assessment and modelling of Moringa oleifera seeds in water purification by whole cell bioreporter.

    Science.gov (United States)

    Al-Anizi, Ali Adnan; Hellyer, Maria Theresa; Zhang, Dayi

    2014-06-01

    Moringa oleifera has been used as a coagulation reagent for drinking water purification, especially in developing countries such as Malawi. This research revealed the cytoxicity and genotoxicity of M. oleifera by Acinetobacter bioreporter. The results indicated that significant cytoxicity effects were observed when the powdered M. oleifera seeds concentration is from 1 to 50 mg/L. Through direct contact, ethanolic-water extraction and hexane extraction, the toxic effects of hydrophobic and hydrophilic components in M. oleifera seeds were distinguished. It suggested that the hydrophobic lipids contributed to the dominant cytoxicity, consequently resulting in the dominant genotoxicity in the water-soluble fraction due to limited dissolution when the M. oleifera seeds granule concentration was from 10 to 1000 mg/L. Based on cytoxicity and genotoxicity model, the LC50 and LC90 of M. oleifera seeds were 8.5 mg/L and 300 mg/L respectively and their genotoxicity was equivalent to 8.3 mg mitomycin C per 1.0 g dry M. oleifera seed. The toxicity of M. oleifera has also remarkable synergistic effects, suggesting whole cell bioreporter as an appropriate and complementary tool to chemical analysis for environmental toxicity assessment.

  11. Accelerating the discovery of antibacterial compounds using pathway-directed whole cell screening.

    Science.gov (United States)

    Matano, Leigh M; Morris, Heidi G; Wood, B McKay; Meredith, Timothy C; Walker, Suzanne

    2016-12-15

    Since the introduction of penicillin into the clinic in 1942, antibiotics have saved the lives of millions of people around the world. While penicillin and other traditional broad spectrum antibiotics were effective as monotherapies, the inexorable spread of antibiotic resistance has made alternative therapeutic approaches necessary. Compound combinations are increasingly seen as attractive options. Such combinations may include: lethal compounds; synthetically lethal compounds; or administering a lethal compound with a nonlethal compound that targets a virulence factor or a resistance factor. Regardless of the therapeutic strategy, high throughput screening is a key approach to discover potential leads. Unfortunately, the discovery of biologically active compounds that inhibit a desired pathway can be a very slow process, and an inordinate amount of time is often spent following up on compounds that do not have the desired biological activity. Here we describe a pathway-directed high throughput screening paradigm that combines the advantages of target-based and whole cell screens while minimizing the disadvantages. By exploiting this paradigm, it is possible to rapidly identify biologically active compounds that inhibit a pathway of interest. We describe some previous successful applications of this paradigm and report the discovery of a new class of d-alanylation inhibitors that may be useful as components of compound combinations to treat methicillin-resistant Staphylococcus aureus (MRSA). Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Substoichiometric hydroxynonenylation of a single protein recapitulates whole-cell-stimulated antioxidant response.

    Science.gov (United States)

    Parvez, Saba; Fu, Yuan; Li, Jiayang; Long, Marcus J C; Lin, Hong-Yu; Lee, Dustin K; Hu, Gene S; Aye, Yimon

    2015-01-14

    Lipid-derived electrophiles (LDEs) that can directly modify proteins have emerged as important small-molecule cues in cellular decision-making. However, because these diffusible LDEs can modify many targets [e.g., >700 cysteines are modified by the well-known LDE 4-hydroxynonenal (HNE)], establishing the functional consequences of LDE modification on individual targets remains devilishly difficult. Whether LDE modifications on a single protein are biologically sufficient to activate discrete redox signaling response downstream also remains untested. Herein, using T-REX (targetable reactive electrophiles and oxidants), an approach aimed at selectively flipping a single redox switch in cells at a precise time, we show that a modest level (∼34%) of HNEylation on a single target is sufficient to elicit the pharmaceutically important antioxidant response element (ARE) activation, and the resultant strength of ARE induction recapitulates that observed from whole-cell electrophilic perturbation. These data provide the first evidence that single-target LDE modifications are important individual events in mammalian physiology.

  13. Use of Tunable Whole-Cell Bioreporters to Assess Bioavailable Cadmium and Remediation Performance in Soils.

    Directory of Open Access Journals (Sweden)

    Youngdae Yoon

    Full Text Available It is important to have tools to measure the bioavailability to assess the risks of pollutants because the bioavailability is defined as the portions of pollutants showing the biological effects on living organisms. This study described the construction of tunable Escherichia coli whole-cell bioreporter (WCB using the promoter region of zinc-inducible operon and its application on contaminated soils. It was verified that this WCB system showed specific and sensitive responses to cadmium rather than zinc in the experimental conditions. It was inferred that Cd(II associates stronger with ZntR, a regulatory protein of zinc-inducible operon, than other metal ions. Moreover, the expression of reporter genes, egfp and mcherry, were proportional to the concentration of cadmium, thereby being a quantitative sensor to monitor bioavailable cadmium. The capability to determine bioavailable cadmium was verified with Cd(II amended LUFA soils, and then the applicability on environmental systems was investigated with field soils collected from smelter area in Korea before and after soil-washing. The total amount of cadmium was decreased after soil washing, while the bioavailability was increased. Consequently, it would be valuable to have tools to assess bioavailability and the effectiveness of soil remediation should be evaluated in the aspect of bioavailability as well as removal efficiency.

  14. Whole-cell biosensor of cellobiose and application to wood decay detection.

    Science.gov (United States)

    Toussaint, Maxime; Bontemps, Cyril; Besserer, Arnaud; Hotel, Laurence; Gérardin, Philippe; Leblond, Pierre

    2016-12-10

    Fungal biodegradation of wood is one of the main threats regarding its use as a material. So far, the detection of this decaying process is empirically assessed by loss of mass, when the fungal attack is advanced and woody structure already damaged. Being able to detect fungal attack on wood in earlier steps is thus of special interest for the wood economy. In this aim, we designed here a new diagnostic tool for wood degradation detection based on the bacterial whole-cell biosensor technology. It was designed in diverting the soil bacteria Streptomyces CebR sensor system devoted to cellobiose detection, a cellulolytic degradation by-product emitted by lignolytic fungi since the onset of wood decaying process. The conserved regulation scheme of the CebR system among Streptomyces allowed constructing a molecular tool easily transferable in different strains or species and enabling the screen for optimal host strains for cellobiose detection. Assays are performed in microplates using one-day culture lysates. Diagnostic is performed within one hour by a spectrophotometric measuring of the cathecol deshydrogenase activity. The selected biosensor was able to detect specifically cellobiose at concentrations similar to those measured in decaying wood and in a spruce leachate attacked by a lignolytic fungus, indicating a high potential of applicability to detect ongoing wood decay process. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Dark Variants of Luminous Bacteria Whole Cell Bioluminescent Optical Fiber Sensor to Genotoxicants

    Institute of Scientific and Technical Information of China (English)

    孙雅量; 周铁波; 过健俐; 李义勇

    2004-01-01

    A stable dark variant separated from photobacterium phosphoreum (A2) was fixed in agar-gel membrane and immobilized onto an exposed end of a fiber-optic linked with bioluminometer. The variant could emit a luminescent signal in the presence of genotoxic agents, such as Mitomycin C (MC). The performance of this whole-cell optical fiber sensor system was examined as a function of several parameters, including gel probe thickness, bacterial cell density, and diameter of the fiber-optic core and working temperature. An optimal response to a model genotoxicant, Mitomycin C, was achieved with agar-bacterial gel membrane: the thickness of gel membrane was about 5 mm; the cell density of bacteria in gel membranewas about 2.0 × 107/ml; the diameter of fiberoptic core was 5.0 mm; the working temperature was 25℃. Under these optimized conditions, the response time was less than 10 h to Mitomycin C, with a lower detection threshold of 0.1 mg/L.

  16. Advanced Materials for the Recognition and Capture of Whole Cells and Microorganisms.

    Science.gov (United States)

    Bole, Amanda L; Manesiotis, Panagiotis

    2016-07-01

    Selective cell recognition and capture has recently attracted significant interest due to its potential importance for clinical, diagnostic, environmental, and security applications. Current methods for cell isolation from complex samples are largely dependent on cell size and density, with limited application scope as many of the target cells do not exhibit appreciable differences in this respect. The most recent and forthcoming developments in the area of selective recognition and capture of whole cells, based on natural receptors, as well as synthetic materials utilising physical and chemical properties of the target cell or microorganism, are highlighted. Particular focus is given to the development of cell complementary surfaces using the cells themselves as templating agents, by means of molecular imprinting, and their combination with sensing platforms for rapid cell detection in complex media. The benefits and challenges of each approach are discussed and a perspective of the future of this research area is given. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Classifying compound mechanism of action for linking whole cell phenotypes to molecular targets.

    Science.gov (United States)

    Bourne, Christina R; Wakeham, Nancy; Bunce, Richard A; Nammalwar, Baskar; Berlin, K Darrell; Barrow, William W

    2012-04-01

    Drug development programs have proven successful when performed at a whole cell level, thus incorporating solubility and permeability into the primary screen. However, linking those results to the target within the cell has been a major setback. The Phenotype Microarray system, marketed and sold by Biolog, seeks to address this need by assessing the phenotype in combination with a variety of chemicals with known mechanism of action (MOA). We have evaluated this system for usefulness in deducing the MOA for three test compounds. To achieve this, we constructed a database with 21 known antimicrobials, which served as a comparison for grouping our unknown MOA compounds. Pearson correlation and Ward linkage calculations were used to generate a dendrogram that produced clustering largely by known MOA, although there were exceptions. Of the three unknown compounds, one was definitively placed as an antifolate. The second and third compounds' MOA were not clearly identified, likely because the unique MOA was not represented within the database. The availability of the database generated in this report for Staphylococcus aureus ATCC 29213 will increase the accessibility of this technique to other investigators. From our analysis, the Phenotype Microarray system can group compounds with clear MOA, but the distinction of unique or broadly acting MOA at this time is less clear. Copyright © 2012 John Wiley & Sons, Ltd.

  18. Discrimination and phylogenomic classification of Bacillus anthracis-cereus-thuringiensis strains based on LC-MS/MS analysis of whole cell protein digests.

    Science.gov (United States)

    Dworzanski, Jacek P; Dickinson, Danielle N; Deshpande, Samir V; Snyder, A Peter; Eckenrode, Brian A

    2010-01-01

    Modern taxonomy, diagnostics, and forensics of bacteria benefit from technologies that provide data for genome-based classification and identification of strains; however, full genome sequencing is still costly, lengthy, and labor intensive. Therefore, other methods are needed to estimate genomic relatedness among strains in an economical and timely manner. Although DNA-DNA hybridization and techniques based on genome fingerprinting or sequencing selected genes like 16S rDNA, gyrB, or rpoB are frequently used as phylogenetic markers, analyses of complete genome sequences showed that global measures of genome relatedness, such as the average genome conservation of shared genes, can provide better strain resolution and give phylogenies congruent with relatedness revealed by traditional phylogenetic markers. Bacterial genomes are characterized by a high gene density; therefore, we investigated the integration of mass spectrometry-based proteomic techniques with statistical methods for phylogenomic classification of bacterial strains. For this purpose, we used a set of well characterized Bacillus cereus group strains isolated from poisoned food to describe a method that relies on liquid chromatography-electrospray ionization-tandem mass spectrometry of tryptic peptides derived from whole cell digests. Peptides were identified and matched to a prototype database (DB) of reference bacteria with fully sequenced genomes to obtain their phylogenetic profiles. These profiles were processed for predicting genomic similarities with DB bacteria estimated by fractions of shared peptides (FSPs). FSPs served as descriptors for each food isolate and were jointly analyzed using hierarchical cluster analysis methods for revealing relatedness among investigated strains. The results showed that phylogenomic classification of tested food isolates was in consonance with results from established genomic methods, thus validating our findings. In conclusion, the proposed approach could be

  19. Concise Whole-Cell Modeling of BKCa-CaV Activity Controlled by Local Coupling and Stoichiometry.

    Science.gov (United States)

    Montefusco, Francesco; Tagliavini, Alessia; Ferrante, Marco; Pedersen, Morten Gram

    2017-06-06

    Large-conductance Ca(2+)-dependent K(+) (BKCa) channels are important regulators of electrical activity. These channels colocalize and form ion channel complexes with voltage-dependent Ca(2+) (CaV) channels. Recent stochastic simulations of the BKCa-CaV complex with 1:1 stoichiometry have given important insight into the local control of BKCa channels by fluctuating nanodomains of Ca(2+). However, such Monte Carlo simulations are computationally expensive, and are therefore not suitable for large-scale simulations of cellular electrical activity. In this work we extend the stochastic model to more realistic BKCa-CaV complexes with 1:n stoichiometry, and analyze the single-complex model with Markov chain theory. From the description of a single BKCa-CaV complex, using arguments based on timescale analysis, we derive a concise model of whole-cell BKCa currents, which can readily be analyzed and inserted into models of cellular electrical activity. We illustrate the usefulness of our results by inserting our BKCa description into previously published whole-cell models, and perform simulations of electrical activity in various cell types, which show that BKCa-CaV stoichiometry can affect whole-cell behavior substantially. Our work provides a simple formulation for the whole-cell BKCa current that respects local interactions in BKCa-CaV complexes, and indicates how local-global coupling of ion channels may affect cell behavior. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  20. Whole-cell microtiter plate screening assay for terminal hydroxylation of fatty acids by P450s.

    Science.gov (United States)

    Weissenborn, Martin J; Notonier, Sandra; Lang, Sarah-Luise; Otte, Konrad B; Herter, Susanne; Turner, Nicholas J; Flitsch, Sabine L; Hauer, Bernhard

    2016-05-04

    A readily available galactose oxidase (GOase) variant was used to develop a whole cell screening assay. This endpoint detection system was applied in a proof-of-concept approach by screening a focussed mutant library. This led to the discovery of the thus far most active P450 Marinobacter aquaeolei mutant catalysing the terminal hydroxylation of fatty acids.

  1. Deoxyribonucleic acid-binding ability of androgen receptors in whole cells: implications for the actions of androgens and antiandrogens

    NARCIS (Netherlands)

    C.W. Kuil (Cor); E. Mulder (Eppo)

    1996-01-01

    textabstractIn whole cells, the effects of several androgens and antiandrogens on the in the induction of DNA binding for the human wild-type androgen receptor (AR) and a mutant receptor ARL (LNCaP mutation; codon 868, Thr to Ala) were examined and related to the transc

  2. Microarray analysis of cytoplasmic versus whole cell RNA reveals a considerable number of missed and false positive mRNAs.

    Science.gov (United States)

    Trask, Heidi W; Cowper-Sal-lari, Richard; Sartor, Maureen A; Gui, Jiang; Heath, Catherine V; Renuka, Janhavi; Higgins, Azara-Jane; Andrews, Peter; Korc, Murray; Moore, Jason H; Tomlinson, Craig R

    2009-10-01

    With no known exceptions, every published microarray study to determine differential mRNA levels in eukaryotes used RNA extracted from whole cells. It is assumed that the use of whole cell RNA in microarray gene expression analysis provides a legitimate profile of steady-state mRNA. Standard labeling methods and the prevailing dogma that mRNA resides almost exclusively in the cytoplasm has led to the long-standing belief that the nuclear RNA contribution is negligible. We report that unadulterated cytoplasmic RNA uncovers differentially expressed mRNAs that otherwise would not have been detected when using whole cell RNA and that the inclusion of nuclear RNA has a large impact on whole cell gene expression microarray results by distorting the mRNA profile to the extent that a substantial number of false positives are generated. We conclude that to produce a valid profile of the steady-state mRNA population, the nuclear component must be excluded, and to arrive at a more realistic view of a cell's gene expression profile, the nuclear and cytoplasmic RNA fractions should be analyzed separately.

  3. RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY

    Science.gov (United States)

    Rainbow Trout Androgen Receptor Alpha And Human Androgen Receptor: Comparisons in the COS Whole Cell Binding Assay Mary C. Cardon, L. Earl Gray, Jr. and Vickie S. WilsonU.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology Division, Research Triangle...

  4. Producing biodiesel from cotton seed oil using Rhizopus oryzae ATTC #34612 whole cell biocatalysts: Culture media and cultivation period optimization

    Science.gov (United States)

    The effect of culture medium composition and cultivation time on biodiesel production by Rhizopus oryzae ATCC #34612 whole cell catalysts, immobilized on novel rigid polyethylene biomass supports, was investigated. Supplementation of the medium with carbon sources led to higher lipase activity and i...

  5. Genomic and Haplotype Comparison of Butanol Producing Bacteria Based on 16S rDNA

    Directory of Open Access Journals (Sweden)

    Ekwan Nofa Wiratno

    2016-04-01

    Full Text Available High butanol demand for transportation fuel triggers butanol production development. Exploration of butanolproducing bacteria using genomic comparison and biogeography will help to develop butanol industry. The objectives of this research were butanol production, genome comparison and haplotype analysis of butanolproducing bacteria from Ranu Pani Lake sediment using 16S rDNA sequences. The highest butanol concentrations were showed by Paenibacillus polymyxa RP 2.2 isolate (10.34 g.L-1, followed by Bacillus methylotrophicus RP 3.2 and B. methylotrophicus RP 7.2 isolate (10.11 g.L-1 and 9.63 g.L-1 respectively. Paenibacillus polymyxa RP 2.2 showed similarity in nucleotide composition (ATGC with B. methylotrophicus RP 3.2, B. methylotrophicus RP 7.2, P. polymyxa CR1, Bacillus amyloliquefaciens NELB-12, and Paenibacillus polymyxa WR-2. Clostridium acetobutylicum ATCC 824 showed similarity in nucleotide composition (ATGC with Clostridium saccharoperbutylacetonicum N1-4, and Clostridium saccharobutylicum Ox29. The lowest G+C content was C. saccharobutylicum Ox29 (51.35%, and the highest was B. methylotrophicus RP 7.2 (55.33%. Conserved region of 16S rDNA (1044 bp were consisted of 17 conserved sequences. The number of Parsimony Informative Site (PIS was 319 spot and single tone was 48 spot. We found in this study that all of butanolproducing bacterial DNA sequences have clustered to 8 haplotypes. Based on the origin of sample, there were three haplotype groups. Bacteria from group A were could produce butanol 8.9-10.34 g.L-1, group B 9.2-14.2 g.L-1 and group C was could produce butanol 0.47 g.L-1. The haplotype analysis of bacteria based on 16S rDNA sequences in this study could predict capability of butanol production.

  6. Distinct and complex bacterial profiles in human periodontitis and health revealed by 16S pyrosequencing.

    Science.gov (United States)

    Griffen, Ann L; Beall, Clifford J; Campbell, James H; Firestone, Noah D; Kumar, Purnima S; Yang, Zamin K; Podar, Mircea; Leys, Eugene J

    2012-06-01

    Periodontitis has a polymicrobial etiology within the framework of a complex microbial ecosystem. With advances in sequencing technologies, comprehensive studies to elucidate bacterial community differences have recently become possible. We used 454 sequencing of 16S rRNA genes to compare subgingival bacterial communities from 29 periodontally healthy controls and 29 subjects with chronic periodontitis. Amplicons from both the V1-2 and V4 regions of the 16S gene were sequenced, yielding 1,393,579 sequences. They were identified by BLAST against a curated oral 16S database, and mapped to 16 phyla, 106 genera, and 596 species. 81% of sequences could be mapped to cultivated species. Differences between health- and periodontitis-associated bacterial communities were observed at all phylogenetic levels, and UniFrac and principal coordinates analysis showed distinct community profiles in health and disease. Community diversity was higher in disease, and 123 species were identified that were significantly more abundant in disease, and 53 in health. Spirochaetes, Synergistetes and Bacteroidetes were more abundant in disease, whereas the Proteobacteria were found at higher levels in healthy controls. Within the phylum Firmicutes, the class Bacilli was health-associated, whereas the Clostridia, Negativicutes and Erysipelotrichia were associated with disease. These results implicate a number of taxa that will be targets for future research. Some, such as Filifactor alocis and many Spirochetes were represented by a large fraction of sequences as compared with previously identified targets. Elucidation of these differences in community composition provides a basis for further understanding the pathogenesis of periodontitis.

  7. Clinical identification of bacteria in human chronic wound infections: culturing vs. 16S ribosomal DNA sequencing

    Directory of Open Access Journals (Sweden)

    Rhoads Daniel D

    2012-11-01

    Full Text Available Abstract Background Chronic wounds affect millions of people and cost billions of dollars in the United States each year. These wounds harbor polymicrobial biofilm communities, which can be difficult to elucidate using culturing methods. Clinical molecular microbiological methods are increasingly being employed to investigate the microbiota of chronic infections, including wounds, as part of standard patient care. However, molecular testing is more sensitive than culturing, which results in markedly different results being reported to clinicians. This study compares the results of aerobic culturing and molecular testing (culture-free 16S ribosomal DNA sequencing, and it examines the relative abundance score that is generated by the molecular test and the usefulness of the relative abundance score in predicting the likelihood that the same organism would be detected by culture. Methods Parallel samples from 51 chronic wounds were studied using aerobic culturing and 16S DNA sequencing for the identification of bacteria. Results One hundred forty-five (145 unique genera were identified using molecular methods, and 68 of these genera were aerotolerant. Fourteen (14 unique genera were identified using aerobic culture methods. One-third (31/92 of the cultures were determined to be Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis with higher relative abundance scores were more likely to be detected by culture as demonstrated with regression modeling. Conclusion Discordance between molecular and culture testing is often observed. However, culture-free 16S ribosomal DNA sequencing and its relative abundance score can provide clinicians with insight into which bacteria are most abundant in a sample and which are most likely to be detected by culture.

  8. Global Perspectives on Activated Sludge Community Composition analyzed using 16S rRNA amplicon sequencing

    DEFF Research Database (Denmark)

    Nierychlo, Marta; Saunders, Aaron Marc; Albertsen, Mads

    communities, and in this study activated sludge sampled from 32 Wastewater Treatment Plants (WWTPs) around the world was described and compared. The top abundant bacteria in the global activated sludge ecosystem were found and the core population shared by multiple samples was investigated. The results......Activated sludge is the most commonly applied bioprocess throughout the world for wastewater treatment. Microorganisms are key to the process, yet our knowledge of their identity and function is still limited. High-througput16S rRNA amplicon sequencing can reliably characterize microbial...

  9. Neonatal Meningitis by Multidrug Resistant Elizabethkingia meningosepticum Identified by 16S Ribosomal RNA Gene Sequencing

    Directory of Open Access Journals (Sweden)

    V. V. Shailaja

    2014-01-01

    Full Text Available Clinical and microbiological profile of 9 neonates with meningitis by Elizabethkingia meningosepticum identified by 16S ribosomal gene sequencing was studied. All the clinical isolates were resistant to cephalosporins, aminoglycosides, trimethoprim-sulfamethoxazole, β-lactam combinations, carbapenems and only one isolate was susceptible to ciprofloxacin. All the isolates were susceptible to vancomycin. Six of nine neonates died even after using vancomycin, based on susceptibility results. E. meningosepticum meningitis in neonates results in high mortality rate. Though the organism is susceptible to vancomycin in vitro, its efficacy in vivo is questionable and it is difficult to determine the most appropriate antibiotic for treating E. meningosepticum meningitis in neonates.

  10. Greengenes, a Chimera-checked 16S rRNA gene database and workbenchcompatible with ARB

    Energy Technology Data Exchange (ETDEWEB)

    DeSantis, Todd Z.; Hugenholtz, Philip; Larsen, Neils; Rojas,Mark; Brodie, Eoin L.; Keller, Keith; Huber, Thomas; Dalevi, Daniel; Hu,Ping; Andersen, Gary L.

    2006-04-10

    A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera-screening, standard alignments and taxonomic classification using multiple published taxonomies. It was revealed that in congruent taxonomic nomenclature exists among curators even at the phylum-level. Putative chimeras were identified in 3 percent of environmental sequences and 0.2 percent of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages within the Archaea and Bacteria.

  11. Mutational robustness of 16S ribosomal RNA, shown by experimental horizontal gene transfer in Escherichia coli

    OpenAIRE

    Kitahara, Kei; Yasutake, Yoshiaki; Miyazaki, Kentaro

    2012-01-01

    The bacterial ribosome consists of three rRNA molecules and 57 proteins and plays a crucial role in translating mRNA-encoded information into proteins. Because of the ribosome’s structural and mechanistic complexity, it is believed that each ribosomal component coevolves to maintain its function. Unlike 5S rRNA, 16S and 23S rRNAs appear to lack mutational robustness, because they form the structural core of the ribosome. However, using Escherichia coli Δ7 (null mutant of operons) as a host, w...

  12. Improved performance of the PacBio SMRT technology for 16S rDNA sequencing.

    Science.gov (United States)

    Mosher, Jennifer J; Bowman, Brett; Bernberg, Erin L; Shevchenko, Olga; Kan, Jinjun; Korlach, Jonas; Kaplan, Louis A

    2014-09-01

    Improved sequencing accuracy was obtained with 16S amplicons from environmental samples and a known pure culture when upgraded Pacific Biosciences (PacBio) hardware and enzymes were used for the single molecule, real-time (SMRT) sequencing platform. The new PacBio RS II system with P4/C2 chemistry, when used with previously constructed libraries (Mosher et al., 2013) surpassed the accuracy of Roche/454 pyrosequencing platform. With accurate read lengths of >1400 base pairs, the PacBio system opens up the possibility of identifying microorganisms to the species level in environmental samples. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. A comparison of rpoB and 16S rRNA as markers in pyrosequencing studies of bacterial diversity

    NARCIS (Netherlands)

    Vos, M.; Quince, C.; Pijl, A.S.; De Hollander, M.; Kowalchuk, G.A.

    2012-01-01

    Background The 16S rRNA gene is the gold standard in molecular surveys of bacterial and archaeal diversity, but it has the disadvantages that it is often multiple-copy, has little resolution below the species level and cannot be readily interpreted in an evolutionary framework. We compared the 16S r

  14. Analysis of 16S libraries of mouse gastrointestinal microflora reveals a large new group of mouse intestinal bacteria

    NARCIS (Netherlands)

    Salzman, NH; de Jong, H; Paterson, Y; Harmsen, HJM; Welling, GW; Bos, NA

    2002-01-01

    Total genomic DNA from samples of intact mouse small intestine, large intestine, caecum and faeces was used as template for PCR amplification of 16S rRNA gene sequences with conserved bacterial primers. Phylogenetic analysis of the amplification products revealed 40 unique 16S rDNA sequences. Of

  15. Microvariation artifacts introduced by PCR and cloning of closely related 16S rRNA gene sequences

    NARCIS (Netherlands)

    Speksnijder, A.G.C.L.; Kowalchuk, G.A.; Jong, S. de; Kline, E.; Stephen, J.R.; Laanbroek, H.J.

    2001-01-01

    A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a m

  16. Microvariation Artifacts Introduced by PCR and Cloning of Closely Related 16S rRNA Gene Sequences

    NARCIS (Netherlands)

    Speksnijder, A.G.C.L.; Kowalchuk, G.A.; Jong, de S.; Kline, E.; Stephen, J.R.; Laanbroek, H.J.

    2001-01-01

    A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a m

  17. Molecular phylogeny of silk-producing insects based on 16S ribosomal RNA and cytochrome oxidase subunit I genes

    Indian Academy of Sciences (India)

    B. Mahendran; S. K. Ghosh; S. C. Kundu

    2006-04-01

    We have examined the molecular-phylogenetic relationships between nonmulberry and mulberry silkwormspecies that belong to the families Saturniidae, Bombycidae and Lasiocampidae using 16S ribosomal RNA (16S rRNA) and cytochrome oxidase subunit I (coxI) gene sequences. Aligned nucleotide sequences of 16S rRNA and coxI from 14 silk-producing species were used for construction of phylogenetic trees by maximum likelihood and maximum parsimony methods. The tree topology on the basis of 16S rRNA supports monophyly for members of Saturniidae and Bombycidae. Weighted parsimony analysis weighted towards transversions relative to transitions (ts, tv4) for coxI resulted in more robust bootstrap support over unweighted parsimony and favours the 16S rRNA tree topology. Combined analysis reflected clear biogeographic pattern, and agrees with morphological and cytological data.

  18. 16S rRNA sequences of uncultivated hot spring cyanobacterial mat inhabitants retrieved as randomly primed cDNA

    Energy Technology Data Exchange (ETDEWEB)

    Weller, R.; Ward, D.M. (Montana State Univ., Bozeman (United States)); Weller, J.W. (Univ. of Montana, Missoula (United States))

    1991-04-01

    Cloning and analysis of cDNAs synthesized from rRNAs is one approach to assess the species composition of natural microbial communities. In some earlier attempts to synthesize cDNA from 16S rRNA (16S rcDNA) from the Octopus Spring cyanobacterial mat, a dominance of short 16S rcDNAs was observed, which appear to have originated only from certain organisms. Priming of cDNA synthesis from small ribosomal subunit RNA with random deoxyhexanucleotides can retrieve longer sequences, more suitable for phylogenetic analysis. Here we report the retrieval of 16S rRNA sequences form three formerly uncultured community members. One sequence type, which was retrieved three times from a total of five sequences analyzed, can be placed in the cyanobacterial phylum. A second sequence type is related to 16S rRNAs from green nonsulfur bacteria. The third sequence type may represent a novel phylogenetic type.

  19. Sequence analysis of mitochondrial 16S ribosomal RNA gene fragment from seven mosquito species

    Indian Academy of Sciences (India)

    Yogesh S Shouche; Milind S Patole

    2000-12-01

    Mosquitoes are vectors for the transmission of many human pathogens that include viruses, nematodes and protozoa. For the understanding of their vectorial capacity, identification of disease carrying and refractory strains is essential. Recently, molecular taxonomic techniques have been utilized for this purpose. Sequence analysis of the mitochondrial 16S rRNA gene has been used for molecular taxonomy in many insects. In this paper, we have analysed a 450 bp hypervariable region of the mitochondrial 16S rRNA gene in three major genera of mosquitoes, Aedes, Anopheles and Culex. The sequence was found to be unusually A + T rich and in substitutions the rate of transversions was higher than the transition rate. A phylogenetic tree was constructed with these sequences. An interesting feature of the sequences was a stretch of Ts that distinguished between Aedes and Culex on the one hand, and Anopheles on the other. This is the first report of mitochondrial rRNA sequences from these medically important genera of mosquitoes.

  20. Specific 16S ribosomal RNA targeted oligonucleotide probe against Clavibacter michiganensis subsp. sepedonicus.

    Science.gov (United States)

    Mirza, M S; Rademaker, J L; Janse, J D; Akkermans, A D

    1993-11-01

    In this article we report on the polymerase chain reaction amplification of a partial 16S rRNA gene from the plant pathogenic bacterium Clavibacter michiganensis subsp. sepedonicus. A partial sequence (about 400 base pairs) of the gene was determined that covered two variable regions important for oligonucleotide probe development. A specific 24mer oligonucleotide probe targeted against the V6 region of 16S rRNA was designed. Specificity of the probe was determined using dot blot hybridization. Under stringent conditions (60 degrees C), the probe hybridized with all 16 Cl. michiganensis subsp. sepedonicus strains tested. Hybridization did not occur with 32 plant pathogenic and saprophytic bacteria used as controls under the same conditions. Under less stringent conditions (55 degrees C) the related Clavibacter michiganensis subsp. insidiosus, Clavibacter michiganensis subsp. nebraskensis, and Clavibacter michiganensis subsp. tesselarius also showed hybridization. At even lower stringency (40 degrees C), all Cl. michiganensis subspecies tested including Clavibacter michiganensis subsp. michiganensis showed hybridization signal, suggesting that under these conditions the probe may be used as a species-specific probe for Cl. michiganensis.

  1. Diagnosis of neonatal sepsis by broad-range 16S real-time polymerase chain reaction.

    Science.gov (United States)

    Ohlin, Andreas; Bäckman, Anders; Ewald, Uwe; Schollin, Jens; Björkqvist, Maria

    2012-01-01

    The standard diagnostic test (blood culture) for suspected neonatal sepsis has limitations in sensitivity and specificity, and 16S polymerase chain reaction (PCR) has been suggested as a new diagnostic tool for neonatal sepsis. To develop and evaluate a new real-time PCR method for detection of bacterial DNA in blood samples collected from infants with suspected neonatal sepsis. Immediately after blood culture, a study sample of 0.5-1.0 ml whole blood was collected and used for a novel 16S real-time PCR assay. All positive samples were sequenced. Detailed case studies were performed in all cases with conflicting results, to verify if PCR could detect pathogens in culture negative sepsis. 368 samples from 317 infants were included. When compared with blood culture, the assay yielded a sensitivity of 79%, a specificity of 90%, a positive predictive value of 59%, and a negative predictive value of 96%. Seven of the 31 samples with a positive PCR result and a negative blood culture had definite or suspected bacterial sepsis. In five samples, PCR (but not blood culture) could detect a pathogen that was present in a blood culture collected more than 24 h prior to the PCR sample. This study presents an evaluation of a new real-time PCR technique that can detect culture-positive sepsis, and suggests that PCR has the potential to detect bacteria in culture-negative samples even after the initiation of intravenous antibiotics. Copyright © 2011 S. Karger AG, Basel.

  2. Identification and characterization of rhizospheric microbial diversity by 16S ribosomal RNA gene sequencing

    Directory of Open Access Journals (Sweden)

    Muhammad Naveed

    2014-09-01

    Full Text Available In the present study, samples of rhizosphere and root nodules were collected from different areas of Pakistan to isolate plant growth promoting rhizobacteria. Identification of bacterial isolates was made by 16S rRNA gene sequence analysis and taxonomical confirmation on EzTaxon Server. The identified bacterial strains were belonged to 5 genera i.e. Ensifer, Bacillus, Pseudomona, Leclercia and Rhizobium. Phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relationship of bacterial strains with the respective genera. Based on phylogenetic analysis, some candidate novel species were also identified. The bacterial strains were also characterized for morphological, physiological, biochemical tests and glucose dehydrogenase (gdh gene that involved in the phosphate solublization using cofactor pyrroloquinolone quinone (PQQ. Seven rhizoshperic and 3 root nodulating stains are positive for gdh gene. Furthermore, this study confirms a novel association between microbes and their hosts like field grown crops, leguminous and non-leguminous plants. It was concluded that a diverse group of bacterial population exist in the rhizosphere and root nodules that might be useful in evaluating the mechanisms behind plant microbial interactions and strains QAU-63 and QAU-68 have sequence similarity of 97 and 95% which might be declared as novel after further taxonomic characterization.

  3. Mitochondrial swinger replication: DNA replication systematically exchanging nucleotides and short 16S ribosomal DNA swinger inserts.

    Science.gov (United States)

    Seligmann, Hervé

    2014-11-01

    Assuming systematic exchanges between nucleotides (swinger RNAs) resolves genomic 'parenthood' of some orphan mitochondrial transcripts. Twenty-three different systematic nucleotide exchanges (bijective transformations) exist. Similarities between transcription and replication suggest occurrence of swinger DNA. GenBank searches for swinger DNA matching the 23 swinger versions of human and mouse mitogenomes detect only vertebrate mitochondrial swinger DNA for swinger type AT+CG (from five different studies, 149 sequences) matching three human and mouse mitochondrial genes: 12S and 16S ribosomal RNAs, and cytochrome oxidase subunit I. Exchange AT+CG conserves self-hybridization properties, putatively explaining swinger biases for rDNA, against protein coding genes. Twenty percent of the regular human mitochondrial 16S rDNA consists of short swinger repeats (from 13 exchanges). Swinger repeats could originate from recombinations between regular and swinger DNA: duplicated mitochondrial genes of the parthenogenetic gecko Heteronotia binoei include fewer short AT+CG swinger repeats than non-duplicated mitochondrial genomes of that species. Presumably, rare recombinations between female and male mitochondrial genes (and in parthenogenetic situations between duplicated genes), favors reverse-mutations of swinger repeat insertions, probably because most inserts affect negatively ribosomal function. Results show that swinger DNA exists, and indicate that swinger polymerization contributes to the genesis of genetic material and polymorphism.

  4. Changes in 16s RNA Gene Microbial Community Profiling by Concentration of Prokaryotic DNA.

    Science.gov (United States)

    Glassing, Angela; Dowd, Scot E; Galandiuk, Susan; Davis, Brian; Jorden, Jeffrey R; Chiodini, Rodrick J

    2015-12-01

    Microbial metagenomics are hindered in clinical tissue samples as a result of the large relative amount of human DNA in relation to microbial DNA acting as competitive inhibitors of downstream applications. We evaluated the LOOXSTER® Enrichment Kit to separate eukaryotic and prokaryotic DNA in submucosal intestinal tissue samples having a low microbial biomass and to determine the effects of enrichment on 16s rRNA microbiota sequencing. The enrichment kit reduced the amount of human DNA in the samples 40-70% resulting in a 3.5-fold increase in the number of 16s bacterial gene sequences detected on the Illumina MiSeq platform. This increase was accompanied by the detection of 41 additional bacterial genera and 94 tentative species. The additional bacterial taxa detected accounted for as much as 25% of the total bacterial population that significantly altered the relative prevalence and composition of the intestinal microbiota. The ability to reduce the competitive inhibition created by human DNA and the concentration of bacterial DNA may allow metagenomics to be performed on complex tissues containing a low bacterial biomass.

  5. Identification and characterization of rhizospheric microbial diversity by 16S ribosomal RNA gene sequencing.

    Science.gov (United States)

    Naveed, Muhammad; Mubeen, Samavia; Khan, SamiUllah; Ahmed, Iftikhar; Khalid, Nauman; Suleria, Hafiz Ansar Rasul; Bano, Asghari; Mumtaz, Abdul Samad

    2014-01-01

    In the present study, samples of rhizosphere and root nodules were collected from different areas of Pakistan to isolate plant growth promoting rhizobacteria. Identification of bacterial isolates was made by 16S rRNA gene sequence analysis and taxonomical confirmation on EzTaxon Server. The identified bacterial strains were belonged to 5 genera i.e. Ensifer, Bacillus, Pseudomona, Leclercia and Rhizobium. Phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relationship of bacterial strains with the respective genera. Based on phylogenetic analysis, some candidate novel species were also identified. The bacterial strains were also characterized for morphological, physiological, biochemical tests and glucose dehydrogenase (gdh) gene that involved in the phosphate solublization using cofactor pyrroloquinolone quinone (PQQ). Seven rhizoshperic and 3 root nodulating stains are positive for gdh gene. Furthermore, this study confirms a novel association between microbes and their hosts like field grown crops, leguminous and non-leguminous plants. It was concluded that a diverse group of bacterial population exist in the rhizosphere and root nodules that might be useful in evaluating the mechanisms behind plant microbial interactions and strains QAU-63 and QAU-68 have sequence similarity of 97 and 95% which might be declared as novel after further taxonomic characterization.

  6. GENE 16S RRNA SEQUENCE PHYLOGENETIC ANALYSIS OF LYSINE PRODUCERS STRAINS

    Directory of Open Access Journals (Sweden)

    G. S. Andriiash

    2014-12-01

    Full Text Available The phylogenetic relationships of strainsproducers of essential amino acids of aspartate family Brevibacterium sp. UCM Ac-674 (Brevibacterium sp. 90, Brevibacterium sp. IMV Ac-5004 (Brevibacterium sp. 90H, Brevibacterium sp. UCM Ac-675 (Brevibacterium sp. E531, mutant strain Brevibacterium sp. IMV B-7447 from the «Collections strains and lines of plants for food and agricultural biotechnology SO “Institute for Food Biotechnology and Genomics” of National Academy of Sciences of Ukraine were investigated. The affiliation strain Brevibacterium sp. IMV B-7447 to the genus Brevibacterium within the sequences of the genes based on 16S rRNA was confirmed. The dendogram of phylogenetic relationships of studied strains and related strains Brevibacterium from database GenBank was constructed. It was shown that by the criterion of homology gene sequences based on 16S rRNA the investigated strains-producers belong to three phylogenetic groups. It was established that the mutant strain Brevibacterium sp. ІMV B-7447 has no analogues in the database GenBank.

  7. Immunoproteomic analysis of human serological antibody responses to vaccination with whole-cell pertussis vaccine (WCV.

    Directory of Open Access Journals (Sweden)

    Yong-Zhang Zhu

    Full Text Available BACKGROUND: Pertussis (whooping cough caused by Bordetella pertussis (B.p, continues to be a serious public health threat. Vaccination is the most economical and effective strategy for preventing and controlling pertussis. However, few systematic investigations of actual human immune responses to pertussis vaccines have been performed. Therefore, we utilized a combination of two-dimensional electrophoresis (2-DE, immunoblotting, and mass spectrometry to reveal the entire antigenic proteome of whole-cell pertussis vaccine (WCV targeted by the human immune system as a first step toward evaluating the repertoire of human humoral immune responses against WCV. METHODOLOGY/PRINCIPAL FINDINGS: Immunoproteomic profiling of total membrane enriched proteins and extracellular proteins of Chinese WCV strain 58003 identified a total of 30 immunoreactive proteins. Seven are known pertussis antigens including Pertactin, Serum resistance protein, chaperonin GroEL and two OMP porins. Sixteen have been documented to be immunogenic in other pathogens but not in B.p, and the immunogenicity of the last seven proteins was found for the first time. Furthermore, by comparison of the human and murine immunoproteomes of B.p, with the exception of four human immunoreactive proteins that were also reactive with mouse immune sera, a unique group of antigens including more than 20 novel immunoreactive proteins that uniquely reacted with human immune serum was confirmed. CONCLUSIONS/SIGNIFICANCE: This study is the first time that the repertoire of human serum antibody responses against WCV was comprehensively investigated, and a small number of previously unidentified antigens of WCV were also found by means of the classic immunoproteomic strategy. Further research on these newly identified predominant antigens of B.p exclusively against humans will not only remarkably accelerate the development of diagnostic biomarkers and subunit vaccines but also provide detailed insight

  8. Whole-cell clamp of dissociated photoreceptors from the eye of Lima scabra.

    Science.gov (United States)

    Nasi, E

    1991-01-01

    Voltage-dependent membrane currents were investigated in enzymatically dissociated photoreceptors of Lima scabra using the whole-cell clamp technique. Depolarizing steps to voltages more positive than -10 mV elicit a transient inward current followed by a delayed, sustained outward current. The outward current is insensitive to replacement of a large fraction of extracellular Cl- with the impermeant anion glucuronate. Superfusion with tetraethylammonium and 4-aminopyridine reversibly abolishes the outward current, and internal perfusion with cesium also suppresses it, indicating that it is mediated by potassium channels. Isolation of the inward current reveals a fast activation kinetics, the peak amplitude occurring as early as 4-5 ms after stimulus onset, and a relatively rapid, though incomplete inactivation. Within the range of voltages examined, spanning up to +90 mV, reversal was not observed. The inward current is not sensitive to tetrodotoxin at concentrations up to 10 microM, and survives replacement of extracellular Na with tetramethylammonium. On the other hand, it is completely eliminated by calcium removal from the perfusing solution, and it is partially blocked by submillimolar concentrations of cadmium, suggesting that it is entirely due to voltage-dependent calcium channels. Analysis of the kinetics and voltage dependence of the isolated calcium current indicates the presence of two components, possibly reflecting the existence of separate populations of channels. Barium and strontium can pass through these channels, though less easily than calcium. Both the activation and the inactivation become significantly more sluggish when these ions serve as the charge carrier. A large fraction of the outward current is activated by preceding calcium influx. Suppression of this calcium-dependent potassium current shows a small residual component resembling the delayed rectifier. In addition, a transient outward current sensitive to 4-aminopyridine (Ia) could

  9. Whole Cell Biosensor Using Anabaena torulosa with Optical Transduction for Environmental Toxicity Evaluation

    Directory of Open Access Journals (Sweden)

    Ling Shing Wong

    2013-01-01

    Full Text Available A whole cell-based biosensor using Anabaena torulosa for the detection of heavy metals (Cu, Pb, and Cd, 2,4-dichlorophenoxyacetate (2,4-D, and chlorpyrifos was constructed. The cyanobacteria were entrapped on a cellulose membrane through filtration. Then, the membrane was dried and fixed into a cylindrical well, which was designed to be attached to an optical probe. The probe was connected to fluorescence spectrometer with optical fibre. The presence of the toxicants was indicated by the change of fluorescence emission, before and after the exposure. The linear detection ranges for Cu, Pb, and Cd were 2.5–10.0 µg/L, 0.5–5.0 µg/L, and 0.5–10.0 µg/L, respectively, while 2,4-D and chlorpyrifos shared similar linear ranges of 0.05–0.75 µg/L. The biosensor showed good sensitivity with the lowest limits of detection (LLD for Cu, Pb, Cd, 2,4-D and chlorpyrifos determined at 1.195 µg/L, 0.100 µg/L, 0.027 µg/L, 0.025 µg/L, and 0.025 µg/L, respectively. The overall reproducibility of the biosensor (n=3 was <±6.35%. The biosensor had been tested with different combinations of toxicants, with the results showing predominantly antagonistic responses. The results confirmed that the biosensor constructed in this report is suitable to be used in quantitative and qualitative detections of heavy metals and pesticides.

  10. Adverse events following primary and secondary immunisation with whole-cell pertussis: a systematic review protocol

    Science.gov (United States)

    Patterson, Jenna; Kagina, Benjamin M; Gold, Michael; Hussey, Gregory D; Muloiwa, Rudzani

    2017-01-01

    Introduction Pertussis is a contagious respiratory illness caused by the bacterium Bordetella pertussis. Two types of vaccines are currently available against the disease: whole-cell pertussis (wP) and acellular pertussis (aP). With the shift of high-income countries from wP to aP as a result of adverse events following immunisation (AEFI), an upsurge in reported cases of pertussis has been noticed. Owing to this, it is proposed to use wP as a prime and aP for boost vaccination strategy. However, a comparison of the AEFI with the first doses of wP and aP are not clearly documented. Methods and analysis The primary outcomes of interest are AEFI with dose 1 of wP, subsequent doses of wP and dose 1 of aP. As a secondary outcome frequency of AEFI with wP will be compared with the AEFI of doses 2 and 3 of wP and dose 1 of aP. Electronic databases will be searched and two authors will screen the titles and abstracts of the output. Full texts will then be independently reviewed by the first author and two other authors. Qualifying studies will then be formally assessed for quality and risk of bias using a scoring tool. Following standardised data extraction, statistical analysis will be carried out using STATA. Where data are available, subgroup analyses will be performed. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines will be followed in reporting the findings of the systematic review and meta-analysis. Ethics and dissemination No ethics approval is required as the systematic review will use only published data already in the public domain. Findings will be disseminated through publication in a peer-reviewed journal. Trial registration number This protocol has been registered with the International Prospective Register of Systematic Reviews (PROSPERO), registration number CRD42016035809. PMID:28122832

  11. HCN Producing Bacteria Enable Sensing Of Non-Bioavailable Hg Species by the Whole Cell Biosensor

    Science.gov (United States)

    Horvat, M.; Rijavec, T.; Koron, N.; Lapanje, A.

    2015-12-01

    Bacteria play an important role in Hg transformation reactions. The production of cyanide (HCN) and other secondary metabolites seems to be key elements involved in these transformations. Current hypotheses link the role of HCN production to growth inhibition of nonHCN producing competitor organisms (role of an antimicrobial agent). Our past investigations showed that HCN production did not correlate with antimicrobial activity and since pK value of HCN is very high (pK = 9,21), it can be expected that most of the produced HCN is removed from the microenvironment. This way, the expected inhibitory concentrations can hardly be reached. Accordingly, we proposed a new concept, where the ability of complexation of transient metals by HCN served as a regulation process for the accessibility of micro-elements. In our study, we focused on the presence of HCN producing bacteria and carried it out in the Hg contaminated environment connected to the Idrija Mercury Mine, Slovenia. We characterised the isolates according to the presence of Hg resistance (HgR), level of HCN production and genetic similarities. In laboratory setups, using our merR whole cell based biosensor, we determined the transformation of low bioavailable Hg0 and HgS forms into bioavailable Hg by these HCN producing bacteria. We observed that HgR strains producing HCN had the highest impact on increased Hg bioavailability. In the proposed ecological strategy HgR HCN producing bacteria increase their competitive edge over non-HgR competitors through the increase of Hg toxicity. Due to their activity, Hg is made available to other organisms as well and thus enters into the ecosystem. Finally, using some of the characteristics of bacteria (e.g. Hg resistance genetic elements), we developed a fully automated sensing approach, combining biosensorics and mechatronics, to measure the bioavailability of Hg in situ.

  12. Properties of the nucleo-olivary pathway: an in vivo whole-cell patch clamp study.

    Directory of Open Access Journals (Sweden)

    Paolo Bazzigaluppi

    Full Text Available The inferior olivary nucleus (IO forms the gateway to the cerebellar cortex and receives feedback information from the cerebellar nuclei (CN, thereby occupying a central position in the olivo-cerebellar loop. Here, we investigated the feedback input from the CN to the IO in vivo in mice using the whole-cell patch-clamp technique. This approach allows us to study how the CN-feedback input is integrated with the activity of olivary neurons, while the olivo-cerebellar system and its connections are intact. Our results show how IO neurons respond to CN stimulation sequentially with: i a short depolarization (EPSP, ii a hyperpolarization (IPSP and iii a rebound depolarization. The latter two phenomena can also be evoked without the EPSPs. The IPSP is sensitive to a GABA(A receptor blocker. The IPSP suppresses suprathreshold and subthreshold activity and is generated mainly by activation of the GABA(A receptors. The rebound depolarization re-initiates and temporarily phase locks the subthreshold oscillations. Lack of electrotonical coupling does not affect the IPSP of individual olivary neurons, nor the sensitivity of its GABA(A receptors to blockers. The GABAergic feedback input from the CN does not only temporarily block the transmission of signals through the IO, it also isolates neurons from the network by shunting the junction current and re-initiates the temporal pattern after a fixed time point. These data suggest that the IO not only functions as a cerebellar controlled gating device, but also operates as a pattern generator for controlling motor timing and/or learning.

  13. Bacterial host and reporter gene optimization for genetically encoded whole cell biosensors.

    Science.gov (United States)

    Brutesco, Catherine; Prévéral, Sandra; Escoffier, Camille; Descamps, Elodie C T; Prudent, Elsa; Cayron, Julien; Dumas, Louis; Ricquebourg, Manon; Adryanczyk-Perrier, Géraldine; de Groot, Arjan; Garcia, Daniel; Rodrigue, Agnès; Pignol, David; Ginet, Nicolas

    2017-01-01

    Whole-cell biosensors based on reporter genes allow detection of toxic metals in water with high selectivity and sensitivity under laboratory conditions; nevertheless, their transfer to a commercial inline water analyzer requires specific adaptation and optimization to field conditions as well as economical considerations. We focused here on both the influence of the bacterial host and the choice of the reporter gene by following the responses of global toxicity biosensors based on constitutive bacterial promoters as well as arsenite biosensors based on the arsenite-inducible Pars promoter. We observed important variations of the bioluminescence emission levels in five different Escherichia coli strains harboring two different lux-based biosensors, suggesting that the best host strain has to be empirically selected for each new biosensor under construction. We also investigated the bioluminescence reporter gene system transferred into Deinococcus deserti, an environmental, desiccation- and radiation-tolerant bacterium that would reduce the manufacturing costs of bacterial biosensors for commercial water analyzers and open the field of biodetection in radioactive environments. We thus successfully obtained a cell survival biosensor and a metal biosensor able to detect a concentration as low as 100 nM of arsenite in D. deserti. We demonstrated that the arsenite biosensor resisted desiccation and remained functional after 7 days stored in air-dried D. deserti cells. We also report here the use of a new near-infrared (NIR) fluorescent reporter candidate, a bacteriophytochrome from the magnetotactic bacterium Magnetospirillum magneticum AMB-1, which showed a NIR fluorescent signal that remained optimal despite increasing sample turbidity, while in similar conditions, a drastic loss of the lux-based biosensors signal was observed.

  14. Scavenging ROS dramatically increase NMDA receptor whole-cell currents in painted turtle cortical neurons.

    Science.gov (United States)

    Dukoff, David James; Hogg, David William; Hawrysh, Peter John; Buck, Leslie Thomas

    2014-09-15

    Oxygen deprivation triggers excitotoxic cell death in mammal neurons through excessive calcium loading via over-activation of N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. This does not occur in the western painted turtle, which overwinters for months without oxygen. Neurological damage is avoided through anoxia-mediated decreases in NMDA and AMPA receptor currents that are dependent upon a modest rise in intracellular Ca(2+) concentrations ([Ca(2+)]i) originating from mitochondria. Anoxia also blocks mitochondrial reactive oxygen species (ROS) generation, which is another potential signaling mechanism to regulate glutamate receptors. To assess the effects of decreased intracellular [ROS] on NMDA and AMPA receptor currents, we scavenged ROS with N-2-mercaptopropionylglycine (MPG) or N-acetylcysteine (NAC). Unlike anoxia, ROS scavengers increased NMDA receptor whole-cell currents by 100%, while hydrogen peroxide decreased currents. AMPA receptor currents and [Ca(2+)]i concentrations were unaffected by ROS manipulation. Because decreases in [ROS] increased NMDA receptor currents, we next asked whether mitochondrial Ca(2+) release prevents receptor potentiation during anoxia. Normoxic activation of mitochondrial ATP-sensitive potassium (mKATP) channels with diazoxide decreased NMDA receptor currents and was unaffected by subsequent ROS scavenging. Diazoxide application following ROS scavenging did not rescue scavenger-mediated increases in NMDA receptor currents. Fluorescent measurement of [Ca(2+)]i and ROS levels demonstrated that [Ca(2+)]i increases before ROS decreases. We conclude that decreases in ROS concentration are not linked to anoxia-mediated decreases in NMDA/AMPA receptor currents but are rather associated with an increase in NMDA receptor currents that is prevented during anoxia by mitochondrial Ca(2+) release.

  15. Whole-cell based label-free capacitive biosensor for rapid nanosize-dependent toxicity detection.

    Science.gov (United States)

    Qureshi, Anjum; Pandey, Ashish; Chouhan, Raghuraj S; Gurbuz, Yasar; Niazi, Javed H

    2015-05-15

    Despite intensive studies on examining the toxicity of nanomaterials (NMs), our current understanding on potential toxicity in relation to size and cellular responses has remained limited. In this work, we have developed a whole-cell based capacitive biosensor (WCB) to determine the biological toxicity of nanoparticles (NPs) using iron oxide (Fe3O4) NPs as models. This WCB chip comprised of an array of capacitor sensors made of gold interdigitated microelectrodes on which living Escherichia coli cells were immobilized. Cells-on-chip was then allowed to interact with different sizes of Fe3O4 NPs (5, 20 and 100 nm) and concentration-depended cellular-responses were measured in terms of change in dielectric properties (capacitance) as a function of applied AC frequency. The WCB response showed smaller-sized Fe3O4 NPs (5 nm) induced maximum change in surface capacitance because of their effective cellular interaction with E. coli cells-on-chip indicating that the cells suffered from severe cellular deformation, which was confirmed by scanning electron microscopic (SEM) examination. Further our results were validated through their cell viability and E. coli responses at the interface of cell-membrane and NPs as a proof-of-concept. WCB response showed a size-dependent shift in maximum response level from 2 µg/ml of 5 nm sized NPs to 4 µg/ml with NP-sizes greater than 20 nm. The developed WCB offered real-time, label-free and noninvasive detection of cellular responses against Fe3O4 NPs' toxicity with speed, simplicity and sensitivity that can be extended to toxicity screening of various other NPs. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Comparative effectiveness of acellular versus whole-cell pertussis vaccines in teenagers.

    Science.gov (United States)

    Klein, Nicola P; Bartlett, Joan; Fireman, Bruce; Rowhani-Rahbar, Ali; Baxter, Roger

    2013-06-01

    During the 1990s, the United States switched from combined diphtheria, tetanus toxoids, whole-cell pertussis (DTwP) vaccines to combined acellular pertussis (DTaP) vaccines because of safety concerns. After a 2010-2011 pertussis outbreak, we sought to evaluate whether disease risk in 10 to 17 year olds differed between those who previously received DTwP from those who received DTaP. A case-control study among individuals born from 1994 to 1999 who received 4 pertussis-containing vaccines during the first 2 years of life at Kaiser Permanente Northern California (KPNC). We separately compared pertussis polymerase chain reaction (PCR)-positive cases with PCR-negative and KPNC-matched controls. We assessed risk of pertussis relative to vaccine type in early childhood (4 DTwPs, mixed DTwP/DTaP, or 4 DTaPs) by using conditional logistic regression stratified for calendar time and adjusted for gender, race, medical clinic, and receipt of reduced antigen content acellular pertussis (Tdap) vaccine. We compared 138 PCR-positive cases with 899 PCR-negative and 54 339 KPNC-matched controls. Teenagers who had received 4 DTwPs were much less likely to be pertussis PCR-positive than those who had received 4 DTaPs (odds ratio 5.63, 95% confidence interval 2.55-12.46) or mixed DTwP/DTaP vaccines (odds ratio 3.77, 95% confidence interval 1.57-9.07). Decreasing number of DTwP doses was significantly associated with increased pertussis risk (P vaccines in childhood were more protected during a pertussis outbreak than were those who received DTaP vaccines.

  17. 16S rRNA甲基化介导的氨基糖苷类耐药%Resistance mechanism against aminoglycosides mediated by 16S rRNA methylation

    Institute of Scientific and Technical Information of China (English)

    张晓文

    2012-01-01

    Aminoglycosid.es have been used for the treatment of a broad range of life -threatening Gram-positive and Grarrmeg-ative bacterial infections. These agents bind to the A site of the 16S rRNA of the bacterial 30S ribosomal subunit and subsequently block its growth through interference with its protein synthesis . 16S rRNA methylation is capable of conferring an extraordinarily high level of resistance against most of the clinically important aminoglycosides . Previous research has shown that this phenomenon is media -ted by some 16S rRNA methylase. Because of the clinical importance of these enzymes , further global dissemination of 16S rRNA methylase genes among pathogenic bacilli will be a cause of great concern in the near future . This article presents an overview on the action mechanism, origin, classification and genetic environment of 16S rRNA methylase.%氨基糖苷类抗生素在治疗革兰阳性和阴性细菌引起的感染中起着重要的作用,可通过与细菌30S核糖体亚基的16S rRNA的A位点结合而阻碍蛋白质的合成.16S rRNA甲基化作用可导致细菌对氨基糖苷类药物高水平耐药,大量研究显示这一现象是由一类16S rRNA甲基化酶所介导的.由于16S rRNA甲基化酶在临床上的重要性,为引起医务人员的重视,文中将从此类酶的作用机制、起源、分类以及基因环境等方面作一综述.

  18. Sequencing of an atypical Brucella strain 16S rDNA%1株非典型布鲁杆菌的16S rDNA序列分析

    Institute of Scientific and Technical Information of China (English)

    刘志国; 王妙; 刘日宏; 崔步云

    2015-01-01

    目的 分析非典型布鲁杆菌的16S rDNA序列,评价16S rDNA序列测定方法鉴定布鲁杆菌的可行性.方法 用常规方法对l株非典型菌株进行初步鉴定;提取菌株的DNA,双向测定16S rDNA全序列,在NCBI对该序列进行Blast比对,用DNAMAN软件进行菌株的16S rDNA序列一致性比对;同时,从GenBank下载与布鲁杆菌有血清学交叉反应菌株的16S rDNA全序列,用MEGA 6.0构建16S rDNA系统发生树.结果 常规鉴定显示,试验菌株为非典型布鲁杆菌.试验菌株的16S rDNA序列与布鲁杆菌基因的相似性为99%,与牛种布鲁杆菌544A、羊种布鲁杆菌16M的16S rDNA序列的一致性为96.99%.系统发生树显示,试验菌株为布鲁杆菌,且与牛种布鲁杆菌544A、羊种布鲁杆菌16M有较为密切的亲缘关系.结论 试验菌株为非典型布鲁杆菌,16S rDNA序列测定是一种简便、快速、准确的非典型布鲁杆菌鉴定方法.%Objective To sequence an atypical Brucella strain 16S rDNA,and to evaluate the feasibility of 16S rDNA sequencing method for identification of Brucella.Methods Preliminary identification of atypical strains was carried out with conventional method.Strain DNA was extracted,and 16S rDNA complete sequence was bidirectional sequenced,and Blast in NCBI and DNAMAN software were used for comparison of the sequence identities of the 16S rDNA.Moreover,16S rDNA complete sequence of the stains those were known to cross-react serologically with Brucella was downloaded from GenBank,MEGA 6.0 was used to construct the phylogenetic tree.Results The conventional identification results revealed that it was an atypical Brucella,the gene similarity between the sequences of the test strain 16S rDNA and Brucella was 99%,between 16S rDNA sequence of Brucella abortus 544A and Brucella melitensis 16M was 96.99%.Phylogenetic tree revealed that the test stain was a Brucella,and closely related to Brucella abortus 544A and Brucella melitensis 16M.Conclusion The

  19. Different organisms associated with heartwater as shown by analysis of 16S ribosomal RNA gene sequences.

    Science.gov (United States)

    Allsopp, M; Visser, E S; du Plessis, J L; Vogel, S W; Allsopp, B A

    1997-08-01

    Cowdria ruminantium is a rickettsial parasite which causes heartwater, a economically important disease of domestic and wild ruminants in tropical and subtropical Africa and parts of the Caribbean. Because existing diagnostic methods are unreliable, we investigated the small-subunit ribosomal RNA (srRNA) gene from heartwater-infected material to characterise the organisms present and to develop specific oligonucleotide probes for polymerase chain reaction (PCR) based diagnosis. DNA was obtained from ticks and ruminants from heartwater-free and heartwater-endemic areas from Cowdria in tissue culture. PCR was carried out using primers designed to amplify only rickettsial srRNA genes, the target region being the highly variable V1 loop. Amplicons were cloned and sequenced; 51% were C. ruminantium sequences corresponding to four genotypes, two of which were identical to previously reported C. ruminantium sequences while the other two were new. The four different Cowdria genotypes can be correlated with different phenotypes. Tissue-culture samples yielded only Cowdria genotype sequences, but an extraordinary heterogeneity of 16S sequences was obtained from field samples. In addition to Cowdria genotypes we found sequences from previously unknown Ehrlichia spp., sequences showing homology to other Rickettsiales and a variety of Pseudomonadaceae. One Ehrlichia sequence was phylogenetically closely related to Ehrlichia platys (Group II Ehrlichia) and one to Ehrlichia canis (Group III Ehrlichia). This latter sequence was from an isolate (Germishuys) made from a naturally infected sheep which, from brain smear examination and pathology, appeared to be suffering from heartwater; nevertheless no Cowdria genotype sequences were found in this isolate. In addition no Cowdria sequences were obtained from uninfected ticks. Complete 16S rRNA gene sequences were determined for two C. ruminantium genotypes and for two previously uncharacterised heartwater-associated Ehrlichia spp

  20. Improved identification of Gordonia, Rhodococcus and Tsukamurella species by 5'-end 16S rRNA gene sequencing.

    Science.gov (United States)

    Wang, Tao; Kong, Fanrong; Chen, Sharon; Xiao, Meng; Sorrell, Tania; Wang, Xiaoyan; Wang, Shuo; Sintchenko, Vitali

    2011-01-01

    The identification of fastidious aerobic Actinomycetes such as Gordonia, Rhodococcus, and Tsukamurella has remained a challenge leading to clinically significant misclassifications. This study is intended to examine the feasibility of partial 5'-end 16S rRNA gene sequencing for the identification of Gordonia, Rhodococcus, and Tsukamurella, and defined potential reference sequences for species from each of these genera. The 16S rRNA gene sequence based identification algorithm for species identification was used and enhanced by aligning test sequences with reference sequences from the List of Prokaryotic Names with Standing in Nomenclature. Conventional PCR based 16S rRNA gene sequencing and the alignment of the isolate 16S rRNA gene sequence with reference sequences accurately identified 100% of clinical strains of aerobic Actinomycetes. While partial 16S rRNA gene sequences of reference type strains matched with the 16S rRNA gene sequences of 19 isolates in our data set, another 13 strains demonstrated a degree of polymorphism with a 1-4 bp difference in the regions of difference. 5'-end 606 bp 16S rRNA gene sequencing, coupled with the assignment of well defined reference sequences to clinically relevant species of bacteria, can be a useful strategy for improving the identification of clinically relevant aerobic Actinomycetes.

  1. [Phylogenetic comparison between Spirulina and Arthrospira based on 16S rRNA and rpoC1 gene].

    Science.gov (United States)

    Wu, Yuemei; Wang, Suying; Dong, Shirui

    2016-02-04

    Based on 16S rRNA and rpoC1 gene sequences, the phylogenetic relationship between Spirulina and Arthrospira were studied and compared. We amplified, sequenced and analyzed 16S rRNA and rpoC1 of 84 strains. Then the phylogenetic trees were constructed and compared. The conserved sites percentage, average G+C content and sequence identity of rpoC1 were 49.7%, 47.7%, 76%-100% respectively, significantly lower than 79.4%, 55.6% and 91%-100% of 16S rRNA, and the heterogeneity degree was higher. The trees generated with two different genes showed similar topologies and thus inferred consistent phylogenetic relationships. Eighty-four experimental strains were divided into 3 groups belonging to 2 genera: F-35 1, F-904-2, F-1070 and TJBC14 were Spirulina and the rest were Arthrospira. Although morphospecies and geographical species could not be distinguished based on 16S rRNA and rpoC1 gene sequences, the bootstrap value of rpoC1 (100%) was higher than that of 16S rRNA (99%). Moreover, clustering effect of rpoC1 for Spirulina and Arthrospirai was better than 16S rRNA. Spirulina and Arthrospira were different genera, rpoC1 gene has more advantage to distinguish the strains in the same genus than that of 16S rRNA gene.

  2. Partial Sequencing of 16S rRNA Gene of Selected Staphylococcus aureus Isolates and its Antibiotic Resistance

    Directory of Open Access Journals (Sweden)

    Harsi Dewantari Kusumaningrum

    2016-08-01

    Full Text Available The choice of primer used in 16S rRNA sequencing for identification of Staphylococcus species found in food is important. This study aimed to characterize Staphylococcus aureus isolates by partial sequencing based on 16S rRNA gene employing primers 16sF, 63F or 1387R. The isolates were isolated from milk, egg dishes and chicken dishes and selected based on the presence of sea gene that responsible for formation of enterotoxin-A. Antibiotic susceptibility of the isolates towards six antibiotics was also tested. The use of 16sF resulted generally in higher identity percentage and query coverage compared to the sequencing by 63F or 1387R. BLAST results of all isolates, sequenced by 16sF, showed 99% homology to complete genome of four S. aureus strains, with different characteristics on enterotoxin production and antibiotic resistance. Considering that all isolates were carrying sea gene, indicated by the occurence of 120 bp amplicon after PCR amplification using primer SEA1/SEA2,  the isolates were most in agreeing to S. aureus subsp. aureus ST288. This study indicated that 4 out of 8 selected isolates were resistant towards streptomycin. The 16S rRNA gene sequencing using 16sF is useful for identification of S. aureus. However, additional analysis such as PCR employing specific gene target, should give a valuable supplementary information, when specific characteristic is expected.

  3. Towards a phylogeny of the genus Vibrio based on 16S rRNA sequences.

    Science.gov (United States)

    Dorsch, M; Lane, D; Stackebrandt, E

    1992-01-01

    The inter- and intrageneric relationships of the genus Vibrio were investigated by performing a comparative analysis of the 16S rRNAs of 10 species, including four pathogenic representatives. The results of immunological and 5S rRNA studies were confirmed in that the genus is a neighboring taxon of the family Enterobacteriaceae. With regard to the intrageneric structure, Vibrio alginolyticus, Vibrio campbellii, Vibrio natriegens, Vibrio harveyi, Vibrio proteolyticus, Vibrio parahaemolyticus, and Vibrio vulnificus form the core of the genus, while Vibrio (Listonella) anguillarum, Vibrio diazotrophicus, and Vibrio hollisae are placed on the outskirts of the genus. Variable regions around positions 80, 180, and 450 could be used as target sites for genus- and species-specific oligonucleotide probes and polymerase chain reaction primers to be used in molecular identification.

  4. An unusual case of seronegative, 16S PCR positive Brucella infection

    Science.gov (United States)

    Backhouse, Lucy; Rawat, David; Naik, Sandhia; Millar, Michael

    2016-01-01

    Introduction: Brucella is a zoonotic infection commonly diagnosed by isolation of the organism from blood culture or positive serological testing. It is an uncommon cause of a pyrexia of unknown origin in the United Kingdom. Case presentation: We describe the case of a 14-year-old girl with no history of travel who presented with pyrexia, weight loss, arthralgia, multiple splenic abscesses and a subsequent pleural effusion, the latter of which isolated a Brucella species on 16S rRNA PCR. The patient responded well to initiation of treatment for brucellosis and on repeat imaging, after 3 months, the splenic abscesses had resolved. Conclusion: This unique case demonstrates uncommon complications of brucellosis and the challenges of diagnosing the organism, the latter of which can be alleviated by the utilization of molecularbased technologies. This patient had a negative serology result for brucellosis, which highlights the need to interpret serology results with caution in non-endemic regions for brucellosis.

  5. Genetic Diversity in Populations of Sepiella maindroni Using 16S rRNA Gene Sequence Analysis

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Part of the 16S rRNA gene is amplified with PCR and sequenced for 5 populations of common Chinese cuttlefish Sepiella maindroni: three from the South China Sea, one from East China Sea and one from Japan. The result shows that a total of 5 nucleotide positions are found to have gaps or insertions of base pairs among these individuals, and 13 positions are examined to be variable in all the sequences, which range from 494 to 509 base pairs. All of the individuals are grouped into 7 haplotypes (h1-h7). No marked genetic difference is observed among those populations. All of the individuals from Nagasaki belong to h1 and the h3 haplotype is found only in the coastal waters of China. AG transition in Nucleotide 255 is suggested to be taken as a kind of genetic marker to identify the populations distributed in East-South China Sea and the Nagasaki waters of Japan.

  6. Preliminary study on mitochondrial 16S rRNA gene sequences and phylogeny of flatfishes (Pleuronectiformes)

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    A 605 bp section of mitochondrial 16S rRNA gene from Paralichthys olivaceus, Pseudorhombus cinnamomeus, Psetta maxima and Kareius bicoloratus, which represent 3 families of Order Pleuronectiformes was amplified by PCR and sequenced to show the molecular systematics of Pleuronectiformes for comparison with related gene sequences of other 6 flatfish downloaded from GenBank. Phylogenetic analysis based on genetic distance from related gene sequences of 10 flatfish showed that this method was ideal to explore the relationship between species, genera and families. Phylogenetic trees set-up is based on neighbor-joining, maximum parsimony and maximum likelihood methods that accords to the general rule of Pleuronectiformes evolution. But they also resulted in some confusion. Unlike data from morphological characters, P. olivaceus clustered with K.bicoloratus, but P. cinnamomeus did not cluster with P. olivaceus, which is worth further studying.

  7. [PCR rDNA 16S used for the etiological diagnosis of blood culture negative endocarditis].

    Science.gov (United States)

    Baty, G; Lanotte, P; Hocqueloux, L; Prazuck, T; Bret, L; Romano, M; Mereghetti, L

    2010-06-01

    We report the case of a 55 year-old man presenting with a double aortic and mitral endocarditis for which resected valve culture was repeatedly negative. Specific PCR made on valves because of highly positive blood tests for Bartonella henselae remained negative. A molecular approach was made with 16S rDNA PCR, followed by sequencing. Bartonella quintana was identified as the etiology of endocarditis. B. quintana, "fastidious" bacteria, even if hard to identify in a laboratory, is often reported as a blood culture negative endocarditis (BCNE) agent. Molecular biology methods have strongly improved the diagnosis of BCNE. We propose a review of the literature focusing on the interest of broad-spectrum PCR on valve for the etiological diagnosis of BCNE.

  8. RISSC: a novel database for ribosomal 16S-23S RNA genes spacer regions.

    Science.gov (United States)

    García-Martínez, J; Bescós, I; Rodríguez-Sala, J J; Rodríguez-Valera, F

    2001-01-01

    A novel database, under the acronym RISSC (Ribosomal Intergenic Spacer Sequence Collection), has been created. It compiles more than 1600 entries of edited DNA sequence data from the 16S-23S ribosomal spacers present in most prokaryotes and organelles (e.g. mitochondria and chloroplasts) and is accessible through the Internet (http://ulises.umh.es/RISSC), where systematic searches for specific words can be conducted, as well as BLAST-type sequence searches. Additionally, a characteristic feature of this region, the presence/absence and nature of tRNA genes within the spacer, is included in all the entries, even when not previously indicated in the original database. All these combined features could provide a useful documentation tool for studies on evolution, identification, typing and strain characterization, among others.

  9. discussion on validity of rana maoershanensis based on partial sequence of 16s rrna gene

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    rana maoershanensis found in mt.maoershan in guangxi,china was reported as a new species in 2007,but there was no molecular data for this frog.the partial sequences (543 bp) of 16s rrna gene from 12 specimens of 3 brown frog species (rana hanluica,r.maoershanensis and r.chensinensis) were analyzed with 17 specimens of 9 species from genbank.the nucleotide sequence divergence between r.maoershanensis and the other brown frog species were 4.5%-6.5%,with 22-30 nucleotide substitutions at this locus.the phylogenetic relationships based on mp,ml,and bayesian inference indicate that the brown frogs from southern china were diverged into three groups (clades a,b and c).r.maoershanensis was clustered together a well-supported subclade (b-l).it is suggested that r.maoershanensis is a valid species.

  10. Diversity of 16S rDNA and environmental factor influencing microorganisms in Malan ice core

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The research on extrempholic microorganisms in glacial low-temperature environment receives more attention than ever before. Due to the successive chronological records in ice core, it is important to initiate microbiological studies on ice core samples. 23 samples from one ice core, drilled from central Qinghai-Tibetan Plateau, were analyzed. The number of total microorganisms and culturable microorganisms in different layers showed that it related with the content of dust in ice. It is suggested that the distribution of microorganisms in ice depends on the transportation of materials during glacier development. The bacteria diversity in Malan Glacier was analyzed by 16S rDNA sequencing methods, which showed that many sequences were similar to known psychrophilic bacteria.

  11. Hosts, distribution and genetic divergence (16S rDNA) of Amblyomma dubitatum (Acari: Ixodidae).

    Science.gov (United States)

    Nava, Santiago; Venzal, José M; Labruna, Marcelo B; Mastropaolo, Mariano; González, Enrique M; Mangold, Atilio J; Guglielmone, Alberto A

    2010-08-01

    We supply information about hosts and distribution of Amblyomma dubitatum. In addition, we carry out an analysis of genetic divergence among specimens of A. dubitatum from different localities and with respect to other Neotropical Amblyomma species, using sequences of 16S rDNA gene. Although specimens of A. dubitatum were collected on several mammal species as cattle horse, Tapirus terrestris, Mazama gouazoubira, Tayassu pecari, Sus scrofa, Cerdocyon thous, Myocastor coypus, Allouata caraya, Glossophaga soricina and man, most records of immature and adult stages of A. dubitatum were made on Hydrochoerus hydrochaeris, making this rodent the principal host for all parasitic stages of this ticks. Cricetidae rodents (Lundomys molitor, Scapteromys tumidus), opossums (Didelphis albiventris) and vizcacha (Lagostomus maximus) also were recorded as hosts for immature stages. All findings of A. dubitatum correspond to localities of Argentina, Brazil, Paraguay and Uruguay, and they were concentrated in the Biogeographical provinces of Pampa, Chaco, Cerrado, Brazilian Atlantic Forest, Parana Forest and Araucaria angustifolia Forest. The distribution of A. dubitatum is narrower than that of its principal host, therefore environmental variables rather than hosts determine the distributional ranges of this tick. The intraspecific genetic divergence among 16S rDNA sequences of A. dubitatum ticks collected in different localities from Argentina, Brazil and Uruguay was in all cases lower than 0.8%, whereas the differences with the remaining Amblyomma species included in the analysis were always bigger than 6.8%. Thus, the taxonomic status of A. dubitatum along its distribution appears to be certain at the specific level.

  12. Phylogenetic analysis of Demodex caprae based on mitochondrial 16S rDNA sequence.

    Science.gov (United States)

    Zhao, Ya-E; Hu, Li; Ma, Jun-Xian

    2013-11-01

    Demodex caprae infests the hair follicles and sebaceous glands of goats worldwide, which not only seriously impairs goat farming, but also causes a big economic loss. However, there are few reports on the DNA level of D. caprae. To reveal the taxonomic position of D. caprae within the genus Demodex, the present study conducted phylogenetic analysis of D. caprae based on mt16S rDNA sequence data. D. caprae adults and eggs were obtained from a skin nodule of the goat suffering demodicidosis. The mt16S rDNA sequences of individual mite were amplified using specific primers, and then cloned, sequenced, and aligned. The sequence divergence, genetic distance, and transition/transversion rate were computed, and the phylogenetic trees in Demodex were reconstructed. Results revealed the 339-bp partial sequences of six D. caprae isolates were obtained, and the sequence identity was 100% among isolates. The pairwise divergences between D. caprae and Demodex canis or Demodex folliculorum or Demodex brevis were 22.2-24.0%, 24.0-24.9%, and 22.9-23.2%, respectively. The corresponding average genetic distances were 2.840, 2.926, and 2.665, and the average transition/transversion rates were 0.70, 0.55, and 0.54, respectively. The divergences, genetic distances, and transition/transversion rates of D. caprae versus the other three species all reached interspecies level. The five phylogenetic trees all presented that D. caprae clustered with D. brevis first, and then with D. canis, D. folliculorum, and Demodex injai in sequence. In conclusion, D. caprae is an independent species, and it is closer to D. brevis than to D. canis, D. folliculorum, or D. injai.

  13. Identification of the microbiota in carious dentin lesions using 16S rRNA gene sequencing.

    Science.gov (United States)

    Obata, Junko; Takeshita, Toru; Shibata, Yukie; Yamanaka, Wataru; Unemori, Masako; Akamine, Akifumi; Yamashita, Yoshihisa

    2014-01-01

    While mutans streptococci have long been assumed to be the specific pathogen responsible for human dental caries, the concept of a complex dental caries-associated microbiota has received significant attention in recent years. Molecular analyses revealed the complexity of the microbiota with the predominance of Lactobacillus and Prevotella in carious dentine lesions. However, characterization of the dentin caries-associated microbiota has not been extensively explored in different ethnicities and races. In the present study, the bacterial communities in the carious dentin of Japanese subjects were analyzed comprehensively with molecular approaches using the16S rRNA gene. Carious dentin lesion samples were collected from 32 subjects aged 4-76 years, and the 16S rRNA genes, amplified from the extracted DNA with universal primers, were sequenced with a pyrosequencer. The bacterial composition was classified into clusters I, II, and III according to the relative abundance (high, middle, low) of Lactobacillus. The bacterial composition in cluster II was composed of relatively high proportions of Olsenella and Propionibacterium or subdominated by heterogeneous genera. The bacterial communities in cluster III were characterized by the predominance of Atopobium, Prevotella, or Propionibacterium with Streptococcus or Actinomyces. Some samples in clusters II and III, mainly related to Atopobium and Propionibacterium, were novel combinations of microbiota in carious dentin lesions and may be characteristic of the Japanese population. Clone library analysis revealed that Atopobium sp. HOT-416 and P. acidifaciens were specific species associated with dentinal caries among these genera in a Japanese population. We summarized the bacterial composition of dentinal carious lesions in a Japanese population using next-generation sequencing and found typical Japanese types with Atopobium or Propionibacterium predominating.

  14. Modulation of Whole-Cell Currents in Plasmodium Falciparum-Infected Human Red Blood Cells by Holding Potential and Serum

    Science.gov (United States)

    Staines, Henry M; Powell, Trevor; Clive Ellory, J; Egée, Stéphane; Lapaix, Franck; Decherf, Gaëtan; Thomas, Serge L Y; Duranton, Christophe; Lang, Florian; Huber, Stephan M

    2003-01-01

    Recent electrophysiological studies have identified novel ion channel activity in the host plasma membrane of Plasmodium falciparum-infected human red blood cells (RBCs). However, conflicting data have been published with regard to the characteristics of induced channel activity measured in the whole-cell configuration of the patch-clamp technique. In an effort to establish the reasons for these discrepancies, we demonstrate here two factors that have been found to modulate whole-cell recordings in malaria-infected RBCs. Firstly, negative holding potentials reduced inward currents (i.e. at negative potentials), although this result was highly complex. Secondly, the addition of human serum increased outward currents (i.e. at positive potentials) by approximately 4-fold and inward currents by approximately 2-fold. These two effects may help to resolve the conflicting data in the literature, although further investigation is required to understand the underlying mechanisms and their physiological relevance in detail. PMID:12937282

  15. Whole-cell recordings of voltage-gated Calcium, Potassium and Sodium currents in acutely isolated hippocampal pyramidal neurons

    Institute of Scientific and Technical Information of China (English)

    Shuyun Huang; Qing Cai; Weitian Liu; Xiaoling Wang; Tao Wang

    2009-01-01

    Objective:To record Calcium, Potassium and Sodium currents in acutely isolated hippocampal pyramidal neurons. Methods:Hip-pocampal CA3 neurons were freshly isolated by 1 mg protease/3 ml SES and mechanical trituration with polished pipettes of progressively smaller tip diameters. Patch clamp technique in whole-cell mode was employed to record voltage-gated channel currents. Results:The procedure dissociated hippocampal neurons, preserving apical dendrites and several basal dendrites, without impairing the electrical characteristics of the neurons. Whole-cell patch clamp configuration was successfully used to record voltage-gated Ca2+ currents, delayed rectifier K+ current and voltage-gated Na+ currents. Conclusion:Protease combined with mechanical trituration may be used for the dissociation of neurons from rat hippocampus. Voltage-gated channels currents could be recorded using a patch clamp technique.

  16. 哈维弧菌16S rRNA基因拷贝数的种内变异%Intra-species variation of 16S rRNA gene copy number of Vibrio harveyi

    Institute of Scientific and Technical Information of China (English)

    郑嘉来; 阎永伟; 唐姝; 杨坤杰; 李长红; 王凯; 张德民

    2015-01-01

    利用荧光定量PCR法测定哈维弧菌( Vibrio harveyi)基因组内16S rRNA基因拷贝数. 基于16S rDNA、pyrH、rpoA和recA4个基因的系统发育学分析鉴定弧菌菌株,然后通过重叠PCR构建包含哈维弧菌模式菌株16S rD-NA和gyrB基因片段的标准质粒,建立了定量PCR测定细胞内16S rRNA基因拷贝数方法,并测定了6株测试菌株和哈维弧菌模式株的16S rRNA基因拷贝数,同时以已知拷贝数的菌株ATCC BAA-1116做对照. 6个测试菌株均为哈维弧菌. 菌株ATCC BAA-1116的16S rRNA基因拷贝数为11个,与已知值相符. 模式菌株MCCC 1H00031T和6个测试菌株SXB-C、SCB-4、NBV00029、NBV00035、NBV00039和NBV00269的拷贝数分别为9、12、13、12、11、13和9个. 利用荧光定量PCR法测定哈维弧菌基因组内16S rRNA基因拷贝数的方法简单可行. 哈维弧菌16S rRNA基因拷贝数的种内变异可能是其适应近岸多变生境的原因之一.%This study aims to determine intra-genomic 16S rRNA gene copy number of Vibrio harveyi strains by a method of fluorescent quantitative PCR. Vibrio strains were identified by multilocussequence analysis based on 16S rDNA, pyrH, rpoA and recA. A standard plasmid, harboring fragments of 16S rDNA and the single copy gene gyrB from V. harveyi type strain, was constructed. A fluorescent quantitative method was established and used to determine the intra-genomic 16S rRNA gene copy number of the tested 6 strains, taken the type strains of V. harveyi and V. harvei ATCC BAA-1116 as reference, whose 16S rRNA gene copy number is well known. All the 6 tested Vibrio strains were identified as V. harveyi. The copy number of 16S rRNA gene in strain ATCC BAA-1116 was 11, consistent with the known values. The copy number of strain MCCC 1H00031T, SXB-C, SCB-4, NBV00029, NBV00035, NBV00039 and NBV00269 was 9, 12, 13, 12, 11, 13 and 9, respectively. Fluorescent quantitative PCR method is simple and reliable for determi-ning 16S rRNA gene copy number

  17. Comparing the Potential for Identification of Lactobacillus spp. of 16S rDNA Variable Regions

    Directory of Open Access Journals (Sweden)

    Diego Mauricio Riaño-Pachón

    2013-07-01

    Full Text Available 0 0 1 248 1368 Universidad de los Andes 11 3 1613 14.0 Normal 0 21 false false false ES-TRAD JA X-NONE 16s rDNA is used for bacterial identification because its variation rate between species allows differentiation. The gene for this ribosomal subunit has 9 variable regions and some of them give more information than others. We were interested in evaluating the potential for species identification of each region and their combinations. We extracted the V1 to V8 regions of 16s rDNA from different strains and species of Lactobacillus and analyzed them using STAP (ss-RNA Taxonomy Assigning Pipeline and RDP (Ribosomal Database Project multiclassifier packages. Phylogenetic trees obtained by maximum likelihood analyses were compared. Classification results show that many regions give the correct genus classification using RDP and STAP, however they are not enough to classify up to the level of species. V5V6 region presents the highest quantity of informative fragments but also present the highest rate of false negatives. V1V3 region presents the highest rate of true positives (species using STAP and the region V5V8 in RDP (genus.The phylogenetic result shows that the reference topology could be obtained using different combination of regions as V1V3 and V1V8.The experimental validation was done using commercial strains from a probiotic tampon. Sequencing analysis show that the V1V3 region gives the same information and result as the complete 16s rDNA; the three isolated strains correspond to the strains indicated in the product. We conclude that the V1V3 region is the minimum required region to classify Lactobacillus spp. in the correct way and this region is useful in metagenomics to analyze probiotics samples  COMPARACIÓN ENTRE EL POTENCIAL DE LAS REGIONES VARIABLES DEL 16S rDNA PARA LA IDENTIFICACIÓN DE Lactobacillus spp (LactobacilliaceaeEl 16s rDNA es utilizado para la identificación bacteriana dada su tasa de variaci

  18. Whole cell biosynthesis of a functional oligosaccharide, 2′-fucosyllactose, using engineered Escherichia coli

    Directory of Open Access Journals (Sweden)

    Lee Won-Heong

    2012-04-01

    Full Text Available Abstract Background 2'-Fucosyllactose (2-FL is a functional oligosaccharide present in human milk which protects against the infection of enteric pathogens. Because 2-FL can be synthesized through the enzymatic fucosylation of lactose with guanosine 5′-diphosphate (GDP-l-fucose by α-1,2-fucosyltransferase (FucT2, an 2-FL producing Escherichia coli can be constructed through overexpressing genes coding for endogenous GDP- l-fucose biosynthetic enzymes and heterologous fucosyltransferase. Results The gene for FucT2 from Helicobacter pylori was introduced to the GDP- l-fucose producing recombinant E. coli BL21 star(DE3 strain. However, only small amount of 2-FL was produced in a batch fermentation because the E. coli BL21star(DE3 strain assimilated lactose instead of converting to 2-FL. As an alternative host, the E. coli JM109(DE3 strain which is incapable of assimilating lactose was chosen as a 2-FL producer. Whole cell biosynthesis of 2-FL from lactose was investigated in a series of batch fermentations using various concentrations of lactose. The results of batch fermentations showed that lactose was slowly assimilated by the engineered E. coli JM109(DE3 strain and 2-FL was synthesized without supplementation of another auxiliary sugar for cell growth. A maximum 2-FL concentration of 1.23 g/l was obtained from a batch fermentation with 14.5 g/l lactose. The experimentally obtained yield (g 2-FL/g lactose corresponded to 20% of the theoretical maximum yield estimated by the elementary flux mode (EFM analysis. Conclusions The experimental 2-FL yield in this study corresponded to about 20% of the theoretical maximum yield, which suggests further modifications via metabolic engineering of a host strain or optimization of fermentation processes might be carried out for improving 2-FL yield. Improvement of microbial production of 2-FL from lactose by engineered E. coli would increase the feasibility of utilizing 2-FL as a prebiotic in various

  19. 福建华溪蟹线粒体DNA COI和16S rRNA基因序列的遗传多样性%Genetic diversity on mitochondrial DNA COI and 16S rRNA of Sinopotamon fukienense

    Institute of Scientific and Technical Information of China (English)

    石林波; 张小燕; 汪雁; 王云龙; 周宪民; 邹节新

    2013-01-01

    目的 探讨福建华溪蟹(Sinopotamonfukienense)的遗传多样性.方法 采用PCR结合DNA测序技术,测定S.fukienense的线粒体COI和16S rRNA基因序列的组成.经比对获得639 bp长度的COI基因序列和526 bp的16S rRNA基因序列,以对比分析S.fukienense的遗传多样性.结果 S.fukienense基于COI基因核苷酸多样性(Pi)为0.048 4,高于其基于16S rRNA基因核苷酸多样性(Pi)为0.021 6.同时,S.fukienense基于COI基因单倍型间的平均遗传距离(P)为0.048,大于其基于16S rRNA基因单倍型间的平均遗传距离(P)0.026.结论 COI序列在分析S.fukienense遗传异变时的作用更优于16S rRNA基因序列.%The aim of this study was to explore the genetic diversity of the freshwater crab S.fukienense based on the gene sequence CO I and 16S rRNA.The genes of CO I and 16S rRNA were amplified with PCR and subsequently sequenced.The base composition and sequence variation of them were analyed with Mega version 4.0 and DnaSP version 4.10.We obtained 639 bp nucleotide sequence of gene COI and 526 bp nucleotide sequence of gene 16S rRNA.The nucleotide diversity based on gene COI (Pi=0.048 4) was higher than that based on 16S rRNA (Pi=0.021 6).At the same time,the average genetic distance of haplotypes based on gene COI (P=0.048) was larger than that based on 16S rRNA (P=0.026).Results suggest that the COI sequence is better than the 16S rRNA sequence in the analysis of genetic mutation for S.fukienense.

  20. 16S rRNA基因测序技术在肝脓疡细菌鉴定中的作用%Usefulness of 16S rRNA Gene Sequencing for Identification of Bacteria from Pyogenic Liver Abscess

    Institute of Scientific and Technical Information of China (English)

    宋伦圭

    2014-01-01

    目的:评价16S核糖体RNA (rRNA)基因测序在肝脓疡中细菌鉴定中的应用价值。方法2012年1月-2013年12月间共20例肝脓疡行经皮置管引流的患者,分别行脓液培养,血培养和16S rRNA基因测序。利用454 GS Junior System对脓液基因组DNA行PCR和16S rRNA基因测序。脓液培养,血液培养和16S rRNA 基因测序结果进行分别评价。结果脓液和血液培养阳性的患者分别是9例(45%)和4例(20%)。16S rRNA基因测序细菌鉴定率为90%,明显高于传统的培养方法。结论16S rRNA基因测序方法较传统的培养方法能更准确和有效对肝脓疡进行细菌鉴定。%Background/Aims To evaluate the usefulness of 16S ribosomal RNA (rRNA) gene sequencing for an accurate and better identification of bacteria from pyogenic liver abscess (PLA). Methodology 20 patients with PLA were included who underwent percutaneous catheter drainage, abscess culture, blood culture and 16S rRNA gene sequencing for isolates from January 2012 to December 2013. Genomic DNAs of abscess fluids were subjected to PCR and sequencing of 16S rRNA gene by on a 454 GS Junior System. The results were evaluated between abscess cultures, blood and 16S rRNA gene sequencing for isolates. Results Abscess and blood cultures were positive in 9 (45%) and 4 (20%) patients, respectively. The 16S rRNA gene sequencing showed with 90% identification of bacteria a significantly greater identification than conventional cultured methods. Conclusion This study showed a greater usefulness of 16S rRNA gene sequencing than conventional cultured methods for accurate and better identification of bacteria from PLA.

  1. Real Time Multiplicative Memory Amplification Mediated by Whole-Cell Scaling of Synaptic Response in Key Neurons

    Science.gov (United States)

    Reuveni, Iris; Ghosh, Sourav; Barkai, Edi

    2017-01-01

    Intense spiking response of a memory-pattern is believed to play a crucial role both in normal learning and pathology, where it can create biased behavior. We recently proposed a novel model for memory amplification where the simultaneous two-fold increase of all excitatory (AMPAR-mediated) and inhibitory (GABAAR-mediated) synapses in a sub-group of cells that constitutes a memory-pattern selectively amplifies this memory. Here we confirm the cellular basis of this model by validating its major predictions in four sets of experiments, and demonstrate its induction via a whole-cell transduction mechanism. Subsequently, using theory and simulations, we show that this whole-cell two-fold increase of all inhibitory and excitatory synapses functions as an instantaneous and multiplicative amplifier of the neurons’ spiking. The amplification mechanism acts through multiplication of the net synaptic current, where it scales both the average and the standard deviation of the current. In the excitation-inhibition balance regime, this scaling creates a linear multiplicative amplifier of the cell’s spiking response. Moreover, the direct scaling of the synaptic input enables the amplification of the spiking response to be synchronized with rapid changes in synaptic input, and to be independent of previous spiking activity. These traits enable instantaneous real-time amplification during brief elevations of excitatory synaptic input. Furthermore, the multiplicative nature of the amplifier ensures that the net effect of the amplification is large mainly when the synaptic input is mostly excitatory. When induced on all cells that comprise a memory-pattern, these whole-cell modifications enable a substantial instantaneous amplification of the memory-pattern when the memory is activated. The amplification mechanism is induced by CaMKII dependent phosphorylation that doubles the conductance of all GABAA and AMPA receptors in a subset of neurons. This whole-cell transduction

  2. Identiifcation of pathogenic microorganism by sequencing 16S rRNA gene%16S rRNA基因序列分析法鉴定病原细菌

    Institute of Scientific and Technical Information of China (English)

    朱飞舟; 陈利玉; 陈汉春

    2013-01-01

    目的:运用16S rRNA 基因序列分析法鉴定14种细菌,为该方法的临床应用奠定基础。方法:提取细菌DNA,采用通用引物PCR扩增16S rRNA 基因片段并测序。将测序结果用Blastn 在线软件在Nucleotide 数据库中进行序列同源性比对,根据序列同源性鉴定病原细菌。结果:12种细菌可以鉴定到“种”,2种细菌可以鉴定到“属”。结论:16S rRNA 基因序列分析是一种有效的病原细菌鉴定方法。%Objective: To identify 14 bacteria by sequencing the 16S rRNA gene and establish the basis for clinical application in the future. Methods: DNA samples of the 14 bacteria were extracted. The 16S rRNA genes were amplified by PCR and sequenced with common primers. The sequences of the 16S rRNA genes were aligned by online software Blastn in nucleotide database. The bacteria were identified according to the homology of their 16S rRNA genes. Results: Twelve bacteria were classified to species, the other 2 bacteria were classified to genus. Conclusion: 16S rRNA gene sequence analysis is useful in identifying pathogenic bacteria.

  3. Target Mechanism-Based Whole-Cell Screening Identifies Bortezomib as an Inhibitor of Caseinolytic Protease in Mycobacteria

    Science.gov (United States)

    Moreira, Wilfried; Ngan, Grace J. Y.; Low, Jian Liang; Poulsen, Anders; Chia, Brian C. S.; Ang, Melgious J. Y.; Yap, Amelia; Fulwood, Justina; Lakshmanan, Umayal; Lim, Jolander; Khoo, Audrey Y. T.; Flotow, Horst; Hill, Jeffrey; Raju, Ravikiran M.; Rubin, Eric J.

    2015-01-01

    ABSTRACT A novel type of antibacterial screening method, a target mechanism-based whole-cell screening method, was developed to combine the advantages of target mechanism- and whole-cell-based approaches. A mycobacterial reporter strain with a synthetic phenotype for caseinolytic protease (ClpP1P2) activity was engineered, allowing the detection of inhibitors of this enzyme inside intact bacilli. A high-throughput screening method identified bortezomib, a human 26S proteasome drug, as a potent inhibitor of ClpP1P2 activity and bacterial growth. A battery of secondary assays was employed to demonstrate that bortezomib indeed exerts its antimicrobial activity via inhibition of ClpP1P2: Down- or upmodulation of the intracellular protease level resulted in hyper- or hyposensitivity of the bacteria, the drug showed specific potentiation of translation error-inducing aminoglycosides, ClpP1P2-specific substrate WhiB1 accumulated upon exposure, and growth inhibition potencies of bortezomib derivatives correlated with ClpP1P2 inhibition potencies. Furthermore, molecular modeling showed that the drug can bind to the catalytic sites of ClpP1P2. This work demonstrates the feasibility of target mechanism-based whole-cell screening, provides chemical validation of ClpP1P2 as a target, and identifies a drug in clinical use as a new lead compound for tuberculosis therapy. PMID:25944857

  4. Modeling and measurement of a whole-cell bioluminescent biosensor based on a single photon avalanche diode.

    Science.gov (United States)

    Daniel, Ramiz; Almog, Ronen; Ron, Amit; Belkin, Shimshon; Diamand, Yosi Shacahm

    2008-12-01

    Whole-cell biosensors are potential candidates for on-line and in situ environmental monitoring. In this work we present a new design of a whole-cell bioluminescence biosensor for water toxicity detection, based on genetically engineered Escherichia coli bacteria, carrying a recA::luxCDABE promoter-reporter fusion. Sensitive optical detection is achieved using a single photon avalanche photodiode (SPAD) working in the Geiger mode. The present work describes a simple mathematical model for the kinetic process of the bioluminescence based SOS toxin response of E. coli bacteria. We find that initially the bioluminescence signal depends on the time square and we show that the spectral intensity of the bioluminescence signal is inverse proportional to the frequency. We get excellent agreement between the theoretical model and the measured light signal. Furthermore, we present experimental results of the bioluminescent signal measurement using a SPAD and a photomultiplier, and demonstrate improvement of the measurement by applying a matched digital filter. Low intensity bioluminescence signals were measured after the whole-cell sensors were exposed to various toxicant concentrations (5, 15 and 20ppm).

  5. QSSPN: dynamic simulation of molecular interaction networks describing gene regulation, signalling and whole-cell metabolism in human cells.

    Science.gov (United States)

    Fisher, Ciarán P; Plant, Nicholas J; Moore, J Bernadette; Kierzek, Andrzej M

    2013-12-15

    Dynamic simulation of genome-scale molecular interaction networks will enable the mechanistic prediction of genotype-phenotype relationships. Despite advances in quantitative biology, full parameterization of whole-cell models is not yet possible. Simulation methods capable of using available qualitative data are required to develop dynamic whole-cell models through an iterative process of modelling and experimental validation. We formulate quasi-steady state Petri nets (QSSPN), a novel method integrating Petri nets and constraint-based analysis to predict the feasibility of qualitative dynamic behaviours in qualitative models of gene regulation, signalling and whole-cell metabolism. We present the first dynamic simulations including regulatory mechanisms and a genome-scale metabolic network in human cell, using bile acid homeostasis in human hepatocytes as a case study. QSSPN simulations reproduce experimentally determined qualitative dynamic behaviours and permit mechanistic analysis of genotype-phenotype relationships. The model and simulation software implemented in C++ are available in supplementary material and at http://sysbio3.fhms.surrey.ac.uk/qsspn/.

  6. Yeast whole-cell biocatalyst constructed by intracellular overproduction of Rhizopus oryzae lipase is applicable to biodiesel fuel production

    Energy Technology Data Exchange (ETDEWEB)

    Matsumoto, T.; Fukuda, H. [Kobe University, Kobe (Japan). Graduate School of Science and Technology, Division of Molecular Science; Takahashi, S.; Ueda, M.; Tanaka, A. [Kyoto University, Kyoto (Japan). Dept. of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering; Kaieda, M.; Kondo, A. [Kobe University, Kobe (Japan). Faculty of Engineering, Dept. of Chemical Science and Engineering

    2001-07-01

    Yeast whole-cell biocatalysts for lipase-catalyzed reactions were constructed by intracellularly overproducing Rhizopus oryzae lipase (ROL) in Saccharomyces cerevisiae MT8-1. The gene encoding lipase from R. oryzae IFO4697 was cloned, and intracellular overproduction systems of a recombinant ROL with a pro-sequence (rPRoROL) were constructed. When rProROL from R. oryzae IFO4697 was produced under the control of the 5'-upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL) at 30 C for 98 h by two-stage cultivation using SDC medium (SD medium with 2% casamino acids) containing 2.0% and 0.5% glucose, intracellular lipase activity reached levels up to 474.5 IU/l. These whole-cell biocatalysts were permeabilized by air-drying and used for the synthesis of methyl esters (MEs), a potential biodiesel fuel, from plant oil and methanol in a solvent-free and water-containing system. The ME content in the reaction mixture was 71 wt% after a 165-h reaction at 37 C with stepwise addition of methanol. These results indicate that an efficient whole-cell biocatalyst can be prepared by intracellular overproduction of lipase in yeast cells and their permeabilization. (orig.)

  7. Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome

    National Research Council Canada - National Science Library

    Nossa, Carlos W; Oberdorf, William E; Yang, Liying; Aas, Jørn A; Paster, Bruce J; Desantis, Todd Z; Brodie, Eoin L; Malamud, Daniel; Poles, Michael A; Pei, Zhiheng

    2010-01-01

    .... A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral, esophageal, and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species...

  8. Experimental study of 16S rRNA gene sequence analysis for identification of atypical bacteria%16S rRNA基因序列分析鉴定非典型细菌的实验研究

    Institute of Scientific and Technical Information of China (English)

    沈亚娟; 夏云

    2013-01-01

    目的 建立以16S rRNA基因序列分析为基础的细菌鉴定方法,并初步将其应用于临床常规细菌的鉴定.方法 选择临床微生物实验室不能准确鉴定的细菌,以16S rRNA为靶序列,在两端保守区设计引物,PCR反应扩增目的 片段,测序后与数据库中已知细菌的16S rRNA序列进行序列比对.结果 13株菌中,有11株与数据库中的已知16S rRNA序列相似性达99.0%以上,成功鉴定到种的水平.结论 16S rRNA基因序列分析的方法可快速、准确地鉴定不典型菌株,可作为细菌常规鉴定的补充方法.%Objective To establish an approach for bacteria identification based on 16S rRNA gene sequence analysis ,and apply it to clinical routine bacteria identification initially .Methods Bacteria which can not be accurately identified in clinical microbiologi -cal laboratory were selected .16S rRNA served as target sequence ,primers were designed to match the conserved region at both ends and target fragments were amplified by means of PCR reaction .After sequencing ,their sequences were compared with 16S rRNA sequence of known bacteria in the database .Results Among 13 strains ,11 strains showed sequence similarity of 99 .0% with 16S rRNA sequence of known bacteria in the database ,and were successfully identified to the species level .Conclusion 16S rRNA gene sequence analysis can be applied to identify atypical bacteria quickly and accurately ,and serve as a supplementary method of routine bacteria identification .

  9. 16S rRNA is Exchangeable in Escherichia coli%外源16S rRNA在大肠杆菌中的可替换性研究

    Institute of Scientific and Technical Information of China (English)

    李晓丹; 杨瑾; 曹慧; 崔中利

    2007-01-01

    16S rRNA对于构建功能性核糖体是十分重要的,人们普遍将其作为原核生物进化中保守的系统发育标志.为了进一步研究16S rRNA的进化,本文将Escherichia coli中最后一个拷贝的16S rRNA基因替换为Bacillus subtilis的16S rRNA 基因,得到了菌株SQ110BSX.菌株SQ110BSX的代时与出发菌株SQ110基本一致,但是SQ110BSX表现出冷敏感性,而且rRNA/蛋白比值为SQ110的148%.实验结果表明,菌株SQ110BSX中的核糖体效率明显下降.由于E.coli和B.subtilis在遗传距离上较远,两者的可替换性证明了16S rRNA的高度保守性.%16S rRNA plays an important role in the functional construction of ribosomes and is considered to be a conserved phylogenic marker of prokaryotic evolution.To elucidate the evolution of 16S rRNA, we succeeded in replacing the 16S rRNA gene of an E.coli strain with all rrn operons deleted with the 16S rRNA gene of Bacillus subtilis.We found that manipulated strain SQ110BSX showed similar characteristics with the starting E.coli strain SQ110 in terms of generation time.Strain SQ110BSX was cold sensitive, and the rRNA/protein ratio of it increased to 148% of the starting strain.These results indicate that ribosome efficiency has decreased.In view of the phylogenic distance between E.coli and B.subtilis, 16S rRNA was demonstrated to have a highly exchangeable property between these two species of different evolution status.

  10. 16S rRNA partial gene sequencing for the differentiation and molecular subtyping of Listeria species.

    Science.gov (United States)

    Hellberg, Rosalee S; Martin, Keely G; Keys, Ashley L; Haney, Christopher J; Shen, Yuelian; Smiley, R Derike

    2013-12-01

    Use of 16S rRNA partial gene sequencing within the regulatory workflow could greatly reduce the time and labor needed for confirmation and subtyping of Listeria monocytogenes. The goal of this study was to build a 16S rRNA partial gene reference library for Listeria spp. and investigate the potential for 16S rRNA molecular subtyping. A total of 86 isolates of Listeria representing L. innocua, L. seeligeri, L. welshimeri, and L. monocytogenes were obtained for use in building the custom library. Seven non-Listeria species and three additional strains of Listeria were obtained for use in exclusivity and food spiking tests. Isolates were sequenced for the partial 16S rRNA gene using the MicroSeq ID 500 Bacterial Identification Kit (Applied Biosystems). High-quality sequences were obtained for 84 of the custom library isolates and 23 unique 16S sequence types were discovered for use in molecular subtyping. All of the exclusivity strains were negative for Listeria and the three Listeria strains used in food spiking were consistently recovered and correctly identified at the species level. The spiking results also allowed for differentiation beyond the species level, as 87% of replicates for one strain and 100% of replicates for the other two strains consistently matched the same 16S type.

  11. Phylogenetic analysis of the Listeria monocytogenes based on sequencing of 16S rRNA and hlyA genes.

    Science.gov (United States)

    Soni, Dharmendra Kumar; Dubey, Suresh Kumar

    2014-12-01

    The discrimination between Listeria monocytogenes and Listeria species has been detected. The 16S rRNA and hlyA were PCR amplified with set of oligonucleotide primers with flank 1,500 and 456 bp fragments, respectively. Based on the differences in 16S rRNA and hlyA genes, a total 80 isolates from different environmental, food and clinical samples confirmed it to be L. monocytogenes. The 16S rRNA sequence similarity suggested that the isolates were similar to the previously reported ones from different habitats by others. The phylogenetic interrelationships of the genus Listeria were investigated by sequencing of 16S rRNA and hlyA gene. The 16S rRNA sequence indicated that genus Listeria is comprised of following closely related but distinct lines of descent, one is the L. monocytogenes species group (including L. innocua, L. ivanovii, L. seeligeri and L. welshimeri) and other, the species L. grayi, L. rocourtiae and L. fleischmannii. The phylogenetic tree based on hlyA gene sequence clearly differentiates between the L. monocytogenes, L. ivanovii and L. seeligeri. In the present study, we identified 80 isolates of L. monocytogenes originating from different clinical, food and environmental samples based on 16S rRNA and hlyA gene sequence similarity.

  12. Updated 16S rRNA-RFLP method for the identification of all currently characterised Arcobacter spp

    Directory of Open Access Journals (Sweden)

    Figueras María José

    2012-12-01

    Full Text Available Abstract Background Arcobacter spp. (family Campylobacteraceae are ubiquitous zoonotic bacteria that are being increasingly recognised as a threat to human health. A previously published 16S rRNA-RFLP Arcobacter spp. identification method produced specific RFLP patterns for the six species described at that time, using a single endonuclease (MseI. The number of characterised Arcobacter species has since risen to 17. The aim of the present study was to update the 16S rRNA-RFLP identification method to include all currently characterised species of Arcobacter. Results Digestion of the 16S rRNA gene with the endonuclease MseI produced clear, distinctive patterns for 10 of the 17 species, while the remaining species shared a common or very similar RFLP pattern. Subsequent digestion of the 16S rRNA gene from these species with the endonucleases MnlI and/or BfaI generated species-specific RFLP patterns. Conclusions 16S rRNA-RFLP analysis identified 17 Arcobacter spp. using either polyacrylamide or agarose gel electrophoresis. Microheterogeneities within the 16S rRNA gene, which interfered with the RFLP identification, were also documented for the first time in this genus, particularly in strains of Arcobacter cryaerophilus isolated from animal faeces and aborted foetuses.

  13. Identification of Pheretima aspergillum by CO Ⅰ and 16S rRNA with DNA Molecular Marker Methods%基于COⅠ与16S rRNA基因对广地龙的DNA分子鉴定研究

    Institute of Scientific and Technical Information of China (English)

    韦健红; 李薇; 吴文如; 喻良文

    2012-01-01

    OBJECTIVE: To establish a rapid, accurate and standardized DNA molecular marker method for the identification of Pheretima aspergillum. METHODS: The CO Ⅰ and 16S rRNA gene sequences of P. aspergillum from 5 different populations were determined using CodonCode Aligner sequence assembly. In addition, the CO Ⅰ and 16S rRNA gene sequences of P. aspergillum were downloaded from GenBank, and intraspecific and interspecific K2P genetic distance between P. aspergillum and counterfeit were calculated with MEGA 4.1 to construct NJ and MP phylogenetic tree. RESULTS: The variation sites and information sites of CO Ⅰ were higher than those of 16S rRNA. There were no insertions and deletions of CO Ⅰ , and there were 4 insertions and deletions of 16S rRNA. The interspecific genetic distances of CO Ⅰ and 16S rRNA sequences were significantly greater than intraspecific ones, and CO Ⅰ and 16S rRNA gene can be identified from the other earthworm species. CONCLUSION: The CO I and 16S rRNA gene can provide reference for the identification of the animal Chinese medicine at the molecular level, and accumulate information for DNA barcode of animal Chinese medicine.%目的:建立一种快速、准确和标准化的广地龙DNA分子标记鉴别方法.方法:测定了5个不同居群广地龙的线粒体细胞色素酶亚单位(CO)Ⅰ和16S rRNA基因序列,采用CodonCode Aligner进行序列拼接,通过下载GenBank地龙原动物的CO Ⅰ与16S rRNA序列,采用MEGA 4.1计算广地龙及其伪品地龙的种内、种间的K2P遗传距离,并基于K2P模型构建NJ和MP树.结果:COⅠ变异位点、信息位点均高于16S rRNA,CO Ⅰ基因无插入和缺失,16S rRNA存在4个插入和缺失.CO Ⅰ和16S rRNA序列种间遗传距离均明显大于种内,CO Ⅰ和16S rRNA基因均能将广地龙从其他地龙或蚯蚓物种鉴别开来.结论:获得的广地龙CO Ⅰ和16S rRNA序列可为动物性中药材地龙的分子水平鉴定提供参考,为动物性中药材DNA条

  14. 基于16S rDNA序列的Wolbachia的检测及分型%Detection and type determination of Wolbachia based on 16S rDNA sequences

    Institute of Scientific and Technical Information of China (English)

    曲哲; 丛斌; 褚栋; 董辉

    2009-01-01

    Wolbachia是广泛分布于节肢动物体内的一类共生细菌.采用16S rDNA特异片段的PCR-RFLP方法对烟粉虱Bemisia tabaci (Gennadius)不同生物型及米蛾Corcyra cephalonica (Stainton)共生菌Wolbachia进行了检测与分型分析.基于wsp基因对烟粉虱共生菌B组Wolbachia以及米蛾共生菌Wolbachia进行了系统树分析,并对相应的Wolbachia16S rDNA特异片段进行了克隆、测序以及序列比对.结果表明:16S rDNA的特异片段经NheⅠ酶切后RFLP图谱可有效检测与鉴别Wolbachia.烟粉虱共生菌Wolbachia的16S rDNA特异片段经VspⅠ酶切后可得到预期RFLP图谱,而米蛾共生菌B组Wolbachia (基于wsp序列分析为B组)则产生不同的RFLP图谱.序列分析表明,Nauru型烟粉虱体内B组Wolbachia的16S rDNA片段序列与已知B组Wolbachia对应序列(DQ278884)同源性为100%;米蛾体内B组Wolbachia 16S rDNA特异片段有碱基变异,并存在于VspⅠ识别位点内,这是导致VspⅠ酶切后RFLP图谱不同的原因.结果提示,B组Wolbachia 16S rDNA特异片段经VspⅠ酶切的RFLP图谱存在多态性.本研究结果可为今后Wolbachia的检测与分型提供借鉴.

  15. 16S rRNA-based detection of oral pathogens in coronary atherosclerotic plaque

    Directory of Open Access Journals (Sweden)

    Mahendra Jaideep

    2010-01-01

    Full Text Available Background: Atherosclerosis develops as a response of the vessel wall to injury. Chronic bacterial infections have been associated with an increased risk for atherosclerosis and coronary artery disease. The ability of oral pathogens to colonize in coronary atheromatous plaque is well known. Aim: The aim of this study was to detect the presence of Treponema denticola, Porphyromonas gingivalis and Campylobacter rectus in the subgingival and atherosclerotic plaques of patients with coronary artery disease. Materials and Methods: Fifty-one patients in the age group of 40-80 years with coronary artery disease were selected for the study. DNA was extracted from the plaque samples. The specific primers for T. denticola, C. rectus and P. gingivalis were used to amplify a part of the 16S rRNA gene by polymerase chain reaction. Statistical Analysis Used: Chi-square analysis, correlation coefficient and prevalence percentage of the microorganisms were carried out for the analysis. Results: Of the 51 patients, T. denticola, C. rectus and P. gingivalis were detected in 49.01%, 21.51% and 45.10% of the atherosclerotic plaque samples. Conclusions: Our study revealed the presence of bacterial DNA of the oral pathogenic microorganisms in coronary atherosclerotic plaques. The presence of the bacterial DNA in the coronary atherosclerotic plaques in significant proportion may suggest the possible relationship between periodontal bacterial infection and genesis of coronary atherosclerosis.

  16. Mitochondrial 16S rRNA sequence variations and phylogeny of the Chinese sisorid catfishes

    Institute of Scientific and Technical Information of China (English)

    GUO Xianguang; ZHANG Yaoguang; HE Shunping; CHEN Yiyu

    2004-01-01

    Partial sequences of mitochondrial 16S rRNA gene were obtained by PCR amplification for comparisons among nine species of glyptosternoid fishes and six species of non-glyptosternoids representing 10 sisorid genera. There are compositional biases in the A-rich unpaired regions and G-rich paired regions. A-G transitions are primarily responsible for the Ts/Tv bias in unpaired regions. The overall substitution rate in unpaired regions is almost two times higher than that in the paired regions. Saturation plots at comparable levels of sequence divergence demonstrate no saturation effects. Phylogenetic analyses using both maximum likelihood and Bayesian methods support the monophyly of Sisoridae. Chinese sisorid catfishes are composed of two major lineages, one represented by (Gagata (Bagarius, Glyptothorax)) and the other by "glyptosternoids + Pseudecheneis".The glyptosternoids may not be a monophyletic group. A previous hypothesis referring to Pseudecheneis as the sister group of monophyletic glyptosternoids, based on morphological evidence, is not supported by the molecular data.Pseudecheneis is shown to be a sister taxon of Glaridoglanis.Pareuchiloglanis might be paraphyletic with Pseudexostoma and Euchiloglanis. Our results also support the hypothesis that Pareuchiloglanis anteanalis might be considered as the synonyms of Pareuchiloglanis sinensis, and genus Euchiloglanis might have only one valid species, Euchiloglanis davidi.

  17. Algae-bacteria association inferred by 16S rDNA similarity in established microalgae cultures.

    Science.gov (United States)

    Schwenk, Dagmar; Nohynek, Liisa; Rischer, Heiko

    2014-06-01

    Forty cultivable, visually distinct bacterial cultures were isolated from four Baltic microalgal cultures Chlorella pyrenoidosa, Scenedesmus obliquus, Isochrysis sp., and Nitzschia microcephala, which have been maintained for several years in the laboratory. Bacterial isolates were characterized with respect to morphology, antibiotic susceptibility, and 16S ribosomal DNA sequence. A total of 17 unique bacterial strains, almost all belonging to one of three families, Rhodobacteraceae, Rhizobiaceae, and Erythrobacteraceae, were subsequently isolated. The majority of isolated bacteria belong to Rhodobacteraceae. Literature review revealed that close relatives of the bacteria isolated in this study are not only often found in marine environments associated with algae, but also in lakes, sediments, and soil. Some of them had been shown to interact with organisms in their surroundings. A Basic Local Alignment Search Tool study indicated that especially bacteria isolated from the Isochrysis sp. culture were highly similar to microalgae-associated bacteria. Two of those isolates, I1 and I6, belong to the Cytophaga-Flavobacterium-Bacteroides phylum, members of which are known to occur in close communities with microalgae. An UniFrac analysis revealed that the bacterial community of Isochrysis sp. significantly differs from the other three communities.

  18. Phylogenetic Relationship of Phosphate Solubilizing Bacteria according to 16S rRNA Genes

    Directory of Open Access Journals (Sweden)

    Mohammad Bagher Javadi Nobandegani

    2015-01-01

    Full Text Available Phosphate solubilizing bacteria (PSB can convert insoluble form of phosphorous to an available form. Applications of PSB as inoculants increase the phosphorus uptake by plant in the field. In this study, isolation and precise identification of PSB were carried out in Malaysian (Serdang oil palm field (University Putra Malaysia. Identification and phylogenetic analysis of 8 better isolates were carried out by 16S rRNA gene sequencing in which as a result five isolates belong to the Beta subdivision of Proteobacteria, one isolate was related to the Gama subdivision of Proteobacteria, and two isolates were related to the Firmicutes. Bacterial isolates of 6upmr, 2upmr, 19upmnr, 10upmr, and 24upmr were identified as Alcaligenes faecalis. Also, bacterial isolates of 20upmnr and 17upmnr were identified as Bacillus cereus and Vagococcus carniphilus, respectively, and bacterial isolates of 31upmr were identified as Serratia plymuthica. Molecular identification and characterization of oil palm strains as the specific phosphate solubilizer can reduce the time and cost of producing effective inoculate (biofertilizer in an oil palm field.

  19. 16S rRNA in identification and typing of Clostridium botulinum%16S rRNA在肉毒梭菌分型鉴定中的应用

    Institute of Scientific and Technical Information of China (English)

    卢卫嘉; 毛晓燕; 熊颖; 张超; 王建锋

    2011-01-01

    Objective To establish a rapid, reliable method for identifying and typing of Clostridium botulinum using 16S rDNA sequencing and phylogenetic tree construction. Methods Clostridium genome templates were extracted from 9 strains, including LCL063, 830110, LC175, LCL155, 66418, N153, 61082, ALASKA, and IWANAIs 16S rRNA genes were amplifed by PCR with the 16S rRNA specific primers, and then the PCR products were cloned to pGEM -T Easy vector and sequenced. Finally, the sequence of 16S rDNA was analyzed by Clustal and Mega program; the phylogenetic trees were constructed by Neighor joining and Maximum parsimony. Results It was found that LCL063, 830110, and LCL175 were BONT/E producing Clostridium butyricums; IWANAI and ALASKA were Clostridium botulinum type E. The results of the present method were consistent with those of the conventional method. Conclusion 16S rRNA sequencing combined with phylogenetic tree analysis is a rapid and accurate method in Clostridium botulinum identification, and the method may serve as a criterion for bacterial typing with the completion of ribosomal RNA data bank.%目的 建立一种快速、可靠的对肉毒梭菌进行分型鉴定的手段.方法 以从LCL063、830110、LC175、LCL155、66418、N153、61082、ALASKA、IWANAI共9株梭菌中提取的基因组DNA为模板,利用16S rRNA特异性引物分别进行PCR扩增并进行T-A克隆转化、测序.通过Clustal和Mega软件分析16S rDNA序列,以NJ法和MP法构建进化树,分析其种属特异性.结果 16S rRNA分型结果可判断出LCL063、830110、LCL175为产E型毒素的酪酸梭菌.IWANAI与ALASKA株为E型肉毒梭菌.与传统分型鉴定得到的结果一致.结论 16S rRNA在肉毒梭菌分型鉴定中具有快速、准确的优势,随着核糖体库的不断完善,有望成为细菌分型鉴定的标准依据.

  20. RFLP Analysis of 16S rRNA Genes of Bacterial Community in Water Sample of a Petroleum Reservoir%油藏水样细菌群落16S rRNA基因的RFLP分析

    Institute of Scientific and Technical Information of China (English)

    陈平; 李辉; 牟伯中

    2005-01-01

    通过16S rRNA基因的限制性酶切片段长度多态性分析(RFLP)方法,考察了油藏水样中细菌群落及多样性.从水样中分离纯化微生物总DNA,选择性扩增细菌16S rRNA基因,并构建16S rDNA克隆文库.337 个16S rDNA克隆片段分别用限制性内切酶HinfⅠ和HaeⅢ酶切分析,得到 74 个操作分类单元 (OTUs) ,其中数量最多的 4 个OTUs共占克隆子总数的73.6%,另外 70 个OTUs的丰度均处于较低水平,有 57 个 OTUs 仅含有 1 个克隆子.结果表明,运用RFLP方法分析16S rDNA克隆片段能够有效评估油藏水样中的细菌群落和多样性.

  1. Cloning and Sequencing of Phytoplasma 16S rRNA Gene From Infected Mulberry in China%桑黄化型萎缩病病原体16S rRNA基因的序列分析

    Institute of Scientific and Technical Information of China (English)

    夏志松; 難波成任

    2004-01-01

    用PCR法克隆了中国桑黄化型萎缩病病原植原体的16S rRNA基因,并进行了序列分析.结果表明:克隆的基因大小为1 372 bp,与日本桑萎缩病病原植原体16S rRNA的同源性高达99.85%,只存在个别碱基的突变.Blastj检索结果显示与中国桑黄化型萎缩病病原体16S rRNA的基因同源性高达99%的有来源于玉米、洋葱、土豆等50多种其它植物的植原体16S rRNA基因,表明植物萎缩病病原体16S rRNA基因具高度保守性.

  2. Application of 16S rRNA Gene Library Method in Bacterium Flora Analysis of Infected Wound%16S rRNA基因文库方法在感染标本菌群分析中的应用

    Institute of Scientific and Technical Information of China (English)

    常玉梅; 刘海燕

    2013-01-01

    目的 探讨16S rRNA基因文库方法对感染标本菌群的鉴定效果.方法 采集70例创伤患者的感染伤口脓液或渗出液标本,使用通用引物扩增标本中所有细菌的16S rRNA基因片段,构建16S rRNA基因文库,挑取阳性克隆测序,分析感染细菌的数量与种类.结果 从150个克隆子中检测7种不同酶切类型的克隆,鉴定出7类微生物,其中屎肠球菌种类最多,占88%,坚强肠球菌比例占2%,另5类序列与非可培养细菌类群的16S rRNA基因序列相似性较高,均占2%.结论 感染标本中屎肠球菌含量丰富,应用16S rRNA基因文库的方法可有效分离到感染标本中的非可培养菌.

  3. The feline oral microbiome: a provisional 16S rRNA gene based taxonomy with full-length reference sequences.

    Science.gov (United States)

    Dewhirst, Floyd E; Klein, Erin A; Bennett, Marie-Louise; Croft, Julie M; Harris, Stephen J; Marshall-Jones, Zoe V

    2015-02-25

    The human oral microbiome is known to play a significant role in human health and disease. While less well studied, the feline oral microbiome is thought to play a similarly important role. To determine roles oral bacteria play in health and disease, one first has to be able to accurately identify bacterial species present. 16S rRNA gene sequence information is widely used for molecular identification of bacteria and is also useful for establishing the taxonomy of novel species. The objective of this research was to obtain full 16S rRNA gene reference sequences for feline oral bacteria, place the sequences in species-level phylotypes, and create a curated 16S rRNA based taxonomy for common feline oral bacteria. Clone libraries were produced using "universal" and phylum-selective PCR primers and DNA from pooled subgingival plaque from healthy and periodontally diseased cats. Bacteria in subgingival samples were also cultivated to obtain isolates. Full-length 16S rDNA sequences were determined for clones and isolates that represent 171 feline oral taxa. A provisional curated taxonomy was developed based on the position of each taxon in 16S rRNA phylogenetic trees. The feline oral microbiome curated taxonomy and 16S rRNA gene reference set will allow investigators to refer to precisely defined bacterial taxa. A provisional name such as "Propionibacterium sp. feline oral taxon FOT-327" is an anchor to which clone, strain or GenBank names or accession numbers can point. Future next-generation-sequencing studies of feline oral bacteria will be able to map reads to taxonomically curated full-length 16S rRNA gene sequences.

  4. International interlaboratory study comparing single organism 16S rRNA gene sequencing data: Beyond consensus sequence comparisons.

    Science.gov (United States)

    Olson, Nathan D; Lund, Steven P; Zook, Justin M; Rojas-Cornejo, Fabiola; Beck, Brian; Foy, Carole; Huggett, Jim; Whale, Alexandra S; Sui, Zhiwei; Baoutina, Anna; Dobeson, Michael; Partis, Lina; Morrow, Jayne B

    2015-03-01

    This study presents the results from an interlaboratory sequencing study for which we developed a novel high-resolution method for comparing data from different sequencing platforms for a multi-copy, paralogous gene. The combination of PCR amplification and 16S ribosomal RNA gene (16S rRNA) sequencing has revolutionized bacteriology by enabling rapid identification, frequently without the need for culture. To assess variability between laboratories in sequencing 16S rRNA, six laboratories sequenced the gene encoding the 16S rRNA from Escherichia coli O157:H7 strain EDL933 and Listeria monocytogenes serovar 4b strain NCTC11994. Participants performed sequencing methods and protocols available in their laboratories: Sanger sequencing, Roche 454 pyrosequencing(®), or Ion Torrent PGM(®). The sequencing data were evaluated on three levels: (1) identity of biologically conserved position, (2) ratio of 16S rRNA gene copies featuring identified variants, and (3) the collection of variant combinations in a set of 16S rRNA gene copies. The same set of biologically conserved positions was identified for each sequencing method. Analytical methods using Bayesian and maximum likelihood statistics were developed to estimate variant copy ratios, which describe the ratio of nucleotides at each identified biologically variable position, as well as the likely set of variant combinations present in 16S rRNA gene copies. Our results indicate that estimated variant copy ratios at biologically variable positions were only reproducible for high throughput sequencing methods. Furthermore, the likely variant combination set was only reproducible with increased sequencing depth and longer read lengths. We also demonstrate novel methods for evaluating variable positions when comparing multi-copy gene sequence data from multiple laboratories generated using multiple sequencing technologies.

  5. 16S rRNA基因检测对新生儿败血症诊断价值的Meta分析%Meta-analysis of value of 16S rRNA gene detection in diagnosis of neonatal sepsis

    Institute of Scientific and Technical Information of China (English)

    王远明; 杜惠容; 陈恒; 张辉

    2012-01-01

    目的 16S rRNA 基因检测是诊断新生儿败血症的方法之一,但是尚无研究综合评价其诊断价值,本研究拟系统评价16S rRNA基因检测在新生儿败血症中的诊断价值.方法 在Cochrane图书馆、Medline、Embase、Science Direct、中国期刊全文数据库、万方、维普等数据库中查找利用16S rRNA基因检测诊断新生儿败血症的文献.QUADAS工具评价纳入文献的质量,Meta-Disc 1.4 软件检验异质性并根据其结果选择相应效应模型计算纳入研究的合并敏感性、特异性等指标,绘制汇总受试者工作特征(SROC)曲线,综合评价16S rRNA基因检测的诊断价值.结果共有8篇文献共计2 543例新生儿纳入本次研究,Meta分析显示16S rRNA基因检测对新生儿败血症的合并诊断价值分别为:敏感度为0.93,特异度为0.97,阳性似然比为25.12,阴性似然比为0.08,诊断比值比为323.40.SROC曲线下面积为0.99.结论 16S rRNA基因检测中对新生儿败血症具有较高的诊断价值,可作为诊断新生儿败血症重要工具.%Objective 16S Rrna gene detection is one of the methods for the diagnosis of neonatal sepsis, but there is not any study evaluating its diagnostic value comprehensively. So this study aims to evaluate the diagnostic value of 16S Rrna gene detection in neonatal sepsis comprehensively. Methods Literature on diagnosis of neonatal sepsis using the 16S Rrna gene detection was searched in Cochrane Library, Medline, Embase, Science Direct, CNKI, Wanfang, VIP and other databases. QUADAS tool was used to evaluate the quality of literature; Meta-Disc 1.4 software was used to test heterogeneity, based on which the relevant effect model was selected to calculate the combined sensitivity, specificity and other indicators of the literature, the summary receiver operating characteristic (SROC) curve was drawn and the diagnostic value of 16S Rrna gene detection was evaluated comprehensively. Results A total of 8 pieces of

  6. Identification of clinical uncommon bacteria by sequence analysis of 16S rRNA gene%基于16S rRNA序列鉴定临床不常见细菌

    Institute of Scientific and Technical Information of China (English)

    吴智刚; 李小蓝; 吴奎海

    2013-01-01

    目的 以16S rRNA基因为靶序列,建立核糖体测序方法鉴定临床不常见细菌.方法 利用通用引物聚合酶链反应(PCR)扩增细菌16S rRNA基因,对PCR产物进行测序,在GenBank中对测序结果进行Blastn分析.结果 10株临床常见细菌测序结果与表型鉴定符合,检测出4株临床不常见细菌.结论 16S rRNA基因测序可以作为鉴定临床不常见细菌的重要方法之一.

  7. Integration of spore-based genetically engineered whole-cell sensing systems into portable centrifugal microfluidic platforms.

    Science.gov (United States)

    Date, Amol; Pasini, Patrizia; Daunert, Sylvia

    2010-09-01

    Bacterial whole-cell biosensing systems provide important information about the bioavailable amount of target analytes. They are characterized by high sensitivity and specificity/selectivity along with rapid response times and amenability to miniaturization as well as high-throughput analysis. Accordingly, they have been employed in various environmental and clinical applications. The use of spore-based sensing systems offers the unique advantage of long-term preservation of the sensing cells by taking advantage of the environmental resistance and ruggedness of bacterial spores. In this work, we have incorporated spore-based whole-cell sensing systems into centrifugal compact disk (CD) microfluidic platforms in order to develop a portable sensing system, which should enable the use of these hardy sensors for fast on-field analysis of compounds of interest. For that, we have employed two spore-based sensing systems for the detection of arsenite and zinc, respectively, and evaluated their analytical performance in the miniaturized microfluidic format. Furthermore, we have tested environmental and clinical samples on the CD microfluidic platforms using the spore-based sensors. Germination of spores and quantitative response to the analyte could be obtained in 2.5-3 h, depending on the sensing system, with detection limits of 1 x 10(-7) M for arsenite and 1 x 10(-6) M for zinc in both serum and fresh water samples. Incorporation of spore-based whole-cell biosensing systems on microfluidic platforms enabled the rapid and sensitive detection of the analytes and is expected to facilitate the on-site use of such sensing systems.

  8. The Production of Biodiesel from Cottonseed Oil Using Rhizopus oryzae Whole Cell Biocatalysts

    Science.gov (United States)

    Athalye, Sneha Kishor

    Biodiesel is an environmentally friendly alternative to fossil fuels which have become increasingly expensive in recent times. An alternate approach to alkaline biodiesel production is needed as catalyst miscibility with the glycerol by-product, generation of large amounts of waste water, and saponification of the feedstock are major disadvantages associated with the process. Lipases are water soluble enzymes which act as catalysts in many lipid based reactions. Reuse of lipases can significantly reduce cost of enzymatic biodiesel production; however retention of lipolytic activity still remains a challenge. Use of microbial cells immobilized on various surfaces like sponge, foam and plastics as biocatalysts instead of extracted enzyme could help overcome this problem. A novel, rigid biomass support with high surface area made from recyclable polyethylene (Bioblok(TM)) was used in this study. Several fungal and bacterial species have been reported to possess appreciable levels of lipase activity. The biomass production and immobilization as well as lipase activity of three different species; Candida rugosa (ATCC #38772), Aspergillus oryzae (ATCC #58299), and Rhizopus oryzae (ATTC #34612) were tested. C. rugosa did not attach well to the support particles while A.oryzae had lower biomass accumulation of 6.1 g (dry cell wt)/L compared to 11.8 g (dry cell wt)/L for R.oryzae. Hence Rhizopus oryzae, fungal specie with cell surface bound lipase was selected for the current study. The study investigated the influence of media composition and growth time of the R.oryzae whole cell biocatalysts, immobilized on the BSPs, for FAME production from cottonseed oil. R.oryzae BSPs grown in basal media supplemented with 1% (w/v) of glucose or oil or both for 48 h, 72 h or 90 h were used in a 36 h transesterification reaction with cottonseed oil and methanol. BSPs grown in both glucose and oil supplemented medium for 72 h had the highest conversion of 22.4% (wt/wt) and a biomass

  9. Beyond 16S rRNA Community Profiling: Intra-Species Diversity in the Gut Microbiota

    Science.gov (United States)

    Ellegaard, Kirsten M.; Engel, Philipp

    2016-01-01

    Interactions with microbes affect many aspects of animal biology, including immune system development, nutrition and health. In vertebrates, the gut microbiota is dominated by a small subset of phyla, but the species composition within these phyla is typically not conserved. Moreover, several recent studies have shown that bacterial species in the gut are composed of a multitude of strains, which frequently co-exist in their host, and may be host-specific. However, since the study of intra-species diversity is challenging, particularly in the setting of complex, host-associated microbial communities, our current understanding of the distribution, evolution and functional relevance of intra-species diversity in the gut is scarce. In order to unravel how genomic diversity translates into phenotypic diversity, community analyses going beyond 16S rRNA profiling, in combination with experimental approaches, are needed. Recently, the honeybee has emerged as a promising model for studying gut bacterial communities, particularly in terms of strain-level diversity. Unlike most other invertebrates, the honeybee gut is colonized by a remarkably consistent and specific core microbiota, which is dominated by only eight bacterial species. As for the vertebrate gut microbiota, these species are composed of highly diverse strains suggesting that similar evolutionary forces shape gut community structures in vertebrates and social insects. In this review, we outline current knowledge on the evolution and functional relevance of strain diversity within the gut microbiota, including recent insights gained from mammals and other animals such as the honeybee. We discuss methodological approaches and propose possible future avenues for studying strain diversity in complex bacterial communities. PMID:27708630

  10. Analysis of the unexplored features of rrs (16S rDNA) of the Genus Clostridium

    Science.gov (United States)

    2011-01-01

    Background Bacterial taxonomy and phylogeny based on rrs (16S rDNA) sequencing is being vigorously pursued. In fact, it has been stated that novel biological findings are driven by comparison and integration of massive data sets. In spite of a large reservoir of rrs sequencing data of 1,237,963 entries, this analysis invariably needs supplementation with other genes. The need is to divide the genetic variability within a taxa or genus at their rrs phylogenetic boundaries and to discover those fundamental features, which will enable the bacteria to naturally fall within them. Within the large bacterial community, Clostridium represents a large genus of around 110 species of significant biotechnological and medical importance. Certain Clostridium strains produce some of the deadliest toxins, which cause heavy economic losses. We have targeted this genus because of its high genetic diversity, which does not allow accurate typing with the available molecular methods. Results Seven hundred sixty five rrs sequences (> 1200 nucleotides, nts) belonging to 110 Clostridium species were analyzed. On the basis of 404 rrs sequences belonging to 15 Clostridium species, we have developed species specific: (i) phylogenetic framework, (ii) signatures (30 nts) and (iii) in silico restriction enzyme (14 Type II REs) digestion patterns. These tools allowed: (i) species level identification of 95 Clostridium sp. which are presently classified up to genus level, (ii) identification of 84 novel Clostridium spp. and (iii) potential reduction in the number of Clostridium species represented by small populations. Conclusions This integrated approach is quite sensitive and can be easily extended as a molecular tool for diagnostic and taxonomic identification of any microbe of importance to food industries and health services. Since rapid and correct identification allows quicker diagnosis and consequently treatment as well, it is likely to lead to reduction in economic losses and mortality

  11. A core human microbiome as viewed through 16S rRNA sequence clusters.

    Directory of Open Access Journals (Sweden)

    Susan M Huse

    Full Text Available We explore the microbiota of 18 body sites in over 200 individuals using sequences amplified V1-V3 and the V3-V5 small subunit ribosomal RNA (16S hypervariable regions as part of the NIH Common Fund Human Microbiome Project. The body sites with the greatest number of core OTUs, defined as OTUs shared amongst 95% or more of the individuals, were the oral sites (saliva, tongue, cheek, gums, and throat followed by the nose, stool, and skin, while the vaginal sites had the fewest number of OTUs shared across subjects. We found that commonalities between samples based on taxonomy could sometimes belie variability at the sub-genus OTU level. This was particularly apparent in the mouth where a given genus can be present in many different oral sites, but the sub-genus OTUs show very distinct site selection, and in the vaginal sites, which are consistently dominated by the Lactobacillus genus but have distinctly different sub-genus V1-V3 OTU populations across subjects. Different body sites show approximately a ten-fold difference in estimated microbial richness, with stool samples having the highest estimated richness, followed by the mouth, throat and gums, then by the skin, nasal and vaginal sites. Richness as measured by the V1-V3 primers was consistently higher than richness measured by V3-V5. We also show that when such a large cohort is analyzed at the genus level, most subjects fit the stool "enterotype" profile, but other subjects are intermediate, blurring the distinction between the enterotypes. When analyzed at the finer-scale, OTU level, there was little or no segregation into stool enterotypes, but in the vagina distinct biotypes were apparent. Finally, we note that even OTUs present in nearly every subject, or that dominate in some samples, showed orders of magnitude variation in relative abundance emphasizing the highly variable nature across individuals.

  12. Investigating bacterial populations in styrene-degrading biofilters by 16S rDNA tag pyrosequencing.

    Science.gov (United States)

    Portune, Kevin J; Pérez, M Carmen; Álvarez-Hornos, F Javier; Gabaldón, Carmen

    2015-01-01

    Microbial biofilms are essential components in the elimination of pollutants within biofilters, yet still little is known regarding the complex relationships between microbial community structure and biodegradation function within these engineered ecosystems. To further explore this relationship, 16S rDNA tag pyrosequencing was applied to samples taken at four time points from a styrene-degrading biofilter undergoing variable operating conditions. Changes in microbial structure were observed between different stages of biofilter operation, and the level of styrene concentration was revealed to be a critical factor affecting these changes. Bacterial genera Azoarcus and Pseudomonas were among the dominant classified genera in the biofilter. Canonical correspondence analysis (CCA) and correlation analysis revealed that the genera Brevundimonas, Hydrogenophaga, and Achromobacter may play important roles in styrene degradation under increasing styrene concentrations. No significant correlations (P > 0.05) could be detected between biofilter operational/functional parameters and biodiversity measurements, although biological heterogeneity within biofilms and/or technical variability within pyrosequencing may have considerably affected these results. Percentages of selected bacterial taxonomic groups detected by fluorescence in situ hybridization (FISH) were compared to results from pyrosequencing in order to assess the effectiveness and limitations of each method for identifying each microbial taxon. Comparison of results revealed discrepancies between the two methods in the detected percentages of numerous taxonomic groups. Biases and technical limitations of both FISH and pyrosequencing, such as the binding of FISH probes to non-target microbial groups and lack of classification of sequences for defined taxonomic groups from pyrosequencing, may partially explain some differences between the two methods.

  13. Sequencing of 16S rDNA of Klebsiella: taxonomic relations within the genus and to other Enterobacteriaceae.

    Science.gov (United States)

    Boye, Kit; Hansen, Dennis S

    2003-02-01

    The 16S rDNAs of 20 strains of Klebsiella were sequenced and used for construction of a phylogenetic tree together with already published Enterobacteriaceae 16S rDNA sequences. The taxonomy within the Klebsiella genus, as reflected by the 16S rDNA tree, was in agreement with existing DNA-DNA hybridisation and numerical taxonomy data, indicating that for Klebsiella, 16S rDNA sequencing is a valid method for identification and taxonomical purposes. Five closely related clusters were found in the Klebsiella genus; Cluster I, K. oxytoca; Cluster II, K. terrigena, Cluster III, K. planticola and K. ornithinolytica; Cluster IV, Enterobacter aerogenes (K. mobilis); and Cluster V, K. pneumoniae. The position of Calymmatobacterium granulomatis within the genus and closest to K. pneumoniae was confirmed. For the species K. oxytoca, data seem to indicate a subdivision into two subspecies. In addition, a biochemically aberrant Klebsiella strain (BEC441) that was included in the analysis could not be assigned to any of the known species, but was found to be closest related to K. oxytoca. Furthermore, the high sequence similarity between the two environmental species K. planticola and K. ornithinolytica does not justify a distinction of the two species. Finally, within a 165-bp stretch of the 16S rDNA sequences, species-specific nucleotides were found.

  14. [Strategy of selecting 16S rRNA hypervariable regions for metagenome-phylogenetic marker genes based analysis].

    Science.gov (United States)

    Zhang, Jun-yi; Zhu, Bing-chuan; Xu, Chao; Ding, Xiao; Li, Jun-feng; Zhang, Xue-gong; Lu, Zu-hong

    2015-11-01

    The advent of next generation sequencing technology enables parallel analysis of the whole microbial community from multiple samples. Particularly, sequencing 16S rRNA hypervariable tags has become the most efficient and cost-effective method for assessing microbial diversity. Due to its short read length of the 2nd-generation sequencing methods that cannot cover the full 16S rRNA genomic region, specific hypervariable regions or V-regions must be selected to act as the proxy. Over the past decade, selection of V-regions has not been consistent in assessing microbial diversity. Here we evaluated the current strategies of selecting 16S rRNA hypervariable regions for surveying microbial diversity. The environmental condition was considered as one of the important factors for selection of 16S rRNA hypervariable regions. We suggested that a pilot study to test different V-regions is required in bacterial diversity studies based on 16S rRNA genes.

  15. Differentiation Between Bacillus thuringiensis and Bacillus cereus by 16S rDNA-PCR and ERIC-PCR

    Institute of Scientific and Technical Information of China (English)

    LI Haitao; LIU Dongming; GAO Jiguo

    2011-01-01

    16S rDNA and ERIC (Enterobacteia Repetitive Intergenic Consensus Sequences) based on PCR method were tested for the effectiveness of the differentiation of B. thuringiensis and B. cereus. 16S rDNA-PCR primers were designed based on the sequence difference in variable regions of B. cereus 16S rDNA and B. thuringiensis 16S rDNA, 16S rDNA-PCR showed no obvious difference between B. cereus and B. thuringiensis. The only difference was that one 1600-bp amplificon could be obtained from all the three B. Cereus strains, and none amplificon from any B. thuringiensis strains. ERIC was optimized based on previous reports. The genonlic DNA was used for the template of ER1C-PCR, and the following DNA fingerprints were analyzed by the agarose gel electrophoresis. The results showed that DNA fingerprint of three B. thuringiensis strains had a unique amplicon less than 100-bp, while DNA fingerprint of three B. cereus" strains had none. Moreover, DNA fingerprint of B. cereus showed a 700-bp amplicon, but didn't have any DNA fingerprints ofB. thuringiensis genome. Therefore, ERIC-PCR technique should be able to be used for the differentiation of B. thuringiensis and B. cereus.

  16. Effects of 16S rDNA sampling on estimates of the number of endosymbiont lineages in sucking lice

    Directory of Open Access Journals (Sweden)

    Julie M. Allen

    2016-07-01

    Full Text Available Phylogenetic trees can reveal the origins of endosymbiotic lineages of bacteria and detect patterns of co-evolution with their hosts. Although taxon sampling can greatly affect phylogenetic and co-evolutionary inference, most hypotheses of endosymbiont relationships are based on few available bacterial sequences. Here we examined how different sampling strategies of Gammaproteobacteria sequences affect estimates of the number of endosymbiont lineages in parasitic sucking lice (Insecta: Phthirapatera: Anoplura. We estimated the number of louse endosymbiont lineages using both newly obtained and previously sequenced 16S rDNA bacterial sequences and more than 42,000 16S rDNA sequences from other Gammaproteobacteria. We also performed parametric and nonparametric bootstrapping experiments to examine the effects of phylogenetic error and uncertainty on these estimates. Sampling of 16S rDNA sequences affects the estimates of endosymbiont diversity in sucking lice until we reach a threshold of genetic diversity, the size of which depends on the sampling strategy. Sampling by maximizing the diversity of 16S rDNA sequences is more efficient than randomly sampling available 16S rDNA sequences. Although simulation results validate estimates of multiple endosymbiont lineages in sucking lice, the bootstrap results suggest that the precise number of endosymbiont origins is still uncertain.

  17. Increased availability of NADH in metabolically engineered baker's yeast improves transaminase-oxidoreductase coupled asymmetric whole-cell bioconversion

    DEFF Research Database (Denmark)

    Knudsen, Jenny Dahl; Hägglöf, Cecilia; Weber, Nora

    2016-01-01

    yeast for transamination-reduction coupled asymmetric one-pot conversion was investigated. RESULTS: A series of active whole-cell biocatalysts were constructed by over-expressing the (S)-selective ω-transaminase (VAMT) from Capsicum chinense together with the NADH-dependent (S)-selective alcohol...... dehydrogenase (SADH) originating from Rhodococcus erythropolis in strains with or without deletion of glycerol-3-phosphate dehydrogenases 1 and 2 (GPD1 and GPD2). The yeast strains were evaluated as catalysts for simultaneous: (a) kinetic resolution of the racemic mixture to (R)-1-phenylethylamine, and (b...

  18. Artificial neural networks for classification in metabolomic studies of whole cells using 1H nuclear magnetic resonance.

    LENUS (Irish Health Repository)

    Brougham, D F

    2011-01-01

    We report the successful classification, by artificial neural networks (ANNs), of (1)H NMR spectroscopic data recorded on whole-cell culture samples of four different lung carcinoma cell lines, which display different drug resistance patterns. The robustness of the approach was demonstrated by its ability to classify the cell line correctly in 100% of cases, despite the demonstrated presence of operator-induced sources of variation, and irrespective of which spectra are used for training and for validation. The study demonstrates the potential of ANN for lung carcinoma classification in realistic situations.

  19. An improved enzyme-linked immunosorbent assay for whole-cell determination of methanogens in samples from anaerobic reactors

    DEFF Research Database (Denmark)

    Sørensen, A.H.; Ahring, B.K.

    1997-01-01

    An enzyme-linked immunosorbent assay was developed for the detection of whole cells of methanogens in samples from anaerobic continuously stirred tank digesters treating slurries of solid waste. The assay was found to allow for quantitative analysis of the most important groups of methanogens......-quality microtiter plates and the addition of dilute hydrochloric acid to the samples. In an experiment on different digester samples, the test demonstrated a unique pattern of different methanogenic strains present in each sample. The limited preparatory work required for the assay and the simple assay design make...

  20. Advance in application of 16S rRNA gene in bacteriology%16S rRNA基因序列分析技术在细菌分类中应用的研究进展

    Institute of Scientific and Technical Information of China (English)

    杨霞; 陈陆; 王川庆

    2008-01-01

    由于16S rRNA基因序列的保守性和存在的普遍性,应用16S rRNA作为分子指标已逐渐成为微生物检测和分类鉴定的一种强有力工具.文章就该基因的特征、研究方法、检测方法及临床应用与研究的新进展等作以简要综述,同时对存在的问题进行了探讨.

  1. 16S rDNA sequence as measure for identification of bacterial isolates in bulk%16S rDNA序列在大批量细菌分离物分子鉴定中的应用研究

    Institute of Scientific and Technical Information of China (English)

    陈源源; 沈微; 樊游; 陈献忠; Suren Singh; 石贵阳; 王正祥

    2013-01-01

    The feasibility of 16S rDNA sequence-based analysis for systemic classification of bacterial isolates was extensively evaluated. The 16S rDNA sequences of 3 726 bacterial strains collected in CICIM-CU were amplified by PCR and sequenced. The sequencing results were compared to reference data available at the GenBank database by using BLAST. Most of bacterial isolates (82.5% ) were assigned to species level successfully. 653 strains (17.5% ) were only could be assigned to genus level. Some closely-related species belonged to genus Bacillus and Geobacillus had highly similar 16S rDNA sequences, making 16S rDNA sequence analysis-based identification problematic. This study showed that 16S rDNA sequences were sufficiently sensitive for identifying most of bacterial isolates. It could be powerfully and ordinarily used as first-round molecular marker to systemic identification of bacterial isolates in bulk.%利用16S rDNA序列同源性分析法对保藏于中国高校工业微生物资源与信息中心(CICIM-CU)的3 726株细菌分离物进行了分子鉴定.实验结果显示:其中3 073株细菌分离物可被成功鉴定至种一级水平,分属于210个种,45个属,占整个细菌分离物的82.5%;其余653株细菌分离物可鉴定到属一级,占整个细菌分离物的17.5%.研究中也发现,16S rDNA序列在芽孢杆菌属(Bacillus)和土芽孢杆菌属(eobacillus)等少数菌属中的部分种间鉴定的敏感性不高.上述结果表明16S rDNA序列在大多数细菌种属鉴定中具有较高的特异性和敏感性,可以作为第一轮分子标识用于大批量细菌分离物的鉴定.

  2. Short-chain flavor ester synthesis in organic media by an E. coli whole-cell biocatalyst expressing a newly characterized heterologous lipase

    National Research Council Canada - National Science Library

    Brault, Guillaume; Shareck, François; Hurtubise, Yves; Lépine, François; Doucet, Nicolas

    2014-01-01

    ...) for overexpression using the pET16b plasmid. Using this recombinant strain as a whole-cell biocatalyst, short chain esters were efficiently synthesized by transesterification and esterification reactions in organic media...

  3. Comparative sequence analysis of 16S rRNA gene of Pasteurella multocida serogroup B isolates from different animal species.

    Science.gov (United States)

    Dey, S; Singh, V P; Kumar, A A; Sharma, B; Srivastava, S K; Singh, Nem

    2007-08-01

    The phylogenetic relationships of five isolates of Pasteurella multocida serotype B:2 belonging to buffalo, cattle, pig, sheep and goat were investigated by comparative sequence analysis of 16S rRNA gene. The 1468bp fragment of 16S rRNA gene sequence comparison showed that the isolates of cattle (PM75), pig (PM49) and sheep (PM82) shared 99.9% homology with the buffalo isolate (vaccine strain P52) whereas, the goat isolate (PM86) shared 99.8% homology with the vaccine strain. The 16S rRNA gene sequences of these isolates were also found monophyletic with type B reference strain NCTC 10323 of P. multocida subsp. multocida. The present study indicated the close relationships of haemorrhagic septicaemia causing P. multocida serotype B:2 isolates of buffalo and cattle with other uncommon hosts (pig, sheep and goat).

  4. 16S rRNA gene sequencing as a tool to study microbial populations in foods and process environments

    DEFF Research Database (Denmark)

    Buschhardt, Tasja; Hansen, Tina Beck; Bahl, Martin Iain

    2015-01-01

    and their role in food safety. During method optimization, we have identified several factors which distort the characterization of microbial populations, including DNA extraction methods, DNA polymerases, and most importantly the analyzed fragment of the 16S rRNA gene. Methods: This study investigated microbial...... reference. Results: Taxonomic assignments and abundances of sequences in the total community and in the Enterobacteriaceae subpopulation were affected by the 16S rRNA gene variable region, DNA extraction methods, and polymerases chosen. However, community compositions were very reproducible when the same......Introduction: Methodological constraints during culturing and biochemical testing have left the true microbiological diversity of foods and process environments unexplored. Culture-independent molecular methods, such as 16S rRNA gene sequencing, may provide deeper insight into microbial communities...

  5. Phylogeny of the cuttlefishes (Mollusca:Cephalopoda) based on mitochondrial COI and 16S rRNA gene sequence data

    Institute of Scientific and Technical Information of China (English)

    LIN Xiangzhi; ZHENG Xiaodong; XIAO Shu; WANG Rucai

    2004-01-01

    To clarify cuttlefish phylogeny, mitochondrial cytochrome c oxidase subunit I (COI) gene and partial 16S rRNA gene are sequenced for 13 cephalopod species. Phylogenetic trees are constructed, with the neighbor-joining method.Coleoids are divided into two main lineages, Decabrachia and Octobrachia. The monophyly of the order Sepioidea,which includes the families Sepiidae, Sepiolidae and Idiosepiidae, is not supported. From the two families of Sepioidea examined, the Sepiolidae are polyphyletic and are excluded from the order. On the basis of 16S rRNA and amino acid of COI gene sequences data, the two genera (Sepiella and Sepia) from the Sepiidae can be distinguished, but do not have a visible boundary using COI gene sequences. The reason is explained. This suggests that the 16S rDNA of cephalopods is a precious tool to analyze taxonomic relationships at the genus level, and COI gene is fitter at a higher taxonomic level (i.e., family).

  6. Effect of mutations in the A site of 16 S rRNA on aminoglycoside antibiotic-ribosome interaction

    DEFF Research Database (Denmark)

    Recht, M I; Douthwaite, S; Dahlquist, K D

    1999-01-01

    Decoding of genetic information occurs upon interaction of an mRNA codon-tRNA anticodon complex with the small subunit of the ribosome. The ribosomal decoding region is associated with highly conserved sequences near the 3' end of 16 S rRNA. The decoding process is perturbed by the aminoglycoside...... of universally conserved nucleotides at 1406 to 1408 and 1494 to 1495 in the decoding region of plasmid-encoded bacterial 16 S rRNA. Phenotypic changes range from the benign effect of U1406-->A or A1408-->G substitutions, to the highly deleterious 1406G and 1495 mutations that assemble into 30 S subunits...... but are defective in forming functional ribosomes. Changes in the local conformation of the decoding region caused by these mutations were identified by chemical probing of isolated 30 S subunits. Ribosomes containing 16 S rRNA with mutations at positions 1408, 1407+1494, or 1495 had reduced affinity...

  7. Influence of organic solvents on catalytic behaviors and cell morphology of whole-cell biocatalysts for synthesis of 5'-arabinocytosine laurate.

    Directory of Open Access Journals (Sweden)

    Meiyan Yang

    Full Text Available A whole-cell based method was developed for the regioselective synthesis of arabinocytosine laurate. Among the seven kinds of bacteria strains tested in the acylation reaction, Pseudomonas fluorescens gave the highest productivity and a higher 5'-regioselectivity than 99%. Compared with pure organic solvents, the use of organic solvent mixtures greatly promoted the yield of the whole-cell catalyzed reaction, but showed little influence on the 5'-regioselectivity. Of all the tested solvent mixtures, the best reaction result was found in isopropyl ether/pyridine followed by isopentanol/pyridine. However, the whole-cells showed much lower thermostability in isopropyl ether/pyridine than in THF-pyridine. To better understand the toxic effects of the organic solvents on P. fluorescens whole-cells and growing cells were further examined. Significant influences of organic solvents on the biomass of the cells were found, which differed depending on the type of solvents used. SEM analysis visually revealed the changes in the surface morphology of whole-cells and growing cells cultured in media containing various organic solvents, in terms of surface smoothness, bulges and changed cell sizes. Results demonstrated that organic toxicity to cell structure played an important role in whole-cell mediated catalysis.

  8. Influence of Organic Solvents on Catalytic Behaviors and Cell Morphology of Whole-Cell Biocatalysts for Synthesis of 5′-Arabinocytosine Laurate

    Science.gov (United States)

    Yang, Meiyan; Wu, Hui; Lian, Yan; Li, Xiaofeng; Lai, Furao; Zhao, Guanglei

    2014-01-01

    A whole-cell based method was developed for the regioselective synthesis of arabinocytosine laurate. Among the seven kinds of bacteria strains tested in the acylation reaction, Pseudomonas fluorescens gave the highest productivity and a higher 5′-regioselectivity than 99%. Compared with pure organic solvents, the use of organic solvent mixtures greatly promoted the yield of the whole-cell catalyzed reaction, but showed little influence on the 5′-regioselectivity. Of all the tested solvent mixtures, the best reaction result was found in isopropyl ether/pyridine followed by isopentanol/pyridine. However, the whole-cells showed much lower thermostability in isopropyl ether/pyridine than in THF-pyridine. To better understand the toxic effects of the organic solvents on P. fluorescens whole-cells and growing cells were further examined. Significant influences of organic solvents on the biomass of the cells were found, which differed depending on the type of solvents used. SEM analysis visually revealed the changes in the surface morphology of whole-cells and growing cells cultured in media containing various organic solvents, in terms of surface smoothness, bulges and changed cell sizes. Results demonstrated that organic toxicity to cell structure played an important role in whole-cell mediated catalysis. PMID:25136983

  9. Influence of organic solvents on catalytic behaviors and cell morphology of whole-cell biocatalysts for synthesis of 5'-arabinocytosine laurate.

    Science.gov (United States)

    Yang, Meiyan; Wu, Hui; Lian, Yan; Li, Xiaofeng; Lai, Furao; Zhao, Guanglei

    2014-01-01

    A whole-cell based method was developed for the regioselective synthesis of arabinocytosine laurate. Among the seven kinds of bacteria strains tested in the acylation reaction, Pseudomonas fluorescens gave the highest productivity and a higher 5'-regioselectivity than 99%. Compared with pure organic solvents, the use of organic solvent mixtures greatly promoted the yield of the whole-cell catalyzed reaction, but showed little influence on the 5'-regioselectivity. Of all the tested solvent mixtures, the best reaction result was found in isopropyl ether/pyridine followed by isopentanol/pyridine. However, the whole-cells showed much lower thermostability in isopropyl ether/pyridine than in THF-pyridine. To better understand the toxic effects of the organic solvents on P. fluorescens whole-cells and growing cells were further examined. Significant influences of organic solvents on the biomass of the cells were found, which differed depending on the type of solvents used. SEM analysis visually revealed the changes in the surface morphology of whole-cells and growing cells cultured in media containing various organic solvents, in terms of surface smoothness, bulges and changed cell sizes. Results demonstrated that organic toxicity to cell structure played an important role in whole-cell mediated catalysis.

  10. PCR Primer Design for 16S rRNAs for Experimental Horizontal Gene Transfer Test in Escherichia coli

    Science.gov (United States)

    Miyazaki, Kentaro; Sato, Mitsuharu; Tsukuda, Miyuki

    2017-01-01

    We recently demonstrated that the Escherichia coli ribosome is robust enough to accommodate foreign 16S rRNAs from diverse gamma- and betaproteobacteria bacteria (Kitahara et al., 2012). Therein, we used the common universal primers Bac8f and UN1541r to obtain a nearly full-length gene. However, we noticed that these primers overlap variable sites at 19[A/C] and 1527[U/C] in Bac8f and UN1541r, respectively, and thus, the amplicon could contain mutations. This is problematic, particularly for the former site, because the 19th nucleotide pairs with the 916th nucleotide, which is a part of the “central pseudoknot” and is critical for function. Therefore, we mutationally investigated the role of the base pair using several 16S rRNAs from gamma- and betaproteobacteria. We found that both the native base pairs (gammaproteobacterial 19A–916U and betaproteobacterial 19C–916G) and the non-native 19A–916G pair retained function, whereas the non-native 19C–916U was defective 16S rRNAs. We next designed a new primer set, Bac1f and UN1542r, so that they do not overlap the potential mismatch sites. 16S rRNA amplicons obtained from the environmental metagenome using the new primer set were dominated by proteobacterial species (~85%). Subsequent functional screening identified various 16S rRNAs from proteobacteria, all of which contained native 19A–916U or 19C–916G base pairs. The primers developed in this study are thus advantageous for functional characterization of foreign 16S rRNA in E. coli with no artifacts.

  11. Expanded versions of the 16S and 23S ribosomal RNA mutation databases (16SMDBexp and 23SMDBexp)

    OpenAIRE

    Triman, K L; Peister, A; Goel, R A

    1998-01-01

    Expanded versions of the Ribosomal RNA Mutation Databases provide lists of mutated positions in 16S and 16S-like ribosomal RNA (16SMDBexp) and 23S and 23S-like ribosomal RNA (23SMDBexp) and the identity of each alteration. Alterations from organisms other than Escherichia coli are reported at positions according to the E.coli numbering system. Information provided for each mutation includes: (i) a brief description of the phenotype(s) associated with each mutation, (ii) whether a mutant pheno...

  12. Defining reference sequences for Nocardia species by similarity and clustering analyses of 16S rRNA gene sequence data.

    Directory of Open Access Journals (Sweden)

    Manal Helal

    Full Text Available BACKGROUND: The intra- and inter-species genetic diversity of bacteria and the absence of 'reference', or the most representative, sequences of individual species present a significant challenge for sequence-based identification. The aims of this study were to determine the utility, and compare the performance of several clustering and classification algorithms to identify the species of 364 sequences of 16S rRNA gene with a defined species in GenBank, and 110 sequences of 16S rRNA gene with no defined species, all within the genus Nocardia. METHODS: A total of 364 16S rRNA gene sequences of Nocardia species were studied. In addition, 110 16S rRNA gene sequences assigned only to the Nocardia genus level at the time of submission to GenBank were used for machine learning classification experiments. Different clustering algorithms were compared with a novel algorithm or the linear mapping (LM of the distance matrix. Principal Components Analysis was used for the dimensionality reduction and visualization. RESULTS: The LM algorithm achieved the highest performance and classified the set of 364 16S rRNA sequences into 80 clusters, the majority of which (83.52% corresponded with the original species. The most representative 16S rRNA sequences for individual Nocardia species have been identified as 'centroids' in respective clusters from which the distances to all other sequences were minimized; 110 16S rRNA gene sequences with identifications recorded only at the genus level were classified using machine learning methods. Simple kNN machine learning demonstrated the highest performance and classified Nocardia species sequences with an accuracy of 92.7% and a mean frequency of 0.578. CONCLUSION: The identification of centroids of 16S rRNA gene sequence clusters using novel distance matrix clustering enables the identification of the most representative sequences for each individual species of Nocardia and allows the quantitation of inter- and intra

  13. Analysis of the genotypes among different strains of common Mycobacteria based on 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer sequences%常见分枝杆菌种内不同株之间16S rRNA基因和16S-23SrRNA ITS序列分析结果的比较

    Institute of Scientific and Technical Information of China (English)

    黄至澄; 徐黔宁; 闫李侠; 陈保文; 王国治

    2011-01-01

    目的 针对常见分枝杆菌不同株对其基因序列进行分析,比较分析结果.方法 利用16S rRNA Gene和16S-23S rRNAITS(转录间隔区序列)分析法分别对97株共7种DSMZ/ATCC引进的常见分枝杆菌进行种内不同株之间基因差异性分析,对比两种分型结果的异同.结果 16S rRNA基因可将13株草分枝杆菌分为3个型别,18株偶发分枝杆菌分为6个型别,17株耻垢分枝杆菌分为4个型别,8株戈登分枝杆菌分为3个型别,9株龟分枝杆菌龟亚种分为3个型别,15株堪萨斯分枝杆菌分为2个型别,17株产鼻疽分枝杆菌分为1个型别;而16S-23S rRNA ITS可依次将上述分枝杆菌分为3个、15个、7个、3个、4个、3个、5个型别.结论 16S rRNA G ene分析和16S-23S rRNA ITS分析均是分枝杆菌基因型分析的可靠方法,此外,16S-23SrRNA ITS的种内多态性高于16S rRNA Gene.

  14. Escherichia coli Vertebral Osteomyelitis Diagnosed According to Broad-range 16S rRNA Gene Polymerase Chain Reaction (PCR).

    Science.gov (United States)

    Shibata, Satoshi; Tanizaki, Ryutaro; Watanabe, Koji; Makabe, Kenta; Shoda, Naoki; Kutsuna, Satoshi; Nagamatsu, Maki; Oka, Shinichi; Ohmagari, Norio

    2015-01-01

    Identifying the causative agent of pyogenic osteomyelitis is often challenging, especially when antibiotics are administered before a biopsy. We herein present a case of osteomyelitis in the cervical vertebrae presenting with progressive paralytic symptoms, in which we successfully identified Escherichia coli from a biopsy specimen using broad-range 16S rRNA gene polymerase chain reaction (PCR) even though sensitive antibiotics had been used for more than 50 days before the biopsy. Broad-range 16S rRNA gene PCR is a useful diagnostic method, especially when prebiopsy antibiotics are unavoidably used for a clinically unstable state.

  15. Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome

    Institute of Scientific and Technical Information of China (English)

    Carlos; W; Nossa; William; E; Oberdorf; Jφrn; A; Aas; Bruce; J; Paster; Todd; Z; DeSantis; Eoin; L; Brodie; Daniel; Malamud; Michael; A; Poles

    2010-01-01

    AIM:To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome.METHODS:A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral,esophageal,and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species.Candidate primers evaluated were from the European rRNA database.To assess the effect of sequence length on accuracy of classifica...

  16. PCR amplification of 16S rDNA from lyophilized cell cultures facilitates studies in molecular systematics

    Science.gov (United States)

    Wisotzkey, J. D.; Jurtshuk, P. Jr; Fox, G. E.

    1990-01-01

    The sequence of the major portion of a Bacillus cycloheptanicus strain SCH(T) 16S rRNA gene is reported. This sequence suggests that B. cycloheptanicus is genetically quite distinct from traditional Bacillus strains (e.g., B. subtilis) and may be properly regarded as belonging to a different genus. The sequence was determined from DNA that was produced by direct amplification of ribosomal DNA from a lyophilized cell pellet with straightforward polymerase chain reaction (PCR) procedures. By obviating the need to revive cell cultures from the lyophile pellet, this approach facilitates rapid 16S rDNA sequencing and thereby advances studies in molecular systematics.

  17. Assessing the Fecal Microbiota: An Optimized Ion Torrent 16S rRNA Gene-Based Analysis Protocol

    Science.gov (United States)

    Foroni, Elena; Duranti, Sabrina; Turroni, Francesca; Lugli, Gabriele Andrea; Sanchez, Borja; Martín, Rebeca; Gueimonde, Miguel; van Sinderen, Douwe; Margolles, Abelardo; Ventura, Marco

    2013-01-01

    Assessing the distribution of 16S rRNA gene sequences within a biological sample represents the current state-of-the-art for determination of human gut microbiota composition. Advances in dissecting the microbial biodiversity of this ecosystem have very much been dependent on the development of novel high-throughput DNA sequencing technologies, like the Ion Torrent. However, the precise representation of this bacterial community may be affected by the protocols used for DNA extraction as well as by the PCR primers employed in the amplification reaction. Here, we describe an optimized protocol for 16S rRNA gene-based profiling of the fecal microbiota. PMID:23869230

  18. Assessing the fecal microbiota: an optimized ion torrent 16S rRNA gene-based analysis protocol.

    Directory of Open Access Journals (Sweden)

    Christian Milani

    Full Text Available Assessing the distribution of 16S rRNA gene sequences within a biological sample represents the current state-of-the-art for determination of human gut microbiota composition. Advances in dissecting the microbial biodiversity of this ecosystem have very much been dependent on the development of novel high-throughput DNA sequencing technologies, like the Ion Torrent. However, the precise representation of this bacterial community may be affected by the protocols used for DNA extraction as well as by the PCR primers employed in the amplification reaction. Here, we describe an optimized protocol for 16S rRNA gene-based profiling of the fecal microbiota.

  19. Comparative analysis of vaginal microbiota sampling using 16S rRNA gene analysis

    Science.gov (United States)

    Kalliala, Ilkka; Nieminen, Pekka; Salonen, Anne

    2017-01-01

    Background Molecular methods such as next-generation sequencing are actively being employed to characterize the vaginal microbiota in health and disease. Previous studies have focused on characterizing the biological variation in the microbiota, and less is known about how factors related to sampling contribute to the results. Our aim was to investigate the impact of a sampling device and anatomical sampling site on the quantitative and qualitative outcomes relevant for vaginal microbiota research. We sampled 10 Finnish women representing diverse clinical characteristics with flocked swabs, the Evalyn® self-sampling device, sterile plastic spatulas and a cervical brush that were used to collect samples from fornix, vaginal wall and cervix. Samples were compared on DNA and protein yield, bacterial load, and microbiota diversity and species composition based on Illumina MiSeq sequencing of the 16S rRNA gene. We quantified the relative contributions of sampling variables versus intrinsic variables in the overall microbiota variation, and evaluated the microbiota profiles using several commonly employed metrics such as alpha and beta diversity as well as abundance of major bacterial genera and species. Results The total DNA yield was strongly dependent on the sampling device and to a lesser extent on the anatomical site of sampling. The sampling strategy did not affect the protein yield or the bacterial load. All tested sampling methods produced highly comparable microbiota profiles based on MiSeq sequencing. The sampling method explained only 2% (p-value = 0.89) of the overall microbiota variation, markedly surpassed by intrinsic factors such as clinical status (microscopy for bacterial vaginosis 53%, p = 0.0001), bleeding (19%, p = 0.0001), and the variation between subjects (11%, p-value 0.0001). Conclusions The results indicate that different sampling strategies yield comparable vaginal microbiota composition and diversity. Hence, past and future vaginal

  20. Comparative analysis of vaginal microbiota sampling using 16S rRNA gene analysis.

    Science.gov (United States)

    Virtanen, Seppo; Kalliala, Ilkka; Nieminen, Pekka; Salonen, Anne

    2017-01-01

    Molecular methods such as next-generation sequencing are actively being employed to characterize the vaginal microbiota in health and disease. Previous studies have focused on characterizing the biological variation in the microbiota, and less is known about how factors related to sampling contribute to the results. Our aim was to investigate the impact of a sampling device and anatomical sampling site on the quantitative and qualitative outcomes relevant for vaginal microbiota research. We sampled 10 Finnish women representing diverse clinical characteristics with flocked swabs, the Evalyn® self-sampling device, sterile plastic spatulas and a cervical brush that were used to collect samples from fornix, vaginal wall and cervix. Samples were compared on DNA and protein yield, bacterial load, and microbiota diversity and species composition based on Illumina MiSeq sequencing of the 16S rRNA gene. We quantified the relative contributions of sampling variables versus intrinsic variables in the overall microbiota variation, and evaluated the microbiota profiles using several commonly employed metrics such as alpha and beta diversity as well as abundance of major bacterial genera and species. The total DNA yield was strongly dependent on the sampling device and to a lesser extent on the anatomical site of sampling. The sampling strategy did not affect the protein yield or the bacterial load. All tested sampling methods produced highly comparable microbiota profiles based on MiSeq sequencing. The sampling method explained only 2% (p-value = 0.89) of the overall microbiota variation, markedly surpassed by intrinsic factors such as clinical status (microscopy for bacterial vaginosis 53%, p = 0.0001), bleeding (19%, p = 0.0001), and the variation between subjects (11%, p-value 0.0001). The results indicate that different sampling strategies yield comparable vaginal microbiota composition and diversity. Hence, past and future vaginal microbiota studies employing different

  1. Production of flavor esters catalyzed by CALB-displaying Pichia pastoris whole-cells in a batch reactor.

    Science.gov (United States)

    Jin, Zi; Ntwali, Janvier; Han, Shuang-Yan; Zheng, Sui-Ping; Lin, Ying

    2012-05-31

    Candida antarctica lipase B (CALB) has been employed as an efficient catalyst in the preparation of many flavor esters. A CALB-displaying yeast whole-cell biocatalyst could be an attractive alternative to commercial immobilized CALB because of its low-cost preparation and high enzymatic activity. We investigated the potential application of CALB-displaying Pichia pastoris cells for the production of flavor esters. The optimal conditions for flavor esters synthesis by this biocatalyst were determined in 50-ml shake flasks. Under optimized conditions, the synthesis of 12 kinds flavor esters were scaled up in a 5-l batch stirred reactor. Among these, the mole conversions of 10 exceeded 95% after reactions for 4h. In addition, this biocatalyst showed good tolerance for high substrates concentration and excellent operational stability. Repeated use of the cells in 10 batches resulted in an activity loss of less than 10%. Thus, CALB-displaying P. pastoris whole cells are robust biocatalysts with potential commercial application in the large-scale production of flavor esters in non-aqueous media. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Development of a novel automated ion channel recording method using "inside-out" whole-cell membranes.

    Science.gov (United States)

    Vasilyev, Dmitry V; Merrill, Thomas L; Bowlby, Mark R

    2005-12-01

    Efforts to develop novel methods for recording from ion channels have been receiving increased attention in recent years. In this study, the authors report a unique "inside-out" whole-cell configuration of patch-clamp recording that has been developed. This method entails adding cells into a standard patch pipette and, with positive pressure, obtaining a gigaseal recording from a cell at the inside tip of the electrode. In this configuration, the cell may be moved through the air, first rupturing part of the cellular membrane and enabling bath access to the intracellular side of the membrane, and then into a series of wells containing differing solutions, enabling robotic control of all the steps in an experiment. The robotic system developed here fully automates the electrophysiological experiments, including gigaseal formation, obtaining whole-cell configuration, data acquisition, and drug application. Proof-of-principle experiments consisting of application of intracellularly acting potassium channel blockers to K+ channel cell lines resulted in a very rapid block, as well as block reversal, of the current. This technique allows compound application directly to the intracellular side of ion channels and enables the dissociation of compound in activities due to cellular barrier limitations. This technique should allow for parallel implementation of recording pipettes and the future development of larger array-based screening methods.

  3. Innovative approaches for effective selection of lipase-producing microorganisms as whole cell catalysts for biodiesel production.

    Science.gov (United States)

    Ciudad, Gustavo; Reyes, Isaac; Azócar, Laura; Briones, Reinaldo; Jorquera, Milko; Wick, Lukas Y; Navia, Rodrigo

    2011-07-01

    The high cost of commercial lipases limits their industrial application in the production of biodiesel or fatty acid methyl esters (FAME). This disadvantage has encouraged the search for lipase-producing microorganisms (LPMs) as potential whole cell catalysts for FAME production. The aim of this study, therefore, was to evaluate innovative procedures for easy selection and testing of LPMs as a low-cost whole cell catalyst, based on catalytic performance, methanol tolerance and physico-chemical cell surface properties. The latter (in particular the cell surface hydrophobicity and charge) were analyzed because of their crucial role in microbial adhesion to surfaces and the concomitant increase in cell immobilization and bioavailability of hydrophobic substrates. Biocatalysis experiments performed in the presence of nutrient, rapeseed oil and methanol were an effective tool for studying and identifying, in just two experiments, the capacity of different LPMs as biocatalysts in organic media, as well as the methanol tolerance of the cell and the lipase. This indicates the potential for using live microorganisms for FAME production. Another finding was that the inhibitory effect of methanol is more significant for lipase activity than LPM growth, indicating that the way in which alcohol is supplied to the reaction is a crucial step in FAME production by biocatalysts. According to these results, the application of these innovative assessments should simplify the search for new strains which are able to effectively catalyze the FAME production process.

  4. Selection and Characterization of Aptamers Using a Modified Whole Cell Bacterium SELEX for the Detection of Salmonella enterica Serovar Typhimurium.

    Science.gov (United States)

    Lavu, Padma Sudha Rani; Mondal, Bhairab; Ramlal, Shylaja; Murali, Harishchandra Sreepathy; Batra, Harsh Vardhan

    2016-06-13

    This study describes the selection of single-stranded DNA (ssDNA) aptamers against Salmonella enterica serovar Typhimurium using a modified whole cell systematic evolution of ligands by exponential enrichment (whole cell SELEX). For evolving specific aptamers, ten rounds of selection to live Salmonella cells, alternating with negative selection against a cocktail of related pathogens, were performed. The resulting highly enriched oligonucleotide pools were sequenced and clustered into eight groups based on primary sequence homology and predicted secondary structure similarity. Fifteen sequences from different groups were selected for further characterization. The binding affinity and specificity of aptamers were determined by fluorescence binding assays. Aptamers (SAL 28, SAL 11, and SAL 26) with dissociation constants of 195 ± 46, 184 ± 43, and 123 ± 23 nM were used to develop a nanogold-based colorimetric detection method and a sedimentation assay. The former showed a better sensitivity limit of 10(2) CFU/mL using aptamer SAL 26. This approach should enable further refinement of diagnostic methods for the detection of Salmonella enterica serovar Typhimurium and of other microbial pathogens.

  5. 16S rRNA二级结构分析在微生物分类鉴定中的应用%Application of Analysis for 16S rRNA Variable Regions of Secondary Structure in Microbiology Classification

    Institute of Scientific and Technical Information of China (English)

    赵婷; 李辉; 程池

    2011-01-01

    We summarized a new approach that uses 16S rRNA variable regions of secondary structure in the classification of microbiologym.The secondary structure of 16S rRNA variable regions of Bacillus subtilis and Bacillus lichenformis were analyzed and compared.The results showed that the pattern analysis can be used as the differentiation of some close related strains at species level.%综述了16S rRNA二级结构图形分析在微生物分类鉴定中的应用,并采用该方法比较了枯草芽孢杆菌(Bacillus subtilis)和地衣芽孢杆菌(Bacillus lichenformis)模式株16S rRNA可变区二级结构的差异,结果表明,该方法可以应用于细菌相近种间的区分。

  6. DNA sequencing reveals limited heterogeneity in the 16S rRNA gene from the rrnB operon among five Mycoplasma hominis isolates

    DEFF Research Database (Denmark)

    Mygind, T; Birkelund, Svend; Christiansen, Gunna

    1998-01-01

    To investigate the intraspecies heterogeneity within the 16S rRNA gene of Mycoplasma hominis, five isolates with diverse antigenic profiles, variable/identical P120 hypervariable domains, and different 16S rRNA gene RFLP patterns were analysed. The 16S rRNA gene from the rrnB operon was amplified...

  7. Identification of Bacillus strains isolated from Yacai by 16S rRNA gene sequencing%利用16S rRNA序列鉴定分离自芽菜中的芽孢杆菌

    Institute of Scientific and Technical Information of China (English)

    张良; 吴华昌; 邓静; 李萍萍; 肖辰; 沈芳

    2012-01-01

    从宜宾芽菜中分离优势菌群,选取4株芽孢杆菌,分别为B1、B2、B3、B4.对4株菌的16S rRNA基因经PCR扩增测序,将测序结果同该属内菌株的16S rRNA序列作多序列比较,并建立芽孢杆菌属的系统发育树.结合细菌形态学生理生化特性鉴定结果,结果表明菌株B1、B3为枯草芽孢杆菌,菌株B2为解淀粉芽孢杆菌,菌株B4为乙酰微小杆菌.%Four dominant Bacillus strains named Bl, B2, B3 and B4 were isolated from Yacai (a kind of Yibin pickles in China). The 16S rRNA genes of these strains were amplified in vitro and sequenced. Then a phylogenetic tree was constructed by multiple alignments of their sequences with other 16S rRNA gene sequences of Bacillus. According to 16S rRNA gene analysis combined with morphological, physiological and biochemical characteristics, B1 and B3 were identified as Bacillus subtilis, B2 was Bacillus amyloliquefaciens and B4 was Exiguobacterium acetylicum.

  8. Group-specific 16S rRNA-targeted oligonucleotide probes to identify thermophilic bacteria in marine hydrothermal vents

    NARCIS (Netherlands)

    Harmsen, HJM; Prieur, D; Jeanthon, C

    1997-01-01

    Four 16S rRNA-targeted oligonucleotide probes were designed for the detection of thermophilic members of the domain Bacteria known to thrive in marine hydrothermal systems, We developed and characterized probes encompassing most of the thermophilic members of the genus Bacillus, most species of the

  9. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and relevance to genomovar assignment

    Science.gov (United States)

    Flavobacterium columnare is the causative agent of columnaris disease which severely impacts channel catfish production in the USA and may be emerging as an important pathogen in the rainbow trout industry. The 16S rRNA gene is a housekeeping gene commonly used for bacterial taxonomy and genotyping...

  10. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and standard protocol for genomovar assignment

    Science.gov (United States)

    Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing this important fish pathogen. Interpretation of restriction patterns can be difficult due to th...

  11. Extensive 16S rRNA gene sequence diversity in Campylobacter hyointestinalis strains: taxonomic and applied implications

    DEFF Research Database (Denmark)

    Harrington, C.S.; On, Stephen L.W.

    1999-01-01

    Phylogenetic relationships of Campylobacter hyointestinalis subspecies were examined by means of 16S rRNA gene sequencing. Sequence similarities among C. hyointestinalis subsp. lawsonii strains exceeded 99.0 %, but values among C. hyointestinalis subsp. hyointestinalis strains ranged from 96.4...

  12. COI is better than 16S rRNA for DNA barcoding Asiatic salamanders (Amphibia: Caudata: Hynobiidae).

    Science.gov (United States)

    Xia, Yun; Gu, Hai-Feng; Peng, Rui; Chen, Qin; Zheng, Yu-Chi; Murphy, Robert W; Zeng, Xiao-Mao

    2012-01-01

    The 5' region of the mitochondrial DNA (mtDNA) gene cytochrome c oxidase I (COI) is the standard marker for DNA barcoding. However, because COI tends to be highly variable in amphibians, sequencing is often challenging. Consequently, another mtDNA gene, 16S rRNA gene, is often advocated for amphibian barcoding. Herein, we directly compare the usefulness of COI and 16S in discriminating species of hynobiid salamanders using 130 individuals. Species identification and classification of these animals, which are endemic to Asia, are often based on morphology only. Analysis of Kimura 2-parameter genetic distances (K2P) documents the mean intraspecific variation for COI and 16S rRNA genes to be 1.4% and 0.3%, respectively. Whereas COI can always identify species, sometimes 16S cannot. Intra- and interspecific genetic divergences occasionally overlap in both markers, thus reducing the value of a barcoding gap to identify genera. Regardless, COI is the better DNA barcoding marker for hynobiids. In addition to the comparison of two potential markers, high levels of intraspecific divergence in COI (>5%) suggest that both Onychodactylus fischeri and Salamandrella keyserlingii might be composites of cryptic species.

  13. Typical freshwater bacteria: an analysis of available 16S rRNA gene sequences from plankton of lakes and rivers

    NARCIS (Netherlands)

    Zwart, G.; Crump, B.C.; Kamst-van Agterveld, M.P.; Hagen, F.; Han, S.K.

    2002-01-01

    In order to identify patterns in bacterial community composition in freshwater habitats, we analyzed the available database of 16S rDNA sequences from freshwater plankton, including 24 new sequences from Parker River (Massachusetts, USA), 42 from Lake Soyang (South Korea) and 148 from Lake IJssel

  14. Campylobacter jejuni, an uncommon cause of splenic abscess diagnosed by 16S rRNA gene sequencing.

    Science.gov (United States)

    Seng, Piseth; Quenard, Fanny; Menard, Amélie; Heyries, Laurent; Stein, Andreas

    2014-12-01

    Splenic abscess is a rare disease that primarily occurs in patients with splenic trauma, endocarditis, sickle cell anemia, or other diseases that compromise the immune system. This report describes a culture-negative splenic abscess in an immunocompetent patient caused by Campylobacter jejuni, as determined by 16S rRNA gene sequencing.

  15. 16S rRNA amplicon sequencing identifies microbiota associated with oral cancer, Human Papilloma Virus infection and surgical treatment

    NARCIS (Netherlands)

    Guerrero-Preston, Rafael; Godoy-Vitorino, Filipa; Jedlicka, Anne; Rodriguez-Hilario, Arnold; Gonzalez, Herminio; Bondy, Jessica; Lawson, Fahcina; Folawiyo, Oluwasina; Michailidi, Christina; Dziedzic, Amanda; Thangavel, Rajagowthamee; Hadar, Tal; Noordhuis, Maartje G.; Westra, William; Koch, Wayne; Sidransky, David

    2016-01-01

    Systemic inflammatory events and localized disease, mediated by the microbiome, may be measured in saliva as head and neck squamous cell carcinoma (HNSCC) diagnostic and prognostic biomonitors. We used a 16S rRNA V3-V5 marker gene approach to compare the saliva microbiome in DNA isolated from Oropha

  16. A Web-Hosted R Workflow to Simplify and Automate the Analysis of 16S NGS Data

    Science.gov (United States)

    Next-Generation Sequencing (NGS) produces large data sets that include tens-of-thousands of sequence reads per sample. For analysis of bacterial diversity, 16S NGS sequences are typically analyzed in a workflow that containing best-of-breed bioinformatics packages that may levera...

  17. Unexpected Diagnosis of Cerebral Toxoplasmosis by 16S and D2 Large-Subunit Ribosomal DNA PCR and Sequencing

    DEFF Research Database (Denmark)

    Kruse, Alexandra Yasmin Collin; Kvich, Lasse Andersson; Eickhardt-Dalbøge, Steffen Robert;

    2015-01-01

    The protozoan parasite Toxoplasma gondii causes severe opportunistic infections. Here, we report an unexpected diagnosis of cerebral toxoplasmosis. T. gondii was diagnosed by 16S and D2 large-subunit (LSU) ribosomal DNA (rDNA) sequencing of a cerebral biopsy specimen and confirmed by T. gondii......-specific PCR and immunohistochemistry. The patient was later diagnosed with HIV/AIDS....

  18. TaxCollector: Modifying Current 16S rRNA Databases for the Rapid Classification at Six Taxonomic Levels

    Directory of Open Access Journals (Sweden)

    Eric W. Triplett

    2010-07-01

    Full Text Available The high level of conservation of 16S ribosomal RNA gene (16S rRNA in all Prokaryotes makes this gene an ideal tool for the rapid identification and classification of these microorganisms. Databases such as the Ribosomal Database Project II (RDP-II and the Greengenes Project offer access to sets of ribosomal RNA sequence databases useful in identification of microbes in a culture-independent analysis of microbial communities. However, these databases do not contain all of the taxonomic levels attached to the published names of the bacterial and archaeal sequences. TaxCollector is a set of scripts developed in Python language that attaches taxonomic information to all 16S rRNA sequences in the RDP-II and Greengenes databases. These modified databases are referred to as TaxCollector databases, which when used in conjunction with BLAST allow for rapid classification of sequences from any environmental or clinical source at six different taxonomic levels, from domain to species. The TaxCollector database prepared from the RDP-II database is an important component of a new 16S rRNA pipeline called PANGEA. The usefulness of TaxCollector databases is demonstrated with two very different datasets obtained using samples from a clinical setting and an agricultural soil. The six TaxCollector scripts are freely available on http://taxcollector.sourceforge.net and on http://www.microgator.org.

  19. Phylogenetic analysis of Thai oyster (Ostreidae) based on partial sequences of the mitochondrial 16S rDNA gene

    DEFF Research Database (Denmark)

    Bussarawit, Somchai; Gravlund, Peter; Glenner, Henrik;

    2006-01-01

    Ten oyster species of the family Ostreidae (Subfamilies Crassostreinae and Lophinae) from Thailand were studied using morphological data and mitochondrial 16S rDNA gene sequences. Additional sequence data from five specimens of Ostreidae and one specimen of Tridacna gigas were downloaded from Gen...

  20. A mutation in the 530 loop of Escherichia coli 16S ribosomal RNA causes resistance to streptomycin.

    Science.gov (United States)

    Melançon, P; Lemieux, C; Brakier-Gingras, L

    1988-10-25

    Oligonucleotide-directed mutagenesis was used to introduce an A to C transversion at position 523 in the 16S ribosomal RNA gene of Escherichia coli rrnB operon cloned in plasmid pKK3535. E. coli cells transformed with the mutated plasmid were resistant to streptomycin. The mutated ribosomes isolated from these cells were not stimulated by streptomycin to misread the message in a poly(U)-directed assay. They were also restrictive to the stimulation of misreading by other error-promoting related aminoglycoside antibiotics such as neomycin, kanamycin or gentamicin, which do not compete for the streptomycin binding site. The 530 loop where the mutation in the 16S rRNA is located has been mapped at the external surface of the 30S subunit, and is therefore distal from the streptomycin binding site at the subunit interface. Our results support the conclusion that the mutation at position 523 in the 16S rRNA does not interfere with the binding of streptomycin, but prevents the drug from inducing conformational changes in the 530 loop which account for its miscoding effect. Since this effect primarily results from a perturbation of the translational proofreading control, our results also provide evidence that the 530 loop of the 16S rRNA is involved in this accuracy control.

  1. ASSESSMENT OF FECAL POLLUTION SOURCES IN PLUM CREEK WATERSHED USING BACTEROIDETES 16S RDNA-BASED ASSAYS

    Science.gov (United States)

    Recently, 16S rDNA Bacteroidetes-targeted PCR assays were developed to discriminate between ruminant and human fecal pollution. These assays are rapid and relatively inexpensive but have been used in a limited number of studies. In this study, we evaluated the efficacy o...

  2. ASSESSMENT OF FECAL POLLUTION SOURCES IN PLUM CREEK WATERSHED USING PCR AND PHYLOGENETIC ANALYSES OF BACTEROIDETES 16S RDNA

    Science.gov (United States)

    Traditional methods for assessing fecal pollution in environmental systems, such as monitoring for fecal coliforms are not capable of discriminating between different sources fecal pollution. Recently, 16S rDNA Bacteroidetes-targeted PCR assays were developed to discriminate betw...

  3. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and relevance to genomovar assignment

    Science.gov (United States)

    Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing this important fish pathogen. Interpretation of restriction patterns can be difficult due to th...

  4. Micelle PCR reduces chimera formation in 16S rRNA profiling of complex microbial DNA mixtures

    NARCIS (Netherlands)

    S.A. Boers (Stefan A.); J.P. Hays (John P.); R. Jansen (Ruud)

    2015-01-01

    textabstract16S rRNA gene profiling has revolutionized the field of microbial ecology. Many researchers in various fields have embraced this technology to investigate bacterial compositions of samples derived from many different ecosystems. However, it is important to acknowledge the current

  5. Improved taxonomic assignment of human intestinal 16S rRNA sequences by a dedicated reference database

    NARCIS (Netherlands)

    Ritari, Jarmo; Salojärvi, Jarkko; Lahti, Leo; Vos, de Willem M.

    2015-01-01

    Background: Current sequencing technology enables taxonomic profiling of microbial ecosystems at high resolution and depth by using the 16S rRNA gene as a phylogenetic marker. Taxonomic assignation of newly acquired data is based on sequence comparisons with comprehensive reference databases to f

  6. 16S rRNA gene sequencing in routine identification of anaerobic bacteria isolated from blood cultures

    DEFF Research Database (Denmark)

    Justesen, Ulrik Stenz; Skov, Marianne Nielsine; Knudsen, Elisa;

    2010-01-01

    A comparison between conventional identification and 16S rRNA gene sequencing of anaerobic bacteria isolated from blood cultures in a routine setting was performed (n = 127). With sequencing, 89% were identified to the species level, versus 52% with conventional identification. The times...

  7. First report of neonatal bacteremia caused by "Haemophilus quentini" diagnosed by 16S rRNA gene sequencing, Italy.

    Science.gov (United States)

    Giufrè, Maria; Cardines, Rita; Degl'Innocenti, Roberto; Cerquetti, Marina

    2015-10-01

    We report the first case of neonatal bacteremia caused by a "Haemophilus quentini" isolate in Italy. The isolate was differentiated from H. influenzae by 16S rRNA sequencing and was characterized by comparison with the wild-type "H. quentini" CCUG 36167. Both isolates carried substitutions in penicillin-binding protein 3 but were susceptible to aminopenicillins.

  8. Direct 16S rRNA gene sequencing of polymicrobial culture-negative samples with analysis of mixed chromatograms

    DEFF Research Database (Denmark)

    Hartmeyer, Gitte N; Justesen, Ulrik S

    2010-01-01

    Two cases involving polymicrobial culture-negative samples were investigated by 16S rRNA gene sequencing, with analysis of mixed chromatograms. Fusobacterium necrophorum, Prevotella intermedia and Streptococcus constellatus were identified from pleural fluid in a patient with Lemierre's syndrome...

  9. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and standard protocol for genomovar assignment.

    Science.gov (United States)

    LaFrentz, B R; Waldbieser, G C; Welch, T J; Shoemaker, C A

    2014-07-01

    Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare, and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing of this important fish pathogen. Interpretation of restriction patterns can be difficult due to the lack of a formal description of the expected number and sizes of DNA fragments generated for each of the described genomovars. In this study, partial 16S rRNA gene sequences (ca. 1250-bp fragment) from isolates representing each described genomovar and isolates generating unique restriction patterns were cloned and sequenced. The results demonstrated that some isolates contained up to three different 16S rRNA genes whose sequences generate different RFLP patterns due to intragenomic heterogeneity within HaeIII restriction sites. The occurrence of HaeIII restriction sites within the portion of the 16S rRNA gene used for typing the F. columnare isolates and intragenomic heterogeneity within these sites explained the restriction patterns observed following RFLP analyses. This research provides a standard protocol for typing isolates of F. columnare by RFLP and a formal description of the expected restriction patterns for the previously described genomovars I, II, II-B and III. Additionally, we describe a new genomovar, I/II.

  10. Group-specific 16S rRNA-targeted oligonucleotide probes to identify thermophilic bacteria in marine hydrothermal vents

    NARCIS (Netherlands)

    Harmsen, HJM; Prieur, D; Jeanthon, C

    1997-01-01

    Four 16S rRNA-targeted oligonucleotide probes were designed for the detection of thermophilic members of the domain Bacteria known to thrive in marine hydrothermal systems, We developed and characterized probes encompassing most of the thermophilic members of the genus Bacillus, most species of the

  11. Phylogenetic relationships in Demodex mites (Acari: Demodicidae) based on mitochondrial 16S rDNA partial sequences.

    Science.gov (United States)

    Zhao, Ya-E; Wu, Li-Ping

    2012-09-01

    To confirm phylogenetic relationships in Demodex mites based on mitochondrial 16S rDNA partial sequences, mtDNA 16S partial sequences of ten isolates of three Demodex species from China were amplified, recombined, and sequenced and then analyzed with two Demodex folliculorum isolates from Spain. Lastly, genetic distance was computed, and phylogenetic tree was reconstructed. MEGA 4.0 analysis showed high sequence identity among 16S rDNA partial sequences of three Demodex species, which were 95.85 % in D. folliculorum, 98.53 % in Demodex canis, and 99.71 % in Demodex brevis. The divergence, genetic distance, and transition/transversions of the three Demodex species reached interspecies level, whereas there was no significant difference of the divergence (1.1 %), genetic distance (0.011), and transition/transversions (3/1) of the two geographic D. folliculorum isolates (Spain and China). Phylogenetic trees reveal that the three Demodex species formed three separate branches of one clade, where D. folliculorum and D. canis gathered first, and then gathered with D. brevis. The two Spain and five China D. folliculorum isolates did not form sister clades. In conclusion, 16S mtDNA are suitable for phylogenetic relationship analysis in low taxa (genus or species), but not for intraspecies determination of Demodex. The differentiation among the three Demodex species has reached interspecies level.

  12. Molecular diagnosis of Kingella kingae pericarditis by amplification and sequencing of the 16S rRNA gene.

    Science.gov (United States)

    Matta, Matta; Wermert, Delphine; Podglajen, Isabelle; Sanchez, Olivier; Buu-Hoï, Annie; Gutmann, Laurent; Meyer, Guy; Mainardi, Jean-Luc

    2007-09-01

    Kingella kingae is a fastidious gram-negative bacillus that is considered an emerging pathogen in pediatric settings but remains less common in adults. Here we describe a case of pericarditis in an immunocompetent adult host. The microorganism was identified directly from the clinical sample by molecular techniques, i.e., 16S rRNA gene amplification and sequencing.

  13. MULTIPLE ENZYME RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS FOR HIGH RESOLUTION DISTINCTION OF PSEUDOMONAS (SENSU STRICTO) 16S RRNA GENES

    Science.gov (United States)

    Pseudomonas specific 16S rDNA PCR amplification and multiple enzyme restriction fragment length polymorphism (MERFLP) analysis using a single digestion mixture of Alu I, Hinf I, Rsa I, and Tru 9I distinguished 150 published sequences and reference strains of authentic Pseudomonas...

  14. Micelle PCR reduces chimera formation in 16S rRNA profiling of complex microbial DNA mixtures

    NARCIS (Netherlands)

    S.A. Boers (Stefan A.); J.P. Hays (John P.); R. Jansen (Ruud)

    2015-01-01

    textabstract16S rRNA gene profiling has revolutionized the field of microbial ecology. Many researchers in various fields have embraced this technology to investigate bacterial compositions of samples derived from many different ecosystems. However, it is important to acknowledge the current limitat

  15. IDENTIFICATION OF ACTIVE BACTERIAL COMMUNITIES IN A MODEL DRINKING WATER BIOFILM SYSTEM USING 16S RRNA-BASED CLONE LIBRARIES

    Science.gov (United States)

    Recent phylogenetic studies have used DNA as the target molecule for the development of environmental 16S rDNA clone libraries. As DNA may persist in the environment, DNA-based libraries cannot be used to identify metabolically active bacteria in water systems. In this study, a...

  16. Improving the microbial community reconstruction at the genus level by multiple 16S rRNA regions.

    Science.gov (United States)

    Wang, Shengqin; Sun, Beili; Tu, Jing; Lu, Zuhong

    2016-06-07

    16S rRNA genes have been widely used for phylogenetic reconstruction and the quantification of microbial diversity through the application of next-generation sequencing technology. However, long-read sequencing is still costly, while short-read sequencing carries less information for complex microbial community profiling; therefore, the applications of high throughput sequencing platforms still remain challenging in microbial community reconstruction analysis. Here, we developed a method to investigate the profile of aligned 16S rRNA gene sequences and to measure the proper region for microbial community reconstruction, as a step in creating a more efficient way to detect microorganism at the genus level. Finally, we found that each genus has its own preferential genus-specific amplicons for a genus assignment, which are not always located in hyper variable regions (HVRs). It was also noted that the rare genera should contribute less than dominant ones to the common profile of the aligned 16S rRNA sequences and have lower affinity to the common universal primer. Therefore, using multiple 16S rRNA regions rather than one "universal" region can significantly improve the ability of microbial community reconstruction. In addition, we found that a short fragment is suitable for most genera identifications, and the proper conserved regions used for primer design are larger than before. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. 16S rRNA amplicon sequencing identifies microbiota associated with oral cancer, Human Papilloma Virus infection and surgical treatment

    NARCIS (Netherlands)

    Guerrero-Preston, Rafael; Godoy-Vitorino, Filipa; Jedlicka, Anne; Rodriguez-Hilario, Arnold; Gonzalez, Herminio; Bondy, Jessica; Lawson, Fahcina; Folawiyo, Oluwasina; Michailidi, Christina; Dziedzic, Amanda; Thangavel, Rajagowthamee; Hadar, Tal; Noordhuis, Maartje G.; Westra, William; Koch, Wayne; Sidransky, David

    2016-01-01

    Systemic inflammatory events and localized disease, mediated by the microbiome, may be measured in saliva as head and neck squamous cell carcinoma (HNSCC) diagnostic and prognostic biomonitors. We used a 16S rRNA V3-V5 marker gene approach to compare the saliva microbiome in DNA isolated from Oropha

  18. Unraveling the outcome of 16S rDNA-based taxonomy analysis through mock data and simulations

    NARCIS (Netherlands)

    May, A.; Abeln, S.; Crielaard, W.; Heringa, J.; Brandt, B.W.

    2014-01-01

    Motivation: 16S rDNA pyrosequencing is a powerful approach that requires extensive usage of computational methods for delineating microbial compositions. Previously, it was shown that outcomes of studies relying on this approach vastly depend on the choice of pre-processing and clustering algorithms

  19. Species identification and profiling of complex microbial communities using shotgun Illumina sequencing of 16S rRNA amplicon sequences.

    Directory of Open Access Journals (Sweden)

    Swee Hoe Ong

    Full Text Available The high throughput and cost-effectiveness afforded by short-read sequencing technologies, in principle, enable researchers to perform 16S rRNA profiling of complex microbial communities at unprecedented depth and resolution. Existing Illumina sequencing protocols are, however, limited by the fraction of the 16S rRNA gene that is interrogated and therefore limit the resolution and quality of the profiling. To address this, we present the design of a novel protocol for shotgun Illumina sequencing of the bacterial 16S rRNA gene, optimized to amplify more than 90% of sequences in the Greengenes database and with the ability to distinguish nearly twice as many species-level OTUs compared to existing protocols. Using several in silico and experimental datasets, we demonstrate that despite the presence of multiple variable and conserved regions, the resulting shotgun sequences can be used to accurately quantify the constituents of complex microbial communities. The reconstruction of a significant fraction of the 16S rRNA gene also enabled high precision (>90% in species-level identification thereby opening up potential application of this approach for clinical microbial characterization.

  20. Comparison of gull-specific assays targeting 16S rRNA gene of Catellicoccus marimammalium and Streptococcus spp.

    Science.gov (United States)

    Gulls have been implicated as a source of fecal contamination in inland and coastal waters. Only one gull-specific assay is currently available (i.e., gull2 qPCR assay). This assay is based on the 16S rRNA gene of Catellicocclls marimammalium and has showed a high level of host-s...

  1. VITCOMIC: visualization tool for taxonomic compositions of microbial communities based on 16S rRNA gene sequences

    Directory of Open Access Journals (Sweden)

    Kurokawa Ken

    2010-06-01

    Full Text Available Abstract Background Understanding the community structure of microbes is typically accomplished by sequencing 16S ribosomal RNA (16S rRNA genes. These community data can be represented by constructing a phylogenetic tree and comparing it with other samples using statistical methods. However, owing to high computational complexity, these methods are insufficient to effectively analyze the millions of sequences produced by new sequencing technologies such as pyrosequencing. Results We introduce a web tool named VITCOMIC (VIsualization tool for Taxonomic COmpositions of MIcrobial Community that can analyze millions of bacterial 16S rRNA gene sequences and calculate the overall taxonomic composition for a microbial community. The 16S rRNA gene sequences of genome-sequenced strains are used as references to identify the nearest relative of each sample sequence. With this information, VITCOMIC plots all sequences in a single figure and indicates relative evolutionary distances. Conclusions VITCOMIC yields a clear representation of the overall taxonomic composition of each sample and facilitates an intuitive understanding of differences in community structure between samples. VITCOMIC is freely available at http://mg.bio.titech.ac.jp/vitcomic/.

  2. Intra-Genomic Heterogeneity in 16S rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses.

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    Jiazhen Chen

    Full Text Available Members of the genera Prevotella, Veillonella and Fusobacterium are the predominant culturable obligate anaerobic bacteria isolated from periodontal abscesses. When determining the cumulative number of clinical anaerobic isolates from periodontal abscesses, ambiguous or overlapping signals were frequently encountered in 16S rRNA gene sequencing chromatograms, resulting in ambiguous identifications. With the exception of the genus Veillonella, the high intra-chromosomal heterogeneity of rrs genes has not been reported.The 16S rRNA genes of 138 clinical, strictly anaerobic isolates and one reference strain were directly sequenced, and the chromatograms were carefully examined. Gene cloning was performed for 22 typical isolates with doublet sequencing signals for the 16S rRNA genes, and four copies of the rrs-ITS genes of 9 Prevotella intermedia isolates were separately amplified by PCR, sequenced and compared. Five conserved housekeeping genes, hsp60, recA, dnaJ, gyrB1 and rpoB from 89 clinical isolates of Prevotella were also amplified by PCR and sequenced for identification and phylogenetic analysis along with 18 Prevotella reference strains.Heterogeneity of 16S rRNA genes was apparent in clinical, strictly anaerobic oral bacteria, particularly in the genera Prevotella and Veillonella. One hundred out of 138 anaerobic strains (72% had intragenomic nucleotide polymorphisms (SNPs in multiple locations, and 13 strains (9.4% had intragenomic insertions or deletions in the 16S rRNA gene. In the genera Prevotella and Veillonella, 75% (67/89 and 100% (19/19 of the strains had SNPs in the 16S rRNA gene, respectively. Gene cloning and separate amplifications of four copies of the rrs-ITS genes confirmed that 2 to 4 heterogeneous 16S rRNA copies existed.Sequence alignment of five housekeeping genes revealed that intra-species nucleotide similarities were very high in the genera Prevotella, ranging from 94.3-100%. However, the inter-species similarities

  3. Intra-Genomic Heterogeneity in 16S rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses

    Science.gov (United States)

    Chen, Jiazhen; Miao, Xinyu; Xu, Meng; He, Junlin; Xie, Yi; Wu, Xingwen; Chen, Gang; Yu, Liying; Zhang, Wenhong

    2015-01-01

    Background Members of the genera Prevotella, Veillonella and Fusobacterium are the predominant culturable obligate anaerobic bacteria isolated from periodontal abscesses. When determining the cumulative number of clinical anaerobic isolates from periodontal abscesses, ambiguous or overlapping signals were frequently encountered in 16S rRNA gene sequencing chromatograms, resulting in ambiguous identifications. With the exception of the genus Veillonella, the high intra-chromosomal heterogeneity of rrs genes has not been reported. Methods The 16S rRNA genes of 138 clinical, strictly anaerobic isolates and one reference strain were directly sequenced, and the chromatograms were carefully examined. Gene cloning was performed for 22 typical isolates with doublet sequencing signals for the 16S rRNA genes, and four copies of the rrs-ITS genes of 9 Prevotella intermedia isolates were separately amplified by PCR, sequenced and compared. Five conserved housekeeping genes, hsp60, recA, dnaJ, gyrB1 and rpoB from 89 clinical isolates of Prevotella were also amplified by PCR and sequenced for identification and phylogenetic analysis along with 18 Prevotella reference strains. Results Heterogeneity of 16S rRNA genes was apparent in clinical, strictly anaerobic oral bacteria, particularly in the genera Prevotella and Veillonella. One hundred out of 138 anaerobic strains (72%) had intragenomic nucleotide polymorphisms (SNPs) in multiple locations, and 13 strains (9.4%) had intragenomic insertions or deletions in the 16S rRNA gene. In the genera Prevotella and Veillonella, 75% (67/89) and 100% (19/19) of the strains had SNPs in the 16S rRNA gene, respectively. Gene cloning and separate amplifications of four copies of the rrs-ITS genes confirmed that 2 to 4 heterogeneous 16S rRNA copies existed. Conclusion Sequence alignment of five housekeeping genes revealed that intra-species nucleotide similarities were very high in the genera Prevotella, ranging from 94.3–100%. However, the

  4. 16S-23S rRNA Gene Intergenic Spacer Region Variability Helps Resolve Closely Related Sphingomonads.

    Science.gov (United States)

    Tokajian, Sima; Issa, Nahla; Salloum, Tamara; Ibrahim, Joe; Farah, Maya

    2016-01-01

    Sphingomonads comprise a physiologically versatile group many of which appear to be adapted to oligotrophic environments, but several also had features in their genomes indicative of host associations. In this study, the extent variability of the 16S-23S rDNA intergenic spacer (ITS) sequences of 14 ATCC reference sphingomonad strains and 23 isolates recovered from drinking water was investigated through PCR amplification and sequencing. Sequencing analysis of the 16S-23S rRNA gene ITS region revealed that the ITS sizes for all studied isolates varied between 415 and 849 bp, while their G+C content was 42.2-57.9 mol%. Five distinct ITS types were identified: ITS(none) (without tRNA genes), ITS(Ala(TGC)), ITS(Ala(TGC)+Ile(GAT)), ITS(Ile(GAT)+Ala(TGC)), and ITS (Ile(GAT)+Pseudo). All of the identified tRNA(Ala(TGC)) molecules consisted of 73 bases, and all of the tRNA(Ile(GAT)) molecules consisted of 74 bases. We also detected striking variability in the size of the ITS region among the various examined isolates. Highest variability was detected within the ITS-2. The importance of this study is that this is the first comparison of the 16S-23S rDNA ITS sequence similarities and tRNA genes from sphingomonads. Collectively the data obtained in this study revealed the heterogeneity and extent of variability within the ITS region compared to the 16S rRNA gene within closely related isolates. Sequence and length polymorphisms within the ITS region along with the ITS types (tRNA-containing or lacking and the type of tRNA) and ITS-2 size and sequence similarities allowed us to overcome the limitation we previously encountered in resolving closely related isolates based on the 16S rRNA gene sequence.

  5. The Identification of Discriminating Patterns from 16S rRNA Gene to Generate Signature for Bacillus Genus.

    Science.gov (United States)

    More, Ravi P; Purohit, Hemant J

    2016-08-01

    The 16S ribosomal RNA (16S rRNA) gene has been widely used for the taxonomic classification of bacteria. A molecular signature is a set of nucleotide patterns, which constitute a regular expression that is specific to each particular taxon. Our main goal was to identify discriminating nucleotide patterns in 16S rRNA gene and then to generate signatures for taxonomic classification. To demonstrate our approach, we used the phylum Firmicutes as a model using representative taxa Bacilli (class), Bacillales (order), Bacillaceae (family), and Bacillus (genus), according to their dominance at each hierarchical taxonomic level. We applied combined composite vector and multiple sequence alignment approaches to generate gene-specific signatures. Further, we mapped all the patterns into the different hypervariable regions of 16S rRNA gene and confirmed the most appropriate distinguishing region as V3-V4 for targeted taxa. We also examined the evolution in discriminating patterns of signatures across taxonomic levels. We assessed the comparative classification accuracy of signatures with other methods (i.e., RDP Classifier, KNN, and SINA). Results revealed that the signatures for taxa Bacilli, Bacillales, Bacillaceae, and Bacillus could correctly classify isolate sequences with sensitivity of 0.99, 0.97, 0.94, and 0.89, respectively, and specificity close to 0.99. We developed signature-based software DNA Barcode Identification (DNA BarID) for taxonomic classification that is available at website http://www.neeri.res.in/DNA_BarID.htm . This pattern-based study provides a deeper understanding of taxon-specific discriminating patterns in 16S rRNA gene with respect to taxonomic classification.

  6. Plastid 16S rRNA gene diversity among eukaryotic picophytoplankton sorted by flow cytometry from the South Pacific Ocean.

    Science.gov (United States)

    Shi, Xiao Li; Lepère, Cécile; Scanlan, David J; Vaulot, Daniel

    2011-04-28

    The genetic diversity of photosynthetic picoeukaryotes was investigated in the South East Pacific Ocean. Genetic libraries of the plastid 16S rRNA gene were constructed on picoeukaryote populations sorted by flow cytometry, using two different primer sets, OXY107F/OXY1313R commonly used to amplify oxygenic organisms, and PLA491F/OXY1313R, biased towards plastids of marine algae. Surprisingly, the two sets revealed quite different photosynthetic picoeukaryote diversity patterns, which were moreover different from what we previously reported using the 18S rRNA nuclear gene as a marker. The first 16S primer set revealed many sequences related to Pelagophyceae and Dictyochophyceae, the second 16S primer set was heavily biased toward Prymnesiophyceae, while 18S sequences were dominated by Prasinophyceae, Chrysophyceae and Haptophyta. Primer mismatches with major algal lineages is probably one reason behind this discrepancy. However, other reasons, such as DNA accessibility or gene copy numbers, may be also critical. Based on plastid 16S rRNA gene sequences, the structure of photosynthetic picoeukaryotes varied along the BIOSOPE transect vertically and horizontally. In oligotrophic regions, Pelagophyceae, Chrysophyceae, and Prymnesiophyceae dominated. Pelagophyceae were prevalent at the DCM depth and Chrysophyceae at the surface. In mesotrophic regions Pelagophyceae were still important but Chlorophyta contribution increased. Phylogenetic analysis revealed a new clade of Prasinophyceae (clade 16S-IX), which seems to be restricted to hyper-oligotrophic stations. Our data suggest that a single gene marker, even as widely used as 18S rRNA, provides a biased view of eukaryotic communities and that the use of several markers is necessary to obtain a complete image.

  7. Plastid 16S rRNA gene diversity among eukaryotic picophytoplankton sorted by flow cytometry from the South Pacific Ocean.

    Directory of Open Access Journals (Sweden)

    Xiao Li Shi

    Full Text Available The genetic diversity of photosynthetic picoeukaryotes was investigated in the South East Pacific Ocean. Genetic libraries of the plastid 16S rRNA gene were constructed on picoeukaryote populations sorted by flow cytometry, using two different primer sets, OXY107F/OXY1313R commonly used to amplify oxygenic organisms, and PLA491F/OXY1313R, biased towards plastids of marine algae. Surprisingly, the two sets revealed quite different photosynthetic picoeukaryote diversity patterns, which were moreover different from what we previously reported using the 18S rRNA nuclear gene as a marker. The first 16S primer set revealed many sequences related to Pelagophyceae and Dictyochophyceae, the second 16S primer set was heavily biased toward Prymnesiophyceae, while 18S sequences were dominated by Prasinophyceae, Chrysophyceae and Haptophyta. Primer mismatches with major algal lineages is probably one reason behind this discrepancy. However, other reasons, such as DNA accessibility or gene copy numbers, may be also critical. Based on plastid 16S rRNA gene sequences, the structure of photosynthetic picoeukaryotes varied along the BIOSOPE transect vertically and horizontally. In oligotrophic regions, Pelagophyceae, Chrysophyceae, and Prymnesiophyceae dominated. Pelagophyceae were prevalent at the DCM depth and Chrysophyceae at the surface. In mesotrophic regions Pelagophyceae were still important but Chlorophyta contribution increased. Phylogenetic analysis revealed a new clade of Prasinophyceae (clade 16S-IX, which seems to be restricted to hyper-oligotrophic stations. Our data suggest that a single gene marker, even as widely used as 18S rRNA, provides a biased view of eukaryotic communities and that the use of several markers is necessary to obtain a complete image.

  8. Design of a molecular method for subspecies specific identification of Klebsiella pneumoniae by using the 16S ribosomal subunit gene

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    Nelson Enrique Arenas

    2009-12-01

    Full Text Available Introduction: Rhinoscleroma is caused by Klebsiella pneumoniae subsp. rhinoscleromatis and the ozena infections caused by K. pneumoniae subsp. ozaenae, both infections affect the upper respiratory tract. In the first clinical phases the symptoms are unspecific, and the disease can be misdiagnosed as a common cold, therefore antimicrobial therapy cannot reach effective results and patients must be following up for several years since the infection became chronic. Objective: To identify Klebsiella subspecies using a specific assay based on amplicons restriction of a gene which encodes 16S subunit ribosomal (rDNA16S. Methodology: Specific restriction patterns were generated; using reported sequences from rDNA16S gene and bioinformatics programs MACAW, PFE, GENEDOC and GENE RUNNER. Amplification and restriction assays were standardized. Results: Predictions in silico allowed us to propose an algorithm for Klebsiella species and subspecies identification. Two reference strains were included and two clinical isolates which were biotyped and identified by the proposed method. rDNA16S gene restriction patterns showed differences regarding the initially identified species for conventional methods. Additionally two patterns of bands were observed for K. pneumoniae subsp. rhinoscleromatis, indicating the polymorphisms presence in the rDNA16S gene. Conclusions: We confirmed the difficulty to identify K. pneumoniae subspecies by conventional methods. Implementation of this technique could allow accurate and rapid differentiation among K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis the aetiological agents of two frequently misdiagnosed infections. Antimicrobial therapy usually could be ineffective, especially in chronic patients. Finally we consider very important to enlarge the study by using more clinical and reference strains.

  9. Design of a molecular method for subspecies specific identification of Klebsiella pneumoniae by using the 16S ribosomal subunit gene.

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    Nelson Enrique Arenas

    2009-12-01

    Full Text Available Introduction: Rhinoscleroma is caused by Klebsiella pneumoniae rhinoscleromatis and the ozena infections caused by K. pneumoniae ozaenae, both infections affect the upper respiratory tract. In the first clinical phases the symptoms are unspecific, and the disease can be misdiagnosed as a common cold, therefore antimicrobial therapy cannot reach effective results and patients must be following up for several years since the infection became chronic. Objective: To identify Klebsiella subspecies using a specific assay based on amplicons restriction of a gene which encodes 16S subunit ribosomal (rDNA16S.Methodology: Specific restriction patterns were generated; using reported sequences from rDNA16S gene and bioinformatics programs MACAW, PFE, GENEDOC and GENE RUNNER. Amplification and restriction assays were standardized. Results: Predictions in silico allowed to propose an algorithm for Klebsiella species and subspecies identification. Two reference strains were included and two clinical isolates which were biotyped and identified by the proposed method. rDNA16S gene restriction patterns showed differences regarding the initially identified species for conventional methods. Additionally two patterns of bands were observed for K. pneumoniae rhinoscleromatis, indicating the polymorphisms presence in the rDNA16S gene. Conclusions: It was confirmed the difficulty to identify K. pneumoniae subspecies by conventional methods. Implementation of this technique could allow an accurate and rapid differentiation among K. pneumoniae ozaenae and K. pneumoniae rhinoscleromatis aetiological agents of two frequently misdiagnosed infections. Antimicrobial therapy usually could be ineffective, especially in chronic patients. Finally it is considered very important to enlarge the study by using more clinical and reference strains.

  10. 16S rRNA terminal restriction fragment length polymorphism for the characterization of the nasopharyngeal microbiota.

    Directory of Open Access Journals (Sweden)

    Silvio D Brugger

    Full Text Available A novel non-culture based 16S rRNA Terminal Restriction Fragment Length Polymorphism (T-RFLP method using the restriction enzymes Tsp509I and Hpy166II was developed for the characterization of the nasopharyngeal microbiota and validated using recently published 454 pyrosequencing data. 16S rRNA gene T-RFLP for 153 clinical nasopharyngeal samples from infants with acute otitis media (AOM revealed 5 Tsp509I and 6 Hpy166II terminal fragments (TFs with a prevalence of >10%. Cloning and sequencing identified all TFs with a prevalence >6% allowing a sufficient description of bacterial community changes for the most important bacterial taxa. The conjugated 7-valent pneumococcal polysaccharide vaccine (PCV-7 and prior antibiotic exposure had significant effects on the bacterial composition in an additive main effects and multiplicative interaction model (AMMI in concordance with the 16S rRNA 454 pyrosequencing data. In addition, the presented T-RFLP method is able to discriminate S. pneumoniae from other members of the Mitis group of streptococci, which therefore allows the identification of one of the most important human respiratory tract pathogens. This is usually not achieved by current high throughput sequencing protocols. In conclusion, the presented 16S rRNA gene T-RFLP method is a highly robust, easy to handle and a cheap alternative to the computationally demanding next-generation sequencing analysis. In case a lot of nasopharyngeal samples have to be characterized, it is suggested to first perform 16S rRNA T-RFLP and only use next generation sequencing if the T-RFLP nasopharyngeal patterns differ or show unknown TFs.

  11. Thinking beside the box: Should we care about the non-coding strand of the 16S rRNA gene?

    Science.gov (United States)

    Garcia-Mazcorro, Jose F; Barcenas-Walls, Jose R

    2016-08-01

    The 16S rRNA gene (16S rDNA) codes for RNA that plays a fundamental role during translation in the ribosome and is used extensively as a marker gene to establish relationships among bacteria. However, the complementary non-coding 16S rDNA (nc16S rDNA) has been ignored. An idea emerged in the course of analyzing bacterial 16S rDNA sequences in search for nucleotide composition and substitution patterns: Does the nc16S rDNA code? If so, what does it code for? More importantly: Does 16S rDNA evolution reflect its own evolution or the evolution of its counterpart nc16S rDNA? The objective of this minireview is to discuss these thoughts. nc strands often encode small RNAs (sRNAs), ancient components of gene regulation. nc16S rDNA sequences from different bacterial groups were used to search for possible matches in the Bacterial Small Regulatory RNA Database. Intriguingly, the sequence of one published sRNA obtained from Legionella pneumophila (GenBank: AE0173541) showed high non-random similarity with nc16S rDNA corresponding in part to the V5 region especially from Legionella and relatives. While the target(s) of this sRNA is unclear at the moment, its mere existence might open up a new chapter in the use of the 16S rDNA to study relationships among bacteria.

  12. The use of 16s rDNA methods in soil microbial ecology Uso de métodos 16S rDNA em ecologia microbiana do solo

    Directory of Open Access Journals (Sweden)

    Andrew Macrae

    2000-06-01

    Full Text Available New and exciting molecular methods, many using the 16S small sub-unit ribosomal nucleic acid molecule, are opening the microbial "black box" in soil. These studies have added much to our knowledge of microbial diversity in soils, and are beginning to advance our understanding of the relationship between this diversity and its function in soil processes. Over the next few years, the knowledge gained from molecular studies will, we hope, lead to improvements in sustainable land management and sustainable exploitation of soil genetic resources. As we enter the third millenium, it is appropriate to review the application of 16S rDNA methods to soil microbiology. This review examines 16S ribosomal DNA (rDNA methods and their application to soil. It mentions their limits and suggests how they may be applied in the future.Novas e excitantes técnicas moleculares muitas usando a fração 16S da subunidade menor da molécula de ácido nucleico ribossomal, estão abrindo a "caixa-preta" da microbiologia do solo. Esses estudos têm acrescentado muito ao nosso conhecimento acerca da diversidade microbiana no solo, e começam a avançar nosso entendimento sobre a relação entre essa diversidade a sua função nos processos no solo. Ao longo dos próximos anos, o conhecimento obtido a partir de técnicas moleculares irão, esperamos, levar a melhoramentos do manejo de áreas sustentáveis da exploração dos recursos genéticos do solo. Com a chegada do terceiro milênio, é apropriado revermos a aplicação das técnicas da fração 16S do rDNA em microbiologia de solo. Esta revisão examina aplicações das técnicas da fração 16S do DNA (RNA no solo, menciona seus limites e sugere como elas poderão ser usadas no futuro.

  13. 机采血小板细菌16s rRNA基因检测的临床研究%Clinical study of bacterial 16s rRNA gene detection in apheresis platelets

    Institute of Scientific and Technical Information of China (English)

    周火根; 池妤

    2009-01-01

    目的 探讨快速、可靠的检测机采血小板(PLT)细菌污染的新方法.方法 用聚合酶链反应(PCR)扩增1 308份献血员机采PLT 16s rRNA基因,以乙型肝炎病毒-脱氧核糖核酸(HBV DNA)、白假丝酵母菌和人类基因组DNA为对照,检测该方法的特异性;采用10倍倍比稀释法进行该方法的敏感性检测.结果 检测1308份献血员机采PLT,其中7份16s rRNA基因为阳性,阳性率为0.54%(7/1 308).而与HBV DNA、白假丝酵母菌和人基因组DNA无交叉反应;PCR最低能检测1.5 × 10~4cfu/L大肠埃希菌.结论 献血员机采PLT板细菌污染的阳性率为0.54%;利用PCR检测献血员机采PLT细菌16s rRNA基因的方法具有特异性、快速性和敏感性高等特点.%Objective To explore a rapid and reliable method for the detection of bacterial contamination in the apheresis platelets. Methods The fragment of bacterial 16s rRNA gene,taken from apheresis platelets of 1 308 blood donors, was amplified by polymerase chain reaction(PCR). Using HBV DNA, Candida albicans and human genome DNA as controls,the specificity was tested. The sensitivity test was performed by the method of 10 gradual dilution. Results 7 of 1 308 alpheresis platelets samples showed 16s rRNA gene positive and the bacterial contamination ratio of apheresis platelets was 0.54%. The cross-reaction with the HBV DNA, Candida albicans and human genome DNA was negative. PCR exhibited sensitivity as low as 1.5 × 10_4 cfu/L Escherichia coli. Conclusions The bacterial detection method of 16s rRNA gene offers the adventages of high specificity, sensitivity and rapidity.

  14. Pseudomonas fluorescens HK44: Lessons Learned from a Model Whole-Cell Bioreporter with a Broad Application History

    Directory of Open Access Journals (Sweden)

    Gary S. Sayler

    2012-02-01

    Full Text Available Initially described in 1990, Pseudomonas fluorescens HK44 served as the first whole-cell bioreporter genetically endowed with a bioluminescent (luxCDABE phenotype directly linked to a catabolic (naphthalene degradative pathway. HK44 was the first genetically engineered microorganism to be released in the field to monitor bioremediation potential. Subsequent to that release, strain HK44 had been introduced into other solids (soils, sands, liquid (water, wastewater, and volatile environments. In these matrices, it has functioned as one of the best characterized chemically-responsive environmental bioreporters and as a model organism for understanding bacterial colonization and transport, cell immobilization strategies, and the kinetics of cellular bioluminescent emission. This review summarizes the characteristics of P. fluorescens HK44 and the extensive range of its applications with special focus on the monitoring of bioremediation processes and biosensing of environmental pollution.

  15. Pseudomonas fluorescens HK44: Lessons Learned from a Model Whole-Cell Bioreporter with a Broad Application History

    Science.gov (United States)

    Trögl, Josef; Chauhan, Archana; Ripp, Steven; Layton, Alice C.; Kuncová, Gabriela; Sayler, Gary S.

    2012-01-01

    Initially described in 1990, Pseudomonas fluorescens HK44 served as the first whole-cell bioreporter genetically endowed with a bioluminescent (luxCDABE) phenotype directly linked to a catabolic (naphthalene degradative) pathway. HK44 was the first genetically engineered microorganism to be released in the field to monitor bioremediation potential. Subsequent to that release, strain HK44 had been introduced into other solids (soils, sands), liquid (water, wastewater), and volatile environments. In these matrices, it has functioned as one of the best characterized chemically-responsive environmental bioreporters and as a model organism for understanding bacterial colonization and transport, cell immobilization strategies, and the kinetics of cellular bioluminescent emission. This review summarizes the characteristics of P. fluorescens HK44 and the extensive range of its applications with special focus on the monitoring of bioremediation processes and biosensing of environmental pollution. PMID:22438725

  16. Comparison of immune responses to a killed bivalent whole cell oral cholera vaccine between endemic and less endemic settings.

    Science.gov (United States)

    Desai, Sachin N; Akalu, Zenebe; Teferi, Mekonnen; Manna, Byomkesh; Teshome, Samuel; Park, Ju Yeon; Yang, Jae Seung; Kim, Deok Ryun; Kanungo, Suman; Digilio, Laura

    2016-02-01

    Studies on safety, immunogenicity and efficacy of the killed, bivalent whole cell oral cholera vaccine (Shanchol) have been conducted in historically endemic settings of Asia. Recent cholera vaccination campaigns in Haiti and Guinea have also demonstrated favourable immunogenicity and effectiveness in nonendemic outbreak settings. We performed a secondary analysis, comparing immune responses of Shanchol from two randomised controlled trials performed in an endemic and a less endemic area (Addis Ababa) during a nonoutbreak setting. While Shanchol may offer some degree of immediate protection in primed populations living in cholera endemic areas, as well as being highly immunogenic in less endemic settings, understanding the characteristics of immune responses in each of these areas is vital in determining ideal dosing strategies that offer the greatest public health impact to populations from areas with varying degrees of cholera endemicity. © 2015 John Wiley & Sons Ltd.

  17. Typing of Typhoidal Salmonella Using Extraction of Water Soluble Whole Cell Proteins and Analysing by SDS-PAGE

    Directory of Open Access Journals (Sweden)

    R. Yousefi Mashouf

    2005-10-01

    Full Text Available Introduction & Objective : Salmonella is one of the most important genus of Enterobacteriacea family. The aim of this study was typing of typhoidal Salmonella by SDS-PAGE and comparing the results with those of serotyping method.Materials and Methods: In this study, 4 reference strains of Salmonella species, 5 reference strains of Enterobacteriacea family and 100 clinical isolates of Salmonella that were previously collected from laboratories of Hamadan medical centers were studied. Serotyping of strains were performed by Biomereux and Difco monovalent antisera. Whole-cell proteins of strains were also separated on 10% poly acrylamide gel. Gels were stained by Coomassie Brilliant Blue and analyzed by densitometry. Results: Of 100 cases of Salmonella species, 43 cases (43% were S. typhi, 20 cases (20% were S. typhymurium, 12 cases (12% were S. para typhi B, 10 cases (10% were S. para typhi C, S. para typhi A 1 case (1% and other cases were non-typhoidal Salmonella. The results of serotyping were compared with the results obtained by SDS-PAGE. Many protein bands from 220 KDa to 18.5 KDa were detected by SDS-PAGE and they were used to differentiate the strains. S. typhi serotypes were divided into 5 sub-species and S. para typhi B and C were divided each into 3 sub-species. Protein profiles of the reference strains of Salmonella were compared with protein profiles of Enterobacteriaceae species and showed some differences in major protein bands, however, they had a very similar protein band in 43 KDa area. Conclusion: Since our data was able to divide Salmonella species to sub-types and differentiate them from Enterobacteriacea species, we concluded that analsying SDS-PAGE profile of water soluble whole-cell proteins can be used for typing of these organisms and it is comparble with serotyping, nevertheless, further researches are needed to establish SDS-PAGE method and to replace it with serotyping method.

  18. 病犬大肠杆菌16S rRNA甲基化酶基因检测%Molecular Detection of 16S rRNA Methylase Genes Among Escherichia coli Strains Isolated from Diseased Dogs

    Institute of Scientific and Technical Information of China (English)

    陈玉霞; 李德喜; 杜向党; 潘玉善; 刘建华; 苑丽; 张素梅

    2011-01-01

    为了解郑州市发病犬大肠杆菌对氨基糖苷类药物高度耐药的16S rRNA甲基化酶流行情况,主要研究测定了分离自河南郑州市宠物医院发病犬的123株大肠杆菌对氨基糖苷类代表药物阿米卡星的敏感性;分别设计6种16S rRNA甲基化酶基因特异性引物,对耐药分离株进行16S rRNA甲基化酶基因PCR扩增检测.检测结果显示,在发病犬大肠杆菌中仅检测到armA和rmtB,其检出率分别为3.25%(4/123)和38.2%(47/123).其中,有3.25%(4/123)的大肠杆菌可同时检测到armA和rmtB.这些结果提示,此地区发病犬大肠杆菌16S rRNA甲基化酶基因以rmtB为主.病犬细菌一旦携带这些耐药基因,可导致对氨基糖苷类药物高度耐药,应引起重视.%In order to investigate the epidemiology of 16S rRNA methylases which mediated the high level resistance to aminoglycosides among Escherichia coli strains isolated from diseased dogs in zhengzhou city of He' nan Province. 123 E. coli strains were isolated from clinic samples in diseased dogs. Antibacterial susceptibility determinations were performed and six types of 16S rRNA methylase genes were detected by PCR. Of six types of 16S rRNA methylase genes, only armA and rmtB were detected and the positive rates among these E. coli strains were 3.25% (4/123)and 38.2% (47/123), respectively. The concurrence rate of armA and rmtB genes in E. coli strains in diseased dogs was 3.25%. These results suggested 16S rRNA methylases among E. coli strains isolated from diseased dogs were prevalent in Zhengzhou City of He' nan Province, with rmtB dominant. One the pathogens in dogs carried these resistant genes, which could result in the high level resistance to aminoglycosides. So, this serious situation should cause concern.

  19. The study of 16S rRNA methylase genes in Enterobacter cloacae%庆大霉素耐药阴沟肠杆菌16S rRNA甲基化酶基因的研究

    Institute of Scientific and Technical Information of China (English)

    李玉珍; 吴燕峰; 李红玉; 杨银梅

    2011-01-01

    目的 了解庆大霉素耐药阴沟肠杆菌中16S rRNA甲基化酶基因的分布情况.方法 筛选临床分离的庆大霉素耐药的阴沟肠杆菌30株.采用聚合酶链反应(PCR)方法扩增16S rRNA甲基化酶五种相关耐药基因armA、rmtA、rmtB、rmtC和rmtD,PCR产物进行电泳分析和测序,抗菌药物的药物敏感性试验采用纸片扩散法进行,Whonet 5.4软件进行数据分析.结果 30株庆大霉素耐药阴沟肠杆菌中16S rRNA甲基化酶基因的检出率为56.7%(17/30),其中30.0%(9/30)检出armA基因、26.7%(8/30)检出rmtB基因,未检出rmtA、rmtC和rmtD基因.30株庆大霉素耐药阴沟肠杆菌对妥布霉素、阿米卡星耐药率分别为93.3%,83.3%;对其他抗菌药物除亚胺培南较敏感外,其余抗菌药物耐药率大于60.0%.结论 庆大霉素耐药阴沟肠杆菌中16S rRNA甲基化酶基因主要是armA和rmtB基因,碳青霉烯类对该菌有较好体外抗菌活性.%Objective To investigate the distribution of 16S rRNA methylase genes in gentamicin-resistant Enterobacter cloacae.Method 30 strains of clinically isolated Enterobacter cloacae were collected.Five 16S rRNA methylase genes including armA, rmtA, rmtB, rmtC and rmtD, were detected by polymerase chain reaction (PCR).The PCR products were confirmed by electrophoresis analysis and sequencing.The Antimicrobial Susceptibility Testing was conducted by disk diffusion method.The data were analyzed by Whonet 5.4 software.Result The relevance ratio of 16S rRNA methylase genes were 56.7% (17/30) including armA genes 30.0% (9/30) and rmtB genes 26.7% (8/30).None of the isolates carried rmtA, rmtC and rmtD genes.The resistence rate of Enterobacter cloacae to amikacin and tobramycin were 93.3% and 83.3% respectively.While being sensitive to imipenem, the strains were resistant to many other antimicrobial drugs with the resistance rate over 60.0%.Conclusion 16S rRNA methylase genes in Enterobacter cloacae are mainly armA and rmtB genes

  20. Identification of Lactic Acid Bacteria Ⅱ 32 by Sequence Analysis of 16S rDNA and RecA - gene%16S rDNA和recA-gene对乳酸菌Ⅱ32的鉴定

    Institute of Scientific and Technical Information of China (English)

    刘长建; 权春善; 范圣第

    2007-01-01

    对乳酸菌Ⅱ32进行了生化实验.以菌株Ⅱ32的总DNA为模板,采用细菌通用的引物,对其16S rDNA进行特异扩增,并进行序列测定,将测定结果与GenBank DNA数据库中已知菌种的16S rDNA序列通过BLAST软件进行分析比较,初步确定该菌株为戊糖乳酸菌、植物乳杆菌或类植物乳杆菌.采用recA-gene约300bp的特异扩增片段最终确定乳酸菌Ⅱ32为类植物乳杆菌.

  1. 16S rRNA甲基化酶在产KPC酶肺炎克雷伯菌中的分布%The distribution of 16S rRNA methylase genes in KPC-producing Klebsiella pneumoniae strains

    Institute of Scientific and Technical Information of China (English)

    陆理英; 张伟丽; 杨青; 周华; 俞云松

    2009-01-01

    目的 了解产肺炎克雷伯菌碳青霉烯酶-2型(KPC-2)肺炎克雷伯菌中16S rRNA甲基化酶基因的分布.方法 收集37株产KPC-2肺炎克雷伯菌,使用琼脂稀释法测定其对阿米卡星、庆大霉素和奈替米星的最小抑菌浓度(MIC),PCR扩增6种16S rRNA甲基化酶基因:armA、rmtA、rmtB、rmtC、rmtD和npmA.结果 产KPC-2肺炎克雷伯菌对阿米卡星、庆大霉素和奈替米星的耐药率均为97.3%(MIC50≥1024μg/mL),其中8株检出arms基因,25株检出rmtB基因,同时检出armA和rmtB基因的有4株,未检测到rmtA、rmtC、rmtD和npmA阳性菌株.16S rRNA甲基化酶基因总阳性率为78.4%(29/37).结论 16S rRNA甲基化酶基因armA和rmtB在产KPC-2肺炎克雷伯菌中广泛分布.%Objective To investigate the distribution of 16S rRNA methylase genes in Klebsiella pneumoniae strains producing Klebsiella pneumoniae ealbapenenase type 2(KPC-2).Methods A total of 37 Klebsiella pneumoniae isolates producing KPC-2 were collected.The minimal inhibitory concentrations (MICs)of these strains to amikacin,gentamyein and netilmicin were determinated by agal dilution method.Six 16S rRNA methylase genes(armA,rmtA,rmtB,rmtC,rmtD and npmA)were detected by PCR.Results The resistant rates to amikacin,gentamycin and netilmicin were 97.3%(MIC50≥1024μg/mL).Among those resistant strains,8 were armr/A positive,25 were rmtB positive,4 were both armA and rmtB positive.and no other 16S rRNA methylase genes were found.The total positive rate of 16S rRNA methylase genes was 78.4%(29/37).Conclusion 16S rRNA methylase genes armA and rmtB ale prevalent in Klebsiella pneumoniae strains producing KPC-2.

  2. Sequence Analysis and Comparison of 16S rRNA, 23S rRNA and 16S/23S Intergenic Spacer Region of Greening Bacterium Associated with Yellowing Disease (Huanglongbing) of Kinnow Mandarin.

    Science.gov (United States)

    Gupta, K N; Baranwal, V K; Haq, Q M R

    2012-03-01

    High incidence (up to 40%) of symptoms of yellowing and yellow mottling was observed in 5-8 years old orchards of kinnow mandarin {Citrus reticulate Balanco ('King' × 'Willow mandarin')} in the Punjab state of India during a survey in January 2007. These symptoms are often confused with nutrient deficiency and other stress related disorders. However, a greening bacterium has been attributed to cause the disease. The disease was graft transmissible and sequencing of 16S rRNA, 16S/23S intergenic spacer region and 23S rRNA of the greening bacterium associated with yellowing disease in kinnow mandarin confirmed it to be Candidatus Liberibacter asiaticus ('Ca L. asiaticus') showing maximum identity of 95.9% with 'Ca L. asiaticus' from USA and Brazil in 16S rRNA. The study indicates definite association of 'Ca L. asiaticus' with yellowing/chlorotic mottling symptoms of greening disease of kinnow mandarin in Punjab state of India.

  3. 我国重要帘蛤科(Veneridae)贝类的16S rRNA序列系统学分析%MOLECULAR PHYLOGENY OF VENERIDAE (MOLLUSCA, BIVALVIA) BASED ON 16S rRNA SEQUENCES

    Institute of Scientific and Technical Information of China (English)

    赵婷; 吴琪; 潘宝平

    2013-01-01

    本文对我国隶属于帘蛤科(Veneridae)10个亚科、17个属、20种贝类的16S rRNA基因片段进行了系统学分析,上述动物的16S rRNA片段长度在438-648bp之间,利用PAUP软件包在对序列比对基础上构建了邻接系统树(NJ)和最大拟然系统树(ML).16S rRNA数据显示,我国帘蛤科贝类由三个主要分支组成,美女蛤亚科中的加夫蛤属(Gafrarium)可能是一个单型属,该属与美女蛤属合并为加夫蛤属比较恰当.帘蛤亚科与雪蛤亚科应属于不连续的分类单元.另外,青蛤亚科与仙女蛤亚科均应作为独立的亚科存在.本文的研究结论与修订后的帘蛤科形态分类观点一致.

  4. Application of bacterial 16S rDNA amplification and sequencing in the classification and identification of bacteria%16S rDNA扩增及测序在细菌鉴定与分类中的应用

    Institute of Scientific and Technical Information of China (English)

    朱诗应; 戚中田

    2013-01-01

    Bacterial 16S rDNA amplification and sequencing is a new tool which has been widely used to identify bacterial species and perform taxonomic studies . The application of this technology for identification of uncultivable bacteria , differentiating species with high DNA sequence similarity and discovering novel bacterial genus and species are introduced in this paper . Future perspective of the method in clinical microbiology laboratories is also discussed .%16S rDNA扩增及测序技术在细菌的鉴定与分类研究中发挥着越来越重要的作用.本文就16S rDNA结构、可变区和保守区部分序列或全序列在临床上细菌鉴定和新细菌识别等方面的研究进展进行综述,并对其在临床实验室中的应用进行展望.

  5. Advances and Applications on Methodology of 16S rRNA Sequencing in Gut Microbiota Analysis%16S rRNA测序技术在肠道微生物中的应用研究进展

    Institute of Scientific and Technical Information of China (English)

    李东萍; 郭明璋; 许文涛

    2015-01-01

    16S rRNA sequencing is one of the high-throughput-sequencing-based methods used in gut microbiota analysis. Almost all the bacterial species in gut microbiota can be quantified through 16S rRNA sequencing, which has made this method into the mainstream. Two issues are very important in the application of 16S rRNA sequencing:sequencing strategy and bioinformatic analysis. In this review, three aspects of the sequencing strategy, including sequencing platform, sequencing region, and data size were discussed. While on bioinformatic analysis, the advance in sequences cluster and annotation, microbiota structure analysis, key taxa screening and functional analysis were reviewed here.%16S rRNA测序是高通量测序依赖的肠道微生物研究方法之一,该方法可以对肠道微生物中的所有菌种进行精确定量,因此正逐渐成为研究肠道微生物菌种丰度变化的主流。肠道微生物16S rRNA测序的应用过程中有两个问题至关重要,一是如何根据需要选择测序方案;二是面对高通量测序得到的海量数据,如何进行生物信息学分析,以得到具有生物学意义的结果。从测序平台、测序片段、测序数据量的选择3个方面讨论了如何选择测序方案,并从序列聚类与注释、群落结构分析、关键分类单位的筛选与功能分析等方面对目前常用的生物信息学分析手段进行综述。

  6. 多重耐药鲍曼不动杆菌16S rRNA甲基化酶研究进展%Advances in studying 16S rRNA methylation enzymes in multiple drug-resistant Acinetobacter baumannii

    Institute of Scientific and Technical Information of China (English)

    刘振茹; 凌保东

    2011-01-01

    Aminoglycoside antibiotics play an important role in the treatment of infections caused by Acinetobacter baumannii. The mechanisms of resistance to this class of antibiotics include mainly the production of aminoglycoside-modifying enzymes, the lack or reduction of expression of certain outer membrane proteins, the increased expression of active drug efflux pumps and the alterations of the ribosome-binding sites. The involvement of 16S rRNA methylation enzymes encoded by plasmids belongs to a newly-identified drug resistance mechanism frequently observed in multidrug-resistant Acinetobacter baumannii isolated in recent years and this often leads to high levels of aminoglycoside resistance. This review focuses on the research progress in 16S rRNA methylation enzymes of multidrug-resistant Acinetobacter baumannii such as the diversity of these enzymes and their role in aminoglycoside resistance.%氨基糖苷类抗生素在治疗鲍曼不动杆菌引起的感染中起着重要的作用.而鲍曼不动杆菌对该类抗生素的耐药机制主要包括产氨基糖苷修饰酶,外膜孔蛋白表达缺失,药物外排泵的表达和核糖体结合位点的改变等.质粒介导的16S rRNA甲基化酶是近年在多重耐药鲍曼不动杆菌中发现的一种新的耐药机制,可导致对氨基糖苷类抗生素的高水平耐药.本文就细菌rRNA的修饰作用、16S rRNA甲基化酶的发现、耐药与传播机制、耐药菌的流行、多重耐药鲍曼不动杆菌16S rRNA甲基化酶基[因的研究进展等方面作一综述.

  7. 16S rDNA在婴幼儿配方乳粉阪崎肠杆菌鉴定中的应用%Application of 16S rDNA in Enterobacter Sakazakii Identiifcation for Infant Formula Milk Powder

    Institute of Scientific and Technical Information of China (English)

    林玉宙

    2015-01-01

    选取近几年从婴幼儿配方乳粉生产过程环境及产品中收集、经国标GB/T 4789.40-2010检验和API20E鉴定为阪崎肠杆菌阳性的菌株16株,利用MicroSEQ ID微生物鉴定系统进行16S rDNA基因测序分析,构建系统发育树,鉴定种属。结果显示,这16份经API20E鉴定为阪崎肠杆菌阳性的菌株,经16S rDNA基因测序证实,其中2份为梨形肠杆菌,1份为克氏柠檬酸杆菌,其余13份样品均为阪崎肠杆菌,且与数据库中标准菌株的同源性达到99.5%以上。16S rDNA基因测序方法在婴幼儿配方乳粉企业进行阪崎肠杆菌鉴定和溯源方面具有广阔的前景。%Sixteen Enterobacter sakazaki strains col ected from the environment and products during infant formula milk powder production process and identiifed to be positive by GB/T 4789.40-2010 as wel as API20E were analyzed by 16S rDNA gene se-quencing using MicroSEQ ID microbial identiifcation system,and the system development tree was built to identify the species. The results showed that two of 16 Enterobacter sakazaki strains were Enterobacter pyrinus,and one was Citrobacter koseri,and the other thirteen were Enterobacter sakazaki and the homology with standard Enterobacter sakazaki strain in the database was more than 99.5%. 16S rDNA gene sequencing method has the broad prospect in the identiifcation and traceability of Enterobacter saka-zaki for infant formula enterprises.

  8. Molecular Phylogeny of Bisexual Artemia Based on 16S rDNA%两性生殖卤虫16S rDNA分子系统学研究

    Institute of Scientific and Technical Information of China (English)

    印红; 关妮; 付育婷

    2011-01-01

    [Objective] The research aimed at investigating the taxonomic position and phylogenetic relationship of bisexual brine shrimps.[Method] 16S rDNA of three species of bisexual Artemia from China was determined; the homologous sequences between them and 11 relative species of Artemia from GenBank were compared; the molecular phylogenetic tree was constructed by Mega Microsoft using Artemiopsis stefanssoni as outgroup. [Result] Artemia persimilis was the primal group in genus Artemia; Artemia franciscana and Artemia monica were the evolved groups; Artemia urmiana, Artemia sinica and other Artemia species from China shared a close genetic relationship. [Conclusion] Based on the 16S rDNA sequence of them, the phylogenetic relationships of these bisexual Artemia species were A. persirmilis→A. urmiana, A. sinica and A. tibetiana→A. tunisiana→A. monica→A. Franciscan.%[目的]对两性生殖卤虫的分类地位和系统发育情况进行探讨.[方法]测定了中国分布的A.sinica tibetiana、未定名的新疆鲸鱼湖卤虫和青海小柴旦卤虫3种两性生殖卤虫的16S rDNA序列,测序结果与Genbank已发表的另外11个两性生殖种卤虫的16S rDNA序列进行分析比较,并选取丰年虫科Artemiopsis stefanssoni为外群,运用MEGA软件中的NJ法构建分子系统树.[结果]A.persimilis是卤虫属中原始的类群,A.franciscana和A.monica为进化类群,A.urmiana与A.s.sinica以及中国的其它种类有密切的亲缘关系.[结论]基于线粒体16S rDNA序列的两性生殖卤虫系统发育关系为:A.persimilis→A.urmiana、A.sinica和A.tibetiana→A.tunisiana→A.monica→A.Franciscan.

  9. 食品中弓形菌16S rRNA特异性扩增检测方法的建立%Development of a specific amplification method of Arcobacter 16S rRNA gene in foods

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    针对弓形菌16SrRNA基因合成1对引物,通过对聚合酶链式反应(PCR)扩增条件的优化,建立了检测弓形菌的PCR方法。3株弓形菌标准菌株PCR产物测序结果与NCBI上公布的弓形菌16S rRNA基因序列进行比对,比对结果表明3株弓形菌测序结果与NCBI上公布的弓形菌16S rRNA基因序列同源性均在99%以上。3株弓形菌标准菌株均特异性地扩增出了长度为1202bp的片段,其他19株不同种类的菌株均无扩增产物出现。55份食品样品用Johnson-Murano肉汤增菌后用此法进行检测,其中6份样品为弓形菌阳性,阳性率为10.9%。上述实验结果表明,方法特异性强、操作简便,节省了检测时间,可用于食品中弓形菌的快速检测。%One pair of primers were used to amplify 16S rRNA sequence of Arcobacter,and a PCR method for detection of Arcobacter was developed with optimization of PCR amplification conditions.PCR products of 3 different Arcobacter type strains were sequenced and compared with 16S rRNA genes of Arcobacter published on NCBI.It's proved that the homology between the PCR products and 16S rRNA genes of Arcobacter published were all above 99%.3 different type strains of Arcobacter produced a 1202 bp amplified band,while all of the other 19 different bacteria didn't produced any band.55 food samples were detected whether Arcobacter present or not by the PCR method following enrichment in Johnson-Murano broth,and 6 samples were detected positive for this microorganism,namely with the positive ratio of 10.9%.The result suggested that the method developed was specific,easy to operate and timesaving,and could be used for rapid detection of Arcobacter in foods.

  10. Salinity inhibits post transcriptional processing of chloroplast 16S rRNA in shoot cultures of jojoba (Simmondsia chinesis).

    Science.gov (United States)

    Mizrahi-Aviv, Ela; Mills, David; Benzioni, Aliza; Bar-Zvi, Dudy

    2005-03-01

    Chloroplast metabolism is rapidly affected by salt stress. Photosynthesis is one of the first processes known to be affected by salinity. Here, we report that salinity inhibits chloroplast post-transcriptional RNA processing. A differentially expressed 680-bp cDNA, containing the 3' sequence of 16S rRNA, transcribed intergenic spacer, exon 1 and intron of tRNA(Ile), was isolated by differential display reverse transcriptase PCR from salt-grown jojoba (Simmondsia chinesis) shoot cultures. Northern blot analysis indicated that although most rRNA appears to be fully processed, partially processed chloroplast 16S rRNA accumulates in salt-grown cultures. Thus, salinity appears to decrease the processing of the rrn transcript. The possible effect of this decreased processing on physiological processes is, as yet, unknown.

  11. Phylogenetic relationships of five species of Dorippinae (Crustacea, Decapoda) revealed by 16S rDNA sequence analysis

    Institute of Scientific and Technical Information of China (English)

    FANYu; LIXinzheng; SONGLinsheng; CAIZhonghua

    2004-01-01

    A molecular phylogeny is presented for the subfamily Dorippinae (including 9 individuals, representing 5 species and 4 genera), based on the sequence data from 16S rRNA gene. Two-cluster test between lineages in these phylogenetic trees has been performed. On the basis of rate constancy, the rate of nucleotide substitutions of 16S rDNA sequence data is estimated as 0.27% per million years. The analysis strongly supports the recognition of the Dorippinae as a monophyletic subfamily. Phylogenetic tree indicates that the subfamily Dorippinae is divided into two main clades, and genus Dorippe appears basal in the subfamily, diverging from other species 36.6 Ma ago. It is also clear that the Heikea is closely related to the genus Neodorippe. The divergence time between them is 15.8 Ma.

  12. Isolation and 16S DNA characterization of soil microorganisms from tropical soils capable of utilizing the herbicides hexazinone and tebuthiuron.

    Science.gov (United States)

    Mostafa, Fadwa I Y; Helling, Charles S

    2003-11-01

    Six non-fermentative bacteria were isolated from Colombian (South America) and Hawaiian (USA) soils after enrichment with minimal medium supplemented with two herbicides, hexazinone (Hex) and tebuthiuron (Teb). Microscopic examination and physiological tests were followed by partial 16S DNA sequence analysis, using the first 527 bp of the 16S rRNA gene for bacterial identification. The isolated microorganisms (and in brackets, the herbicide that each degraded) were identified as: from Colombia. Methylobacterium organophilum [Teb], Paenibacillus pabuli [Teb], and Micrmbacterium foliorum [Hex]; and from Hawaii, Methylobacterium radiotolerans [Teb], Paenibacillus illinoisensis [Hex], and Rhodococcus equi [Hex]. The findings further explain how these herbicides, which have potential for illicit coca (Erythroxylum sp.) control, dissipate following their application to tropical soils.

  13. Phylogenetic relationship of 16 Oedipodidae species (Insecta: Orthoptera) based on the 16S rRNA gene sequences

    Institute of Scientific and Technical Information of China (English)

    HUI-MENG LU; YUAN HUANG

    2006-01-01

    The sequences of the mitochondrial 16S rRNA gene of 16 Oedipodidae species were amplified and sequenced. All sequences were aligned and analyzed and the phyloge netic relationships were inferred. The properties of 16S gene in Oedipodidae showed typical patterns of many insects such as a high A+T content and variable distance-dependent transition/transversion ratios. The 0.2 weight for sites of loops may be advisable for phylogeny reconstruction using the maximum parsimony method. The phylogenetic analysis results do not support the current subfamily classification systems of Oedipodidae. Bryodemellinae and Bryodeminae are closely related and should be merged as one subfamily. The status of Oedipodinae and Locustinae is also problematic.

  14. Flow Cytometry-Assisted Cloning of Specific Sequence Motifs from Complex 16S rRNA Gene Libraries

    DEFF Research Database (Denmark)

    Nielsen, Jeppe Lund; Schramm, Andreas; Bernhard, Anne E.

    2004-01-01

      FLOW CYTOMETRY-ASSISTED CLONING OF SPECIFIC SEQUENCE MOTIFS FROM COMPLEX 16S RRNA GENE LIBRARIES Jeppe L. Nielsen,1 Andreas Schramm,1,2 Anne E. Bernhard,1 Gerrit J. van den Engh,3 and David A. Stahl1* Department of Civil and Environmental Engineering, University of Washington,1 and Institute...... for Systems Biology,3 Seattle, Washington, and Department of Ecological Microbiology, University of Bayreuth, Bayreuth, Germany2 A flow cytometry method was developed for rapid screening and recovery of cloned DNA containing common sequence motifs. This approach, termed fluorescence-activated cell sorting......-assisted cloning, was used to recover sequences affiliated with a unique lineage within the Bacteroidetes not abundant in a clone library of environmental 16S rRNA genes.  ...

  15. Analysis of 16S rRNA amplicon sequencing options on the Roche/454 next-generation titanium sequencing platform.

    Directory of Open Access Journals (Sweden)

    Hideyuki Tamaki

    Full Text Available BACKGROUND: 16S rRNA gene pyrosequencing approach has revolutionized studies in microbial ecology. While primer selection and short read length can affect the resulting microbial community profile, little is known about the influence of pyrosequencing methods on the sequencing throughput and the outcome of microbial community analyses. The aim of this study is to compare differences in output, ease, and cost among three different amplicon pyrosequencing methods for the Roche/454 Titanium platform METHODOLOGY/PRINCIPAL FINDINGS: The following three pyrosequencing methods for 16S rRNA genes were selected in this study: Method-1 (standard method is the recommended method for bi-directional sequencing using the LIB-A kit; Method-2 is a new option designed in this study for unidirectional sequencing with the LIB-A kit; and Method-3 uses the LIB-L kit for unidirectional sequencing. In our comparison among these three methods using 10 different environmental samples, Method-2 and Method-3 produced 1.5-1.6 times more useable reads than the standard method (Method-1, after quality-based trimming, and did not compromise the outcome of microbial community analyses. Specifically, Method-3 is the most cost-effective unidirectional amplicon sequencing method as it provided the most reads and required the least effort in consumables management. CONCLUSIONS: Our findings clearly demonstrated that alternative pyrosequencing methods for 16S rRNA genes could drastically affect sequencing output (e.g. number of reads before and after trimming but have little effect on the outcomes of microbial community analysis. This finding is important for both researchers and sequencing facilities utilizing 16S rRNA gene pyrosequencing for microbial ecological studies.

  16. Cilantro microbiome before and after nonselective pre-enrichment for Salmonella using 16S rRNA and metagenomic sequencing

    OpenAIRE

    Jarvis, Karen G.; White, James R; Grim, Christopher J.; Ewing, Laura; Ottesen, Andrea R; Beaubrun, Junia Jean-Gilles; Pettengill, James B.; Brown, Eric; Hanes, Darcy E.

    2015-01-01

    Background Salmonella enterica is a common cause of foodborne gastroenteritis in the United States and is associated with outbreaks in fresh produce such as cilantro. Salmonella culture-based detection methods are complex and time consuming, and improvments to increase detection sensitivity will benefit consumers. In this study, we used 16S rRNA sequencing to determine the microbiome of cilantro. We also investigated changes to the microbial community prior to and after a 24-hour nonselective...

  17. Differentiation of Closely Related Carnobacterium Food Isolates Based on 16S-23S Ribosomal DNA Intergenic Spacer Region Polymorphism

    OpenAIRE

    Kabadjova, Petia; Dousset, Xavier; Le Cam, Virginie; Prevost, Hervé

    2002-01-01

    A novel strategy for identification of Carnobacterium food isolates based on restriction fragment length polymorphism (RFLP) of PCR-amplified 16S-23S ribosomal intergenic spacer regions (ISRs) was developed. PCR amplification from all Carnobacterium strains studied always yielded three ISR amplicons, which were designated the small ISR (S-ISR), the medium ISR (M-ISR), and the large ISR (L-ISR). The lengths of these ISRs varied from one species to another. Carnobacterium divergens NCDO 2763T a...

  18. The structure of the archaebacterial ribosomal protein S7 and its possible interaction with 16S rRNA.

    Science.gov (United States)

    Hosaka, H; Yao, M; Kimura, M; Tanaka, I

    2001-11-01

    Ribosomal protein S7 is one of the ubiquitous components of the small subunit of the ribosome. It is a 16S rRNA-binding protein positioned close to the exit of the tRNA, and it plays a role in initiating assembly of the head of the 30S subunit. Previous structural analyses of eubacterial S7 have shown that it has a stable alpha-helix core and a flexible beta-arm. Unlike these eubacterial proteins, archaebacterial or eukaryotic S7 has an N-terminal extension of approximately 60 residues. The crystal structure of S7 from archaebacterium Pyrococcus horikoshii (PhoS7) has been determined at 2.1 A resolution. The final model of PhoS7 consists of six major alpha-helices, a short 3(10)-helix and two beta-stands. The major part (residues 18-45) of the N-terminal extension of PhoS7 reinforces the alpha-helical core by well-extended hydrophobic interactions, while the other part (residues 46-63) is not visible in the crystal and is possibly fixed only by interacting with 16S rRNA. These differences in the N-terminal extension as well as in the insertion (between alpha1 and alpha2) of the archaebacterial S7 structure from eubacterial S7 are such that they do not necessitate a major change in the structure of the currently available eubacterial 16S rRNA. Some of the inserted chains might pass through gaps formed by helices of the 16S rRNA.

  19. YebU is a m5C methyltransferase specific for 16 S rRNA nucleotide 1407

    DEFF Research Database (Denmark)

    Andersen, Niels Møller; Douthwaite, Stephen

    2006-01-01

    The rRNAs in Escherichia coli contain methylations at 24 nucleotides, which collectively are important for ribosome function. Three of these methylations are m5C modifications located at nucleotides C967 and C1407 in 16S rRNA and at nucleotide C1962 in 23S rRNA. Bacterial rRNA modifications gener...... methyltransferase gene rsmF, and that the nomenclature system be extended to include the rRNA methyltransferases that still await identification....

  20. Detection and quantification of Dehalogenimonas and "Dehalococcoides" populations via PCR-based protocols targeting 16S rRNA genes.

    Science.gov (United States)

    Yan, Jun; Rash, Brian A; Rainey, Fred A; Moe, William M

    2009-12-01

    Members of the haloalkane dechlorinating genus Dehalogenimonas are distantly related to "Dehalococcoides" but share high homology in some variable regions of their 16S rRNA gene sequences. In this study, primers and PCR protocols intended to uniquely target Dehalococcoides were reevaluated, and primers and PCR protocols intended to uniquely target Dehalogenimonas were developed and tested. Use of the genus-specific primers revealed the presence of both bacterial groups in groundwater at a Louisiana Superfund site.

  1. Deep sequencing of 16S rRNA gene to efficiently assess bacterial richness and the rare biosphere in soils

    OpenAIRE

    Terrat, Sébastien; Dequiedt, Samuel; Bachar, Dipankar; Christen, Richard; Mougel, Christophe; Lelievre, Mélanie; Maron, Pierre-Alain; Plassart, Pierre; Wincker, Patrick; Cruaud, C; Jolivet, Claudy; Arrouays, Dominique; Bispo, Antonio; Lemanceau, Philippe; Ranjard, Lionel

    2012-01-01

    Soil is one of the most important reservoirs of microbiological diversity on our planet and, above all, one of the last bastions of such biodiversity. Since two decades, numerous molecular tools have been developed to assess accurately this huge diversity directly from soil DNA. In this context, 16S rRNA gene is widely used to study microbial community taxonomic diversity, as it can be easily amplified by PCR, and large databases are available relating obtained sequences to bacteria...

  2. Predictive microbiology combined with metagenomic analysis targeted on the 16S rDNA : A new approach for food quality

    OpenAIRE

    Delhalle, Laurent; Ellouze, Mariem; Taminiau, Bernard; Korsak Koulagenko, Nicolas; Nezer, Carine; Daube,Georges

    2013-01-01

    OBJECTIVES The food spoilage process is mainly caused by alteration micro-organisms and classical culture-based methods have therefore been used to assess the microbiological quality of food. These techniques are simple to implement but may not be relevant to understand the modifications of the microbial ecology which occur in the food product in response to different changes in the environmental conditions. Metagenomic analysis targeted on 16S ribosomal DNA can bring about a solution to t...

  3. 16s rRNA Identification of Pediococcus spp. from Broiler and Studies of Adherence Ability on Immobilized Mucus

    OpenAIRE

    Ema Damayanti; Lies Mira Yusiati; Achmad Dinoto

    2015-01-01

    The objectives of this research were to study taxonomical status of lactic acid bacteria (LAB) isolated from broiler and adherence ability on mucus in vitro. Molecular analysis was performed by analyzing 16S rRNA gene using universal primer. The adherence assay on mucus was carried out using microplate method with total plate count (TPC), absorbance (A550) and confirmed by scanning electron microscopy (SEM). The results of this studies revealed that three of LAB isolates have closed relation ...

  4. Asaia bogorensis peritonitis identified by 16S ribosomal RNA sequence analysis in a patient receiving peritoneal dialysis.

    Science.gov (United States)

    Snyder, Richard W; Ruhe, Jorg; Kobrin, Sidney; Wasserstein, Alan; Doline, Christa; Nachamkin, Irving; Lipschutz, Joshua H

    2004-08-01

    Here the authors report a case of refractory peritonitis leading to multiple hospitalizations and the loss of peritoneal dialysis access in a patient on automated peritoneal dialysis, caused by Asaia bogorensis, a bacterium not previously described as a human pathogen. This organism was identified by sequence analysis of the 16S ribosomal RNA gene. Unusual microbial agents may cause peritonitis, and molecular microbiological techniques are important tools for identifying these agents.

  5. Identification of bacteria directly from positive blood culture samples by DNA pyrosequencing of the 16S rRNA gene

    OpenAIRE

    2012-01-01

    Rapid identification of the causative bacteria of sepsis in patients can contribute to the selection of appropriate antibiotics and improvement of patients' prognosis. Genotypic identification is an emerging technology that may provide an alternative method to, or complement, established phenotypic identification procedures. We evaluated a rapid protocol for bacterial identification based on PCR and pyrosequencing of the V1 and V3 regions of the 16S rRNA gene using DNA extracted directly from...

  6. Usefulness of 16S rDNA sequencing for the diagnosis of infective endocarditis caused by Corynebacterium diphtheriae.

    Science.gov (United States)

    Pathipati, Padmaja; Menon, Thangam; Kumar, Naveen; Francis, Thara; Sekar, Prem; Cherian, Kotturathu Mammen

    2012-08-01

    We report a rare case of infective endocarditis caused by Corynebacterium diphtheriae in an 8-year-old boy, 2 years after a right ventricular outflow tract reconstruction with a bovine Contegra valved conduit. The patient recovered well after an RV-PA conduit enblock explantation and replacement with an aortic homograft with antibiotic treatment. All bacteriological cultures of excised tissue and blood were negative. The aetiological agent was identified as C. diphtheriae subsp. gravis by 16s rDNA sequencing.

  7. Assessing hog lagoon waste contamination in the Cape Fear Watershed using Bacteroidetes 16S rRNA gene pyrosequencing.

    Science.gov (United States)

    Arfken, Ann M; Song, Bongkeun; Mallin, Michael A

    2015-09-01

    Hog lagoons can be major sources of waste and nutrient contamination to watersheds adjacent to pig farms. Fecal source tracking methods targeting Bacteroidetes 16S rRNA genes in pig fecal matter may underestimate or fail to detect hog lagoon contamination in riverine environments. In order to detect hog lagoon wastewater contamination in the Cape Fear Watershed, where a large number of hog farms are present, we conducted pyrosequencing analyses of Bacteroidetes 16S rRNA genes in hog lagoon waste and identified new hog lagoon-specific marker sequences. Additional pyrosequencing analyses of Bacteroidetes 16S rRNA genes were conducted with surface water samples collected at 4 sites during 5 months in the Cape Fear Watershed. Using an operational taxonomic unit (OTU) identity cutoff value of 97 %, these newly identified hog lagoon markers were found in 3 of the river samples, while only 1 sample contained the pig fecal marker. In the sample containing the pig fecal marker, there was a relatively high percentage (14.1 %) of the hog lagoon markers and a low pig fecal marker relative abundance of 0.4 % in the Bacteroidetes 16S rRNA gene sequences. This suggests that hog lagoon contamination must be somewhat significant in order for pig fecal markers to be detected, and low levels of hog lagoon contamination cannot be detected targeting only pig-specific fecal markers. Thus, new hog lagoon markers have a better detection capacity for lagoon waste contamination, and in conjunction with a pig fecal marker, provide a more comprehensive and accurate detection of hog lagoon waste contamination in susceptible watersheds.

  8. FILOGENETIK POPULASI UDANG JERBUNG (Fenneropenaeus merguiensis de Man DI INDONESIA BERDASARKAN SEKUENS 16S-rRNA DNA MITOKONDRIA

    Directory of Open Access Journals (Sweden)

    Eni Kusrini

    2016-11-01

    Full Text Available Penelitian ini dilakukan untuk mengetahui hubungan kekerabatan stok udang jerbung Indonesia sebagai informasi dasar bagi program pemuliaan. Udang jerbung uji berasal dari Pantai Bengkulu, Selat Sunda (Banten, Pantai Cilacap (Jawa Tengah, Selat Lombok (NTB, dan Pontianak (Kalimantan Barat. Amplifikasi PCR dan sekuensing daerah 16S-rRNA DNA mitokondria dilakukan menggunakan primer 5’-CGCCTGTTTAAC-AAAAACAT-3’ dan 5’-CCGGTCTGAACTCAGATCATGT-3’. Hasil analisis homologi susunan nukleotida 16S-rRNA DNA mitokondria menunjukkan bahwa udang jerbung yang digunakan dalam penelitian merupakan Fenneropenaeus merguiensis. Hasil analisis kekerabatan menunjukkan bahwa 5 populasi udang jerbung uji dapat dibagi menjadi 2 kelompok besar, yaitu kelompok Kalimantan Barat dan kelompok Bengkulu-Banten-Jawa Tengah-NTB. Populasi udang jerbung Kalimantan dan Bengkulu masing-masing memiliki sekuens spesifik, yaitu ACTGACT dan C-GAC di terminal 5. Sekuens tersebut mungkin dapat digunakan sebagai penanda dalam program pemuliaan udang jerbung Indonesia. The experiment was conducted to understand the family relationship of banana prawn in Indonesia and to provide basic information for breeding program. Prawns were obtained from Bengkulu Coast, Sunda Strait (Banten, Cilacap Coast (Central Java, Lombok Strait (West Nusa Tenggara, Pontianak Coast (West Kalimantan. PCR amplification and sequencing of 16S-rRNA mitochondrial DNA region were performed using 5’-CGCCTGTTTAAC-AAAAACAT-3’ and 5’-CCGGTCTGAACTCAGATCATGT-3’. Analysis of homology sequences of 16S-rRNA mtDNA showed that banana prawn used in this study was Fenneropenaeus merguiensis. Result of family relationship analysis indicated that five populations of banana prawn can be divided into two groups, i.e. West Kalimantan and Bengkulu-Banten-Central Java-NTB groups. Banana prawns from West Kalimantan and Bengkulu have specific sequences at 5’ terminal, ACTGACT and C-GAC, respectively. Those sequences can

  9. Diversity of Archaea in Icelandic hot springs based on 16S rRNA and chaperonin genes.

    Science.gov (United States)

    Mirete, Salvador; de Figueras, Carolina G; González-Pastor, Jose E

    2011-07-01

    The diversity of archaeal communities growing in four hot springs (65-90 °C, pH 6.5) was assessed with 16S rRNA gene primers specific for the domain Archaea. Overall, mainly uncultured members of the Desulfurococcales, the Thermoproteales and the Korarchaeota, were identified. Based on this diversity, a set of chaperonin heat-shock protein (Hsp60) gene sequences from different archaeal species were aligned to design two degenerate primer sets for the amplification of the chaperonin gene: Ths and Kor (which can also detect the korarchaeotal chaperonin gene from one of the samples). A phylogenetic tree was constructed using the chaperonin sequences retrieved and other sequences from cultured representatives. The Alpha and Beta paralogs of the chaperonin gene were observed within the main clades and orthologs among them. Cultivated representatives from these clades were assigned to either paralog in the chaperonin tree. Uncultured representatives observed in the 16S rRNA gene analysis were found to be related to the Desulfurococcales. The topologies of the 16S rRNA gene and chaperonin phylogenetic trees were compared, and similar phylogenetic relationships were observed. Our results suggest that the chaperonin Hsp60 gene may be used as a phylogenetic marker for the clades found in this extreme environment.

  10. Identification of forensically important beetles (Coleoptera: Histeridae) in China based on 16S rRNA and Cyt b.

    Science.gov (United States)

    Su, R N; Guo, Y D; Xie, D; Peng, Y L; Cai, J F; Hua, F; Sheng, L H

    2013-09-01

    Exact identification of an insect sample is usually the first essential step in a forensic entomological analysis. However, the morphological similarity of beetles in the level of species usually poses a challenge for forensic scientists within their routine work. As a supplementary to traditional morphological method, molecular genetics identification turns out to be simple and time-saving. A molecular identification method involving a 288-bp segment of the 16S ribosomal RNA (16S rRNA) gene and a 334-bp segment of the cytochrome b (Cyt b) gene from 23 histerid beetles specimens, collected from 7 locations in 6 Chinese provinces, was evaluated. The 16S rRNA and Cyt b genes are sequenced to examine the ability of the region, resolve species identities and enrich the local databases. The monophyletic branches of the phylogenetic tree showed the potential of the markers in identifying beetles within families. Combined analysis is a more accurate approach for species identication than independent analysis.

  11. Microbial diversity in the sputum of a cystic fibrosis patient studied with 16S rDNA pyrosequencing.

    Science.gov (United States)

    Armougom, F; Bittar, F; Stremler, N; Rolain, J-M; Robert, C; Dubus, J-C; Sarles, J; Raoult, D; La Scola, B

    2009-09-01

    Recent studies using 16S rRNA gene amplification followed by clonal Sanger sequencing in cystic fibrosis demonstrated that cultured microorganisms are only part of the infecting flora. The purpose of this paper was to compare pyrosequencing and clonal Sanger sequencing on sputum. The sputum of a patient with cystic fibrosis was analysed by culture, Sanger clone sequencing and pyrosequencing after 16S rRNA gene amplification. A total of 4,499 sequencing reads were obtained, which could be attributed to six consensus sequences, but the length of reads leads to fastidious data analysis. Compared to clonal Sanger sequencing and to cultivation results, pyrosequencing recovers greater species richness and gives a more reliable estimate of the relative abundance of bacterial species. The 16S pyrosequencing approach expands our knowledge of the microbial diversity of cystic fibrosis sputum. The current lack of phylogenetic resolution at the species level for the GS 20 sequencing reads will be overcome with the next generation of pyrosequencing apparatus.

  12. Studies on the ability of partially iodinated 16S RNA to participate in 30S ribosome assembly.

    Science.gov (United States)

    Schendel, P L; Craven, G R

    1976-11-01

    Deproteinated 16S RNA was iodinated at pH 5.0 in an aqueous solution containing TlCl3 plus KI for 1-5 hours at 42 degrees C. Under these conditions 33 moles of iodine are incorporated per mole of RNA. As judged by sucrose gradient sedimentation, the iodinated RNA does not exhibit any large alteration in conformation as compared to unmodified 16S. The iodinated RNA was examined for its ability to reconstitute with total 30S proteins. Sedimentation velocity analysis reveals that the reconstituted subunit has a sedimentation constant of approximately 20S. In addition, protein analysis of particles reconstituted with 16S RNA iodinated for 5 hours indicates that proteins S2, S10, S13, S14, S15, S17, S18, S19, and S21 are no longer able to participate in the 30S assembly process and that proteins S6, S16 and S20 are present in reduced amounts. The ramifications of these results concerning protein-RNA and RNA-RNA interactions occurring in ribosome assembly are discussed.

  13. 16S rDNA序列分析法鉴定鸡源肠道乳酸菌

    Institute of Scientific and Technical Information of China (English)

    向双云; 周珍辉; 关文怡; 张浩; 段素云; 马建民

    2016-01-01

    从北京郊区某散养鸡场健康成年鸡肠道中分离纯化得到12株未知菌,分别提取DNA,采用16S rDNA扩增通用引物,经PCR扩增后,测定其16S rDNA序列,测序结果与Gen Bank数据库中的序列作对比,根据序列相似性,利用MEGA软件,对12株未知菌序列构建进化树.经鉴定5株为乳酸杆菌、3株为唾液乳杆菌、2株为肠球菌、1株为罗伊氏乳杆菌及1株为葡萄球菌.结果表明,16S rDNA序列分析技术与传统方法相比具有简便和快速等优点,提高鉴定效率,可进行乳酸菌种级水平鉴定,适合用于实验室内对乳酸菌的鉴定.

  14. Complete ecological isolation and cryptic diversity in Polynucleobacter bacteria not resolved by 16S rRNA gene sequences.

    Science.gov (United States)

    Hahn, Martin W; Jezberová, Jitka; Koll, Ulrike; Saueressig-Beck, Tanja; Schmidt, Johanna

    2016-07-01

    Transplantation experiments and genome comparisons were used to determine if lineages of planktonic Polynucleobacter almost indistinguishable by their 16S ribosomal RNA (rRNA) sequences differ distinctively in their ecophysiological and genomic traits. The results of three transplantation experiments differing in complexity of biotic interactions revealed complete ecological isolation between some of the lineages. This pattern fits well to the previously detected environmental distribution of lineages along chemical gradients, as well as to differences in gene content putatively providing adaptation to chemically distinct habitats. Patterns of distribution of iron transporter genes across 209 Polynucleobacter strains obtained from freshwater systems and representing a broad pH spectrum further emphasize differences in habitat-specific adaptations. Genome comparisons of six strains sharing ⩾99% 16S rRNA similarities suggested that each strain represents a distinct species. Comparison of sequence diversity among genomes with sequence diversity among 240 cultivated Polynucleobacter strains indicated a large cryptic species complex not resolvable by 16S rRNA sequences. The revealed ecological isolation and cryptic diversity in Polynucleobacter bacteria is crucial in the interpretation of diversity studies on freshwater bacterioplankton based on ribosomal sequences.

  15. Clinical Fusobacterium mortiferum Isolates Cluster with Undifferentiated Clostridium rectum Species Based on 16S rRNA Gene Phylogenetic Analysis.

    Science.gov (United States)

    Lee, Yangsoon; Eun, Chang Soo; Han, Dong Soo

    2016-05-01

    The most commonly encountered clinical Fusobacterium species are F. nucleatum and F. necrophorum; other Fusobacteria, such as F. mortiferum and F. varium, have occasionally been isolated from human specimens. Clostridium rectum is a gram-positive species characterized as a straight bacillus with oval sub-terminal spores. The close 16S rRNA gene sequence relationship of C. rectum with the genus Fusobacterium is unexpected given their very different phenotypic characteristics. Between 2014 and 2015, a total of 19 Fusobacterium isolates were recovered from the colonic tissue of 10 patients at a university hospital. All isolates were identified based on 16S rRNA gene sequencing. The phylogenetic relationship among these isolates was estimated using the neighbor-joining method and the Molecular Evolutionary Genetic Analysis (MEGA) version 6. Based on phylogenetic analysis, the F. mortiferum isolates clustered into two groups - F. mortiferum DSM 19809 (group I) and F. mortiferum ATCC 25557 (group II) - even though they are of the same species. Furthermore, the F. mortiferum DSM 19809 (group I) showed a close phylogenetic relationship with C. rectum, even though C. rectum is classified as a gram-positive spore-producing bacillus. C. rectum is clearly unrelated to the genus Clostridium as it shows highest 16S rRNA gene sequence similarity with species from the genus Fusobacterium Therefore, additional methods such as Gram staining and other biochemical methods should be performed for Fusobacterium identification.

  16. IMNGS: A comprehensive open resource of processed 16S rRNA microbial profiles for ecology and diversity studies.

    Science.gov (United States)

    Lagkouvardos, Ilias; Joseph, Divya; Kapfhammer, Martin; Giritli, Sabahattin; Horn, Matthias; Haller, Dirk; Clavel, Thomas

    2016-09-23

    The SRA (Sequence Read Archive) serves as primary depository for massive amounts of Next Generation Sequencing data, and currently host over 100,000 16S rRNA gene amplicon-based microbial profiles from various host habitats and environments. This number is increasing rapidly and there is a dire need for approaches to utilize this pool of knowledge. Here we created IMNGS (Integrated Microbial Next Generation Sequencing), an innovative platform that uniformly and systematically screens for and processes all prokaryotic 16S rRNA gene amplicon datasets available in SRA and uses them to build sample-specific sequence databases and OTU-based profiles. Via a web interface, this integrative sequence resource can easily be queried by users. We show examples of how the approach allows testing the ecological importance of specific microorganisms in different hosts or ecosystems, and performing targeted diversity studies for selected taxonomic groups. The platform also offers a complete workflow for de novo analysis of users' own raw 16S rRNA gene amplicon datasets for the sake of comparison with existing data. IMNGS can be accessed at www.imngs.org.

  17. Structure of ERA in Complex with the 3 End of 16s rRNBA Implications for Ribosome Biogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Tu, C.; Zhou, X; Tropea, J; Austin, B; Waugh, D; Court, D; Ji, X

    2009-01-01

    ERA, composed of an N-terminal GTPase domain followed by an RNA-binding KH domain, is essential for bacterial cell viability. It binds to 16S rRNA and the 30S ribosomal subunit. However, its RNA-binding site, the functional relationship between the two domains, and its role in ribosome biogenesis remain unclear. We have determined two crystal structures of ERA, a binary complex with GDP and a ternary complex with a GTP-analog and the 1531AUCACCUCCUUA1542 sequence at the 3? end of 16S rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein. We show that GTP binding is a prerequisite for RNA recognition by ERA and that RNA recognition stimulates its GTP-hydrolyzing activity. Based on these and other data, we propose a functional cycle of ERA, suggesting that the protein serves as a chaperone for processing and maturation of 16S rRNA and a checkpoint for assembly of the 30S ribosomal subunit. The AUCA sequence is highly conserved among bacteria, archaea, and eukaryotes, whereas the CCUCC, known as the anti-Shine-Dalgarno sequence, is conserved in noneukaryotes only. Therefore, these data suggest a common mechanism for a highly conserved ERA function in all three kingdoms of life by recognizing the AUCA, with a 'twist' for noneukaryotic ERA proteins by also recognizing the CCUCC.

  18. Structure of ERA in complex with the 3′ end of 16S rRNA: Implications for ribosome biogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Tu, Chao; Zhou, Xiaomei; Tropea, Joseph E.; Austin, Brian P.; Waugh, David S.; Court, Donald L.; Ji, Xinhua; (NCI)

    2009-10-09

    ERA, composed of an N-terminal GTPase domain followed by an RNA-binding KH domain, is essential for bacterial cell viability. It binds to 16S rRNA and the 30S ribosomal subunit. However, its RNA-binding site, the functional relationship between the two domains, and its role in ribosome biogenesis remain unclear. We have determined two crystal structures of ERA, a binary complex with GDP and a ternary complex with a GTP-analog and the {sub 1531}AUCACCUCCUUA{sub 1542} sequence at the 3' end of 16S rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein. We show that GTP binding is a prerequisite for RNA recognition by ERA and that RNA recognition stimulates its GTP-hydrolyzing activity. Based on these and other data, we propose a functional cycle of ERA, suggesting that the protein serves as a chaperone for processing and maturation of 16S rRNA and a checkpoint for assembly of the 30S ribosomal subunit. The AUCA sequence is highly conserved among bacteria, archaea, and eukaryotes, whereas the CCUCC, known as the anti-Shine-Dalgarno sequence, is conserved in noneukaryotes only. Therefore, these data suggest a common mechanism for a highly conserved ERA function in all three kingdoms of life by recognizing the AUCA, with a 'twist' for noneukaryotic ERA proteins by also recognizing the CCUCC.

  19. Diversity of 16S ribosomal DNA-defined bacterial population in acid rock drainage from Japanese pyrite mine.

    Science.gov (United States)

    Okabayashi, Ai; Wakai, Satoshi; Kanao, Tadayoshi; Sugio, Tsuyoshi; Kamimura, Kazuo

    2005-12-01

    Four acidophilic bacteria (YARDs1-4) were isolated from an acid rock drainage (ARD) from Yanahara mine, Okayama prefecture, Japan. The physiological and 16S rDNA sequence analyses revealed that YARD1 was closely affiliated with Acidithiobacillus ferrooxidans, YARD2 was an Acidiphilium-like bacterium, and YARD3 and YARD4 were sulfur-oxidizing bacteria with a relatively close relationship to A. ferrooxidans in the phylogenetic analysis. A molecular approach based on the construction of a 16S rDNA clone library was used to investigate the microbial population of the ARD. Small-subunit rRNA genes were PCR amplified, subsequently cloned and screened for variation by a restriction fragment length polymorphism (RFLP) analysis. A total of 284 clones were grouped into 133 operational taxonomic units (OTUs) by the RFLP analysis. Among them, an OTU showing the same RFLP pattern as those of the isolates from the ARD was not detected. The phylogenetic analysis based on the 16S rDNA sequences from 10 major OTUs and their close relatives revealed that 4 OTUs containing 32.1% of the total clones were loosely affiliated with Verrucomicrobia, 2 OTUs containing 6.6% of the total clones were loosely affiliated with Chloribi, and other OTUs were affiliated with Actinobacteria, Nitrospirae, and beta-Proteobacteria.

  20. A comparison of rpoB and 16S rRNA as markers in pyrosequencing studies of bacterial diversity.

    Directory of Open Access Journals (Sweden)

    Michiel Vos

    Full Text Available BACKGROUND: The 16S rRNA gene is the gold standard in molecular surveys of bacterial and archaeal diversity, but it has the disadvantages that it is often multiple-copy, has little resolution below the species level and cannot be readily interpreted in an evolutionary framework. We compared the 16S rRNA marker with the single-copy, protein-coding rpoB marker by amplifying and sequencing both from a single soil sample. Because the higher genetic resolution of the rpoB gene prohibits its use as a universal marker, we employed consensus-degenerate primers targeting the Proteobacteria. METHODOLOGY/PRINCIPAL FINDINGS: Pyrosequencing can be problematic because of the poor resolution of homopolymer runs. As these erroneous runs disrupt the reading frame of protein-coding sequences, removal of sequences containing nonsense mutations was found to be a valuable filter in addition to flowgram-based denoising. Although both markers gave similar estimates of total diversity, the rpoB marker revealed more species, requiring an order of magnitude fewer reads to obtain 90% of the true diversity. The application of population genetic methods was demonstrated on a particularly abundant sequence cluster. CONCLUSIONS/SIGNIFICANCE: The rpoB marker can be a complement to the 16S rRNA marker for high throughput microbial diversity studies focusing on specific taxonomic groups. Additional error filtering is possible and tests for recombination or selection can be employed.

  1. New screening software shows that most recent large 16S rRNA gene clone libraries contain chimeras.

    Science.gov (United States)

    Ashelford, Kevin E; Chuzhanova, Nadia A; Fry, John C; Jones, Antonia J; Weightman, Andrew J

    2006-09-01

    A new computer program, called Mallard, is presented for screening entire 16S rRNA gene libraries of up to 1,000 sequences for chimeras and other artifacts. Written in the Java computer language and capable of running on all major operating systems, the program provides a novel graphical approach for visualizing phylogenetic relationships among 16S rRNA gene sequences. To illustrate its use, we analyzed most of the large libraries of cloned bacterial 16S rRNA gene sequences submitted to the public repository during 2005. Defining a large library as one containing 100 or more sequences of 1,200 bases or greater, we screened 25 of the 28 libraries and found that all but three contained substantial anomalies. Overall, 543 anomalous sequences were found. The average anomaly content per clone library was 9.0%, 4% higher than that previously estimated for the public repository overall. In addition, 90.8% of anomalies had characteristic chimeric patterns, a rise of 25.4% over that found previously. One library alone was found to contain 54 chimeras, representing 45.8% of its content. These figures far exceed previous estimates of artifacts within public repositories and further highlight the urgent need for all researchers to adequately screen their libraries prior to submission. Mallard is freely available from our website at http://www.cardiff.ac.uk/biosi/research/biosoft/.

  2. Metagenomic Analysis of Slovak Bryndza Cheese Using Next-Generation 16S rDNA Amplicon Sequencing

    Directory of Open Access Journals (Sweden)

    Planý Matej

    2016-06-01

    Full Text Available Knowledge about diversity and taxonomic structure of the microbial population present in traditional fermented foods plays a key role in starter culture selection, safety improvement and quality enhancement of the end product. Aim of this study was to investigate microbial consortia composition in Slovak bryndza cheese. For this purpose, we used culture-independent approach based on 16S rDNA amplicon sequencing using next generation sequencing platform. Results obtained by the analysis of three commercial (produced on industrial scale in winter season and one traditional (artisanal, most valued, produced in May Slovak bryndza cheese sample were compared. A diverse prokaryotic microflora composed mostly of the genera Lactococcus, Streptococcus, Lactobacillus, and Enterococcus was identified. Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris were the dominant taxons in all tested samples. Second most abundant species, detected in all bryndza cheeses, were Lactococcus fujiensis and Lactococcus taiwanensis, independently by two different approaches, using different reference 16S rRNA genes databases (Greengenes and NCBI respectively. They have been detected in bryndza cheese samples in substantial amount for the first time. The narrowest microbial diversity was observed in a sample made with a starter culture from pasteurised milk. Metagenomic analysis by high-throughput sequencing using 16S rRNA genes seems to be a powerful tool for studying the structure of the microbial population in cheeses.

  3. Display of a thermostable lipase on the surface of a solvent-resistant bacterium, Pseudomonas putida GM730, and its applications in whole-cell biocatalysis

    Directory of Open Access Journals (Sweden)

    Kwon Seok-Joon

    2006-04-01

    Full Text Available Abstract Background Whole-cell biocatalysis in organic solvents has been widely applied to industrial bioprocesses. In two-phase water-solvent processes, substrate conversion yields and volumetric productivities can be limited by the toxicity of solvents to host cells and by the low mass transfer rates of the substrates from the solvent phase to the whole-cell biocatalysts in water. Results To solve the problem of solvent toxicity, we immobilized a thermostable lipase (TliA from Pseudomonas fluorescens on the cell surface of a solvent-resistant bacterium, Pseudomonas putida GM730. Surface immobilization of enzymes eliminates the mass-transfer limitation imposed by the cell wall and membranes. TliA was successfully immobilized on the surface of P. putida cells using the ice-nucleation protein (INP anchoring motif from Pseudomonas syrinage. The surface location was confirmed by flow cytometry, protease accessibility and whole-cell enzyme activity using a membrane-impermeable substrate. Three hundred and fifty units of whole-cell hydrolytic activity per gram dry cell mass were obtained when the enzyme was immobilized with a shorter INP anchoring motif (INPNC. The surface-immobilized TliA retained full enzyme activity in a two-phase water-isooctane reaction system after incubation at 37°C for 12 h, while the activity of the free form enzyme decreased to 65% of its initial value. Whole cells presenting immobilized TliA were shown to catalyze three representative lipase reactions: hydrolysis of olive oil, synthesis of triacylglycerol and chiral resolution. Conclusion In vivo surface immobilization of enzymes on solvent-resistant bacteria was demonstrated, and appears to be useful for a variety of whole-cell bioconversions in the presence of organic solvents.

  4. Effect of schedule on reactogenicity and antibody persistence of acellular and whole-cell pertussis vaccines: value of laboratory tests as predictors of clinical performance.

    Science.gov (United States)

    Miller, E; Ashworth, L A; Redhead, K; Thornton, C; Waight, P A; Coleman, T

    1997-01-01

    The performance of four acellular pertussis vaccines containing between two and five pertussis antigens combined with diphtheria and tetanus toxoids was compared with that of British whole-cell diphtheria/tetanus/pertussis (DTP) vaccine both in laboratory assays for potency, toxicity and immunogenicity, and for reactogenicity and immunogenicity in infants. Clinical responses were evaluated in double blind randomized Phase II trials using 3/5/9 month and 2/3/4 month schedules. The acellular DTPs had much lower toxicity than whole-cell DTP in laboratory tests and were significantly less pyrogenic than whole-cell DTP under both schedules. Local reactions were not consistently lower in acellular than whole-cell vaccinees and varied with the source of the diphtheria and tetanus antigens used. Differences in endotoxin level and content of active pertussis toxin (PT) between acellular DTP vaccines were not clinically significant. The reactogenicity advantage of the acellular vaccines was substantially reduced under the 2/3/4 month schedule due to the reduced reactogenicity of the whole-cell DTP vaccine when given at a younger age. There was no relationship between antigen content measured in micrograms per dose and ELISA antibody responses to filamentous haemagglutinin (FHA) and PT in infants, nor was murine immunogenicity predictive of immunogenicity in humans. Antibody response to PT was attenuated in the whole-cell group under the 2/3/4 month schedule but was unaffected in the group receiving acellular vaccines with individually purified components; antibody response to pertactin (69 kDa antigen) was similar in recipients of the whole-cell and component acellular vaccines under the 2/3/4 month schedule. PT antibody persistence until 4-5 years of age was significantly better in recipients of the component acellular than either the whole-cell vaccine or the co-purified acellular vaccine under the 3/5/9 month schedule. However, diphtheria antitoxin levels were reduced in

  5. Development of an Analysis Pipeline Characterizing Multiple Hypervariable Regions of 16S rRNA Using Mock Samples.

    Directory of Open Access Journals (Sweden)

    Jennifer J Barb

    Full Text Available There is much speculation on which hypervariable region provides the highest bacterial specificity in 16S rRNA sequencing. The optimum solution to prevent bias and to obtain a comprehensive view of complex bacterial communities would be to sequence the entire 16S rRNA gene; however, this is not possible with second generation standard library design and short-read next-generation sequencing technology.This paper examines a new process using seven hypervariable or V regions of the 16S rRNA (six amplicons: V2, V3, V4, V6-7, V8, and V9 processed simultaneously on the Ion Torrent Personal Genome Machine (Life Technologies, Grand Island, NY. Four mock samples were amplified using the 16S Ion Metagenomics Kit™ (Life Technologies and their sequencing data is subjected to a novel analytical pipeline.Results are presented at family and genus level. The Kullback-Leibler divergence (DKL, a measure of the departure of the computed from the nominal bacterial distribution in the mock samples, was used to infer which region performed best at the family and genus levels. Three different hypervariable regions, V2, V4, and V6-7, produced the lowest divergence compared to the known mock sample. The V9 region gave the highest (worst average DKL while the V4 gave the lowest (best average DKL. In addition to having a high DKL, the V9 region in both the forward and reverse directions performed the worst finding only 17% and 53% of the known family level and 12% and 47% of the genus level bacteria, while results from the forward and reverse V4 region identified all 17 family level bacteria.The results of our analysis have shown that our sequencing methods using 6 hypervariable regions of the 16S rRNA and subsequent analysis is valid. This method also allowed for the assessment of how well each of the variable regions might perform simultaneously. Our findings will provide the basis for future work intended to assess microbial abundance at different time points

  6. Loop-mediated isothermal amplification assay for 16S rRNA methylase genes in Gram-negative bacteria.

    Science.gov (United States)

    Nagasawa, Mitsuaki; Kaku, Mitsuo; Kamachi, Kazunari; Shibayama, Keigo; Arakawa, Yoshichika; Yamaguchi, Keizo; Ishii, Yoshikazu

    2014-10-01

    Using the loop-mediated isothermal amplification (LAMP) method, we developed a rapid assay for detection of 16S rRNA methylase genes (rmtA, rmtB, and armA), and investigated 16S rRNA methylase-producing strains among clinical isolates. Primer Explorer V3 software was used to design the LAMP primers. LAMP primers were prepared for each gene, including two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB). Detection was performed with the Loopamp DNA amplification kit. For all three genes (rmtA, rmtB, and armA), 10(2) copies/tube could be detected with a reaction time of 60 min. When nine bacterial species (65 strains saved in National Institute of Infectious Diseases) were tested, which had been confirmed to possess rmtA, rmtB, or armA by PCR and DNA sequencing, the genes were detected correctly in these bacteria with no false negative or false positive results. Among 8447 clinical isolates isolated at 36 medical institutions, the LAMP method was conducted for 191 strains that were resistant to aminoglycosides based on the results of antimicrobial susceptibility tests. Eight strains were found to produce 16S rRNA methylase (0.09%), with rmtB being identified in three strains (0.06%) of 4929 isolates of Enterobacteriaceae, rmtA in three strains (0.10%) of 3284 isolates of Pseudomonas aeruginosa, and armA in two strains (0.85%) of 234 isolates of Acinetobacter spp. At present, the incidence of strains possessing 16S rRNA methylase genes is very low in Japan. However, when Gram-negative bacteria showing high resistance to aminoglycosides are isolated by clinical laboratories, it seems very important to investigate the status of 16S rRNA methylase gene-harboring bacilli and monitor their trends among Japanese clinical settings.

  7. Flow Cytometry-assisted Cloning of Specific Sequence Motifs fromComplex 16S ribosomal RNA Gene Libraries.

    Energy Technology Data Exchange (ETDEWEB)

    Nielsen, J.L.; Schramm, A.; Bernhard, A.E.; van den Engh, G.J.; Stahl, D.A.

    2004-07-21

    A flow cytometry method was developed for rapid screeningand recovery of cloned DNA containing common sequence motifs. Thisapproach, termed fluorescence-activated cell sorting-assisted cloning,was used to recover sequences affiliated with a unique lineage within theBacteroidetes not abundant in a clone library of environmental 16S rRNAgenes. Retrieval and sequence analysis of phylogenetically informativegenes has become a standard cultivation-independent technique toinvestigate microbial diversity in nature (7, 18). Genes encoding the 16SrRNA, because of the relative ease of their selective amplification, havebeen most frequently employed for general diversity surveys (16).Environmental studies have also focused on specific subpopulationsaffiliated with a phylogenetic group or identified by genes encodingspecific metabolic functions (e.g., ammonia oxidation, sulfaterespiration, and nitrate reduction) (8,15,20). However, specificpopulations may be of low abundance (1,23), or the genes encodingspecific metabolic functions may be insufficiently conserved to providepriming sites for general PCR amplification. Three general approacheshave been used to obtain 16S rRNA sequence information from low-abundancepopulations: screening hundreds to thousands of clones in a general 16SrRNA gene library (21), flow cytometric sorting of a subpopulation ofenvironmentally derived cells labeled by fluorescent in situhybridization (FISH) (27), or selective PCR amplification using primersspecific for the subpopulation (2,23). While the first approach is simplytime-consuming and tedious, the second has been restricted to fairlylarge and strongly fluorescent cells from aquatic samples (5, 27). Thethird approach often generates fragments of only a few hundred bases dueto the limited number of specific priming sites. Partial sequenceinformation often degrades analysis, obscuring or distorting thephylogenetic placement of the new sequences (11, 20). A more robustcharacterization of environ

  8. Phylogenetic relationships of chemoautotrophic bacterial symbionts of Solemya velum say (Mollusca: Bivalvia) determined by 16S rRNA gene sequence analysis.

    OpenAIRE

    Eisen, J. A.; Smith, S W; Cavanaugh, Colleen Marie

    1992-01-01

    The protobranch bivalve Solemya velum Say (Mollusca: Bivalvia) houses chemoautotrophic symbionts intracellularly within its gills. These symbionts were characterized through sequencing of polymerase chain reaction-amplified 16S rRNA coding regions and hybridization of an Escherichia coli gene probe to S. velum genomic DNA restriction fragments. The symbionts appeared to have only one copy of the 16S rRNA gene. The lack of variability in the 16S sequence and hybridization patterns within and b...

  9. Research progress in exogenous 16S rRNA methylase%外源性16srRNA甲基化酶研究进展

    Institute of Scientific and Technical Information of China (English)

    费秋萍; 张顺; 蔡挺; 胡珊珊; 吴春丽

    2016-01-01

    外源性16S rRNA甲基化酶能介导细菌对多种氨基糖苷类抗菌药物高水平耐药,目前已发现10种外源性16S rRNA甲基化酶;该综述分析了外源性16S rRNA甲基化酶与两类内源性16S rRNA甲基化酶的联系,发现外源性16S rRNA甲基化酶可阻碍特定的内源性16S rRNA甲基化酶的甲基化作用,影响细菌的生长及对抗菌药物的敏感性,对基因环境的分析表明外源性16S rRNA甲基化酶基因常与其他耐药基因位于同一可移动基因元件上,耐药机制复杂,应继续监测外源性16S rRNA甲基化酶的流行情况,深入探讨其耐药机制的形成,以期早日改善临床致病菌耐药情况。%Exogenous 16S rRNA methylase can mediate a mechanism of bacteria with high level resistance to many aminoglycosides ,and ten kinds of exogenous 16S rRNA methylase have already been reported .This essay has an‐alyzed the association of exogenous 16S rRNA methylase with two kinds of endogenous 16S rRNA methylase .The result showed that exogenous 16S rRNA methylase could defect the growth of strains and change the strain's sus‐ceptibility to antibiotics by hindering the methylation of specific endogenous 16S rRNA methylase .The analysis of genetic environment suggested that exogenous 16S rRNA methylase produced a complex resistance mechanism as their encoding genes are mostly locating on transferable plasmids with other resistance determinants .It required us to have a further monitoring of the exogenous 16S rRNA methylase and seek for the resistance mechanism to mini‐mize the occurrence of drug resistance .

  10. Adaptive immune response to whole cell pertussis vaccine reflects vaccine quality: A possible complementation to the Pertussis Serological Potency test.

    Science.gov (United States)

    Hoonakker, M E; Verhagen, L M; van der Maas, L; Metz, B; Uittenbogaard, J P; van de Waterbeemd, B; van Els, C A C M; van Eden, W; Hendriksen, C F M; Sloots, A; Han, W G H

    2016-08-17

    Whole cell Bordetella pertussis (wP) vaccines are still used in many countries to protect against the respiratory disease pertussis. The potency of whole-cell pertussis vaccine lots is determined by an intracerebral challenge test (the Kendrick test). This test is criticized due to lack of immunological relevance of the read-out after an intracerebral challenge with B. pertussis. The alternative in vivo test, which assesses specific antibody levels in serum after wP vaccination, is the Pertussis Serological Potency test (PSPT). Although the PSPT focuses on a parameter that contributes to protection, the protective immune mechanisms after wP vaccination includes more elements than specific antibody responses only. In this study, additional parameters were investigated, i.e. circulating pro-inflammatory cytokines, antibody specificity and T helper cell responses and it was evaluated whether they can be used as complementary readout parameters in the PSPT to assess wP lot quality. By deliberate manipulation of the vaccine preparation procedure, a panel of high, intermediate and low quality wP vaccines were made. The results revealed that these vaccines induced similar IL-6 and IP10 levels in serum 4h after vaccination (innate responses) and similar antibody levels directed against the entire bacterium. In contrast, the induced antibody specificity to distinct wP antigens differed after vaccination with high, intermediate and low quality wP vaccines. In addition, the magnitude of wP-induced Th cell responses (Th17, Th1 and Th2) was reduced after vaccination with a wP vaccine of low quality. T cell responses and antibody specificity are therefore correlates of qualitative differences in the investigated vaccines, while the current parameter of the PSPT alone was not sensitive enough to distinguish between vaccines of different qualities. This study demonstrates that assessment of the magnitude of Th cell responses and the antigen specificity of antibodies induced by w

  11. The photochemical and fluorescence properties of whole cells, spheroplasts and spheroplast particles from the blue-green alga Phormidium luridum.

    Science.gov (United States)

    Tel-or, E; Malkin, S

    1977-02-07

    The photochemical activities and fluorescence properties of cells, spheroplasts and spheroplast particles from the blue-green alga Phormidium luridum were compared. The photochemical activities were measured in a whole range of wavelengths and expressed as quantum yield spectra (quantum yield vs. wavelength). The following reactions were measured. Photosynthesis (O2 evolution) in whole cells; Hill reaction (O2 evolution) with Fe(CN)63- and NADP as electron acceptors (Photosystem II and photosystem II + Photosystem I reactions); electron transfer from reduced 2,6-dichlorophenolindophenol to diquat (Photosystem I reaction). The fluorescence properties were emission spectra, quantum yield spectra and the induction pattern. On the basis of comparison between the quantum yield spectra and the pigments compositions the relative contribution of each pigment to each photosystem was estimated. In normal cells and spheroplasts it was found that Photosystem I (Photosystem II) contains about 90% (10%) of the chlorophyll a, 90% (10%) of the carotenoids and 15% (85%) of the phycocyanin. In spheroplast particles there is a reorganization of the pigments; they loose a certain fraction (about half) of the phycocyanin but the remaining phycocyanin attaches itself exclusively to Photosystem I (!). This is reflected by the loss of Photosystem II activity, a flat quantum yield vs. wavelength dependence and a loss of the fluorescence induction. The fluorescence quantum yield spectra conform qualitatively to the above conclusion. More quantitative estimation shows that only a fraction (20--40%) of the chlorophyll of Photosystem II is fluorescent. Total emission spectrum and the ratio of variable to constant fluorescence are in agreement with this conclusion. The fluorescence emission spectrum shows characteristic differences between the constant and variable components. The variable fluorescence comes exclusively from chlorophyll a; the constant fluorescence is contributed, in addition

  12. 16S rDNA测序快速鉴定废水生物处理系统目标细菌%Use of 16S rDNA sequencing for quickly determining target bacteria in biological wastewater treatment system

    Institute of Scientific and Technical Information of China (English)

    李永峰; 任南琪; 杨传平; 陈瑛; 郑国香; 胡立杰

    2005-01-01

    在废水生物处理系统中建立分子生物学技术快速鉴定有关微生物,具有十分重要的意义.以发酵法生物制氢系统的活性污泥分离培养的厌氧发酵细菌为研究对象,采用16S rDNA碱基测序分子生物学技术,通过生物信息学数据库NCBI将DNA序列输入、比对,可以直接鉴定从废水生物处理系统中分离培养的细菌进行种属科的系统发育学地位的判定,从而简化了细菌鉴定程序.通过16S rDNA测序技术,进行了分离产氢细菌的分子生物学鉴定,发现生物制氢厌氧活性污泥中所分离的细菌可能存在的菌属有:Lactobacillus,Clostridium,Klebsiella,Propionibacterium和可能新发现的1个新属Biohydrogenbacterium等5个菌属的菌种,其中新属Biohydrogenbacterium的菌种都以利用碳水化合物生产氢气为其主要特征.16S rDNA测序技术为废水生物处理系统的细菌学研究提供了方便、准确和经济的技术基础.

  13. Bacterial 16S rDNA sequence analysis of Siberian tiger faecal flora%东北虎粪细菌区系的16S rRNA基因序列分析

    Institute of Scientific and Technical Information of China (English)

    图雅; 朱伟云; 陆承平

    2005-01-01

    为研究东北虎粪微生物区系建立了东北虎粪细菌的16S rDNA文库.通过EcoRⅠ和HindⅢ分别对阳性克隆进行酶切分析,从东北虎的16S rDNA文库中分别获得了15个具有酶切差异的克隆.BLAST分析结果显示,在15个克隆中,10个克隆与梭菌属成员有97%以上的同源性,其中有6个序列与诺维梭菌A型(Clostridium novyi type A)有99%的同源性,为诺维梭菌A型;4个序列与猪粪细菌RT-18B(Swine manure bacterium RT-18B)有97%的同源性,为消化链球菌属(Peptostreptococcus)成员.其它序列与GenBank中登录的序列同源性低于97%,为5种未培养细菌,其中4种16S rRNA基因序列分别与Clostridium pascui 、破伤风梭菌E88(Clostridium tetani E88)、梭菌(Clostridium sp.)14505及产气荚膜梭菌(Clostridiumperfringens)有94%~95%的相似性.第5种与肉杆菌(Carnobacterium sp.)R-7279株有94%的同源性.

  14. Bacterial diversity of soil under eucalyptus assessed by 16S rDNA sequencing analysis Diversidade bacteriana de solo sob eucaliptos obtida por seqüenciamento do 16S rDNA

    Directory of Open Access Journals (Sweden)

    Érico Leandro da Silveira

    2006-10-01

    Full Text Available Studies on the impact of Eucalyptus spp. on Brazilian soils have focused on soil chemical properties and isolating interesting microbial organisms. Few studies have focused on microbial diversity and ecology in Brazil due to limited coverage of traditional cultivation and isolation methods. Molecular microbial ecology methods based on PCR amplified 16S rDNA have enriched the knowledge of soils microbial biodiversity. The objective of this work was to compare and estimate the bacterial diversity of sympatric communities within soils from two areas, a native forest (NFA and an eucalyptus arboretum (EAA. PCR primers, whose target soil metagenomic 16S rDNA were used to amplify soil DNA, were cloned using pGEM-T and sequenced to determine bacterial diversity. From the NFA soil 134 clones were analyzed, while 116 clones were analyzed from the EAA soil samples. The sequences were compared with those online at the GenBank. Phylogenetic analyses revealed differences between the soil types and high diversity in both communities. Soil from the Eucalyptus spp. arboretum was found to have a greater bacterial diversity than the soil investigated from the native forest area.Estudos sobre impacto do Eucalyptus spp. em solos brasileiros têm focalizado propriedades químicas do solo e isolamento de microrganismos de interesse. No Brasil há pouco enfoque em ecologia e diversidade microbiana, devido às limitações dos métodos tradicionais de cultivo e isolamento. A utilização de métodos moleculares no estudo da ecologia microbiana baseados na amplificação por PCR do 16S rDNA têm enriquecido o conhecimento da biodiversidade microbiana dos solos. O objetivo deste trabalho foi comparar e estimar a diversidade bacteriana de comunidades simpátricas em solos de duas áreas: uma floresta nativa (NFA e outra adjacente com arboreto de eucaliptos (EAA. Oligonucleotídeos iniciadores foram utilizados para amplificar o 16S rDNA metagenômico do solo, o qual foi

  15. 基于16S rDNA序列和RFLP分析的病鳗分离菌株鉴定%Identification of Bacteria Isolated from Diseased Eels Using 16S rDNA PCR / RFLP Analysis

    Institute of Scientific and Technical Information of China (English)

    郭松林; 关瑞章; 冯建军; 杨求华

    2012-01-01

    Using 16S rDNA gene sequences and restriction fragment length polymorphism (RFLP) a- nalysis, the study initially identified 30 strains of bacteria isolated from European eel (Anguilla anguilla) , Japanese eel (Anguilla japonica) and American eel (Anguilla rostrata) according to the homology compari- son with bacterial 16S rDNA gene sequence which had been submitted into the C, eneBank database. The re- sults showed that the 30 bacteria could be broadly divided into 7 genera, including Aeromonas sp. , Pseudo- monas sp. , P/es/omonas sp. , Klebsh:lla sp. , Citrobacter sp. , Acinetobacter sp. and Enterobacter sp. Re- sults of the study indicated some of the isolates could be suspected as pathogenic strains which caused diseases to eels.%结合16S rDNA基因序列和限制性片段长度多态性(RFLP)的分析方法,通过与GenBank库中已递交的细菌16S rDNA基因序列进行同源性比较,对分离自发病鳗鲡(欧洲鳗鲡,日本鳗鲡和美洲鳗鲡)的30株细菌进行初步鉴定和分类.结果表明,这些细菌可大致分为气单胞菌属(Aeromonas sp.)、假单胞菌属(Pseudomonas sp.)、邻单胞菌属(Plesiomonas sp.)、克雷伯氏菌属(Klebsiella sp.)、柠檬酸杆菌属(Citrobacter sp.)、不动杆菌属(Acinetobacter sp.)和肠杆菌属(Enterobacter sp.)等7个菌属.分析认为,部分分离菌株可能是引起鳗鲡病害的疑似致病性菌株.

  16. 霍乱弧菌毒力基因检测与16S rRNA基因分型研究%Toxigene detection and 16S rRNA gene typing of Vibrio cholerae

    Institute of Scientific and Technical Information of China (English)

    张政; 朱水荣

    2006-01-01

    目的:了解浙江省霍乱弧菌毒力基因携带情况和16S rRNA基因型状况,为霍乱防治提供科学依据.方法:利用16S rRNA基因探针分析了浙江省不同时期分离的88株霍乱弧菌经Bgl Ⅰ消化的16S rRNA基因限制性酶谱,以多重PCR法对140株霍乱弧菌进行6种毒力相关基因(ctxA、rtxA、Ace、TcpA、Cri、Zot)检测分析.结果:发现各菌株的杂交片断范围为2~12 Kb,每个菌株有6~9条杂交带不等.88株霍乱菌株可分为9个16S rRNA基因型(ribotype,RT),其中埃尔托型霍乱弧菌(el tor vibrio cholerae,EVC)可分为6个RT,O139群霍乱弧菌(vibrio cholerae O139,VC O139)分为3个RT;所有VC O139菌株均携带3种以上毒力基因,86.3%的菌株携带全部6种毒力基因,所有EVC流行株均携带2种以上毒力基因,79.1%的菌株携带全部6种毒力基因.结论:认为霍乱流行菌株基因型的变迁可能是引起新一次流行的原因.结合毒素基因检测结果,我们认为VC O139与EVC在遗传特征上有相近的特点,但又有所区别.

  17. 不同刺槐种源根瘤菌16S rDNA的PCR-RFLP分析%PCR-RFLP Analysis of 16S rDNA of Rhizobia Isolated from Different Provenances of Robinia pseudoacacia

    Institute of Scientific and Technical Information of China (English)

    张泽坤; 韩骞; 杨敏生; 王进茂

    2012-01-01

    为了研究不同刺槐种源根瘤茵的遗传多样性,采用4种限制性内切酶对分离自保定和高邑的38株不同刺槐种源根瘤茵进行16S rDNA PCR-RFLP多态性分析,共产生26种16SrDNA遗传图谱类型.UPGMA聚类分析结果显示,所有供试菌株分为4大类,第3类又分为3个亚类.16S rDNA全序列分析表明,代表菌株GY-2与S.morelense LMG20571序列相似性达到99.6%,在系统发育分类地位上属于Sinorhizobium.刺槐根瘤菌遗传多样性丰富,遗传差异受地理环境影响较大,与寄主种源相关性不明显.%A study was performed to analyze the genetic diversity of rhizobia isolated from different provenances of Robinia pseud-oacacia. PCR-RFLP polymorphism analysis of 16S rDNA of 38 rhizobia grown in Baoding and Gaoyi was made with four restriction endonucleases. A total of 26 geneticmap patterns were generated from the tested strains. UPGMA cluster analysis indicates that all strains can be clustered into four groups, and the third group into three subgroups. 16S rDNA sequence analysis indicates that the representative strain GY-2 has a similarity level of 99. 6% with Sinorhizobium morelense LMG20571, and belongs to Sinorhizobium in the phylogenetic classification status. Results show that the rhizobia isolated from R. pseudoacacia reveal a high level of genetic diversity, and genetic differences are influenced by geographical environment, but its correlation with host provenance is not obvious.

  18. Immunization with lipopolysaccharide-deficient whole cells provides protective immunity in an experimental mouse model of Acinetobacter baumannii infection.

    Science.gov (United States)

    García-Quintanilla, Meritxell; Pulido, Marina R; Pachón, Jerónimo; McConnell, Michael J

    2014-01-01

    The increasing clinical importance of infections caused by multidrug resistant Acinetobacter baumannii warrants the development of novel approaches for prevention and treatment. In this context, vaccination of certain patient populations may contribute to reducing the morbidity and mortality caused by this pathogen. Vaccines against Gram-negative bacteria based on inactivated bacterial cells are highly immunogenic and have been shown to produce protective immunity against a number of bacterial species. However, the high endotoxin levels present in these vaccines due to the presence of lipopolysaccharide complicates their use in human vaccination. In the present study, we used a laboratory-derived strain of A. baumannii that completely lacks lipopolysaccharide due to a mutation in the lpxD gene (IB010), one of the genes involved in the first steps of lipopolysaccharide biosynthesis, for vaccination. We demonstrate that IB010 has greatly reduced endotoxin content (infection tissue bacterial loads and significantly lower serum levels of the pro-inflammatory cytokines IL-1β, TNF-α and IL-6 compared to control mice in a mouse model of disseminated A. baumannii infection. Importantly, immunized mice were protected from infection with the ATCC 19606 strain and an A. baumannii clinical isolate. These data suggest that immunization with inactivated A. baumannii whole cells deficient in lipopolysaccharide could serve as the basis for a vaccine for the prevention of infection caused by A. baumannii.

  19. In vitro cytokine profiles and viability of different human cells treated with whole cell lysate of Mycobacterium avium subsp. paratuberculosis

    Directory of Open Access Journals (Sweden)

    Rani Pittu

    2012-09-01

    Full Text Available Abstract Mycobacterium avium subsp. paratuberculosis (MAP is a zoonotic pathogen, a very slow growing bacterium which is difficult to isolate and passage in conventional laboratory culture. Although its association with Johne’s disease or paratuberculosis of cattle is well established, it has been only putatively linked to Crohn’s disease in humans. Further, MAP has been recently suggested to be a trigger for other autoimmune diseases such as type-1 diabetes mellitus (T1DM. Recently, some studies have indicated that exposure to MAP is associated with elevated levels of antibodies against MAP lysate although the exact mechanism and significance of the same remains unclear. Further, the cytokine profiles relevant in MAP associated diseases of humans and their exact role in the pathophysiology are not clearly known. We performed in vitro cytokine analyses after exposing different cultured human cells to the whole cell lysate of MAP and found that MAP lysate induces secretion of cytokines IL-1β, IL-6, IL-8, IL-10 and TNF-α by human peripheral blood mononuclear cells (PBMCs. Also, it induces secretion of IL-8 by cultured human stomach adenocarcinoma cells (AGS and PANC-1(human pancreatic carcinoma cell line cells. We also found that MAP lysate induced cytotoxicity in PANC-1cells. Collectively, these results provide a much needed base-line data set of cytokines broadly signifying a MAP induced cellular response by human cells.

  20. Highly efficient biodiesel production by a whole-cell biocatalyst employing a system with high lipase expression in Aspergillus oryzae.

    Science.gov (United States)

    Takaya, Tomohiro; Koda, Risa; Adachi, Daisuke; Nakashima, Kazunori; Wada, Junpei; Bogaki, Takayuki; Ogino, Chiaki; Kondo, Akihiko

    2011-05-01

    In the present study, a system with high lipase expression in Aspergillus oryzae was developed using an improved enolase promoter (P-enoA124) and the 5' untranslated region of a heat-shock protein (Hsp-UTR). P-enoA142 enhanced the transcriptional level of a heterologous lipase gene and Hsp-UTR improved its translational efficiency. Fusarium heterosporum lipase (FHL) was inserted into a pSENSU-FHL expression vector harboring P-enoA142 and Hsp-UTR and was transformed into an A. oryzae NS4 strain. Transformants possessing pSENSU-FHL in single (pSENSU-FHL#1) and double copies (pSENSU-FHL#2) were selected to evaluate the lipase activity of the whole-cell biocatalyst. The two strains, pSENSU-FHL#1 and #2, showed excellent lipase activity in hydrolysis compared with the strain transformed with conventional expression vector pNAN8142-FHL. Furthermore, by using pSENSU-FHL#2, methanolysis could proceed much more effectively without deactivation, which allowed a swift addition of methanol to the reaction mixture, thereby reducing reaction time.

  1. Single-channel and whole-cell recordings of mitogen-regulated inward currents in human cloned helper T lymphocytes.

    Science.gov (United States)

    Kuno, M; Goronzy, J; Weyand, C M; Gardner, P

    Cytoplasmic free Ca2+ [( Ca2+]i) appears to be an important signal for DNA synthesis in early stages of lymphocyte activation. In spite of many experimental studies which employ fluorescent Ca2+ indicator dye to demonstrate an early increase of [Ca2+]i in T-lymphocytes after stimulation with lectins, specific antigens, and monoclonal antibodies to T-lymphocyte receptors, the mechanism responsible for the rise of [Ca2+]i is unknown. We have used the extracellular patch clamp technique to investigate this mechanism. Unitary inward currents, mediated by Ca2+ or Ba2+, were recorded in the membrane of T-lymphocytes. The inward current channel was characterized by a conductance of 7 pS and extrapolated reversal potential (Erev) 110 mV positive to resting potential (Vr). While gating kinetic parameters were not affected by membrane potential changes, the probability of channel opening markedly increased upon activation of the T-lymphocyte by the mitogenic lectin, phytohaemagglutinin (PHA). PHA also evoked a cadmium-sensitive, inward Ba2+ current on whole-cell clamp. We suggest that this mitogen-regulated channel introduces Ca2+ into the cytoplasm upon activation and represents a new class of voltage-independent Ca2+ channels.

  2. Biotechnological production of fucosylated human milk oligosaccharides: Prokaryotic fucosyltransferases and their use in biocatalytic cascades or whole cell conversion systems.

    Science.gov (United States)

    Petschacher, Barbara; Nidetzky, Bernd

    2016-10-10

    Human milk oligosaccharides (HMOs) constitute a class of complex carbohydrates unique to mother's milk and are strongly correlated to the health benefits of breastfeeding in infants. HMOs are important as functional ingredients of advanced infant formula and have attracted broad interest for use in health-related human nutrition. About 50% of the HMOs structures contain l-fucosyl residues, which are introduced into nascent oligosaccharides by enzymatic transfer from GDP-l-fucose. To overcome limitation in the current availability of fucosylated HMOs, biotechnological approaches for their production have been developed. Functional expression of the fucosyltransferase(s) and effective supply of GDP-l-fucose, respectively, are both bottlenecks of the biocatalytic routes of synthesis. Strategies of in vitro and in vivo production of fucosylated HMOs are reviewed here. Besides metabolic engineering for enhanced HMO production in whole cells, the focus is on the characteristics and the heterologous overexpression of prokaryotic α1,2- and α1,3/4-fucosyltransferases. Up to 20g/L of fucosylated HMOs were obtained in optimized production systems. Optimized expression enabled recovery of purified fucosyltransferases in a yield of up to 45mg/L culture for α1,2-fucosyltransferases and of up to 200mg protein/L culture for α1,3/4-fucosyltransferases.

  3. Identification of protective pneumococcal T(H17 antigens from the soluble fraction of a killed whole cell vaccine.

    Directory of Open Access Journals (Sweden)

    Kristin L Moffitt

    Full Text Available Mucosal or parenteral immunization with a killed unencapsulated pneumococcal whole cell antigen (WCA with an adjuvant protects mice from colonization by a T(H17 CD4+ cell-mediated mechanism. Using preparative SDS gels, we separated the soluble proteins that compose the WCA in order to identify fractions that were immunogenic and protective. We screened these fractions for their ability to stimulate IL-17A secretion from splenocytes obtained from mice immunized with WCA and adjuvant. We identified 12 proteins within the stimulatory fractions by mass spectrometry; these proteins were then cloned, recombinantly expressed and purified using an Escherichia coli expression system. The ability of these proteins to induce IL-17A secretion was then evaluated by stimulation of mouse splenocytes. Of the four most stimulatory proteins, three were protective in a mouse pneumococcal serotype 6B colonization model. This work thus describes a method for identifying immunogenic proteins from the soluble fraction of pneumococcus and shows that several of the proteins identified protect mice from colonization when used as mucosal vaccines. We propose that, by providing protection against pneumococcal colonization, one or more of these proteins may serve as components of a multivalent pneumococcal vaccine.

  4. Efficient immobilization of mushroom tyrosinase utilizing whole cells from Agaricus bisporus and its application for degradation of bisphenol A.

    Science.gov (United States)

    Kampmann, Markus; Boll, Stefan; Kossuch, Jan; Bielecki, Julia; Uhl, Stefan; Kleiner, Beatrice; Wichmann, Rolf

    2014-06-15

    A simple and efficient procedure for preparation and immobilization of tyrosinase enzyme was developed utilizing whole cells from the edible mushroom Agaricus bisporus, without the need for enzyme purification. Tyrosinase activity in the cell preparation remained constant during storage at 21 °C for at least six months. The cells were entrapped in chitosan and alginate matrix capsules and characterized with respect to their resulting tyrosinase activity. A modification of the alginate with colloidal silica enhanced the activity due to retention of both cells and tyrosinase from fractured cells, which otherwise leached from matrix capsules. The observed activity was similar to the activity that was obtained with immobilized isolated tyrosinase in the same material. Mushroom cells in water were susceptible to rapid inactivation, whereas the immobilized cells maintained 73% of their initial activity after 30 days of storage in water. Application in repeated batch experiments resulted in almost 100% conversion of endocrine disrupting bisphenol A (BPA) for 11 days, under stirring conditions, and 50-60% conversion after 20 days, without stirring under continuous usage. The results represent the longest yet reported application of immobilized tyrosinase for degradation of BPA in environmental water samples.

  5. Whole-cell biocatalytic and de novo production of alkanes from free fatty acids in Saccharomyces cerevisiae.

    Science.gov (United States)

    Foo, Jee Loon; Susanto, Adelia Vicanatalita; Keasling, Jay D; Leong, Susanna Su Jan; Chang, Matthew Wook

    2017-01-01

    Rapid global industrialization in the past decades has led to extensive utilization of fossil fuels, which resulted in pressing environmental problems due to excessive carbon emission. This prompted increasing interest in developing advanced biofuels with higher energy density to substitute fossil fuels and bio-alkane has gained attention as an ideal drop-in fuel candidate. Production of alkanes in bacteria has been widely studied but studies on the utilization of the robust yeast host, Saccharomyces cerevisiae, for alkane biosynthesis have been lacking. In this proof-of-principle study, we present the unprecedented engineering of S. cerevisiae for conversion of free fatty acids to alkanes. A fatty acid α-dioxygenase from Oryza sativa (rice) was expressed in S. cerevisiae to transform C12-18 free fatty acids to C11-17 aldehydes. Co-expression of a cyanobacterial aldehyde deformylating oxygenase converted the aldehydes to the desired alkanes. We demonstrated the versatility of the pathway by performing whole-cell biocatalytic conversion of exogenous free fatty acid feedstocks into alkanes as well as introducing the pathway into a free fatty acid overproducer for de novo production of alkanes from simple sugar. The results from this work are anticipated to advance the development of yeast hosts for alkane production. Biotechnol. Bioeng. 2017;114: 232-237. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

  6. Reaction and strain engineering for improved stereo-selective whole-cell reduction of a bicyclic diketone.

    Science.gov (United States)

    Johanson, Ted; Carlquist, Magnus; Olsson, Cecilia; Rudolf, Andreas; Frejd, Torbjörn; Gorwa-Grauslund, Marie F

    2008-01-01

    Reduction of bicyclo[2.2.2]octane-2,6-dione to (1R, 4S, 6S)-6-hydroxy-bicyclo[2.2.2]octane-2-one by whole cells of Saccharomyces cerevisiae was improved using an engineered recombinant strain and process design. The substrate inhibition followed a Han-Levenspiel model showing an effective concentration window between 12 and 22 g/l, in which the activity was kept above 95%. Yeast growth stage, substrate concentration and a stable pH were shown to be important parameters for effective conversion. The over-expression of the reductase gene YDR368w significantly improved diastereoselectivity compared to previously reported results. Using strain TMB4110 expressing YDR368w in batch reduction with pH control, complete conversion of 40 g/l (290 mM) substrate was achieved with 97% diastereomeric excess (de) and >99 enantiomeric excess (ee), allowing isolation of the optically pure ketoalcohol in 84% yield.

  7. A numerical approach to ion channel modelling using whole-cell voltage-clamp recordings and a genetic algorithm.

    Directory of Open Access Journals (Sweden)

    Meron Gurkiewicz

    2007-08-01

    Full Text Available The activity of trans-membrane proteins such as ion channels is the essence of neuronal transmission. The currently most accurate method for determining ion channel kinetic mechanisms is single-channel recording and analysis. Yet, the limitations and complexities in interpreting single-channel recordings discourage many physiologists from using them. Here we show that a genetic search algorithm in combination with a gradient descent algorithm can be used to fit whole-cell voltage-clamp data to kinetic models with a high degree of accuracy. Previously, ion channel stimulation traces were analyzed one at a time, the results of these analyses being combined to produce a picture of channel kinetics. Here the entire set of traces from all stimulation protocols are analysed simultaneously. The algorithm was initially tested on simulated current traces produced by several Hodgkin-Huxley-like and Markov chain models of voltage-gated potassium and sodium channels. Currents were also produced by simulating levels of noise expected from actual patch recordings. Finally, the algorithm was used for finding the kinetic parameters of several voltage-gated sodium and potassium channels models by matching its results to data recorded from layer 5 pyramidal neurons of the rat cortex in the nucleated outside-out patch configuration. The minimization scheme gives electrophysiologists a tool for reproducing and simulating voltage-gated ion channel kinetics at the cellular level.

  8. Whole-cell biocatalytic production of variously substituted β-aryl- and β-heteroaryl-β-amino acids.

    Science.gov (United States)

    Ratnayake, Nishanka Dilini; Theisen, Chelsea; Walter, Tyler; Walker, Kevin D

    2016-01-10

    Biologically-active β-peptides and pharmaceuticals that contain key β-amino acids are emerging as target therapeutics; thus, synthetic strategies to make substituted, enantiopure β-amino acids are increasing. Here, we use whole-cell Escherichia coli (OD600 ∼ 35) engineered to express a Pantoea agglomerans phenylalanine aminomutase (PaPAM) biocatalyst. In either 5 mL, 100mL, or 1L of M9 minimal medium containing α-phenylalanine (20mM), the cells produced ∼ 1.4 mg mL(-1) of β-phenylalanine in each volume. Representative pilot-scale 5-mL cultures, fermentation reactions converted 18 variously substituted α-arylalanines to their (S)-β-aryl-β-amino acids in vivo and were not toxic to cells at mid- to late-stage growth. The β-aryl-β-amino acids made ranged from 0.043 mg (p-nitro-β-phenylalanine, 4% converted yield) to 1.2mg (m-bromo-β-phenylalanine, 96% converted yield) over 6h in 5 mL. The substituted β-amino acids made herein can be used in redox and Stille-coupling reactions to make synthetic building blocks, or as bioisosteres in drug design.

  9. In vitro innate immune cell based models to assess whole cell Bordetella pertussis vaccine quality: a proof of principle.

    Science.gov (United States)

    Hoonakker, M E; Verhagen, L M; Hendriksen, C F M; van Els, C A C M; Vandebriel, R J; Sloots, A; Han, W G H

    2015-03-01

    Lot release testing of vaccines is primarily based on animal models that are costly, time-consuming and sometimes of questionable relevance. In order to reduce animal use, functional in vitro assays are being explored as an alternative approach for the current lot release testing paradigm. In this study, we present an evaluation of APC platforms assessing innate immune activation by whole cell Bordetella pertussis (wP) vaccines. Primary monocytes, monocyte-derived DC (moDC) and human monocyte/DC cell lines (MonoMac6 and MUTZ-3) were compared for their capacity to respond to wP vaccines of varying quality. To produce such vaccines, the production process of wP was manipulated, resulting in wP vaccines covering a range of in vivo potencies. The responses of MUTZ-3 cells and primary monocytes to these vaccines were marginal and these models were therefore considered inappropriate. Importantly, moDC and MonoMac6 cells responded to the wP vaccines and discriminated between vaccines of varying quality, although slight variations in the responses to wP vaccines of similar quality were also observed. This study provides a proof of principle for the use of in vitro APC platforms as part of a new strategy to assess wP vaccine lot consistency, though careful standardisation of assay conditions is necessary.

  10. Applied catastrophic phase inversion: a continuous non-centrifugal phase separation step in biphasic whole-cell biocatalysis.

    Science.gov (United States)

    Glonke, Sebastian; Sadowski, Gabriele; Brandenbusch, Christoph

    2016-11-01

    Biphasic whole-cell biotransformations are known to be efficient alternatives to common chemical synthesis routes, especially for the production of, e.g. apolar enantiopure organic compounds. They provide high stereoselectivity combined with high product concentrations owing to the presence of an organic phase serving as substrate reservoir and product sink. Industrial implementation suffers from the formation of stable Pickering emulsions caused by the presence of cells. State-of-the-art downstream processing includes inefficient strategies such as excessive centrifugation, use of de-emulsifiers or thermal stress. In contrast, using the catastrophic phase inversion (CPI) phenomenon (sudden switch of emulsion type caused by addition of dispersed phase), Pickering-type emulsions can be destabilized efficiently. Within this work a model system using bis(2-ethylhexyl) phthalate (BEHP) as organic phase in combination with E. coli, JM101 was successfully separated using a continuous mixer settler setup. Compared to the state-of-the-art centrifugal separations, this process allows complete phase separation with no detectable water content or cells in the organic phase with no utilities/additives required. Furthermore, the concentration of the product is not affected by the separation. It is therefore a simple applicable method that can be used for separation of stable Pickering-type emulsions based on the knowledge of the point of inversion.

  11. Biodegradation of methyl parathion by whole cells of marine-derived fungi Aspergillus sydowii and Penicillium decaturense.

    Science.gov (United States)

    Alvarenga, Natália; Birolli, Willian G; Seleghim, Mirna H R; Porto, André L M

    2014-12-01

    Seven marine fungi strains (Aspergillus sydowii CBMAI 934, A. sydowii CBMAI 935, A. sydowii CBMAI 1241, Penicillium decaturense CBMAI 1234, Penicillium raistrickii CBMAI 931, P. raistrickii CBMAI 1235, and Trichoderma sp. CBMAI 932) were screened by their growth in the presence of methyl parathion (MP) in a solid culture medium. The strains with best growth were A. sydowii CBMAI 935 and P. decaturense CBMAI 1234. Biodegradation reactions were performed in 10, 20 and 30d in a malt extract liquid medium containing commercial MP and whole cells of A. sydowii CBMAI 935 and P. decaturense CBMAI 1234. In 20d, A. sydowii CBMAI 935 was able to degrade all pesticide, whereas P. decaturense CBMAI 1234 promoted a complete degradation in 30d. A. sydowii CBMAI 935 and P. decaturense CBMAI 1234 could degrade the product of the MP enzymatic hydrolysis, p-nitrophenol, on average of 51 and 40% respectively. Both strains used MP as a sole source of carbon and provided satisfactory results. Metabolites detected in the medium showed that the presumable reaction pathway occurred through the activation of MP to its more toxic form, methyl paraoxon, which was further degraded to p-nitrophenol.

  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. Highly efficient biodiesel production by a whole-cell biocatalyst employing a system with high lipase expression in Aspergillus oryzae

    Energy Technology Data Exchange (ETDEWEB)

    Takaya, Tomohiro; Koda, Risa; Adachi, Daisuke; Ogino, Chiaki; Kondo, Akihiko [Kobe Univ. (Japan). Dept. of Chemical Science and Engineering; Nakashima, Kazunori [Kobe Univ. (Japan). Organization of Advanced Science and Technology; Wada, Junpei; Bogaki, Takayuki [Ozeki Co., Nishinomiya-shi, Hyogo (Japan)

    2011-05-15

    In the present study, a system with high lipase expression in Aspergillus oryzae was developed using an improved enolase promoter (P-enoA124) and the 5{sup '} untranslated region of a heat-shock protein (Hsp-UTR). P-enoA142 enhanced the transcriptional level of a heterologous lipase gene and Hsp-UTR improved its translational efficiency. Fusarium heterosporum lipase (FHL) was inserted into a pSENSU-FHL expression vector harboring P-enoA142 and Hsp-UTR and was transformed into an A. oryzae NS4 strain. Transformants possessing pSENSU-FHL in single (pSENSU-FHL1) and double copies (pSENSU-FHL2) were selected to evaluate the lipase activity of the whole-cell biocatalyst. The two strains, pSENSU-FHL1 and 2, showed excellent lipase activity in hydrolysis compared with the strain transformed with conventional expression vector pNAN8142-FHL. Furthermore, by using pSENSU-FHL2, methanolysis could proceed much more effectively without deactivation, which allowed a swift addition of methanol to the reaction mixture, thereby reducing reaction time. (orig.)

  14. Construction of an extended range whole-cell tetracycline biosensor by use of the tet(M) resistance gene

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Hansen, Lars Hestbjerg; Sørensen, Søren Johannes

    2005-01-01

    An extended range whole-cell tetracycline biosensor strain was constructed by insertion of the tet(M) gene, encoding tetracycline resistance by ribosomal protection, into plasmid pTGFP2, which contains a transcriptional fusion between a tetracycline regulated promoter and the green fluorescent pr...

  15. Intracellular concentration map of magnesium in whole cells by combined use of X-ray fluorescence microscopy and atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Lagomarsino, Stefano, E-mail: stefano.lagomarsino@cnr.it [IPCF-CNR -UOS Roma c/o Dip Fisica Universita' ' Sapienza' , P.le A. Moro, 2 Rome (Italy); Physics Department, Universita' Sapienza, P.le A. Moro, 2 Rome (Italy); Iotti, Stefano [Dipartimento di Medicina Interna, dell' Invecchiamento e Malattie Nefrologiche Universita di Bologna, Via Massarenti, 9 40138 Bologna (Italy); Istituto Nazionale Biostrutture e Biosistemi - Rome (Italy); Farruggia, Giovanna [Dipartimento di Biochimica ' G. Moruzzi' Universita di Bologna, Via Irnerio, 48 40126 Bologna (Italy); Cedola, Alessia [IFN-CNR - V. Cineto Romano, 42 00156 Rome (Italy); Trapani, Valentina [Istituto di Patologia Generale - Universita Cattolica del Sacro Cuore - Facolta di Medicina ' A. Gemelli' L.go F. Vito, 1 00168 Rome (Italy); Fratini, Michela [IFN-CNR - V. Cineto Romano, 42 00156 Rome (Italy); Bukreeva, Inna [IFN-CNR - V. Cineto Romano, 42 00156 Rome (Italy); Shubnikov Institute of Crystallography, Leninskii prospekt 59, Moscow, 119333 (Russian Federation); Notargiacomo, Andrea [IFN-CNR - V. Cineto Romano, 42 00156 Rome (Italy); Mastrototaro, Lucia [Istituto di Patologia Generale - Universita Cattolica del Sacro Cuore - Facolta di Medicina ' A. Gemelli' L.go F. Vito, 1 00168 Rome (Italy); Marraccini, Chiara [Dipartimento di Medicina Interna, dell' Invecchiamento e Malattie Nefrologiche Universita di Bologna, Via Massarenti, 9 40138 Bologna (Italy); and others

    2011-11-15

    We report a novel experimental approach to derive quantitative concentration map of light elements in whole cells by combining two complementary nano-probe methods: X-ray fluorescence microscopy (XRFM) and atomic force microscopy (AFM). The concentration is derived by normalizing point-by-point the elemental (here Mg) spatial distribution obtained by XRFM, by the thickness measured using AFM. The considerable difference between the elemental distribution and the concentration maps indicates that this procedure is essential to obtain reliable information on the role and function of elements in whole cells. - Highlights: Black-Right-Pointing-Pointer X-ray fluorescence and AFM have been measured on the same de-hydrated whole cells. Black-Right-Pointing-Pointer The element distribution has been normalized point-by-point by the cell thickness. Black-Right-Pointing-Pointer The element (Mg) concentration map has been obtained on a whole cell. Black-Right-Pointing-Pointer The element concentration map is quite different from the distribution map. Black-Right-Pointing-Pointer Higher Mg concentration is found in the cell periphery.

  16. A robust whole-cell biocatalyst that introduces a thermo- and solvent-tolerant lipase into Aspergillus oryzae cells: characterization and application to enzymatic biodiesel production.

    Science.gov (United States)

    Adachi, Daisuke; Koh, FookHee; Hama, Shinji; Ogino, Chiaki; Kondo, Akihiko

    2013-05-10

    To develop a robust whole-cell biocatalyst that works well at moderately high temperature (40-50°C) with organic solvents, a thermostable lipase from Geobacillus thermocatenulatus (BTL2) was introduced into an Aspergillus oryzae whole-cell biocatalyst. The lipase-hydrolytic activity of the immobilized A. oryzae (r-BTL) was highest at 50°C and was maintained even after an incubation of 24-h at 60°C. In addition, r-BTL was highly tolerant to 30% (v/v) organic solvents (dimethyl carbonate, ethanol, methanol, 2-propanol or acetone). The attractive characteristics of r-BTL also worked efficiently on palm oil methanolysis, resulting in a nearly 100% conversion at elevated temperature from 40 to 50°C. Moreover, r-BTL catalyzed methanolysis at a high methanol concentration without a significant loss of lipase activity. In particular, when 2 molar equivalents of methanol were added 2 times, a methyl ester content of more than 90% was achieved; the yield was higher than those of conventional whole-cell biocatalyst and commercial Candida antarctica lipase (Novozym 435). On the basis of the results regarding the excellent lipase characteristics and efficient biodiesel production, the developed whole-cell biocatalyst would be a promising biocatalyst in a broad range of applications including biodiesel production.

  17. Safety and Immunogenicity of an Inactivated Whole Cell Plus Recombinant B Subunit (WC/RBS) Cholera Vaccine in Healthy Adult Peruvian Military Volunteers.

    Science.gov (United States)

    1992-11-30

    AD-A260 586 IFB0 919931 MIPR NO: 92MM2532W TITLE: SAFETY AND IMMUNOGENICITY OF AN INACTIVATED WHOLE CELL PLUS RECOMBINANT B SUBUNIT (WCIRBS) COLERA ...NUMBERS Safety and Immunogenicity of an Inactivated Whole MIPR No. Cell Plus Recombinant B Subunit (WC/RBS) Colera 92MM2532 Vaccine in Healthy Adult

  18. Different IgG-subclass distributions after whole-cell and acellular pertussis infant primary vaccinations in healthy and pertussis infected children

    NARCIS (Netherlands)

    Hendrikx, Lotte H.; Schure, Rose-Minke; Ozturk, Kemal; de Rond, Lia G. H.; de Greeff, S. C.; Sanders, Elisabeth A. M.; Berbers, Guy A. M.; Buisman, Anne-Marie

    2011-01-01

    The distribution of IgG-subclasses provides insight in the immunological mechanisms of protection against whooping cough. We investigated the effect of Dutch whole-cell pertussis and acellular pertussis vaccines administered in infancy on the IgG-subclass distributions in healthy children aged 12 mo

  19. Enzymatic oxidation of cephalosporin C using whole cells of the yeast Triginopsis variabilis within a "cross-flow filter-reactor".

    Science.gov (United States)

    Vicenzi, J T; Hansen, G J

    1993-04-01

    An economical process for the enzymatic oxidation of cephalosporin C to glutaryl-7-ACA was developed at a pilot plant scale. The process utilized nonviable whole cells of the yeast Triginopsis variabilis containing high levels of D-amino acid oxidase. Prior to use, the whole cells were permeabilized with a 25% acetone/water solution which enhanced their apparent activity by 20- to 50-fold. After permeabilization, the whole cells were incubated at pH 11, which served to selectively deactivate catalase which was present in very large quantities. Deactivation of catalase was critical to achieving high reaction yields. The whole cells were utilized within a "cross-flow filter-reactor" which allowed easy and economical recycle of the cells for repeated use. The overall yield of glutaryl-7-ACA from cephalosporin C was 90-95%. The overall productivity of the yeast was 13 kg cephalosporin C oxidized per kilogram yeast (dry basis). The reaction was run at a concentration of 40 g cephalosporin CL-1 and the overall reactor productivity was 11 g glutaryl-7-ACA l-1 h-1. The process has been thoroughly demonstrated on a 35-l scale, and it should be directly scaleable to 10,000 l or more.

  20. Use of a Whole-Cell Biosensor and Flow Cytometry to Detect AHL Production by an Indigenous Soil Community During Decomposition

    DEFF Research Database (Denmark)

    Burmølle, Mette; Hansen, Lars Hestbjerg; Sørensen, Søren Johannes

    2005-01-01

    Quorum sensing, mediated by acylated homoserine lactones (AHLs), is well described for pure culture bacteria, but few studies report detection of AHL compounds in natural bacterial habitats. In this study, we detect AHL production during a degradation process in soil by use of whole-cell biosensor...

  1. Acute necrotizing encephalopathy secondary to diphtheria, tetanus toxoid and whole-cell pertussis vaccination: diffusion-weighted imaging and proton MR spectroscopy findings

    Energy Technology Data Exchange (ETDEWEB)

    Aydin, Hale; Ozgul, Esra; Agildere, Ahmet Muhtesem [Baskent University Hospital, Department of Radiology, Ankara (Turkey)

    2010-07-15

    We present a previously healthy 6-month-old boy who was admitted to our hospital with lethargy, hypotonia and focal clonic seizures 6 days following diptheria, tetanus toxoid and whole-cell pertussis vaccination. A diagnosis of acute necrotising encephalopathy was made with the aid of MRI, including diffusion-weighted imaging and proton MR spectroscopy. (orig.)

  2. Lyme disease caused by Borrelia burgdorferi with two homeologous 16S rRNA genes: a case report

    Directory of Open Access Journals (Sweden)

    Lee SH

    2016-04-01

    Full Text Available Sin Hang Lee,1,21Pathology Department, Milford Hospital, Milford, CT, USA; 2Milford Molecular Diagnostics, Milford, CT, USA Abstract: Lyme disease (LD, the most common tick-borne disease in North America, is believed to be caused exclusively by Borrelia burgdorferi sensu stricto and is usually diagnosed by clinical evaluation and serologic assays. As reported previously in a peer-reviewed article, a 13-year-old boy living in the Northeast of the USA was initially diagnosed with LD based on evaluation of his clinical presentations and on serologic test results. The patient was treated with a course of oral doxycycline for 28 days, and the symptoms resolved. A year later, the boy developed a series of unusual symptoms and did not attend school for 1 year. A LD specialist reviewed the case and found the serologic test band patterns nondiagnostic of LD. The boy was admitted to a psychiatric hospital. After discharge from the psychiatric hospital, a polymerase chain reaction test performed in a winter month when the boy was 16 years old showed a low density of B. burgdorferi sensu lato in the blood of the patient, confirmed by partial 16S rRNA (ribosomal RNA gene sequencing. Subsequent DNA sequencing analysis presented in this report demonstrated that the spirochete isolate was a novel strain of B. burgdorferi with two homeologous 16S rRNA genes, which has never been reported in the world literature. This case report shows that direct DNA sequencing is a valuable tool for reliable molecular diagnosis of Lyme and related borrelioses, as well as for studies of the diversity of the causative agents of LD because LD patients infected by a rare or novel borrelial variant may produce an antibody pattern that can be different from the pattern characteristic of an infection caused by a typical B. burgdorferi sensu stricto strain. Keywords: Lyme disease, Borrelia burgdorferi, homeologous 16S rRNA genes, DNA sequencing

  3. Cilantro microbiome before and after nonselective pre-enrichment for Salmonella using 16S rRNA and metagenomic sequencing.

    Science.gov (United States)

    Jarvis, Karen G; White, James R; Grim, Christopher J; Ewing, Laura; Ottesen, Andrea R; Beaubrun, Junia Jean-Gilles; Pettengill, James B; Brown, Eric; Hanes, Darcy E

    2015-08-12

    Salmonella enterica is a common cause of foodborne gastroenteritis in the United States and is associated with outbreaks in fresh produce such as cilantro. Salmonella culture-based detection methods are complex and time consuming, and improvments to increase detection sensitivity will benefit consumers. In this study, we used 16S rRNA sequencing to determine the microbiome of cilantro. We also investigated changes to the microbial community prior to and after a 24-hour nonselective pre-enrichment culture step commonly used by laboratory analysts to resuscitate microorganisms in foods suspected of contamination with pathogens. Cilantro samples were processed for Salmonella detection according to the method in the United States Food and Drug Administration Bacteriological Analytical Manual. Genomic DNA was extracted from culture supernatants prior to and after a 24-hour nonselective pre-enrichment step and 454 pyrosequencing was performed on 16S rRNA amplicon libraries. A database of Enterobacteriaceae 16S rRNA sequences was created, and used to screen the libraries for Salmonella, as some samples were known to be culture positive. Additionally, culture positive cilantro samples were examined for the presence of Salmonella using shotgun metagenomics on the Illumina MiSeq. Time zero uncultured samples had an abundance of Proteobacteria while the 24-hour enriched samples were composed mostly of Gram-positive Firmicutes. Shotgun metagenomic sequencing of Salmonella culture positive cilantro samples revealed variable degrees of Salmonella contamination among the sequenced samples. Our cilantro study demonstrates the use of high-throughput sequencing to reveal the microbiome of cilantro, and how the microbiome changes during the culture-based protocols employed by food safety laboratories to detect foodborne pathogens. Finding that culturing the cilantro shifts the microbiome to a predominance of Firmicutes suggests that changing our culture-based methods will improve

  4. Diversity of thermophiles in a Malaysian hot spring determined using 16S rRNA and shotgun metagenome sequencing

    Directory of Open Access Journals (Sweden)

    Chia Sing eChan

    2015-03-01

    Full Text Available The Sungai Klah (SK hot spring is the second hottest geothermal spring in Malaysia. This hot spring is a shallow, 150-meter-long, fast-flowing stream, with temperatures varying from 50 to 110°C and a pH range of 7.0 to 9.0. Hidden within a wooded area, the SK hot spring is continually fed by plant litter, resulting in a relatively high degree of total organic content (TOC. In this study, a sample taken from the middle of the stream was analyzed at the 16S rRNA V3−V4 region by amplicon metagenome sequencing. Over 35 phyla were detected by analyzing the 16S rRNA data. Firmicutes and Proteobacteria represented approximately 57% of the microbiome. Approximately 70% of the detected thermophiles were strict anaerobes; however, Hydrogenobacter spp., obligate chemolithotrophic thermophiles, represented one of the major taxa. Several thermophilic photosynthetic microorganisms and acidothermophiles were also detected. Most of the phyla identified by 16S rRNA were also found using the shotgun metagenome approaches. The carbon, sulfur, and nitrogen metabolism within the SK hot spring community were evaluated by shotgun metagenome sequencing, and the data revealed diversity in terms of metabolic activity and dynamics. This hot spring has a rich diversified phylogenetic community partly due to its natural environment (plant litter, high TOC, and a shallow stream and geochemical parameters (broad temperature and pH range. It is speculated that symbiotic relationships occur between the members of the community.

  5. Microbial Dark Matter: Unusual intervening sequences in 16S rRNA genes of candidate phyla from the deep subsurface

    Energy Technology Data Exchange (ETDEWEB)

    Jarett, Jessica; Stepanauskas, Ramunas; Kieft, Thomas; Onstott, Tullis; Woyke, Tanja

    2014-03-17

    The Microbial Dark Matter project has sequenced genomes from over 200 single cells from candidate phyla, greatly expanding our knowledge of the ecology, inferred metabolism, and evolution of these widely distributed, yet poorly understood lineages. The second phase of this project aims to sequence an additional 800 single cells from known as well as potentially novel candidate phyla derived from a variety of environments. In order to identify whole genome amplified single cells, screening based on phylogenetic placement of 16S rRNA gene sequences is being conducted. Briefly, derived 16S rRNA gene sequences are aligned to a custom version of the Greengenes reference database and added to a reference tree in ARB using parsimony. In multiple samples from deep subsurface habitats but not from other habitats, a large number of sequences proved difficult to align and therefore to place in the tree. Based on comparisons to reference sequences and structural alignments using SSU-ALIGN, many of these ?difficult? sequences appear to originate from candidate phyla, and contain intervening sequences (IVSs) within the 16S rRNA genes. These IVSs are short (39 - 79 nt) and do not appear to be self-splicing or to contain open reading frames. IVSs were found in the loop regions of stem-loop structures in several different taxonomic groups. Phylogenetic placement of sequences is strongly affected by IVSs; two out of three groups investigated were classified as different phyla after their removal. Based on data from samples screened in this project, IVSs appear to be more common in microbes occurring in deep subsurface habitats, although the reasons for this remain elusive.

  6. Detection of transient bacteraemia following dental extractions by 16S rDNA pyrosequencing: a pilot study.

    Directory of Open Access Journals (Sweden)

    Alfonso Benítez-Páez

    Full Text Available OBJECTIVE: The current manuscript aims to determine the prevalence, duration and bacterial diversity of bacteraemia following dental extractions using conventional culture-dependent methods and 16S rDNA pyrosequencing. METHODS: The study group included 8 patients undergoing dental extractions under general anaesthesia. Peripheral venous blood samples were collected at baseline, 30 seconds and 15 minutes after the dental extractions. Blood samples were analysed for bacteraemia applying conventional microbiological cultures under aerobic and anaerobic conditions as well as pyrosequencing using universal bacterial primers that target the 16S ribosomal DNA gene. RESULTS: Transient bacteremia was detected by culture-based methods in one sample at baseline time, in eight samples at 30 seconds, and in six samples at 15 minutes after surgical procedure; whereas bacteraemia was detected only in five blood samples at 30 seconds after dental extraction by using pyrosequencing. By applying conventional microbiological methods, a single microbial species was detected in six patients, and Streptococcus viridans was the most frequently cultured identified bacterium. By using pyrosequencing approaches however, the estimated blood microbial diversity after dental extractions was 13.4±1.7 bacterial families and 22.8±1.1 genera per sample. CONCLUSION: The application of 16S rDNA pyrosequencing underestimated the prevalence and duration of bacteraemia following dental extractions, presumably due to not reaching the minimum DNA required for PCR amplification. However, this molecular technique, unlike conventional culture-dependent methods, revealed an extraordinarily high bacterial diversity of post-extraction bacteraemia. We propose that microorganisms recovered by culture may be only the tip of an iceberg of a really diverse microbiota whose viability and potential pathogenicity should be further studied.

  7. Distribution, hosts, 16S rDNA sequences and phylogenetic position of the Neotropical tick Amblyomma parvum (Acari: Ixodidae).

    Science.gov (United States)

    Nava, S; Szabó, M P J; Mangold, A J; Guglielmone, A A

    2008-07-01

    The hosts, distribution, intraspecific genetic variation and phylogenetic position of Amblyomma parvum (Acari: Ixodidae) have recently been re-assessed. Data on this tick's hosts and distribution were obtained not only from existing literature but also from unpublished records. Sequences of the ticks' mitochondrial 16S ribosomal DNA (rDNA) were used to evaluate genetic variation among specimens of A. parvum from different localities in Argentina and Brazil, and to explore the phylogenetic relationships between this tick and other Amblyomma species. Although several species of domestic and wild mammal act as hosts for adult A. parvum, most collected adults of this species have come from cattle and goats. Caviid rodents of the subfamily Caviinae appear to be the hosts for the immature stages. So far, A. parvum has been detected in 12 Neotropical biogeographical provinces (Chaco, Cerrado, Eastern Central America, Venezuelan Coast, Pantanal, Parana Forest, Caatinga, Chiapas, Venezuelan Llanos, Monte, Western Panamanian Isthmus, and Roraima) but the Chaco province has provided significantly more specimens than any other (P<0.0001). The 16S rDNA sequences showed just 0.0%-1.1% divergence among the Argentinean A. parvum investigated and no more than 0.2% divergence among the Brazilian specimens. The observed divergence between the Argentinean and Brazilian specimens was, however, greater (3.0%-3.7%). Although there is now molecular and morphological evidence to indicate that A. parvum, A. pseudoparvum, A. auricularium and A. pseudoconcolor are members of a natural group, previous subgeneric classifications do not reflect this grouping. The subgeneric status of these tick species therefore needs to be re-evaluated. The 16S-rDNA-based evaluation of divergence indicates that the gene flow between Argentinean and Brazilian 'A. parvum' is very limited and that the Argentinean 'A. parvum' may be a different species to the Brazilian.

  8. Heterogeneity within the gram-positive anaerobic cocci demonstrated by analysis of 16S-23S intergenic ribosomal RNA polymorphisms.

    Science.gov (United States)

    Hill, K E; Davies, C E; Wilson, M J; Stephens, P; Lewis, M A O; Hall, V; Brazier, J; Thomas, D W

    2002-11-01

    Peptostreptococci are gram-positive, strictly anaerobic bacteria which, although regarded as members of the commensal human microflora, are also frequently isolated from sites of clinical infection. The study of this diverse group of opportunist pathogens has been hindered by an inadequate taxonomy and the lack of a valid identification scheme. Recent re-classification of the Peptostreptococcus family into five distinct genus groups has helped to clarify the situation. However, this has been on the basis of 16S rRNA sequence determinations, which are both time-consuming and expensive. The aim of the present study was to evaluate the use of PCR-amplified ribosomal DNA spacer polymorphisms for the rapid differentiation of the currently recognised taxa within the group of anaerobic gram-positive cocci. A collection comprising 19 reference strains with representatives of each of the 15 species, two close relatives and two of the well-characterised groups, together with 38 test strains was studied. All strains were identified to species group level by phenotypic means. Amplification of the 16S-23S intergenic spacer region (ISR) with universal primers produced distinct banding patterns for all the 19 reference strains and the patterns could be differentiated easily visually. However, of the 38 test strains, less than half could be speciated from ISR analysis alone. Only five groups produced correlating banding patterns for all members tested (Peptoniphilus lacrimalis, P. ivorii, Anaerococcus octavius, Peptostreptococcus anaerobius and Micromonas micros). For other species, either the type strain differed significantly from other species members (e.g., A. hydrogenalis) or there appeared to be considerable intra-species variation (e.g., A. vaginalis). Partial 16S rRNA gene sequences for the 'trisimilis' and 'betaGAL' groups showed that both are most closely related to the Anaerococcus group. This work highlights the heterogeneous nature of a number of Peptostreptococcus

  9. Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system.

    Science.gov (United States)

    Schloss, Patrick D; Jenior, Matthew L; Koumpouras, Charles C; Westcott, Sarah L; Highlander, Sarah K

    2016-01-01

    Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina's MiSeq, have allowed researchers to obtain millions of high quality but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3-V5, V1-V3, V1-V5, V1-V6, and V1-V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1-V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina's MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.

  10. Multicenter quality assessment of 16S ribosomal DNA-sequencing for microbiome analyses reveals high inter-center variability.

    Science.gov (United States)

    Hiergeist, Andreas; Reischl, Udo; Gessner, Andrè

    2016-08-01

    The composition of human as well as animal microbiota has increasingly gained in interest since metabolites and structural components of endogenous microorganisms fundamentally influence all aspects of host physiology. Since many of the bacteria are still unculturable, molecular techniques such as high-throughput sequencing have dramatically increased our knowledge of microbial communities. The majority of microbiome studies published thus far are based on bacterial 16S ribosomal RNA (rRNA) gene sequencing, so that they can, at least in principle, be compared to determine the role of the microbiome composition for host metabolism and physiology, developmental processes, as well as different diseases. However, differences in DNA preparation and purification, 16S rDNA PCR amplification, sequencing procedures and platforms, as well as bioinformatic analysis and quality control measures may strongly affect the microbiome composition results obtained in different laboratories. To systematically evaluate the comparability of results and identify the most influential methodological factors affecting these differences, identical human stool sample replicates spiked with quantified marker bacteria, and their subsequent DNA sequences were analyzed by nine different centers in an external quality assessment (EQA). While high intra-center reproducibility was observed in repetitive tests, significant inter-center differences of reported microbiota composition were obtained. All steps of the complex analysis workflow significantly influenced microbiome profiles, but the magnitude of variation caused by PCR primers for 16S rDNA amplification was clearly the largest. In order to advance microbiome research to a more standardized and routine medical diagnostic procedure, it is essential to establish uniform standard operating procedures throughout laboratories and to initiate regular proficiency testing.

  11. Using DGGE and 16S rRNA gene sequence analysis to evaluate changes in oral bacterial composition.

    Science.gov (United States)

    Chen, Zhou; Trivedi, Harsh M; Chhun, Nok; Barnes, Virginia M; Saxena, Deepak; Xu, Tao; Li, Yihong

    2011-01-01

    To investigate whether a standard dental prophylaxis followed by tooth brushing with an antibacterial dentifrice will affect the oral bacterial community, as determined by denaturing gradient gel electrophoresis (DGGE) combined with 16S rRNA gene sequence analysis. Twenty-four healthy adults were instructed to brush their teeth using commercial dentifrice for 1 week during a washout period. An initial set of pooled supragingival plaque samples was collected from each participant at baseline (0 h) before prophylaxis treatment. The subjects were given a clinical examination and dental prophylaxis and asked to brush for 1 min with a dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride (Colgate Total). On the following day, a second set of pooled supragingival plaque samples (24 h) was collected. Total bacterial genomic DNA was isolated from the samples. Differences in the microbial composition before and after the prophylactic procedure and tooth brushing were assessed by comparing the DGGE profiles and 16S rRNA gene segments sequence analysis. Two distinct clusters of DGGE profiles were found, suggesting that a shift in the microbial composition had occurred 24 h after the prophylaxis and brushing. A detailed sequencing analysis of 16S rRNA gene segments further identified 6 phyla and 29 genera, including known and unknown bacterial species. Importantly, an increase in bacterial diversity was observed after 24 h, including members of the Streptococcaceae family, Prevotella, Corynebacterium, TM7 and other commensal bacteria. The results suggest that the use of a standard prophylaxis followed by the use of the dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride may promote a healthier composition within the oral bacterial community.

  12. Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system

    Directory of Open Access Journals (Sweden)

    Patrick D. Schloss

    2016-03-01

    Full Text Available Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina’s MiSeq, have allowed researchers to obtain millions of high quality but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3–V5, V1–V3, V1–V5, V1–V6, and V1–V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1–V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina’s MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.

  13. 16S rRNA Gene Sequence Analysis and Fermentation Products Identification of Amycolatopsis sp.FJNU1011%Amycolatopsis sp. FJNU1011的16S rRNA基因序列分析和发酵产物鉴定

    Institute of Scientific and Technical Information of China (English)

    李力; 刘小玲; 黄连琴; 黄建忠

    2013-01-01

    从土壤中分离1株具有抗革兰氏阳性菌和抗肿瘤活性的放线菌,其16S rRNA的序列聚类分析结果表明该菌可能是拟无枝酸菌属中的一个新种,定命为Amycolatopsis sp.FJNU1011.通过液相色谱质谱联用仪对其发酵提取物进行分析,发现不仅含有5种抗肿瘤作用kigamicins的组分,并且含有新的kigamicins结构类似物.%A strain isolated from soil has the inhibitory activities against G + bacterial and tumor. The sequence analysis of 16S rRNA gene showed that the strain is a species of amycolatop sis, named Amycolatopsis sp. FJNU1011. With the analysis by HPLC-MS, Amycolatopsis sp. FJNU1011 can produce not only kigamicins, but also one new uncharacterized analogue of kigam icins.

  14. 16s rDNA文库法分析番石榴果实蝇共生菌组成%Composition of symbiotic bacteria associated with Bactrocera correcta analyzed by 16s rDNA libraries

    Institute of Scientific and Technical Information of China (English)

    戴阳; 李志红; 柳丽君; 吴佳教; 邓裕亮

    2012-01-01

    [Objective] The composition of symbiotic bacteria associated with Guava fruit fly, Bacirocera correcta (Bezzi), was investigated to provide the basis for further study on physiological function of the symbionts and co-evolution between bacteria and the host. [Method] 16s rDNA libraries of laboratory population for B. correcta were constructed, and BLAST analysis of bacterial composition and abundance was carried out in this study. [Result] The dominant symbionts in B. correcta male adults were Pseudomonas aeruginosa and Lactococcus lactis, while symbiotic bacteria in female adults were Enterobacter bacteria. [Conclusion] Various bacteria are present in the Guava fruit fly, B. correcta.%[目的]探索分析番石榴果实蝇[ Bactrocera correcta (Bezzi)]的共生菌组成,为进一步探索共生菌的生理功能以及与宿主的协同进化关系奠定基础.[方法]构建番石榴果实蝇室内种群共生菌的16s rDNA文库,BLAST分析其共生菌的组成以及丰度.[结果]室内雄虫共生菌主要为Pseudomonas aeruginosa和Lactococcus lactis,雌虫共生菌主要为Enterobacter属的细菌.[结论]本研究结果证明番石榴果实蝇存在多种共生菌.

  15. Research on the PCR Amplication to 16S rRNA Gene of Oil Microorganisms%石油微生物16S rRNA基因PCR扩增研究

    Institute of Scientific and Technical Information of China (English)

    卞立红; 曲丽娜; 汪洋; 张虹; 黄永红

    2010-01-01

    掌握石油微生物的16S rRNA基因保守区的扩增方法是先进分子水平鉴定微生物种类一种有效的实验方法.通过研究利用分子生物技术提高对石油微生物的了解,进行未来对石油的开采,摸索出石油微生物的DNA提取方法,16S rRNA基因的PCR扩增反应条件的研究进而初步鉴定菌种类型.本文是以大庆采油三厂分离纯化的石油微生物为实验材料,摸索合适的石油微生物基因组DNA的提取方法及合适的PCR扩增条件和反应体系,在本实验室后续开展石油微生物在分子水平方面的实践指导中有重要意义.

  16. Obtaining representative community profiles of anaerobic digesters through optimisation of 16S rRNA amplicon sequencing protocols

    DEFF Research Database (Denmark)

    Kirkegaard, Rasmus Hansen; McIlroy, Simon Jon; Karst, Søren Michael

    A reliable and reproducible method for identification and quantification of the microorganisms involved in biogas production is important for the study and understanding of the microbial communities responsible for the function of anaerobic digester systems. DNA based identification using 16S r...... of the community composition . As such sample specific optimisation and standardisation of DNA extraction, as well PCR primer selection, are essential to minimising the potential for such biases. The aim of this study was to develop a protocol for optimized community profiling of anaerobic digesters. The Fast...

  17. Optimisation of 16S rDNA amplicon sequencing protocols for microbial community profiling of anaerobic digesters

    DEFF Research Database (Denmark)

    Kirkegaard, Rasmus Hansen; McIlroy, Simon Jon; Larsen, Poul

    A reliable and reproducible method for identification and quantification of the microorganisms involved in biogas production is important for the study and understanding of the microbial communities responsible for the function of anaerobic digester systems. DNA based identification using 16S r...... of the community composition. As such sample specific optimisation and standardisation of DNA extraction, as well PCR primer selection, are essential to minimising the potential for such biases. The aim of this study was to develop a protocol for optimized community profiling of anaerobic digesters. The Fast...

  18. A mutation in the 530 loop of Escherichia coli 16S ribosomal RNA causes resistance to streptomycin.

    OpenAIRE

    1988-01-01

    Oligonucleotide-directed mutagenesis was used to introduce an A to C transversion at position 523 in the 16S ribosomal RNA gene of Escherichia coli rrnB operon cloned in plasmid pKK3535. E. coli cells transformed with the mutated plasmid were resistant to streptomycin. The mutated ribosomes isolated from these cells were not stimulated by streptomycin to misread the message in a poly(U)-directed assay. They were also restrictive to the stimulation of misreading by other error-promoting relate...

  19. Terminal restriction fragment length polymorphism (T-RFLP) profiling of bacterial 16S rRNA genes.

    Science.gov (United States)

    Osborne, Catherine A

    2014-01-01

    T-RFLP profiling is a very effective method for comparing many samples in an environmental microbiology study, because fingerprints of microbial diversity can be generated in a sensitive, reproducible, and cost-effective manner. This protocol describes the steps required to generate T-RFLP profiles of the dominant members of a bacterial community, by PCR amplification of the bacterial 16S rRNA genes and three restriction endonuclease digests to generate three different profiles for each sample. The generation of multiple profiles per sample provides enough information to confidently differentiate rich environmental bacterial communities.

  20. Sampling of intestinal microbiota and targeted amplification of bacterial 16S rRNA genes for microbial ecologic analysis.

    Science.gov (United States)

    Tong, Maomeng; Jacobs, Jonathan P; McHardy, Ian H; Braun, Jonathan

    2014-11-03

    Dysbiosis of host-associated commensal microbiota is emerging as an important factor in risk and phenotype of immunologic, metabolic, and behavioral diseases. Accurate analysis of microbial composition and functional state in humans or mice requires appropriate collection and pre-processing of biospecimens. Methods to sample luminal and mucosal microbiota from human or mouse intestines and to profile microbial phylogenetic composition using 16S rRNA sequencing are presented here. Data generated using the methods in this unit can be used for downstream quantitative analysis of microbial ecology.

  1. 16S and 23S plastid rDNA phylogenies of Prototheca species and their auxanographic phenotypes1

    Science.gov (United States)

    Ewing, Aren; Brubaker, Shane; Somanchi, Aravind; Yu, Esther; Rudenko, George; Reyes, Nina; Espina, Karen; Grossman, Arthur; Franklin, Scott

    2014-01-01

    Because algae have become more accepted as sources of human nutrition, phylogenetic analysis can help resolve the taxonomy of taxa that have not been well studied. This can help establish algal evolutionary relationships. Here, we compare Auxenochlorella protothecoides and 23 strains of Prototheca based on their complete 16S and partial 23S plastid rDNA sequences along with nutrient utilization (auxanographic) profiles. These data demonstrate that some of the species groupings are not in agreement with the molecular phylogenetic analyses and that auxanographic profiles are poor predictors of phylogenetic relationships. PMID:25937672

  2. Linear programming model to construct phylogenetic network for 16S rRNA sequences of photosynthetic organisms and influenza viruses.

    Science.gov (United States)

    Mathur, Rinku; Adlakha, Neeru

    2014-06-01

    Phylogenetic trees give the information about the vertical relationships of ancestors and descendants but phylogenetic networks are used to visualize the horizontal relationships among the different organisms. In order to predict reticulate events there is a need to construct phylogenetic networks. Here, a Linear Programming (LP) model has been developed for the construction of phylogenetic network. The model is validated by using data sets of chloroplast of 16S rRNA sequences of photosynthetic organisms and Influenza A/H5N1 viruses. Results obtained are in agreement with those obtained by earlier researchers.

  3. 沼泽红假单胞菌16S rDNA PCR快速检测方法研究%Study on the Detection Method of Rhodopseudomonas Palustris with 16S rDNA PCR

    Institute of Scientific and Technical Information of China (English)

    王妍力; 耿亚亚; 郝葆青

    2016-01-01

    Objective:according to the species attributes and highly conservation of 16S rDNA sequence of photosynthetic bacteria, the specific primers were designed, and the PCR reaction con-ditions were optimized, in order to explore the identifying methods to Rhodopseudomonas palustris. Method: the chromosomal DNA was extracted from Rhodopseudomonas palustris cell, and it was iso-lated and purified. The 16S rDNA gene sequences of the Pseudomonas genus, Escherichia coil and Nocardiopsis sp came from the GenBank respectively, and the gene sequence fragments of variable region were analyzed and compared with each other for the design of the species-specific primers. Several factors that may affect the PCR amplification were analyzed, and also some pre-tests were done in order to determine the best conditions for the PCR amplification. And then the PCR ampli-fication was done, the PCR products were cloned and sequenced. Meanwhile morphological and bio-chemical characteristics of Rhodopseudomonas palustris were tested. Conclusion:the method was de-veloped to detect Rhodopseudomonas palustris by PCR amplification with species-specific primers, which had the properties of strong specificity, high sensitivity, good repeatability, high maneuver-ability and low price. The whole process from extracting the DNA to the PCR amplification was about 3 h for identifying the strains of Rhodopseudomonas palustris.%目的:依据反映光合菌物种属性和高度保守性的16S rDNA序列,设计其特异性引物,优化并确定PCR反应条件,探索沼泽红假单胞菌快速鉴别的方法。方法:提取沼泽红假单胞菌菌体染色体DNA,分离和纯化;从GenBank分别获取假单胞菌属、大肠杆菌和诺卡氏菌属的16S rDNA基因序列,分析比较并确定其可变区的基因序列片段,设计种属特异引物;对影响PCR扩增的因素进行分析和预试,确定PCR扩增的最佳条件;随后进行PCR扩增试验,对其产

  4. Identification of sulfate reducers and Syntrophobacter sp. in anaerobic granular sludge by fatty-acid biomarkers and 16S rRNA probing

    NARCIS (Netherlands)

    Oude Elferink, S.J.W.H.; Boschker, H.T.S.; Stams, A.J.M.

    1998-01-01

    The sulfate-reducing bacterial sludge population in anaerobic bioreactors, treating different types of wastewater in the presence or absence of sulfate, was evaluated by polar-lipid fatty acid (PLFA) analyses, and by 16S rRNA dot-blot hybridizations using specific 16S rRNA-targeted oligonucleotide p

  5. Usefulness of the MicroSeq 500 16S rDNA bacterial identification system for identification of anaerobic Gram positive bacilli isolated from blood cultures

    National Research Council Canada - National Science Library

    Lau, S K P; Ng, K H L; Woo, P C Y; Yip, K-T; Fung, A M Y; Woo, G K S; Chan, K-M; Que, T-L; Yuen, K-Y

    2006-01-01

    Using full 16S ribosomal RNA (rRNA) gene sequencing as the gold standard, 20 non-duplicating anaerobic Gram positive bacilli isolated from blood cultures were analysed by the MicroSeq 500 16S rDNA bacterial identification system...

  6. Development of a Multiplex PCR Method for Detection of the Genes Encoding 16S rRNA, Coagulase, Methicillin Resistance and Enterotoxins in Staphylococcus aureus

    Science.gov (United States)

    A multiplex PCR method was developed for simultaneous detection of the genes encoding methicillin resistance (mecA), staphylococcal enterotoxins A, B and C (sea, seb and sec), coagulase (coa) and 16S rRNA. The primers for amplification of the 16S rRNA gene were specific for Staphylococcus spp., and ...

  7. Comparison of COBAS AMPLICOR Neissefia gonorrhoeae PCR, including confirmation with N-gonorrhoeae-specific 16S rRNA PCR, with traditional culture

    NARCIS (Netherlands)

    Luijt, DS; Bos, PAJ; van Zwet, AA; Vader, PCV; Schirm, J

    A total of 3,023 clinical specimens were tested for Neisseria gonorrhoeae by using COBAS AMPLICOR (CA) PCR and confirmation of positives by N. gonorrhoeae-specific 16S rRNA PCR. The sensitivity of CA plus 16S rRNA PCR was 98.8%, compared to 68.2% for culture. Confirmation of CA positives increased

  8. QUANTITATIVE FLUORESCENCE IN-SITU HYBRIDIZATION OF BIFIDOBACTERIUM SPP WITH GENUS-SPECIFIC 16S RIBOSOMAL-RNA-TARGETED PROBES AND ITS APPLICATION IN FECAL SAMPLES

    NARCIS (Netherlands)

    LANGENDIJK, PS; SCHUT, F; JANSEN, GJ; RAANGS, GC; KAMPHUIS, GR; WILKINSON, MHF; WELLING, GW

    1995-01-01

    Three 16S rRNA hybridization probes were developed and tested for genus-specific detection of Bifidobacterium species in the human fecal flora. Variable regions V2, V4, and VS of the 16S rRNA contained sequences unique to this genus and proved applicable as target sites for oligodeoxynucleotide prob

  9. Identification of sulfate reducers and Syntrophobacter sp. in anaerobic granular sludge by fatty-acid biomarkers and 16S rRNA probing

    NARCIS (Netherlands)

    Oude Elferink, S.J.W.H.; Boschker, H.T.S.; Stams, A.J.M.

    1998-01-01

    The sulfate-reducing bacterial sludge population in anaerobic bioreactors, treating different types of wastewater in the presence or absence of sulfate, was evaluated by polar-lipid fatty acid (PLFA) analyses, and by 16S rRNA dot-blot hybridizations using specific 16S rRNA-targeted oligonucleotide p

  10. Comparison of COBAS AMPLICOR Neisseria gonorrhoeae PCR, including confirmation with N. gonorrhoeae-specific 16S rRNA PCR, with traditional culture

    NARCIS (Netherlands)

    Luijt, D.S.; Bos, P.A.; van Zwet, A.A.; Voorst-Vader, P.C.; Schirm, J.

    2005-01-01

    : A total of 3,023 clinical specimens were tested for Neisseria gonorrhoeae by using COBAS AMPLICOR (CA) PCR and confirmation of positives by N. gonorrhoeae-specific 16S rRNA PCR. The sensitivity of CA plus 16S rRNA PCR was 98.8%, compared to 68.2% for culture. Confirmation of CA positives increased

  11. Comparison of COBAS AMPLICOR Neissefia gonorrhoeae PCR, including confirmation with N-gonorrhoeae-specific 16S rRNA PCR, with traditional culture

    NARCIS (Netherlands)

    Luijt, DS; Bos, PAJ; van Zwet, AA; Vader, PCV; Schirm, J

    2005-01-01

    A total of 3,023 clinical specimens were tested for Neisseria gonorrhoeae by using COBAS AMPLICOR (CA) PCR and confirmation of positives by N. gonorrhoeae-specific 16