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Sample records for bacterial metagenomic library

  1. Characterization of new bacterial catabolic genes and mobile genetic elements by high throughput genetic screening of a soil metagenomic library.

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    Jacquiod, Samuel; Demanèche, Sandrine; Franqueville, Laure; Ausec, Luka; Xu, Zhuofei; Delmont, Tom O; Dunon, Vincent; Cagnon, Christine; Mandic-Mulec, Ines; Vogel, Timothy M; Simonet, Pascal

    2014-11-20

    A mix of oligonucleotide probes was used to hybridize soil metagenomic DNA from a fosmid clone library spotted on high density membranes. The pooled radio-labeled probes were designed to target genes encoding glycoside hydrolases GH18, dehalogenases, bacterial laccases and mobile genetic elements (integrases from integrons and insertion sequences). Positive hybridizing spots were affiliated to the corresponding clones in the library and the metagenomic inserts were sequenced. After assembly and annotation, new coding DNA sequences related to genes of interest were identified with low protein similarity against the closest hits in databases. This work highlights the sensitivity of DNA/DNA hybridization techniques as an effective and complementary way to recover novel genes from large metagenomic clone libraries. This study also supports that some of the identified catabolic genes might be associated with horizontal transfer events.

  2. Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen suppressive soil

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    Hjort, K.; Bergstrom, M.; Adesina, M.F.; Jansson, J.K.; Smalla, K.; Sjoling, S.

    2009-09-01

    Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal-restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal-restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF{sup 103} of the isolate, Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.

  3. Screening of metagenomic and genomic libraries reveals three classes of bacterial enzymes that overcome the toxicity of acrylate.

    Science.gov (United States)

    Curson, Andrew R J; Burns, Oliver J; Voget, Sonja; Daniel, Rolf; Todd, Jonathan D; McInnis, Kathryn; Wexler, Margaret; Johnston, Andrew W B

    2014-01-01

    Acrylate is produced in significant quantities through the microbial cleavage of the highly abundant marine osmoprotectant dimethylsulfoniopropionate, an important process in the marine sulfur cycle. Acrylate can inhibit bacterial growth, likely through its conversion to the highly toxic molecule acrylyl-CoA. Previous work identified an acrylyl-CoA reductase, encoded by the gene acuI, as being important for conferring on bacteria the ability to grow in the presence of acrylate. However, some bacteria lack acuI, and, conversely, many bacteria that may not encounter acrylate in their regular environments do contain this gene. We therefore sought to identify new genes that might confer tolerance to acrylate. To do this, we used functional screening of metagenomic and genomic libraries to identify novel genes that corrected an E. coli mutant that was defective in acuI, and was therefore hyper-sensitive to acrylate. The metagenomic libraries yielded two types of genes that overcame this toxicity. The majority encoded enzymes resembling AcuI, but with significant sequence divergence among each other and previously ratified AcuI enzymes. One other metagenomic gene, arkA, had very close relatives in Bacillus and related bacteria, and is predicted to encode an enoyl-acyl carrier protein reductase, in the same family as FabK, which catalyses the final step in fatty-acid biosynthesis in some pathogenic Firmicute bacteria. A genomic library of Novosphingobium, a metabolically versatile alphaproteobacterium that lacks both acuI and arkA, yielded vutD and vutE, two genes that, together, conferred acrylate resistance. These encode sequential steps in the oxidative catabolism of valine in a pathway in which, significantly, methacrylyl-CoA is a toxic intermediate. These findings expand the range of bacteria for which the acuI gene encodes a functional acrylyl-CoA reductase, and also identify novel enzymes that can similarly function in conferring acrylate resistance, likely, again

  4. Screening of metagenomic and genomic libraries reveals three classes of bacterial enzymes that overcome the toxicity of acrylate.

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    Andrew R J Curson

    Full Text Available Acrylate is produced in significant quantities through the microbial cleavage of the highly abundant marine osmoprotectant dimethylsulfoniopropionate, an important process in the marine sulfur cycle. Acrylate can inhibit bacterial growth, likely through its conversion to the highly toxic molecule acrylyl-CoA. Previous work identified an acrylyl-CoA reductase, encoded by the gene acuI, as being important for conferring on bacteria the ability to grow in the presence of acrylate. However, some bacteria lack acuI, and, conversely, many bacteria that may not encounter acrylate in their regular environments do contain this gene. We therefore sought to identify new genes that might confer tolerance to acrylate. To do this, we used functional screening of metagenomic and genomic libraries to identify novel genes that corrected an E. coli mutant that was defective in acuI, and was therefore hyper-sensitive to acrylate. The metagenomic libraries yielded two types of genes that overcame this toxicity. The majority encoded enzymes resembling AcuI, but with significant sequence divergence among each other and previously ratified AcuI enzymes. One other metagenomic gene, arkA, had very close relatives in Bacillus and related bacteria, and is predicted to encode an enoyl-acyl carrier protein reductase, in the same family as FabK, which catalyses the final step in fatty-acid biosynthesis in some pathogenic Firmicute bacteria. A genomic library of Novosphingobium, a metabolically versatile alphaproteobacterium that lacks both acuI and arkA, yielded vutD and vutE, two genes that, together, conferred acrylate resistance. These encode sequential steps in the oxidative catabolism of valine in a pathway in which, significantly, methacrylyl-CoA is a toxic intermediate. These findings expand the range of bacteria for which the acuI gene encodes a functional acrylyl-CoA reductase, and also identify novel enzymes that can similarly function in conferring acrylate

  5. Identification of novel glycosyl hydrolases with cellulolytic activity against crystalline cellulose from metagenomic libraries constructed from bacterial enrichment cultures.

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    Mori, Toshio; Kamei, Ichiro; Hirai, Hirofumi; Kondo, Ryuichiro

    2014-01-01

    To obtain cellulases that are capable of degrading crystalline cellulose and cedar wood, metagenomic libraries were constructed from raw soil sample which was covered to pile of cedar wood sawdust or from its enrichment cultures. The efficiency of screening of metagenomic library was improved more than 3 times by repeating enrichment cultivation using crystalline cellulose as a carbon source, compared with the library constructed from raw soil. Four cellulase genes were obtained from the metagenomic libraries that were constructed from the total genome extracted from an enrichment culture that used crystalline cellulose as a carbon source. A cellulase gene and a xylanase gene were obtained from the enrichment culture that used unbleached kraft pulp as a carbon source. The culture supernatants of Escherichia coli expressing three clones that were derived from the enrichment culture that used crystalline cellulose showed activity against crystalline cellulose. In addition, these three enzyme solutions generated a reducing sugar from cedar wood powder. From these results, the construction of a metagenomic library from cultures that were repetition enriched using crystalline cellulose demonstrated that this technique is a powerful tool for obtaining cellulases that have activity toward crystalline cellulose.

  6. A plasmid RK2-based broad-host-range cloning vector useful for transfer of metagenomic libraries to a variety of bacterial species.

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    Aakvik, Trine; Degnes, Kristin Fløgstad; Dahlsrud, Rannveig; Schmidt, Frank; Dam, Ragnar; Yu, Lihua; Völker, Uwe; Ellingsen, Trond Erling; Valla, Svein

    2009-06-01

    The majority of microorganisms in natural environments are difficult to cultivate, but their genes can be studied via metagenome libraries. To enhance the chances that these genes become expressed we here report the construction of a broad-host-range plasmid vector (pRS44) for fosmid and bacterial artificial chromosome (BAC) cloning. pRS44 can be efficiently transferred to numerous hosts by conjugation. It replicates in such hosts via the plasmid RK2 origin of replication, while in Escherichia coli it replicates via the plasmid F origin. The vector was found to be remarkably stable due to the insertion of an additional stability element (parDE). The copy number of pRS44 is adjustable, allowing for easy modifications of gene expression levels. A fosmid metagenomic library consisting of 20 000 clones and BAC clones with insert sizes up to 200 kb were constructed. The 16S rRNA gene analysis of the fosmid library DNA confirmed that it represents a variety of microbial species. The entire fosmid library and the selected BAC clones were transferred to Pseudomonas fluorescens and Xanthomonas campestris (fosmids only), and heterologous proteins from the fosmid library were confirmed to be expressed in P. fluorescens. To our knowledge no other reported vector system has a comparable potential for functional screening across species barriers.

  7. Open resource metagenomics: a model for sharing metagenomic libraries.

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    Neufeld, J D; Engel, K; Cheng, J; Moreno-Hagelsieb, G; Rose, D R; Charles, T C

    2011-11-30

    Both sequence-based and activity-based exploitation of environmental DNA have provided unprecedented access to the genomic content of cultivated and uncultivated microorganisms. Although researchers deposit microbial strains in culture collections and DNA sequences in databases, activity-based metagenomic studies typically only publish sequences from the hits retrieved from specific screens. Physical metagenomic libraries, conceptually similar to entire sequence datasets, are usually not straightforward to obtain by interested parties subsequent to publication. In order to facilitate unrestricted distribution of metagenomic libraries, we propose the adoption of open resource metagenomics, in line with the trend towards open access publishing, and similar to culture- and mutant-strain collections that have been the backbone of traditional microbiology and microbial genetics. The concept of open resource metagenomics includes preparation of physical DNA libraries, preferably in versatile vectors that facilitate screening in a diversity of host organisms, and pooling of clones so that single aliquots containing complete libraries can be easily distributed upon request. Database deposition of associated metadata and sequence data for each library provides researchers with information to select the most appropriate libraries for further research projects. As a starting point, we have established the Canadian MetaMicroBiome Library (CM(2)BL [1]). The CM(2)BL is a publicly accessible collection of cosmid libraries containing environmental DNA from soils collected from across Canada, spanning multiple biomes. The libraries were constructed such that the cloned DNA can be easily transferred to Gateway® compliant vectors, facilitating functional screening in virtually any surrogate microbial host for which there are available plasmid vectors. The libraries, which we are placing in the public domain, will be distributed upon request without restriction to members of both the

  8. Preparation of fosmid libraries and functional metagenomic analysis of microbial community DNA.

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    Martínez, Asunción; Osburne, Marcia S

    2013-01-01

    One of the most important challenges in contemporary microbial ecology is to assign a functional role to the large number of novel genes discovered through large-scale sequencing of natural microbial communities that lack similarity to genes of known function. Functional screening of metagenomic libraries, that is, screening environmental DNA clones for the ability to confer an activity of interest to a heterologous bacterial host, is a promising approach for bridging the gap between metagenomic DNA sequencing and functional characterization. Here, we describe methods for isolating environmental DNA and constructing metagenomic fosmid libraries, as well as methods for designing and implementing successful functional screens of such libraries.

  9. Large-scale screens of metagenomic libraries.

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    Pham, Vinh D; Palden, Tsultrim; DeLong, Edward F

    2007-01-01

    Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

  10. Construction and screening of marine metagenomic libraries.

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    Weiland, Nancy; Löscher, Carolin; Metzger, Rebekka; Schmitz, Ruth

    2010-01-01

    Marine microbial communities are highly diverse and have evolved during extended evolutionary processes of physiological adaptations under the influence of a variety of ecological conditions and selection pressures. They harbor an enormous diversity of microbes with still unknown and probably new physiological characteristics. Besides, the surfaces of marine multicellular organisms are typically covered by a consortium of epibiotic bacteria and act as barriers, where diverse interactions between microorganisms and hosts take place. Thus, microbial diversity in the water column of the oceans and the microbial consortia on marine tissues of multicellular organisms are rich sources for isolating novel bioactive compounds and genes. Here we describe the sampling, construction of large-insert metagenomic libraries from marine habitats and exemplarily one function based screen of metagenomic clones.

  11. Phylogeny-function analysis of (meta)genomic libraries: screening for expression of ribosomal RNA genes by large-insert library fluorescent in situ hybridization (LIL-FISH)

    NARCIS (Netherlands)

    Leveau, J.H.J.; Gerards, S.; De Boer, W.; Van Veen, J.A.

    2004-01-01

    We assessed the utility of fluorescent in situ hybridization (FISH) in the screening of clone libraries of (meta)genomic or environmental DNA for the presence and expression of bacterial ribosomal RNA (rRNA) genes. To establish proof-of-principle, we constructed a fosmid-based library in Escherichia

  12. Functional Metagenomics: Construction and High-Throughput Screening of Fosmid Libraries for Discovery of Novel Carbohydrate-Active Enzymes.

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    Ufarté, Lisa; Bozonnet, Sophie; Laville, Elisabeth; Cecchini, Davide A; Pizzut-Serin, Sandra; Jacquiod, Samuel; Demanèche, Sandrine; Simonet, Pascal; Franqueville, Laure; Veronese, Gabrielle Potocki

    2016-01-01

    Activity-based metagenomics is one of the most efficient approaches to boost the discovery of novel biocatalysts from the huge reservoir of uncultivated bacteria. In this chapter, we describe a highly generic procedure of metagenomic library construction and high-throughput screening for carbohydrate-active enzymes. Applicable to any bacterial ecosystem, it enables the swift identification of functional enzymes that are highly efficient, alone or acting in synergy, to break down polysaccharides and oligosaccharides.

  13. Antibacterial enzymes from the functional screening of metagenomic libraries hosted in Ralstonia metallidurans.

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    Iqbal, Hala A; Craig, Jeffrey W; Brady, Sean F

    2014-05-01

    Phenotype-based screening of bacterial metagenomic libraries provides an avenue for the discovery of novel genes, enzymes, and metabolites that have a variety of potential clinical and industrial uses. Here, we report the identification of a functionally diverse collection of antibacterially active enzymes from the phenotypic screening of 700 000 cosmid clones prepared from Arizona soil DNA and hosted in Ralstonia metallidurans. Environmental DNA clones surrounded by zones of growth inhibition in a bacterial overlay assay were found, through bioinformatics and functional analyses, to encode enzymes with predicted peptidase, lipase, and glycolytic activities conferring antibiosis. The antibacterial activities observed in our R. metallidurans-based assay could not be replicated with the same clones in screens using Escherichia coli as a heterologous host, suggesting that the large-scale screening of metagenomic libraries for antibiosis using phylogenetically diverse hosts should be a productive strategy for identifying enzymes with functionally diverse antibacterial activities.

  14. Multisubstrate isotope labeling and metagenomic analysis of active soil bacterial communities.

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    Verastegui, Y; Cheng, J; Engel, K; Kolczynski, D; Mortimer, S; Lavigne, J; Montalibet, J; Romantsov, T; Hall, M; McConkey, B J; Rose, D R; Tomashek, J J; Scott, B R; Charles, T C; Neufeld, J D

    2014-07-15

    Soil microbial diversity represents the largest global reservoir of novel microorganisms and enzymes. In this study, we coupled functional metagenomics and DNA stable-isotope probing (DNA-SIP) using multiple plant-derived carbon substrates and diverse soils to characterize active soil bacterial communities and their glycoside hydrolase genes, which have value for industrial applications. We incubated samples from three disparate Canadian soils (tundra, temperate rainforest, and agricultural) with five native carbon ((12)C) or stable-isotope-labeled ((13)C) carbohydrates (glucose, cellobiose, xylose, arabinose, and cellulose). Indicator species analysis revealed high specificity and fidelity for many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Actinomycetales (Salinibacterium), Rhizobiales (Devosia), Rhodospirillales (Telmatospirillum), and Caulobacterales (Phenylobacterium and Asticcacaulis) were bacterial indicator species for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (Phenylobacterium) were associated with metabolism of cellulose, and Alphaproteobacteria were associated with the metabolism of arabinose; members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycoside hydrolase gene representation within the pooled heavy DNA. By screening 2,876 cloned fragments derived from the (13)C-labeled DNA isolated from soils incubated with cellulose, we demonstrate the power of combining DNA-SIP, multiple-displacement amplification (MDA), and functional metagenomics by efficiently isolating multiple clones with activity on carboxymethyl cellulose and fluorogenic proxy substrates for carbohydrate-active enzymes. Importance: The ability to identify genes based on function, instead of sequence homology, allows the discovery of genes that would not be identified through sequence alone. This

  15. Metagenomic Analysis of Bacterial Communities of Antarctic Surface Snow.

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    Lopatina, Anna; Medvedeva, Sofia; Shmakov, Sergey; Logacheva, Maria D; Krylenkov, Vjacheslav; Severinov, Konstantin

    2016-01-01

    The diversity of bacteria present in surface snow around four Russian stations in Eastern Antarctica was studied by high throughput sequencing of amplified 16S rRNA gene fragments and shotgun metagenomic sequencing. Considerable class- and genus-level variation between the samples was revealed indicating a presence of inter-site diversity of bacteria in Antarctic snow. Flavobacterium was a major genus in one sampling site and was also detected in other sites. The diversity of flavobacterial type II-C CRISPR spacers in the samples was investigated by metagenome sequencing. Thousands of unique spacers were revealed with less than 35% overlap between the sampling sites, indicating an enormous natural variety of flavobacterial CRISPR spacers and, by extension, high level of adaptive activity of the corresponding CRISPR-Cas system. None of the spacers matched known spacers of flavobacterial isolates from the Northern hemisphere. Moreover, the percentage of spacers with matches with Antarctic metagenomic sequences obtained in this work was significantly higher than with sequences from much larger publically available environmental metagenomic database. The results indicate that despite the overall very high level of diversity, Antarctic Flavobacteria comprise a separate pool that experiences pressures from mobile genetic elements different from those present in other parts of the world. The results also establish analysis of metagenomic CRISPR spacer content as a powerful tool to study bacterial populations diversity.

  16. Metagenomic Analysis of Bacterial Communities of Antarctic Surface Snow

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    Anna eLopatina

    2016-03-01

    Full Text Available The diversity of bacteria present in surface snow around four Russian stations in Eastern Antarctica was studied by high throughput sequencing of amplified 16S rRNA gene fragments and shotgun metagenomic sequencing. Considerable class- and genus-level variation between the samples was revealed indicating a presence of inter-site diversity of bacteria in Antarctic snow. Flavobacterium was a major genus in one sampling site and was also detected in other sites. The diversity of flavobacterial type II-C CRISPR spacers in the samples was investigated by metagenome sequencing. Thousands of unique spacers were revealed with less than 35% overlap between the sampling sites, indicating an enormous natural variety of flavobacterial CRISPR spacers and, by extension, high level of adaptive activity of the corresponding CRISPR-Cas system. None of the spacers matched known spacers of flavobacterial isolates from the Northern hemisphere. Moreover, the percentage of spacers with matches with Antarctic metagenomic sequences obtained in this work was significantly higher than with sequences from much larger publically available environmental metagenomic database. The results indicate that despite the overall very high level of diversity, Antarctic Flavobacteria comprise a separate pool that experiences pressures from mobile genetic elements different from those present in other parts of the world. The results also establish analysis of metagenomic CRISPR spacer content as a powerful tool to study bacterial populations diversity.

  17. Screening metagenomic libraries for laccase activities.

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    Ferrer, Manuel; Beloqui, Ana; Golyshin, Peter N

    2010-01-01

    Laccases are multi-copper oxidoreductases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) able to oxidise a wide variety of phenolic and non-phenolic compounds. They are useful enzymes for a variety of applications, including bioremediation and craft pulp bio-bleaching as the most significant ones. There is a considerable interest to find new laccases through the exploration of biological diversity. Laccases have been found in plants, insects, and bacteria but predominantly in fungi: these enzymes have been documented in about 60 fungal strains. Microbial diversity constitutes a largely unexplored treasure chest with new laccases with a good potential for basic science and biotechnology. At present, due to our inability to cultivate most microbes, the only means of accessing the resources of the microbial world is to harvest genetic resources ("metagenomes"), which can further on be subjected to extensive screening programs. In this chapter, we provide an overview of screening methods to identify laccase-encoding genes from environmental resources.

  18. Cyclodipeptides from metagenomic library of a japanese marine sponge

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    He, Rui; Wang, Bochu; Zhub, Liancai, E-mail: wangbc2000@126.com [Bioengineering College, Chongqing University, Chongqing, (China); Wang, Manyuan [School of Traditional Chinese Medicine, Capital University of Medical Sciences, Beijing (China); Wakimoto, Toshiyuki; Abe, Ikuro, E-mail: abei@mol.f.u-tokyo.ac.jp [Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo (Japan)

    2013-12-01

    Culture-independent metagenomics is an attractive and promising approach to explore unique bioactive small molecules from marine sponges harboring uncultured symbiotic microbes. Therefore, we conducted functional screening of the metagenomic library constructed from the Japanese marine sponge Discodermia calyx. Bioassay-guided fractionation of plate culture extract of antibacterial clone pDC113 afforded eleven cyclodipeptides: Cyclo(l-Thr-l-Leu) (1), Cyclo(l-Val-d-Pro) (2), Cyclo(l-Ile-d-Pro) (3), Cyclo(l-Leu-l-Pro) (4), Cyclo(l-Val-l-Leu) (5), Cyclo(l-Leu-l-Ile) (6), Cyclo(l-Leu-l-Leu) (7), Cyclo(l-Phe-l-Tyr) (8), Cyclo(l-Trp-l-Pro) (9), Cyclo(l-Val-l-Trp) (10) and Cyclo(l-Ile-l-Trp) (11). To the best of our knowledge, these are first cyclodepeptides isolated from metagenomic library. Sequence analysis suggested that isolated cyclodipeptides were not synthesized by nonribosomal peptide synthetases and there was no significant indication of cyclodipeptide synthetases. (author)

  19. [Bacterial genomics and metagenomics: clinical applications and medical relevance].

    Science.gov (United States)

    Diene, S M; Bertelli, C; Pillonel, T; Schrenzel, J; Greub, G

    2014-11-12

    New sequencing technologies provide in a short time and at low cost high amount of genomic sequences useful for applications such as: a) development of diagnostic PCRs and/or serological tests; b) detection of virulence factors (virulome) or genes/SNPs associated with resistance to antibiotics (resistome) and c) investigation of transmission and dissemination of bacterial pathogens. Thus, bacterial genomics of medical importance is useful to clinical microbiologists, to infectious diseases specialists as well as to epidemiologists. Determining the microbial composition of a sample by metagenomics is another application of new sequencing technologies, useful to understand the impact of bacteria on various non-infectious diseases such as obesity, asthma, or diabetes. Genomics and metagenomics will likely become a specialized diagnostic analysis.

  20. Applying Shannon's information theory to bacterial and phage genomes and metagenomes.

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    Akhter, Sajia; Bailey, Barbara A; Salamon, Peter; Aziz, Ramy K; Edwards, Robert A

    2013-01-01

    All sequence data contain inherent information that can be measured by Shannon's uncertainty theory. Such measurement is valuable in evaluating large data sets, such as metagenomic libraries, to prioritize their analysis and annotation, thus saving computational resources. Here, Shannon's index of complete phage and bacterial genomes was examined. The information content of a genome was found to be highly dependent on the genome length, GC content, and sequence word size. In metagenomic sequences, the amount of information correlated with the number of matches found by comparison to sequence databases. A sequence with more information (higher uncertainty) has a higher probability of being significantly similar to other sequences in the database. Measuring uncertainty may be used for rapid screening for sequences with matches in available database, prioritizing computational resources, and indicating which sequences with no known similarities are likely to be important for more detailed analysis.

  1. Applying Shannon's information theory to bacterial and phage genomes and metagenomes

    Science.gov (United States)

    Akhter, Sajia; Bailey, Barbara A.; Salamon, Peter; Aziz, Ramy K.; Edwards, Robert A.

    2013-01-01

    All sequence data contain inherent information that can be measured by Shannon's uncertainty theory. Such measurement is valuable in evaluating large data sets, such as metagenomic libraries, to prioritize their analysis and annotation, thus saving computational resources. Here, Shannon's index of complete phage and bacterial genomes was examined. The information content of a genome was found to be highly dependent on the genome length, GC content, and sequence word size. In metagenomic sequences, the amount of information correlated with the number of matches found by comparison to sequence databases. A sequence with more information (higher uncertainty) has a higher probability of being significantly similar to other sequences in the database. Measuring uncertainty may be used for rapid screening for sequences with matches in available database, prioritizing computational resources, and indicating which sequences with no known similarities are likely to be important for more detailed analysis. PMID:23301154

  2. Applying Shannon's information theory to bacterial and phage genomes and metagenomes

    Science.gov (United States)

    Akhter, Sajia; Bailey, Barbara A.; Salamon, Peter; Aziz, Ramy K.; Edwards, Robert A.

    2013-01-01

    All sequence data contain inherent information that can be measured by Shannon's uncertainty theory. Such measurement is valuable in evaluating large data sets, such as metagenomic libraries, to prioritize their analysis and annotation, thus saving computational resources. Here, Shannon's index of complete phage and bacterial genomes was examined. The information content of a genome was found to be highly dependent on the genome length, GC content, and sequence word size. In metagenomic sequences, the amount of information correlated with the number of matches found by comparison to sequence databases. A sequence with more information (higher uncertainty) has a higher probability of being significantly similar to other sequences in the database. Measuring uncertainty may be used for rapid screening for sequences with matches in available database, prioritizing computational resources, and indicating which sequences with no known similarities are likely to be important for more detailed analysis.

  3. Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics

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    Low, Serene; Raina, Jean-Baptiste; Skarshewski, Adam; Le, Xuyen H.; Butler, Margaret K.; Stocker, Roman; Seymour, Justin; Tyson, Gene W.

    2016-01-01

    High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. Here we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diverse Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (∼100–1,000 microbial cells) to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities. We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics. PMID:27688978

  4. Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics

    Directory of Open Access Journals (Sweden)

    Christian Rinke

    2016-09-01

    Full Text Available High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. Here we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diverse Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (∼100–1,000 microbial cells to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities. We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics.

  5. Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics.

    Science.gov (United States)

    Rinke, Christian; Low, Serene; Woodcroft, Ben J; Raina, Jean-Baptiste; Skarshewski, Adam; Le, Xuyen H; Butler, Margaret K; Stocker, Roman; Seymour, Justin; Tyson, Gene W; Hugenholtz, Philip

    2016-01-01

    High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. Here we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diverse Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (∼100-1,000 microbial cells) to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities. We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics.

  6. A comparison of random sequence reads versus 16S rDNA sequences for estimating the biodiversity of a metagenomic library.

    Science.gov (United States)

    Manichanh, Chaysavanh; Chapple, Charles E; Frangeul, Lionel; Gloux, Karine; Guigo, Roderic; Dore, Joel

    2008-09-01

    The construction of metagenomic libraries has permitted the study of microorganisms resistant to isolation and the analysis of 16S rDNA sequences has been used for over two decades to examine bacterial biodiversity. Here, we show that the analysis of random sequence reads (RSRs) instead of 16S is a suitable shortcut to estimate the biodiversity of a bacterial community from metagenomic libraries. We generated 10,010 RSRs from a metagenomic library of microorganisms found in human faecal samples. Then searched them using the program BLASTN against a prokaryotic sequence database to assign a taxon to each RSR. The results were compared with those obtained by screening and analysing the clones containing 16S rDNA sequences in the whole library. We found that the biodiversity observed by RSR analysis is consistent with that obtained by 16S rDNA. We also show that RSRs are suitable to compare the biodiversity between different metagenomic libraries. RSRs can thus provide a good estimate of the biodiversity of a metagenomic library and, as an alternative to 16S, this approach is both faster and cheaper.

  7. Preparation of metagenomic libraries from naturally occurring marine viruses.

    Science.gov (United States)

    Solonenko, Sergei A; Sullivan, Matthew B

    2013-01-01

    Microbes are now well recognized as major drivers of the biogeochemical cycling that fuels the Earth, and their viruses (phages) are known to be abundant and important in microbial mortality, horizontal gene transfer, and modulating microbial metabolic output. Investigation of environmental phages has been frustrated by an inability to culture the vast majority of naturally occurring diversity coupled with the lack of robust, quantitative, culture-independent methods for studying this uncultured majority. However, for double-stranded DNA phages, a quantitative viral metagenomic sample-to-sequence workflow now exists. Here, we review these advances with special emphasis on the technical details of preparing DNA sequencing libraries for metagenomic sequencing from environmentally relevant low-input DNA samples. Library preparation steps broadly involve manipulating the sample DNA by fragmentation, end repair and adaptor ligation, size fractionation, and amplification. One critical area of future research and development is parallel advances for alternate nucleic acid types such as single-stranded DNA and RNA viruses that are also abundant in nature. Combinations of recent advances in fragmentation (e.g., acoustic shearing and tagmentation), ligation reactions (adaptor-to-template ratio reference table availability), size fractionation (non-gel-sizing), and amplification (linear amplification for deep sequencing and linker amplification protocols) enhance our ability to generate quantitatively representative metagenomic datasets from low-input DNA samples. Such datasets are already providing new insights into the role of viruses in marine systems and will continue to do so as new environments are explored and synergies and paradigms emerge from large-scale comparative analyses.

  8. Functional Screening of Antibiotic Resistance Genes from a Representative Metagenomic Library of Food Fermenting Microbiota

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    Chiara Devirgiliis

    2014-01-01

    Full Text Available Lactic acid bacteria (LAB represent the predominant microbiota in fermented foods. Foodborne LAB have received increasing attention as potential reservoir of antibiotic resistance (AR determinants, which may be horizontally transferred to opportunistic pathogens. We have previously reported isolation of AR LAB from the raw ingredients of a fermented cheese, while AR genes could be detected in the final, marketed product only by PCR amplification, thus pointing at the need for more sensitive microbial isolation techniques. We turned therefore to construction of a metagenomic library containing microbial DNA extracted directly from the food matrix. To maximize yield and purity and to ensure that genomic complexity of the library was representative of the original bacterial population, we defined a suitable protocol for total DNA extraction from cheese which can also be applied to other lipid-rich foods. Functional library screening on different antibiotics allowed recovery of ampicillin and kanamycin resistant clones originating from Streptococcus salivarius subsp. thermophilus and Lactobacillus helveticus genomes. We report molecular characterization of the cloned inserts, which were fully sequenced and shown to confer AR phenotype to recipient bacteria. We also show that metagenomics can be applied to food microbiota to identify underrepresented species carrying specific genes of interest.

  9. Metagenomic and metaproteomic insights into bacterial communities in leaf-cutter ant fungus gardens.

    Science.gov (United States)

    Aylward, Frank O; Burnum, Kristin E; Scott, Jarrod J; Suen, Garret; Tringe, Susannah G; Adams, Sandra M; Barry, Kerrie W; Nicora, Carrie D; Piehowski, Paul D; Purvine, Samuel O; Starrett, Gabriel J; Goodwin, Lynne A; Smith, Richard D; Lipton, Mary S; Currie, Cameron R

    2012-09-01

    Herbivores gain access to nutrients stored in plant biomass largely by harnessing the metabolic activities of microbes. Leaf-cutter ants of the genus Atta are a hallmark example; these dominant neotropical herbivores cultivate symbiotic fungus gardens on large quantities of fresh plant forage. As the external digestive system of the ants, fungus gardens facilitate the production and sustenance of millions of workers. Using metagenomic and metaproteomic techniques, we characterize the bacterial diversity and physiological potential of fungus gardens from two species of Atta. Our analysis of over 1.2 Gbp of community metagenomic sequence and three 16S pyrotag libraries reveals that in addition to harboring the dominant fungal crop, these ecosystems contain abundant populations of Enterobacteriaceae, including the genera Enterobacter, Pantoea, Klebsiella, Citrobacter and Escherichia. We show that these bacterial communities possess genes associated with lignocellulose degradation and diverse biosynthetic pathways, suggesting that they play a role in nutrient cycling by converting the nitrogen-poor forage of the ants into B-vitamins, amino acids and other cellular components. Our metaproteomic analysis confirms that bacterial glycosyl hydrolases and proteins with putative biosynthetic functions are produced in both field-collected and laboratory-reared colonies. These results are consistent with the hypothesis that fungus gardens are specialized fungus-bacteria communities that convert plant material into energy for their ant hosts. Together with recent investigations into the microbial symbionts of vertebrates, our work underscores the importance of microbial communities in the ecology and evolution of herbivorous metazoans.

  10. Rapid DNA Library Construction for Functional Genomic and Metagenomic Screening▿ †

    OpenAIRE

    2007-01-01

    A rapid protocol was developed for constructing plasmid libraries from small quantities of genomic/metagenomic DNA. The technique utilizes linker amplification with topoisomerase cloning and allows for inducible transcription in Escherichia coli. As proof of principle, several anti-Bacillus lysins were cloned from bacteriophage genomes and an aerolysin was cloned from a metagenomic sample.

  11. Dynamics of Sequence -Discrete Bacterial Populations Inferred Using Metagenomes

    Energy Technology Data Exchange (ETDEWEB)

    Stevens, Sarah; Bendall, Matthew; Kang, Dongwan; Froula, Jeff; Egan, Rob; Chan, Leong-Keat; Tringe, Susannah; McMahon, Katherine; Malmstrom, Rex

    2014-03-14

    From a multi-year metagenomic time series of two dissimilar Wisconsin lakes we have assembled dozens of genomes using a novel approach that bins contigs into distinct genome based on sequence composition, e.g. kmer frequencies, and contig coverage patterns at various times points. Next, we investigated how these genomes, which represent sequence-discrete bacterial populations, evolved over time and used the time series to discover the population dynamics. For example, we explored changes in single nucleotide polymorphism (SNP) frequencies as well as patterns of gene gain and loss in multiple populations. Interestingly, SNP diversity was purged at nearly every genome position in some populations during the course of this study, suggesting these populations may have experienced genome-wide selective sweeps. This represents the first direct, time-resolved observations of periodic selection in natural populations, a key process predicted by the ecotype model of bacterial diversification.

  12. Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries

    Directory of Open Access Journals (Sweden)

    Mari eNyyssönen

    2013-09-01

    Full Text Available Recent advances in sequencing technologies generate new predictions and hypotheses about the functional roles of environmental microorganisms. Yet, until we can test these predictions at a scale that matches our ability to generate them, most of them will remain as hypotheses. Function-based mining of metagenomic libraries can provide direct linkages between genes, metabolic traits and microbial taxa and thus bridge this gap between sequence data generation and functional predictions. Here we developed high-throughput screening assays for function-based characterization of activities involved in plant polymer decomposition from environmental metagenomic libraries. The multiplexed assays use fluorogenic and chromogenic substrates, combine automated liquid handling and use a genetically modified expression host to enable simultaneous screening of 12,160 clones for 14 activities in a total of 170,240 reactions. Using this platform we identified 374 (0.26 % cellulose, hemicellulose, chitin, starch, phosphate and protein hydrolyzing clones from fosmid libraries prepared from decomposing leaf litter. Sequencing on the Illumina MiSeq platform, followed by assembly and gene prediction of a subset of 95 fosmid clones, identified a broad range of bacterial phyla, including Actinobacteria, Bacteroidetes, multiple Proteobacteria sub-phyla in addition to some Fungi. Carbohydrate-active enzyme genes from 20 different glycoside hydrolase families were detected. Using tetranucleotide frequency binning of fosmid sequences, multiple enzyme activities from distinct fosmids were linked, demonstrating how biochemically-confirmed functional traits in environmental metagenomes may be attributed to groups of specific organisms. Overall, our results demonstrate how functional screening of metagenomic libraries can be used to connect microbial functionality to community composition and, as a result, complement large-scale metagenomic sequencing efforts.

  13. Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries.

    Science.gov (United States)

    Nyyssönen, Mari; Tran, Huu M; Karaoz, Ulas; Weihe, Claudia; Hadi, Masood Z; Martiny, Jennifer B H; Martiny, Adam C; Brodie, Eoin L

    2013-01-01

    Recent advances in sequencing technologies generate new predictions and hypotheses about the functional roles of environmental microorganisms. Yet, until we can test these predictions at a scale that matches our ability to generate them, most of them will remain as hypotheses. Function-based mining of metagenomic libraries can provide direct linkages between genes, metabolic traits and microbial taxa and thus bridge this gap between sequence data generation and functional predictions. Here we developed high-throughput screening assays for function-based characterization of activities involved in plant polymer decomposition from environmental metagenomic libraries. The multiplexed assays use fluorogenic and chromogenic substrates, combine automated liquid handling and use a genetically modified expression host to enable simultaneous screening of 12,160 clones for 14 activities in a total of 170,240 reactions. Using this platform we identified 374 (0.26%) cellulose, hemicellulose, chitin, starch, phosphate and protein hydrolyzing clones from fosmid libraries prepared from decomposing leaf litter. Sequencing on the Illumina MiSeq platform, followed by assembly and gene prediction of a subset of 95 fosmid clones, identified a broad range of bacterial phyla, including Actinobacteria, Bacteroidetes, multiple Proteobacteria sub-phyla in addition to some Fungi. Carbohydrate-active enzyme genes from 20 different glycoside hydrolase (GH) families were detected. Using tetranucleotide frequency (TNF) binning of fosmid sequences, multiple enzyme activities from distinct fosmids were linked, demonstrating how biochemically-confirmed functional traits in environmental metagenomes may be attributed to groups of specific organisms. Overall, our results demonstrate how functional screening of metagenomic libraries can be used to connect microbial functionality to community composition and, as a result, complement large-scale metagenomic sequencing efforts.

  14. Isolation of xylose isomerases by sequence- and function-based screening from a soil metagenomic library

    Directory of Open Access Journals (Sweden)

    Parachin Nádia

    2011-05-01

    Full Text Available Abstract Background Xylose isomerase (XI catalyses the isomerisation of xylose to xylulose in bacteria and some fungi. Currently, only a limited number of XI genes have been functionally expressed in Saccharomyces cerevisiae, the microorganism of choice for lignocellulosic ethanol production. The objective of the present study was to search for novel XI genes in the vastly diverse microbial habitat present in soil. As the exploitation of microbial diversity is impaired by the ability to cultivate soil microorganisms under standard laboratory conditions, a metagenomic approach, consisting of total DNA extraction from a given environment followed by cloning of DNA into suitable vectors, was undertaken. Results A soil metagenomic library was constructed and two screening methods based on protein sequence similarity and enzyme activity were investigated to isolate novel XI encoding genes. These two screening approaches identified the xym1 and xym2 genes, respectively. Sequence and phylogenetic analyses revealed that the genes shared 67% similarity and belonged to different bacterial groups. When xym1 and xym2 were overexpressed in a xylA-deficient Escherichia coli strain, similar growth rates to those in which the Piromyces XI gene was expressed were obtained. However, expression in S. cerevisiae resulted in only one-fourth the growth rate of that obtained for the strain expressing the Piromyces XI gene. Conclusions For the first time, the screening of a soil metagenomic library in E. coli resulted in the successful isolation of two active XIs. However, the discrepancy between XI enzyme performance in E. coli and S. cerevisiae suggests that future screening for XI activity from soil should be pursued directly using yeast as a host.

  15. Comparison of large-insert, small-insert and pyrosequencing libraries for metagenomic analysis

    OpenAIRE

    2012-01-01

    The development of DNA sequencing methods for characterizing microbial communities has evolved rapidly over the past decades. To evaluate more traditional, as well as newer methodologies for DNA library preparation and sequencing, we compared fosmid, short-insert shotgun and 454 pyrosequencing libraries prepared from the same metagenomic DNA samples. GC content was elevated in all fosmid libraries, compared with shotgun and 454 libraries. Taxonomic composition of the different libraries sugge...

  16. Metagenomic and metaproteomic insights into bacterial communities in leaf-cutter ant fungus gardens

    Energy Technology Data Exchange (ETDEWEB)

    Aylward, Frank O.; Burnum, Kristin E.; Scott, Jarrod J.; Suen, Garret; Tringe, Susannah G.; Adams, Sandra M.; Barry, Kerrie W.; Nicora, Carrie D.; Piehowski, Paul D.; Purvine, Samuel O.; Starrett, Gabriel J.; Goodwin, Lynne A.; Smith, Richard D.; Lipton, Mary S.; Currie, Cameron R.

    2012-09-01

    Herbivores gain access to nutrients stored in plant biomass largely by harnessing the metabolic activities of microbes. Leaf-cutter ants of the genus Atta are a hallmark example; these dominant Neotropical herbivores cultivate symbiotic fungus gardens on massive quantities of fresh plant forage. As the external digestive system of the ants, fungus gardens facilitate the production and sustenance of millions of workers in mature Atta colonies. Here we use metagenomic, and metaproteomic techniques to characterize the bacterial diversity and overall physiological potential of fungus gardens from two species of Atta. Our analysis of over 1.2 Gbp of community metagenomic sequence and three 16S pyrotag libraries reveals that, in addition to harboring the dominant fungal crop, these ecosystems contain abundant populations of Enterobacteriaceae, including the genera Enterobacter, Pantoea, Klebsiella, Citrobacter, and Escherichia. We show that these bacterial communities possess genes commonly associated with lignocellulose degradation, and likely participate in the processing of plant biomass. Additionally, we demonstrate that bacteria in these environments encode a diverse suite of biosynthetic pathways, and that they may enrich the nitrogen-poor forage of the ants with B-vitamins, amino acids, and proteins. These results are consistent with the hypothesis that fungus gardens are highly-specialized fungus-bacteria communities that efficiently convert plant material into usable energy for their ant hosts. Together with recent investigations into the microbial symbionts of vertebrates, our work underscores the importance of microbial communities to the ecology and evolution of herbivorous metazoans.

  17. Bacillus subtilis as a tool for screening soil metagenomic libraries for antimicrobial activities.

    Science.gov (United States)

    Biver, Sophie; Steels, Sébastien; Portetelle, Daniel; Vandenbol, Micheline

    2013-06-28

    Finding new antimicrobial activities by functional metagenomics has been shown to depend on the heterologous host used to express the foreign DNA. Therefore, efforts are devoted to developing new tools for constructing metagenomic libraries in shuttle vectors replicatable in phylogenetically distinct hosts. Here we evaluated the use of the Escherichia coli-Bacillus subtilis shuttle vector pHT01 to construct a forest-soil metagenomic library. This library was screened in both hosts for antimicrobial activities against four opportunistic bacteria: Proteus vulgaris, Bacillus cereus, Staphylococcus epidermidis, and Micrococcus luteus. A new antibacterial activity against B. cereus was found upon screening in B. subtilis. The new antimicrobial agent, sensitive to proteinase K, was not active when the corresponding DNA fragment was expressed in E. coli. Our results validate the use of pHT01 as a shuttle vector and B. subtilis as a host to isolate new activities by functional metagenomics.

  18. Gram negative shuttle BAC vector for heterologous expression of metagenomic libraries.

    Science.gov (United States)

    Kakirde, Kavita S; Wild, Jadwiga; Godiska, Ronald; Mead, David A; Wiggins, Andrew G; Goodman, Robert M; Szybalski, Waclaw; Liles, Mark R

    2011-04-15

    Bacterial artificial chromosome (BAC) vectors enable stable cloning of large DNA fragments from single genomes or microbial assemblages. A novel shuttle BAC vector was constructed that permits replication of BAC clones in diverse Gram-negative species. The "Gram-negative shuttle BAC" vector (pGNS-BAC) uses the F replicon for stable single-copy replication in E. coli and the broad-host-range RK2 mini-replicon for high-copy replication in diverse Gram-negative bacteria. As with other BAC vectors containing the oriV origin, this vector is capable of an arabinose-inducible increase in plasmid copy number. Resistance to both gentamicin and chloramphenicol is encoded on pGNS-BAC, permitting selection for the plasmid in diverse bacterial species. The oriT from an IncP plasmid was cloned into pGNS-BAC to enable conjugal transfer, thereby allowing both electroporation and conjugation of pGNS-BAC DNA into bacterial hosts. A soil metagenomic library was constructed in pGNS-BAC-1 (the first version of the vector, lacking gentamicin resistance and oriT), and recombinant clones were demonstrated to replicate in diverse Gram-negative hosts, including Escherichia coli, Pseudomonas spp., Salmonella enterica, Serratia marcescens, Vibrio vulnificus and Enterobacter nimipressuralis. This shuttle BAC vector can be utilized to clone genomic DNA from diverse sources, and then transfer it into diverse Gram-negative bacterial species to facilitate heterologous expression of recombinant pathways.

  19. Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries.

    Science.gov (United States)

    Terrón-González, L; Medina, C; Limón-Mortés, M C; Santero, E

    2013-01-01

    The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic gene expression, while the other approach uses the lambda phage transcription anti-termination protein N to limit transcription termination. A metagenomic library was constructed and functionally screened to identify genes conferring carbenicillin resistance to E. coli. The use of these enhanced expression systems resulted in a 6-fold increase in the frequency of carbenicillin resistant clones. Subcloning and sequence analysis showed that, besides β-lactamases, efflux pumps are not only able contribute to carbenicillin resistance but may in fact be sufficient by themselves to convey carbenicillin resistance.

  20. Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics

    NARCIS (Netherlands)

    Colin, Pierre-Yves; Kintses, Balint; Gielen, Fabrice; Miton, Charlotte M; Fischer, Gerhard; Mohamed, Mark F; Hyvönen, Marko; Morgavi, Diego P; Janssen, Dick B; Hollfelder, Florian

    2015-01-01

    Unculturable bacterial communities provide a rich source of biocatalysts, but their experimental discovery by functional metagenomics is difficult, because the odds are stacked against the experimentor. Here we demonstrate functional screening of a million-membered metagenomic library in microfluidi

  1. Construction and validation of two metagenomic DNA libraries from Cerrado soil with high clay content.

    Science.gov (United States)

    de Castro, Alinne Pereira; Quirino, Betania Ferraz; Allen, Heather; Williamson, Lynn L; Handelsman, Jo; Krüger, Ricardo Henrique

    2011-11-01

    A challenge of metagenomic studies is in the extraction and purification of DNA from environmental samples. The soils of the Cerrado region of Brazil present several technical difficulties to DNA extraction: high clay content (>55% w/w), low pH (4.7) and high iron levels (146 ppm). Here we describe for the first time the efficient recovery and purification of microbial DNA associated with these unusual soil characteristics and the construction and validation of two metagenomic libraries: a 150,000 clones library with insert size of approximately 8 kb and a 65,000 clones library with insert size of approximately 35 kb. The construction of these metagenomic libraries will allow the biotechnological exploitation of the microbial community present in the soil from this endangered biome.

  2. Biochemical Characterization of a Family 15 Carbohydrate Esterase from a Bacterial Marine Arctic Metagenome.

    Directory of Open Access Journals (Sweden)

    Concetta De Santi

    Full Text Available The glucuronoyl esterase enzymes of wood-degrading fungi (Carbohydrate Esterase family 15; CE15 form part of the hemicellulolytic and cellulolytic enzyme systems that break down plant biomass, and have possible applications in biotechnology. Homologous enzymes are predicted in the genomes of several bacteria, however these have been much less studied than their fungal counterparts. Here we describe the recombinant production and biochemical characterization of a bacterial CE15 enzyme denoted MZ0003, which was identified by in silico screening of a prokaryotic metagenome library derived from marine Arctic sediment. MZ0003 has high similarity to several uncharacterized gene products of polysaccharide-degrading bacterial species, and phylogenetic analysis indicates a deep evolutionary split between these CE15s and fungal homologs.MZ0003 appears to differ from previously-studied CE15s in some aspects. Some glucuronoyl esterase activity could be measured by qualitative thin-layer chromatography which confirms its assignment as a CE15, however MZ0003 can also hydrolyze a range of other esters, including p-nitrophenyl acetate, which is not acted upon by some fungal homologs. The structure of MZ0003 also appears to differ as it is predicted to have several large loop regions that are absent in previously studied CE15s, and a combination of homology-based modelling and site-directed mutagenesis indicate its catalytic residues deviate from the conserved Ser-His-Glu triad of many fungal CE15s. Taken together, these results indicate that potentially unexplored diversity exists among bacterial CE15s, and this may be accessed by investigation of the microbial metagenome. The combination of low activity on typical glucuronoyl esterase substrates, and the lack of glucuronic acid esters in the marine environment suggest that the physiological substrate of MZ0003 and its homologs is likely to be different from that of related fungal enzymes.

  3. A high throughput screen for biomining cellulase activity from metagenomic libraries.

    Science.gov (United States)

    Mewis, Keith; Taupp, Marcus; Hallam, Steven J

    2011-02-01

    Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production (1). Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils (2), buffalo rumen (3) and the termite hind-gut (4) using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood (5)). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio (6). Other methods have been reported (7,8) but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate (9). Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction (10). The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

  4. Metagenomic analysis of bacterial and archaeal assemblages in the soil-mousse surrounding a geothermal spring

    Directory of Open Access Journals (Sweden)

    Sonu Bhatia

    2015-09-01

    Full Text Available The soil-mousse surrounding a geothermal spring was analyzed for bacterial and archaeal diversity using 16S rRNA gene amplicon metagenomic sequencing which revealed the presence of 18 bacterial phyla distributed across 109 families and 219 genera. Firmicutes, Actinobacteria, and the Deinococcus-Thermus group were the predominant bacterial assemblages with Crenarchaeota and Thaumarchaeota as the main archaeal assemblages in this largely understudied geothermal habitat. Several metagenome sequences remained taxonomically unassigned suggesting the presence of a repertoire of hitherto undescribed microbes in this geothermal soil-mousse econiche.

  5. Est16, a New Esterase Isolated from a Metagenomic Library of a Microbial Consortium Specializing in Diesel Oil Degradation.

    Directory of Open Access Journals (Sweden)

    Mariana Rangel Pereira

    Full Text Available Lipolytic enzymes have attracted attention from a global market because they show enormous biotechnological potential for applications such as detergent production, leather processing, cosmetics production, and use in perfumes and biodiesel. Due to the intense demand for biocatalysts, a metagenomic approach provides methods of identifying new enzymes. In this study, an esterase designated as Est16 was selected from 4224 clones of a fosmid metagenomic library, revealing an 87% amino acid identity with an esterase/lipase (accession number ADM63076.1 from an uncultured bacterium. Phylogenetic studies showed that the enzyme belongs to family V of bacterial lipolytic enzymes and has sequence and structural similarities with an aryl-esterase from Pseudomonas fluorescens and a patented Anti-Kazlauskas lipase (patent number US20050153404. The protein was expressed and purified as a highly soluble, thermally stable enzyme that showed a preference for basic pH. Est16 exhibited activity toward a wide range of substrates and the highest catalytic efficiency against p-nitrophenyl butyrate and p-nitrophenyl valerate. Est16 also showed tolerance to the presence of organic solvents, detergents and metals. Based on molecular modeling, we showed that the large alpha-beta domain is conserved in the patented enzymes but not the substrate pocket. Here, it was demonstrated that a metagenomic approach is suitable for discovering the lipolytic enzyme diversity and that Est16 has the biotechnological potential for use in industrial processes.

  6. Versatile broad-host-range cosmids for construction of high quality metagenomic libraries.

    Science.gov (United States)

    Cheng, Jiujun; Pinnell, Lee; Engel, Katja; Neufeld, Josh D; Charles, Trevor C

    2014-04-01

    We constructed IncP broad-host-range Gateway® entry cosmids pJC8 and pJC24, which replicate in diverse Proteobacteria. We demonstrate the functionality of these vectors by extracting, purifying, and size-selecting metagenomic DNA from agricultural corn and wheat soils, followed by cloning into pJC8. Metagenomic DNA libraries of 8×10(4) (corn soil) and 9×10(6) (wheat soil) clones were generated for functional screening. The DNA cloned in these libraries can be transferred from these recombinant cosmids to Gateway® destination vectors for specialized screening purposes. Those library clones are available from the Canadian MetaMicroBiome Library project (http://www.cm2bl.org/).

  7. Construction and screening of a functional metagenomic library to identify novel enzymes produced by Antarctic bacteria

    Institute of Scientific and Technical Information of China (English)

    Ignacio Ferrés; Vanesa Amarelle; Francisco Noya; Elena Fabiano

    2015-01-01

    A metagenomic fosmid library of approximately 52 000 clones was constructed to identify functional genes encoding cold-adapted enzymes. Metagenomic DNA was extracted from a sample of glacial meltwater, collected on the Antarctic Peninsula during the ANTARKOS XXIX Expedition during the austral summer of 2012–2013. Each clone contained an insert of about 35–40 kb, so the library represented almost 2 Gb of genetic information from metagenomic DNA. Activity-driven screening was used to detect the cold-adapted functions expressed by the library. Fifty lipase/esterase and two cellulase-producing clones were isolated, and two clones able to grow on Avicel® as the sole carbon source. Interestingly, three clones formed a brown precipitate in the presence of manganese (II). Accumulation of manganese oxides was determined with a leucoberbelin blue assay, indicating that these three clones had manganese-oxidizing activity. To the best of our knowledge, this is the first report of a manganese oxidase activity detected with a functional metagenomic strategy.

  8. Amplification methods bias metagenomic libraries of uncultured single-stranded and double-stranded DNA viruses.

    Science.gov (United States)

    Kim, Kyoung-Ho; Bae, Jin-Woo

    2011-11-01

    Investigation of viruses in the environment often requires the amplification of viral DNA before sequencing of viral metagenomes. In this study, two of the most widely used amplification methods, the linker amplified shotgun library (LASL) and multiple displacement amplification (MDA) methods, were applied to a sample from the seawater surface. Viral DNA was extracted from viruses concentrated by tangential flow filtration and amplified by these two methods. 454 pyrosequencing was used to read the metagenomic sequences from different libraries. The resulting taxonomic classifications of the viruses, their functional assignments, and assembly patterns differed substantially depending on the amplification method. Only double-stranded DNA viruses were retrieved from the LASL, whereas most sequences in the MDA library were from single-stranded DNA viruses, and double-stranded DNA viral sequences were minorities. Thus, the two amplification methods reveal different aspects of viral diversity.

  9. Expression of heterologous sigma factors enables functional screening of metagenomic and heterologous genomic libraries.

    Science.gov (United States)

    Gaida, Stefan M; Sandoval, Nicholas R; Nicolaou, Sergios A; Chen, Yili; Venkataramanan, Keerthi P; Papoutsakis, Eleftherios T

    2015-05-06

    A key limitation in using heterologous genomic or metagenomic libraries in functional genomics and genome engineering is the low expression of heterologous genes in screening hosts, such as Escherichia coli. To overcome this limitation, here we generate E. coli strains capable of recognizing heterologous promoters by expressing heterologous sigma factors. Among seven sigma factors tested, RpoD from Lactobacillus plantarum (Lpl) appears to be able of initiating transcription from all sources of DNA. Using the promoter GFP-trap concept, we successfully screen several heterologous and metagenomic DNA libraries, thus enlarging the genomic space that can be functionally sampled in E. coli. For an application, we show that screening fosmid-based Lpl genomic libraries in an E. coli strain with a chromosomally integrated Lpl rpoD enables the identification of Lpl genetic determinants imparting strong ethanol tolerance in E. coli. Transcriptome analysis confirms increased expression of heterologous genes in the engineered strain.

  10. High yield of functional metagenomic library from mangroves constructed in fosmid vector.

    Science.gov (United States)

    Gonçalves, A C S; dos Santos, A C F; dos Santos, T F; Pessoa, T B A; Dias, J C T; Rezende, R P

    2015-10-02

    In the present study, metagenomic technique and fosmid vectors were used to construct a library of clones for exploring the biotechnological potential of mangrove soils by isolation of functional genes encoding hydrolytic enzymes. The library was built with genomic DNA from the soil samples of mangrove sediments and the functional screening of 1824 clones (~64 Mbp) was performed to detect the hydrolytic activity specific for cellulases, amylases (at acidic, neutral and basic pH), lipases/esterases, proteases, and nitrilases. Significant numbers of clones, positive for the tested enzyme activities were obtained. Our results indicate the importance and biotechnological potential of mangrove soils especially when compared to those obtained using other soil metagenomic libraries.

  11. Functional screening of metagenome and genome libraries for detection of novel flavonoid-modifying enzymes.

    Science.gov (United States)

    Rabausch, U; Juergensen, J; Ilmberger, N; Böhnke, S; Fischer, S; Schubach, B; Schulte, M; Streit, W R

    2013-08-01

    The functional detection of novel enzymes other than hydrolases from metagenomes is limited since only a very few reliable screening procedures are available that allow the rapid screening of large clone libraries. For the discovery of flavonoid-modifying enzymes in genome and metagenome clone libraries, we have developed a new screening system based on high-performance thin-layer chromatography (HPTLC). This metagenome extract thin-layer chromatography analysis (META) allows the rapid detection of glycosyltransferase (GT) and also other flavonoid-modifying activities. The developed screening method is highly sensitive, and an amount of 4 ng of modified flavonoid molecules can be detected. This novel technology was validated against a control library of 1,920 fosmid clones generated from a single Bacillus cereus isolate and then used to analyze more than 38,000 clones derived from two different metagenomic preparations. Thereby we identified two novel UDP glycosyltransferase (UGT) genes. The metagenome-derived gtfC gene encoded a 52-kDa protein, and the deduced amino acid sequence was weakly similar to sequences of putative UGTs from Fibrisoma and Dyadobacter. GtfC mediated the transfer of different hexose moieties and exhibited high activities on flavones, flavonols, flavanones, and stilbenes and also accepted isoflavones and chalcones. From the control library we identified a novel macroside glycosyltransferase (MGT) with a calculated molecular mass of 46 kDa. The deduced amino acid sequence was highly similar to sequences of MGTs from Bacillus thuringiensis. Recombinant MgtB transferred the sugar residue from UDP-glucose effectively to flavones, flavonols, isoflavones, and flavanones. Moreover, MgtB exhibited high activity on larger flavonoid molecules such as tiliroside.

  12. Mining the metagenome of activated biomass of an industrial wastewater treatment plant by a novel method.

    Science.gov (United States)

    Sharma, Nandita; Tanksale, Himgouri; Kapley, Atya; Purohit, Hemant J

    2012-12-01

    Metagenomic libraries herald the era of magnifying the microbial world, tapping into the vast metabolic potential of uncultivated microbes, and enhancing the rate of discovery of novel genes and pathways. In this paper, we describe a method that facilitates the extraction of metagenomic DNA from activated sludge of an industrial wastewater treatment plant and its use in mining the metagenome via library construction. The efficiency of this method was demonstrated by the large representation of the bacterial genome in the constructed metagenomic libraries and by the functional clones obtained. The BAC library represented 95.6 times the bacterial genome, while, the pUC library represented 41.7 times the bacterial genome. Twelve clones in the BAC library demonstrated lipolytic activity, while four clones demonstrated dioxygenase activity. Four clones in pUC library tested positive for cellulase activity. This method, using FTA cards, not only can be used for library construction, but can also store the metagenome at room temperature.

  13. Comparative analysis of acidobacterial genomic fragments from terrestrial and aquatic metagenomic libraries, with emphasis on acidobacteria subdivision 6.

    Science.gov (United States)

    Kielak, Anna M; van Veen, Johannes A; Kowalchuk, George A

    2010-10-01

    The bacterial phylum Acidobacteria has a widespread distribution and is one of the most common and diverse phyla in soil habitats. However, members of this phylum have often been recalcitrant to cultivation methods, hampering the study of this presumably important bacterial group. In this study, we used a cultivation-independent metagenomic approach to recover genomic information from soilborne members of this phylum. A soil metagenomic fosmid library was screened by PCR targeting acidobacterial 16S rRNA genes, facilitating the recovery of 17 positive clones. Recovered inserts appeared to originate from a range of Acidobacteria subdivisions, with dominance of subdivision 6 (10 clones). Upon full-length insert sequencing, gene annotation identified a total of 350 open reading frames (ORFs), representing a broad range of functions. Remarkably, six inserts from subdivision 6 contained a region of gene synteny, containing genes involved in purine de novo biosynthesis and encoding tRNA synthetase and conserved hypothetical proteins. Similar genomic regions had previously been observed in several environmental clones recovered from soil and marine sediments, facilitating comparisons with respect to gene organization and evolution. Comparative analyses revealed a general dichotomy between marine and terrestrial genes in both phylogeny and G+C content. Although the significance of this homologous gene cluster across subdivision 6 members is not known, it appears to be a common feature within a large percentage of all acidobacterial genomic fragments recovered from both of these environments.

  14. Construction of a Metagenomic DNA Library of Sponge Symbionts and Screening of Antibacterial Metabolites

    Institute of Scientific and Technical Information of China (English)

    CHEN Juan; ZHU Tianjiao; LI Dehai; CUI Chengbin; FANG Yuchun; LIU Hongbing; LIU Peipei; GU Qianqun; ZHU Weiming

    2006-01-01

    To study the bioactive metabolites produced by sponge-derived uncultured symbionts, a metagenomic DNA library of the symbionts of sponge Gelliodes gracilis was constructed. The average size of DNA inserts in the library was 20 kb. This library was screened for antibiotic activity using paper disc assaying. Two clones displayed the antibacterial activity against Micrococcus tetragenus. The metabolites of these two clones were analyzed through HPLC. The result showed that their metabolites were quite different from those of the host E. coli DH5α and the host containing vector pHZ132. This study may present a new approach to exploring bioactive metabolites of sponge symbionts.

  15. Bacterial Human Virulence Genes across Diverse Habitats As Assessed by In silico Analysis of Environmental Metagenomes

    DEFF Research Database (Denmark)

    Søborg, Ditte A; Hendriksen, Niels B; Kilian, Mogens;

    2016-01-01

    and glacial ice. Homologs to 16 bacterial human virulence genes, involved in urinary tract infections, gastrointestinal diseases, skin diseases, and wound and systemic infections, showed global ubiquity. A principal component analysis did not demonstrate clear trends across the metagenomes with respect...

  16. Metagenomic evaluation of bacterial and archaeal diversity in the geothermal hot springs of manikaran, India.

    Science.gov (United States)

    Bhatia, Sonu; Batra, Navneet; Pathak, Ashish; Green, Stefan J; Joshi, Amit; Chauhan, Ashvini

    2015-02-19

    Bacterial and archaeal diversity in geothermal spring water were investigated using 16S rRNA gene amplicon metagenomic sequencing. This revealed the dominance of Firmicutes, Aquificae, and the Deinococcus-Thermus group in this thermophilic environment. A number of sequences remained taxonomically unresolved, indicating the presence of potentially novel microbes in this unique habitat.

  17. Preparation of high-molecular weight DNA and metagenomic libraries from soils and hot springs.

    Science.gov (United States)

    Reigstad, Laila J; Bartossek, Rita; Schleper, Christa

    2011-01-01

    Metagenomics has become an important tool for the characterization of microorganisms, as it is independent of their enrichment or cultivation in the laboratory. Its application has led to the discovery of metabolisms from widespread, yet uncharacterized organisms such as the ammonia-oxidizing archaea. Different approaches ranging from the generation of short sequence reads by direct use of high-throughput sequencing technologies to the construction and sequencing of large-insert DNA libraries are being employed. For these purposes, DNA of high quality needs to be prepared from an environmental sample, which is a particular challenge for soils and sediments. Here we describe the methods used for the isolation of high-molecular weight (hmw) DNA from soil and hot spring samples, the subsequent production of large-insert metagenomic libraries, and the analysis of the resulting genomic fragments. Detailed step-by-step procedures include (1) how to isolate good-quality hmw DNA from soils and mud; (2) how to prepare the DNA for cloning; (3) how to efficiently establish, grow, pick, replicate, and store the large-insert metagenomic fosmid library; and finally, (4) how to screen the library for genes of interest.

  18. Construction and validation of metagenomic DNA libraries from landfarm soil microorganisms.

    Science.gov (United States)

    Pessoa, T B A; de Souza, S S; Cerqueira, A F; Rezende, R P; Pirovani, C P; Dias, J C T

    2013-06-28

    Landfarming biodegradation is a strategy used by the petrochemical industry to reduce pollutants in petroleum-contaminated soil. We constructed 2 metagenomic libraries from landfarming soil in order to determine the pathway used for mineralization of benzene and to examine protein expression of the bacteria in these soils. The DNA of landfarm soil, collected from Ilhéus, BA, Brazil, was extracted and a metagenomic library was constructed with the Copy Control(TM) Fosmid Library Production Kit, which clones 25-45-kb DNA fragments. The clones were selected for their ability to express enzymes capable of cleaving aromatic compounds. These clones were grown in Luria-Bertani broth plus L-arabinose, benzene, and chloramphenicol as induction substances; they were tested for activity in the catechol cleavage pathway, an intermediate step in benzene degradation. Nine clones were positive for ortho-cleavage and one was positive for meta-cleavage. Protein band patterns determined by SDS-polyacrylamide gel electrophoresis differed in bacteria grown on induced versus non-induced media (Luria-Bertani broth). We concluded that the DNA of landfarm soil is an important source of genes involved in mineralization of xenobiotic compounds, which are common in gasoline and oil spills. Metagenomic library allows identification of non-culturable microorganisms that have potential in the bioremediation of contaminated sites.

  19. Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.

    Directory of Open Access Journals (Sweden)

    Kathy N Lam

    Full Text Available High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metagenomics. In this area of research, environmental DNA is typically cloned to generate large-insert libraries from which individual clones are isolated, based on specific activities of interest. Sequence data are required for complete characterization of such clones, but the sequencing of a large set of clones requires individual barcode-based sample preparation; this can become costly, as the cost of clone barcoding scales linearly with the number of clones processed, and thus sequencing a large number of metagenomic clones often remains cost-prohibitive. We investigated a hybrid Sanger/Illumina pooled sequencing strategy that omits barcoding altogether, and we evaluated this strategy by comparing the pooled sequencing results to reference sequence data obtained from traditional barcode-based sequencing of the same set of clones. Using identity and coverage metrics in our evaluation, we show that pooled sequencing can generate high-quality sequence data, without producing problematic chimeras. Though caveats of a pooled strategy exist and further optimization of the method is required to improve recovery of complete clone sequences and to avoid circumstances that generate unrecoverable clone sequences, our results demonstrate that pooled sequencing represents an effective and low-cost alternative for sequencing large sets of metagenomic clones.

  20. Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.

    Science.gov (United States)

    Lam, Kathy N; Hall, Michael W; Engel, Katja; Vey, Gregory; Cheng, Jiujun; Neufeld, Josh D; Charles, Trevor C

    2014-01-01

    High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metagenomics. In this area of research, environmental DNA is typically cloned to generate large-insert libraries from which individual clones are isolated, based on specific activities of interest. Sequence data are required for complete characterization of such clones, but the sequencing of a large set of clones requires individual barcode-based sample preparation; this can become costly, as the cost of clone barcoding scales linearly with the number of clones processed, and thus sequencing a large number of metagenomic clones often remains cost-prohibitive. We investigated a hybrid Sanger/Illumina pooled sequencing strategy that omits barcoding altogether, and we evaluated this strategy by comparing the pooled sequencing results to reference sequence data obtained from traditional barcode-based sequencing of the same set of clones. Using identity and coverage metrics in our evaluation, we show that pooled sequencing can generate high-quality sequence data, without producing problematic chimeras. Though caveats of a pooled strategy exist and further optimization of the method is required to improve recovery of complete clone sequences and to avoid circumstances that generate unrecoverable clone sequences, our results demonstrate that pooled sequencing represents an effective and low-cost alternative for sequencing large sets of metagenomic clones.

  1. The extraordinary potential of metagenomic tools for food microbiology: an example with bacterial microbiota of raw and pasteurized milk cheeses

    OpenAIRE

    Delhalle, Laurent; Nezer, Carine; Taminiau, Bernard; Darcis, Amelie; Daube,Georges

    2012-01-01

    Among the culture-independent techniques, ultra-sequencing has contributed to place metagenomic analysis as the best alternative to study complex microbiota. During the last three years, metagenomic studies were used essentially for environmental samples but it could be used also to analyse bacterial populations of food samples. This work describes the application of this technique to study the bacterial population of different types of soft cheeses. Among these, three of them are a typical B...

  2. Synthetic biology approaches to improve biocatalyst identification in metagenomic library screening.

    Science.gov (United States)

    Guazzaroni, María-Eugenia; Silva-Rocha, Rafael; Ward, Richard John

    2015-01-01

    There is a growing demand for enzymes with improved catalytic performance or tolerance to process-specific parameters, and biotechnology plays a crucial role in the development of biocatalysts for use in industry, agriculture, medicine and energy generation. Metagenomics takes advantage of the wealth of genetic and biochemical diversity present in the genomes of microorganisms found in environmental samples, and provides a set of new technologies directed towards screening for new catalytic activities from environmental samples with potential biotechnology applications. However, biased and low level of expression of heterologous proteins in Escherichia coli together with the use of non-optimal cloning vectors for the construction of metagenomic libraries generally results in an extremely low success rate for enzyme identification. The bottleneck arising from inefficient screening of enzymatic activities has been addressed from several perspectives; however, the limitations related to biased expression in heterologous hosts cannot be overcome by using a single approach, but rather requires the synergetic implementation of multiple methodologies. Here, we review some of the principal constraints regarding the discovery of new enzymes in metagenomic libraries and discuss how these might be resolved by using synthetic biology methods.

  3. Improvement of the representation of bifidobacteria in fecal microbiota metagenomic libraries by application of the cpn60 universal primer cocktail.

    Science.gov (United States)

    Hill, Janet E; Fernando, W M Ursla; Zello, Gordon A; Tyler, Robert T; Dahl, Wendy J; Van Kessel, Andrew G

    2010-07-01

    Actinobacteria, particularly bifidobacteria, are widely observed to be underrepresented in metagenomic studies of microbial communities. We have compared human fecal microbiota clone libraries based on 16S rRNA and cpn60 PCR products. Taxonomic profiles were similar except that the cpn60 libraries contained large numbers of bifidobacterial sequences.

  4. Substrate-induced gene expression screening: a method for high-throughput screening of metagenome libraries.

    Science.gov (United States)

    Uchiyama, Taku; Miyazaki, Kentaro

    2010-01-01

    The SIGEX (substrate-induced gene expression) method is a novel approach for the screening of gene (genome) libraries. In addition to the commonly used function- and sequence-driven approaches to screening, SIGEX provides a third option; in SIGEX, positives are identified using a reporter gene, and the library is constructed using an "operon-trap" vector. This vector contains the reporter gene immediately downstream of the cloning site for the genomic insert so that the expression of the inserted gene(s) is coupled with that of the reporter gene. This system is especially suitable for screening catabolic genes that are induced in response to metabolically relevant compounds, such as substrates. If expression of the inserted gene(s) is activated in response to the addition of these compounds, then positive clones can be identified based on the reporter signal. The most effective selection is obtained by the use of a FACS (fluorescence-activated cell sorter) in conjunction with a FACS-compatible fluorescent reporter protein, such as GFP (green fluorescent protein). Activity-based screening of metagenomic libraries often suffers from low sensitivity and low throughput. In contrast, the high throughput, high sensitivity, and versatility of SIGEX make it a particularly suitable method for screening metagenomic libraries.

  5. An improved protocol for DNA extraction from alkaline soil and sediment samples for constructing metagenomic libraries.

    Science.gov (United States)

    Verma, Digvijay; Satyanarayana, T

    2011-09-01

    An improved single-step protocol has been developed for extracting pure community humic substance-free DNA from alkaline soils and sediments. The method is based on direct cell lysis in the presence of powdered activated charcoal and polyvinylpolypyrrolidone followed by precipitation with polyethyleneglycol and isopropanol. The strategy allows simultaneous isolation and purification of DNA while minimizing the loss of DNA with respect to other available protocols for metagenomic DNA extraction. Moreover, the purity levels are significant, which are difficult to attain with any of the methods reported in the literature for DNA extraction from soils. The DNA thus extracted was free from humic substances and, therefore, could be processed for restriction digestion, PCR amplification as well as for the construction of metagenomic libraries.

  6. Function-Based Metagenomic Library Screening and Heterologous Expression Strategy for Genes Encoding Phosphatase Activity.

    Science.gov (United States)

    Villamizar, Genis A Castillo; Nacke, Heiko; Daniel, Rolf

    2017-01-01

    The release of phosphate from inorganic and organic phosphorus compounds can be mediated enzymatically. Phosphate-releasing enzymes, comprising acid and alkaline phosphatases, are recognized as useful biocatalysts in applications such as plant and animal nutrition, bioremediation and diagnostic analysis. Metagenomic approaches provide access to novel phosphatase-encoding genes. Here, we describe a function-based screening approach for rapid identification of genes conferring phosphatase activity from small-insert and large-insert metagenomic libraries derived from various environments. This approach bears the potential for discovery of entirely novel phosphatase families or subfamilies and members of known enzyme classes hydrolyzing phosphomonoester bonds such as phytases. In addition, we provide a strategy for efficient heterologous phosphatase gene expression.

  7. Characterization of a Novel Amylolytic Enzyme Encoded by a Gene from a Soil-Derived Metagenomic Library

    OpenAIRE

    2004-01-01

    It has been estimated that less than 1% of the microorganisms in nature can be cultivated by conventional techniques. Thus, the classical approach of isolating enzymes from pure cultures allows the analysis of only a subset of the total naturally occurring microbiota in environmental samples enriched in microorganisms. To isolate useful microbial enzymes from uncultured soil microorganisms, a metagenome was isolated from soil samples, and a metagenomic library was constructed by using the pUC...

  8. 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

  9. Global metagenomic survey reveals a new bacterial candidate phylum in geothermal springs.

    Science.gov (United States)

    Eloe-Fadrosh, Emiley A; Paez-Espino, David; Jarett, Jessica; Dunfield, Peter F; Hedlund, Brian P; Dekas, Anne E; Grasby, Stephen E; Brady, Allyson L; Dong, Hailiang; Briggs, Brandon R; Li, Wen-Jun; Goudeau, Danielle; Malmstrom, Rex; Pati, Amrita; Pett-Ridge, Jennifer; Rubin, Edward M; Woyke, Tanja; Kyrpides, Nikos C; Ivanova, Natalia N

    2016-01-27

    Analysis of the increasing wealth of metagenomic data collected from diverse environments can lead to the discovery of novel branches on the tree of life. Here we analyse 5.2 Tb of metagenomic data collected globally to discover a novel bacterial phylum ('Candidatus Kryptonia') found exclusively in high-temperature pH-neutral geothermal springs. This lineage had remained hidden as a taxonomic 'blind spot' because of mismatches in the primers commonly used for ribosomal gene surveys. Genome reconstruction from metagenomic data combined with single-cell genomics results in several high-quality genomes representing four genera from the new phylum. Metabolic reconstruction indicates a heterotrophic lifestyle with conspicuous nutritional deficiencies, suggesting the need for metabolic complementarity with other microbes. Co-occurrence patterns identifies a number of putative partners, including an uncultured Armatimonadetes lineage. The discovery of Kryptonia within previously studied geothermal springs underscores the importance of globally sampled metagenomic data in detection of microbial novelty, and highlights the extraordinary diversity of microbial life still awaiting discovery.

  10. Phylogeny-function analysis of (meta)genomic libraries: screening for expression of ribosomal RNA genes by large-insert library fluorescent in situ hybridization (LIL-FISH).

    Science.gov (United States)

    Leveau, Johan H J; Gerards, Saskia; de Boer, Wietse; van Veen, Johannes A

    2004-09-01

    We assessed the utility of fluorescent in situ hybridization (FISH) in the screening of clone libraries of (meta)genomic or environmental DNA for the presence and expression of bacterial ribosomal RNA (rRNA) genes. To establish proof-of-principle, we constructed a fosmid-based library in Escherichia coli of large-sized genomic DNA fragments of the mycophagous soil bacterium Collimonas fungivorans, and hybridized 768 library clones with the Collimonas-specific fluorescent probe CTE998-1015. Critical to the success of this approach (which we refer to as large-insert library FISH or LIL-FISH) was the ability to induce fosmid copy number, the exponential growth status of library clones in the FISH assay and the use of a simple pooling strategy to reduce the number of hybridizations. Twelve out of 768 E. coli clones were suspected to harbour and express Collimonas 16S rRNA genes based on their hybridization to CTE998-1015. This was confirmed by the finding that all 12 clones were also identified in an independent polymerase chain reaction-based screening of the same 768 clones using a primer set for the specific detection of Collimonas 16S ribosomal DNA (rDNA). Fosmids isolated from these clones were grouped by restriction analysis into two distinct contigs, confirming that C. fungivorans harbours at least two 16S rRNA genes. For one contig, representing 1-2% of the genome, the nucleotide sequence was determined, providing us with a narrow but informative view of Collimonas genome structure and content.

  11. SCREENING OF PROTEASE ENZYME BY CONSTRUCTION OF METAGENOMIC LIBRARY FROM MARINE SOIL SEDIMENTS

    Directory of Open Access Journals (Sweden)

    R.PRABAVATHI

    2012-07-01

    Full Text Available Nonetheless, the cultivable microorganisms constituting these resources correspond to only a small fraction of the microbial diversity less than 1% of the microorganisms in various environments are readily cultivable (Amann et al., 1995. This limits the range of a search for new biocatalysts for the bioprocess industry, so the use of complex communities and the effort to overcome the problem of noncultivability attract not only scientific attention but also biotechnological innovation. Methods have been developed and used toovercome the non-cultivability of environmental microorganisms for biotechnology, namely cloning and the expression of metagenomes in suitable expression hosts. Proteases are present in all living forms as they are involved in various metabolic processes. They are mainly involved in hydrolysis of the peptide bonds (Gupta et al., 2002. Proteases find a wide range of applications in food, pharmaceutical, leather and textile, detergent, diagnostics industries and also in waste management. In order to discover new proteases from metagenomiclibraries, we screened for proteolytic activity from a constructed metagenomic library by direct cloning of environmental DNA of large DNA inserts. A novel gene encoding proteolytic enzyme was picked up,sequenced, expressed in E. coli and characterized. Several microbial proteases from the culturable organisms have been characterized. However, very few proteases have been identified through culture independent metagenomic approach.

  12. Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones.

    Science.gov (United States)

    Ginolhac, Aurélien; Jarrin, Cyrille; Gillet, Benjamin; Robe, Patrick; Pujic, Petar; Tuphile, Karine; Bertrand, Hélène; Vogel, Timothy M; Perrière, Guy; Simonet, Pascal; Nalin, Renaud

    2004-09-01

    The metagenomic approach provides direct access to diverse unexplored genomes, especially from uncultivated bacteria in a given environment. This diversity can conceal many new biosynthetic pathways. Type I polyketide synthases (PKSI) are modular enzymes involved in the biosynthesis of many natural products of industrial interest. Among the PKSI domains, the ketosynthase domain (KS) was used to screen a large soil metagenomic library containing more than 100,000 clones to detect those containing PKS genes. Over 60,000 clones were screened, and 139 clones containing KS domains were detected. A 700-bp fragment of the KS domain was sequenced for 40 of 139 randomly chosen clones. None of the 40 protein sequences were identical to those found in public databases, and nucleic sequences were not redundant. Phylogenetic analyses were performed on the protein sequences of three metagenomic clones to select the clones which one can predict to produce new compounds. Two PKS-positive clones do not belong to any of the 23 published PKSI included in the analysis, encouraging further analyses on these two clones identified by the selection process.

  13. Product-induced gene expression, a product-responsive reporter assay used to screen metagenomic libraries for enzyme-encoding genes.

    Science.gov (United States)

    Uchiyama, Taku; Miyazaki, Kentaro

    2010-11-01

    A reporter assay-based screening method for enzymes, which we named product-induced gene expression (PIGEX), was developed and used to screen a metagenomic library for amidases. A benzoate-responsive transcriptional activator, BenR, was placed upstream of the gene encoding green fluorescent protein and used as a sensor. Escherichia coli sensor cells carrying the benR-gfp gene cassette fluoresced in response to benzoate concentrations as low as 10 μM but were completely unresponsive to the substrate benzamide. An E. coli metagenomic library consisting of 96,000 clones was grown in 96-well format in LB medium containing benzamide. The library cells were then cocultivated with sensor cells. Eleven amidase genes were recovered from 143 fluorescent wells; eight of these genes were homologous to known bacterial amidase genes while three were novel genes. In addition to their activity toward benzamide, the enzymes were active toward various substrates, including d- and l-amino acid amides, and displayed enantioselectivity. Thus, we demonstrated that PIGEX is an effective approach for screening novel enzymes based on product detection.

  14. Targeting modular polyketide synthases with iteratively acting acyltransferases from metagenomes of uncultured bacterial consortia.

    Science.gov (United States)

    Piel, Jörn; Hui, Dequan; Fusetani, Nobuhiro; Matsunaga, Shigeki

    2004-09-01

    Bacterial type I polyketide synthases (PKSs) produce a wide range of biomedically important secondary metabolites. These enzymes possess a modular structure that can be genetically re-engineered to yield novel drug candidates not found in nature. Recently, we have reported the putative pederin PKS from an uncultured bacterial symbiont of Paederus fuscipes beetles. It belongs to an architecturally unusual PKS group, the members of which contain iteratively acting acyltransferases that are not integrated into the PKS modules but are encoded by isolated genes. As these systems are rare, often contain additional unusual features and are of smaller size than regular PKSs, the development of a method for the targeted isolation of new group members would be of great interest. Here, we present a phylogenetic approach to identify these systems rapidly in highly complex metagenomic DNA samples. To demonstrate its practical value, we located two pederin-type PKS systems putatively involved in the biosynthesis of antitumour polyketides in the metagenomic DNA of beetles, sponges and their uncultivated bacterial symbionts.

  15. Rapid identification of genes encoding DNA polymerases by function-based screening of metagenomic libraries derived from glacial ice.

    Science.gov (United States)

    Simon, Carola; Herath, Judith; Rockstroh, Stephanie; Daniel, Rolf

    2009-05-01

    Small-insert and large-insert metagenomic libraries were constructed from glacial ice of the Northern Schneeferner, which is located on the Zugspitzplatt in Germany. Subsequently, these libraries were screened for the presence of DNA polymerase-encoding genes by complementation of an Escherichia coli polA mutant. Nine novel genes encoding complete DNA polymerase I proteins or domains typical of these proteins were recovered.

  16. Rapid Identification of Genes Encoding DNA Polymerases by Function-Based Screening of Metagenomic Libraries Derived from Glacial Ice▿

    OpenAIRE

    2009-01-01

    Small-insert and large-insert metagenomic libraries were constructed from glacial ice of the Northern Schneeferner, which is located on the Zugspitzplatt in Germany. Subsequently, these libraries were screened for the presence of DNA polymerase-encoding genes by complementation of an Escherichia coli polA mutant. Nine novel genes encoding complete DNA polymerase I proteins or domains typical of these proteins were recovered.

  17. Expression and characterization of a new esterase with GCSAG motif from a permafrost metagenomic library.

    Science.gov (United States)

    Petrovskaya, Lada E; Novototskaya-Vlasova, Ksenia A; Spirina, Elena V; Durdenko, Ekaterina V; Lomakina, Galina Yu; Zavialova, Maria G; Nikolaev, Evgeny N; Rivkina, Elizaveta M

    2016-05-01

    As a result of construction and screening of a metagenomic library prepared from a permafrost-derived microcosm, we have isolated a novel gene coding for a putative lipolytic enzyme that belongs to the hormone-sensitive lipase family. It encodes a polypeptide of 343 amino acid residues whose amino acid sequence displays maximum likelihood with uncharacterized proteins from Sphingomonas species. A putative catalytic serine residue of PMGL2 resides in a new variant of a recently discovered GTSAG sequence in which a Thr residue is replaced by a Cys residue (GCSAG). The recombinant PMGL2 was produced in Escherichia coli cells and purified by Ni-affinity chromatography. The resulting protein preferably utilizes short-chain p-nitrophenyl esters (C4 and C8) and therefore is an esterase. It possesses maximum activity at 45°C in slightly alkaline conditions and has limited thermostability at higher temperatures. Activity of PMGL2 is stimulated in the presence of 0.25-1.5 M NaCl indicating the good salt tolerance of the new enzyme. Mass spectrometric analysis demonstrated that N-terminal methionine in PMGL2 is processed and cysteine residues do not form a disulfide bond. The results of the study demonstrate the significance of the permafrost environment as a unique genetic reservoir and its potential for metagenomic exploration.

  18. Novel Lipolytic Enzymes Identified from Metagenomic Library of Deep-Sea Sediment

    Directory of Open Access Journals (Sweden)

    Jeong Ho Jeon

    2011-01-01

    Full Text Available Metagenomic library was constructed from a deep-sea sediment sample and screened for lipolytic activity. Open-reading frames of six positive clones showed only 33–58% amino acid identities to the known proteins. One of them was assigned to a new group while others were grouped into Families I and V or EstD Family. By employing a combination of approaches such as removing the signal sequence, coexpression of chaperone genes, and low temperature induction, we obtained five soluble recombinant proteins in Escherichia coli. The purified enzymes had optimum temperatures of 30–35°C and the cold-activity property. Among them, one enzyme showed lipase activity by preferentially hydrolyzing p-nitrophenyl palmitate and p-nitrophenyl stearate and high salt resistance with up to 4 M NaCl. Our research demonstrates the feasibility of developing novel lipolytic enzymes from marine environments by the combination of functional metagenomic approach and protein expression technology.

  19. Biocatalysts and small molecule products from metagenomic studies.

    Science.gov (United States)

    Iqbal, Hala A; Feng, Zhiyang; Brady, Sean F

    2012-04-01

    The vast majority of bacteria present in environmental samples have never been cultured and therefore have not been exploited for the ability to produce useful biocatalysts or collections of biocatalysts generating interesting small molecules. Metagenomic libraries constructed using DNA extracted directly from natural bacterial communities offer access to the genetic information present in the genomes of these as yet uncultured bacteria. This review highlights recent efforts to recover both discrete enzymes and small molecules from metagenomic libraries.

  20. Phylogenetic and metagenomic analyses of substrate-dependent bacterial temporal dynamics in microbial fuel cells.

    Directory of Open Access Journals (Sweden)

    Husen Zhang

    Full Text Available Understanding the microbial community structure and genetic potential of anode biofilms is key to improve extracellular electron transfers in microbial fuel cells. We investigated effect of substrate and temporal dynamics of anodic biofilm communities using phylogenetic and metagenomic approaches in parallel with electrochemical characterizations. The startup non-steady state anodic bacterial structures were compared for a simple substrate, acetate, and for a complex substrate, landfill leachate, using a single-chamber air-cathode microbial fuel cell. Principal coordinate analysis showed that distinct community structures were formed with each substrate type. The bacterial diversity measured as Shannon index decreased with time in acetate cycles, and was restored with the introduction of leachate. The change of diversity was accompanied by an opposite trend in the relative abundance of Geobacter-affiliated phylotypes, which were acclimated to over 40% of total Bacteria at the end of acetate-fed conditions then declined in the leachate cycles. The transition from acetate to leachate caused a decrease in output power density from 243±13 mW/m2 to 140±11 mW/m2, accompanied by a decrease in Coulombic electron recovery from 18±3% to 9±3%. The leachate cycles selected protein-degrading phylotypes within phylum Synergistetes. Metagenomic shotgun sequencing showed that leachate-fed communities had higher cell motility genes including bacterial chemotaxis and flagellar assembly, and increased gene abundance related to metal resistance, antibiotic resistance, and quorum sensing. These differentially represented genes suggested an altered anodic biofilm community in response to additional substrates and stress from the complex landfill leachate.

  1. Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries

    OpenAIRE

    2013-01-01

    The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic ge...

  2. Analysis of a viral metagenomic library from 200 m depth in Monterey Bay, California constructed by direct shotgun cloning

    Directory of Open Access Journals (Sweden)

    Preston Christina M

    2011-06-01

    Full Text Available Abstract Background Viruses have a profound influence on both the ecology and evolution of marine plankton, but the genetic diversity of viral assemblages, particularly those in deeper ocean waters, remains poorly described. Here we report on the construction and analysis of a viral metagenome prepared from below the euphotic zone in a temperate, eutrophic bay of coastal California. Methods We purified viruses from approximately one cubic meter of seawater collected from 200m depth in Monterey Bay, CA. DNA was extracted from the virus fraction, sheared, and cloned with no prior amplification into a plasmid vector and propagated in E. coli to produce the MBv200m library. Random clones were sequenced by the Sanger method. Sequences were assembled then compared to sequences in GenBank and to other viral metagenomic libraries using BLAST analyses. Results Only 26% of the 881 sequences remaining after assembly had significant (E ≤ 0.001 BLAST hits to sequences in the GenBank nr database, with most being matches to bacteria (15% and viruses (8%. When BLAST analysis included environmental sequences, 74% of sequences in the MBv200m library had a significant match. Most of these hits (70% were to microbial metagenome sequences and only 0.7% were to sequences from viral metagenomes. Of the 121 sequences with a significant hit to a known virus, 94% matched bacteriophages (Families Podo-, Sipho-, and Myoviridae and 6% matched viruses of eukaryotes in the Family Phycodnaviridae (5 sequences or the Mimivirus (2 sequences. The largest percentages of hits to viral genes of known function were to those involved in DNA modification (25% or structural genes (17%. Based on reciprocal BLAST analyses, the MBv200m library appeared to be most similar to viral metagenomes from two other bays and least similar to a viral metagenome from the Arctic Ocean. Conclusions Direct cloning of DNA from diverse marine viruses was feasible and resulted in a distribution of virus

  3. Novel, deep-branching heterotrophic bacterial populations recovered from thermal spring metagenomes

    Directory of Open Access Journals (Sweden)

    Daniel R Colman

    2016-03-01

    Full Text Available Thermal spring ecosystems are a valuable resource for the discovery of novel hyperthermophilic Bacteria and Archaea, and harbor deeply-branching lineages that provide insight regarding the nature of early microbial life. We characterized bacterial populations in two circumneutral (pH ~ 8 Yellowstone National Park thermal (T ~ 80 oC spring filamentous ‘streamer’ communities using random metagenomic DNA sequence to investigate the metabolic potential of these novel populations. Four de novo assemblies representing three abundant, deeply-branching bacterial phylotypes were recovered. Analysis of conserved phylogenetic marker genes indicated that two of the phylotypes represent separate groups of an uncharacterized phylum (for which we propose the candidate phylum name ‘Pyropristinus’. The third new phylotype falls within the proposed Calescamantes phylum. Metabolic reconstructions of the 'Pyropristinus' and Calescamantes populations showed that these organisms appear to be chemoorganoheterotrophs, and have the genomic potential for aerobic respiration and oxidative phosphorylation via archaeal-like V-type, and bacterial F-type ATPases, respectively. A survey of similar phylotypes (> 97% nt identity within 16S rRNA gene datasets suggest that the newly described organisms are restricted to terrestrial thermal springs ranging from 70 - 90 oC and pH values of ~ 7 - 9. The characterization of these lineages is important for understanding the diversity of deeply-branching bacterial phyla, and their functional role in high-temperature circumneutral ‘streamer’ communities.

  4. Genome-wide Selective Sweeps in Natural Bacterial Populations Revealed by Time-series Metagenomics

    Energy Technology Data Exchange (ETDEWEB)

    Chan, Leong-Keat; Bendall, Matthew L.; Malfatti, Stephanie; Schwientek, Patrick; Tremblay, Julien; Schackwitz, Wendy; Martin, Joel; Pati, Amrita; Bushnell, Brian; Foster, Brian; Kang, Dongwan; Tringe, Susannah G.; Bertilsson, Stefan; Moran, Mary Ann; Shade, Ashley; Newton, Ryan J.; Stevens, Sarah; McMahon, Katherine D.; Malmstrom, Rex R.

    2014-06-18

    Multiple evolutionary models have been proposed to explain the formation of genetically and ecologically distinct bacterial groups. Time-series metagenomics enables direct observation of evolutionary processes in natural populations, and if applied over a sufficiently long time frame, this approach could capture events such as gene-specific or genome-wide selective sweeps. Direct observations of either process could help resolve how distinct groups form in natural microbial assemblages. Here, from a three-year metagenomic study of a freshwater lake, we explore changes in single nucleotide polymorphism (SNP) frequencies and patterns of gene gain and loss in populations of Chlorobiaceae and Methylophilaceae. SNP analyses revealed substantial genetic heterogeneity within these populations, although the degree of heterogeneity varied considerably among closely related, co-occurring Methylophilaceae populations. SNP allele frequencies, as well as the relative abundance of certain genes, changed dramatically over time in each population. Interestingly, SNP diversity was purged at nearly every genome position in one of the Chlorobiaceae populations over the course of three years, while at the same time multiple genes either swept through or were swept from this population. These patterns were consistent with a genome-wide selective sweep, a process predicted by the ‘ecotype model’ of diversification, but not previously observed in natural populations.

  5. Genome-wide Selective Sweeps in Natural Bacterial Populations Revealed by Time-series Metagenomics

    Energy Technology Data Exchange (ETDEWEB)

    Chan, Leong-Keat; Bendall, Matthew L.; Malfatti, Stephanie; Schwientek, Patrick; Tremblay, Julien; Schackwitz, Wendy; Martin, Joel; Pati, Amrita; Bushnell, Brian; Foster, Brian; Kang, Dongwan; Tringe, Susannah G.; Bertilsson, Stefan; Moran, Mary Ann; Shade, Ashley; Newton, Ryan J.; Stevens, Sarah; McMcahon, Katherine D.; Mamlstrom, Rex R.

    2014-05-12

    Multiple evolutionary models have been proposed to explain the formation of genetically and ecologically distinct bacterial groups. Time-series metagenomics enables direct observation of evolutionary processes in natural populations, and if applied over a sufficiently long time frame, this approach could capture events such as gene-specific or genome-wide selective sweeps. Direct observations of either process could help resolve how distinct groups form in natural microbial assemblages. Here, from a three-year metagenomic study of a freshwater lake, we explore changes in single nucleotide polymorphism (SNP) frequencies and patterns of gene gain and loss in populations of Chlorobiaceae and Methylophilaceae. SNP analyses revealed substantial genetic heterogeneity within these populations, although the degree of heterogeneity varied considerably among closely related, co-occurring Methylophilaceae populations. SNP allele frequencies, as well as the relative abundance of certain genes, changed dramatically over time in each population. Interestingly, SNP diversity was purged at nearly every genome position in one of the Chlorobiaceae populations over the course of three years, while at the same time multiple genes either swept through or were swept from this population. These patterns were consistent with a genome-wide selective sweep, a process predicted by the ecotype model? of diversification, but not previously observed in natural populations.

  6. Comparative metagenomic and rRNA microbial diversity characterization using archaeal and bacterial synthetic communities.

    Science.gov (United States)

    Shakya, Migun; Quince, Christopher; Campbell, James H; Yang, Zamin K; Schadt, Christopher W; Podar, Mircea

    2013-06-01

    Next-generation sequencing has dramatically changed the landscape of microbial ecology, large-scale and in-depth diversity studies being now widely accessible. However, determining the accuracy of taxonomic and quantitative inferences and comparing results obtained with different approaches are complicated by incongruence of experimental and computational data types and also by lack of knowledge of the true ecological diversity. Here we used highly diverse bacterial and archaeal synthetic communities assembled from pure genomic DNAs to compare inferences from metagenomic and SSU rRNA amplicon sequencing. Both Illumina and 454 metagenomic data outperformed amplicon sequencing in quantifying the community composition, but the outcome was dependent on analysis parameters and platform. New approaches in processing and classifying amplicons can reconstruct the taxonomic composition of the community with high reproducibility within primer sets, but all tested primers sets lead to significant taxon-specific biases. Controlled synthetic communities assembled to broadly mimic the phylogenetic richness in target environments can provide important validation for fine-tuning experimental and computational parameters used to characterize natural communities.

  7. Analysis of bacterial xylose isomerase gene diversity using gene-targeted metagenomics.

    Science.gov (United States)

    Nurdiani, Dini; Ito, Michihiro; Maruyama, Toru; Terahara, Takeshi; Mori, Tetsushi; Ugawa, Shin; Takeyama, Haruko

    2015-08-01

    Bacterial xylose isomerases (XI) are promising resources for efficient biofuel production from xylose in lignocellulosic biomass. Here, we investigated xylose isomerase gene (xylA) diversity in three soil metagenomes differing in plant vegetation and geographical location, using an amplicon pyrosequencing approach and two newly-designed primer sets. A total of 158,555 reads from three metagenomic DNA replicates for each soil sample were classified into 1127 phylotypes, detected in triplicate and defined by 90% amino acid identity. The phylotype coverage was estimated to be within the range of 84.0-92.7%. The xylA gene phylotypes obtained were phylogenetically distributed across the two known xylA groups. They shared 49-100% identities with their closest-related XI sequences in GenBank. Phylotypes demonstrating analysis, suggesting soil-specific xylA genotypes and taxonomic compositions. The differences among xylA members and their compositions in the soil were strongly correlated with 16S rRNA variation between soil samples, also assessed by amplicon pyrosequencing. This is the first report of xylA diversity in environmental samples assessed by amplicon pyrosequencing. Our data provide information regarding xylA diversity in nature, and can be a basis for the screening of novel xylA genotypes for practical applications.

  8. Detection and isolation of selected genes of interest from metagenomic libraries by a DNA microarray approach.

    Science.gov (United States)

    Pathak, Gopal P; Gärtner, Wolfgang

    2010-01-01

    A DNA microarray-based approach is described for screening metagenomic libraries for the presence of selected genes. The protocol is exemplified for the identification of flavin-binding, blue-light-sensitive biological photoreceptors (BL), based on a homology search in already sequenced, annotated genomes. The microarray carried 149 different 54-mer oligonucleotides, derived from consensus sequences of BL photoreceptors. The array could readily identify targets carrying 4% sequence mismatch, and allowed unambiguous identification of a positive cosmid clone of as little as 10 ng against a background of 25 μg of cosmid DNA. The protocol allows screening up to 1,200 library clones in concentrations as low as ca. 20 ng, each with a ca. 40 kb insert size readily in a single batch. Calibration and control conditions are outlined. This protocol, when applied to the thermophilic fraction of a soil sample, yielded the identification and functional characterization of a novel, BL-encoding gene that showed a 58% similarity to a known, BL-encoding gene from Kineococcus radiotolerans SRS30216 (similarity values refer to the respective LOV domains).

  9. A novel family VII esterase with industrial potential from compost metagenomic library

    Directory of Open Access Journals (Sweden)

    Oh Tae-Kwang

    2011-05-01

    Full Text Available Abstract Background Among the vast microbial genomic resources now available, most microbes are unculturable in the laboratory. A culture-independent metagenomic approach is a novel technique that circumvents this culture limitation. For the screening of novel lipolytic enzymes, a metagenomic library was constructed from compost, and the clone of estCS2 was selected for lipolytic properties on a tributyrin-containing medium. Results The estCS2 sequence encodes a protein of 570 amino acid residues, with a predicted molecular mass of 63 kDa, and based on amino acid identity it most closely matches (45% the carboxylesterase from Haliangium ochraceum DSM 14365. EstCS2 belong to family VII, according to the lipolytic enzyme classification proposed by Arpigny and Jaeger, and it retains the catalytic triad Ser245-Glu363-His466 that is typical of an α/β hydrolase. The Ser245 residue in the catalytic triad of EstCS2 is located in the consensus active site motif GXSXG. The EstCS2 exhibits strong activity toward p-nitrophenyl caproate (C6, and it is stable up to 60°C with an optimal enzymatic activity at 55°C. The maximal activity is observed at pH 9, and it remains active between pH 6-10. EstCS2 shows remarkable stability in up to 50% (v/v dimethyl sulfoxide (DMSO or dimethylformamide (DMF. The enzyme has the ability to cleave sterically hindered esters of tertiary alcohol, as well as to degrade polyurethanes, which are widely used in various industries. Conclusions The high stability of EstCS2 in organic solvents and its activity towards esters of ketoprofen and tertiary alcohols, and in polyurethane suggests that it has potential uses for many applications in biotransformation and bioremediation.

  10. Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries

    OpenAIRE

    2013-01-01

    Recent advances in sequencing technologies generate new predictions and hypotheses about the functional roles of environmental microorganisms. Yet, until we can test these predictions at a scale that matches our ability to generate them, most of them will remain as hypotheses. Function-based mining of metagenomic libraries can provide direct linkages between genes, metabolic traits and microbial taxa and thus bridge this gap between sequence data generation and functional predictions. Here we...

  11. Isolation and characterization of metalloproteases with a novel domain structure by construction and screening of metagenomic libraries.

    Science.gov (United States)

    Waschkowitz, Tanja; Rockstroh, Stephanie; Daniel, Rolf

    2009-04-01

    Small-insert metagenomic libraries from four samples were constructed by a topoisomerase-based and a T4 DNA ligase-based approach. Direct comparison of both approaches revealed that application of the topoisomerase-based method resulted in a higher number of insert-containing clones per microg of environmental DNA used for cloning and a larger average insert size. Subsequently, the constructed libraries were partially screened for the presence of genes conferring proteolytic activity. The function-driven screen was based on the ability of the library-containing Escherichia coli clones to form halos on skim milk-containing agar plates. The screening of 80,000 E. coli clones yielded four positive clones. Two of the plasmids (pTW2 and pTW3) recovered from positive clones conferred strong proteolytic activity and were studied further. Analysis of the entire insert sequences of pTW2 (28,113 bp) and pTW3 (19,956 bp) suggested that the DNA fragments were derived from members of the genus Xanthomonas. Each of the plasmids harbored one gene (2,589 bp) encoding a metalloprotease (mprA, pTW2; mprB, pTW3). Sequence and biochemical analyses revealed that MprA and MprB are similar extracellular proteases belonging to the M4 family of metallopeptidases (thermolysin-like family). Both enzymes possessed a unique modular structure and consisted of four regions: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The architecture of the latter region, which was characterized by the presence of two prepeptidase C-terminal domains and one proprotein convertase P domain, is novel for bacterial metalloproteases. Studies with derivatives of MprA and MprB revealed that the C-terminal extension is not essential for protease activity. The optimum pH and temperature of both proteases were 8.0 and 65 degrees C, respectively, when casein was used as substrate.

  12. Metagenomic profile of the bacterial communities associated with Ixodes ricinus ticks.

    Directory of Open Access Journals (Sweden)

    Giovanna Carpi

    Full Text Available Assessment of the microbial diversity residing in arthropod vectors of medical importance is crucial for monitoring endemic infections, for surveillance of newly emerging zoonotic pathogens, and for unraveling the associated bacteria within its host. The tick Ixodes ricinus is recognized as the primary European vector of disease-causing bacteria in humans. Despite I. ricinus being of great public health relevance, its microbial communities remain largely unexplored to date. Here we evaluate the pathogen-load and the microbiome in single adult I. ricinus by using 454- and Illumina-based metagenomic approaches. Genomic DNA-derived sequences were taxonomically profiled using a computational approach based on the BWA algorithm, allowing for the identification of known tick-borne pathogens at the strain level and the putative tick core microbiome. Additionally, we assessed and compared the bacterial taxonomic profile in nymphal and adult I. ricinus pools collected from two distinct geographic regions in Northern Italy by means of V6-16S rRNA amplicon pyrosequencing and community based ecological analysis. A total of 108 genera belonging to representatives of all bacterial phyla were detected and a rapid qualitative assessment for pathogenic bacteria, such as Borrelia, Rickettsia and Candidatus Neoehrlichia, and for other bacteria with mutualistic relationship or undetermined function, such as Wolbachia and Rickettsiella, was possible. Interestingly, the ecological analysis revealed that the bacterial community structure differed between the examined geographic regions and tick life stages. This finding suggests that the environmental context (abiotic and biotic factors and host-selection behaviors affect their microbiome.Our data provide the most complete picture to date of the bacterial communities present within I. ricinus under natural conditions by using high-throughput sequencing technologies. This study further demonstrates a novel detection

  13. New lipolytic enzymes identified by screening two metagenomic libraries derived from the soil of a winter wheat field

    Directory of Open Access Journals (Sweden)

    Stroobants, A.

    2015-01-01

    Full Text Available Description of the subject. Lipolytic enzymes are widely distributed and fulfil important physiological functions in the microorganisms inhabiting diverse environments. Soils are rich, diversified environments containing microbial communities that remain largely unknown. Objectives. This work aimed to discover new lipolytic enzymes. Method. New enzymes were found by functional screening of two seasonal metagenomic libraries (a winter and a spring library constructed from an agricultural soil. Screens were performed on 2xYT medium supplemented with 3% lipase reagent. Results. Nineteen positive clones were isolated. Analysis of the corresponding inserts led to identifying 23 putative lipolytic enzymes (13 for the winter library and 10 for the spring library displaying between 31% and 62% identity to known enzymes and belonging to seven different families. Conclusions. As enzymes show low identity to known enzymes, the encoded enzymes may display novel biochemical features.

  14. Isolation and characterization of a non-specific endoglucanase from a metagenomic library of goat rumen.

    Science.gov (United States)

    Cheng, Jianbo; Huang, Shuai; Jiang, Haiqin; Zhang, Yunhai; Li, Lvmu; Wang, Juhua; Fan, Caiyun

    2016-01-01

    A cellulase gene (cel28a) was isolated from a rumen microbial metagenome library of goat rumen microorganisms, cloned into E. coli, and expressed in active form. The gene has a length of 1596 bp obtained using a genome walking Kit and encodes a protein of 509 amino acids with a calculated MW of 55 kDa. The deduced amino acid sequence was homologous with cellulases belonging to the glycosyl hydrolase family 5 (GH5). The expressed protein showed activity toward carboxymethylcellulose (CMC) and xylan, suggesting non-specific endoglucanase activity. The optimal conditions for endoglucanase and xylanase activities were 50 °C and pH 5.0. The metal ions (Ca(2+), Fe(2+), Mn(2+) and Co(2+)) stimulated the cellulase activity of cel28a, while the other metal ions and chemicals (Ni(2+), Mg(2+), Zn(2+), Cu(2+), SDS and EDTA) inhibited the cellulase activity. Further examination of substrate preference showed a higher activity with CMC, oat spelt xylan and birchwood xylan than with filter paper and microcrystalline cellulose, again suggesting that the protein was an endoglucanase with xylanase activity.

  15. Isolation and characterization of two serine proteases from metagenomic libraries of the Gobi and Death Valley deserts.

    Science.gov (United States)

    Neveu, Julie; Regeard, Christophe; DuBow, Michael S

    2011-08-01

    The screening of environmental DNA metagenome libraries for functional activities can provide an important source of new molecules and enzymes. In this study, we identified 17 potential protease-producing clones from two metagenomic libraries derived from samples of surface sand from the Gobi and Death Valley deserts. Two of the proteases, DV1 and M30, were purified and biochemically examined. These two proteases displayed a molecular mass of 41.5 kDa and 45.7 kDa, respectively, on SDS polyacrylamide gels. Alignments with known protease sequences showed less than 55% amino acid sequence identity. These two serine proteases appear to belong to the subtilisin (S8A) family and displayed several unique biochemical properties. Protease DV1 had an optimum pH of 8 and an optimal activity at 55°C, while protease M30 had an optimum pH >11 and optimal activity at 40°C. The properties of these enzymes make them potentially useful for biotechnological applications and again demonstrate that metagenomic approaches can be useful, especially when coupled with the study of novel environments such as deserts.

  16. Improvement of the Representation of Bifidobacteria in Fecal Microbiota Metagenomic Libraries by Application of the cpn60 Universal Primer Cocktail ▿

    OpenAIRE

    2010-01-01

    Actinobacteria, particularly bifidobacteria, are widely observed to be underrepresented in metagenomic studies of microbial communities. We have compared human fecal microbiota clone libraries based on 16S rRNA and cpn60 PCR products. Taxonomic profiles were similar except that the cpn60 libraries contained large numbers of bifidobacterial sequences.

  17. A novel salt-tolerant chitobiosidase discovered by genetic screening of a metagenomic library derived from chitin-amended agricultural soil

    NARCIS (Netherlands)

    Cretoiu, Mariana Silvia; Berini, Francesca; Kielak, Anna Maria; Marinelli, Flavia; van Elsas, Jan Dirk

    2015-01-01

    Here, we report on the construction of a metagenomic library from a chitin-amended disease-suppressive agricultural soil and its screening for genes that encode novel chitinolytic enzymes. The library, constructed in fosmids in an Escherichia coli host, comprised 145,000 clones containing inserts of

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-03-16

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

  19. A broad pH range and processive chitinase from a metagenome library

    Directory of Open Access Journals (Sweden)

    S.S. Thimoteo

    Full Text Available Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4 present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N′-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.

  20. Synthesis and evaluation of a series of 6-chloro-4-methylumbelliferyl glycosides as fluorogenic reagents for screening metagenomic libraries for glycosidase activity.

    Science.gov (United States)

    Chen, Hong-Ming; Armstrong, Zachary; Hallam, Steven J; Withers, Stephen G

    2016-02-08

    Screening of large enzyme libraries such as those derived from metagenomic sources requires sensitive substrates. Fluorogenic glycosides typically offer the best sensitivity but typically must be used in a stopped format to generate good signal. Use of fluorescent phenols of pKa metagenomic libraries yielded a "hit rate" of 1 in 60. Hits were then readily deconvoluted with the individual substrates in a single plate to identify specific activities within each clone. The use of such a collection of substrates greatly accelerates the screening process.

  1. Mapping gene clusters within arrayed metagenomic libraries to expand the structural diversity of biomedically relevant natural products.

    Science.gov (United States)

    Owen, Jeremy G; Reddy, Boojala Vijay B; Ternei, Melinda A; Charlop-Powers, Zachary; Calle, Paula Y; Kim, Jeffrey H; Brady, Sean F

    2013-07-16

    Complex microbial ecosystems contain large reservoirs of unexplored biosynthetic diversity. Here we provide an experimental framework and data analysis tool to facilitate the targeted discovery of natural-product biosynthetic gene clusters from the environment. Multiplex sequencing of barcoded PCR amplicons is followed by sequence similarity directed data parsing to identify sequences bearing close resemblance to biosynthetically or biomedically interesting gene clusters. Amplicons are then mapped onto arrayed metagenomic libraries to guide the recovery of targeted gene clusters. When applied to adenylation- and ketosynthase-domain amplicons derived from saturating soil DNA libraries, our analysis pipeline led to the recovery of biosynthetic clusters predicted to encode for previously uncharacterized glycopeptide- and lipopeptide-like antibiotics; thiocoraline-, azinomycin-, and bleomycin-like antitumor agents; and a rapamycin-like immunosuppressant. The utility of the approach is demonstrated by using recovered eDNA sequences to generate glycopeptide derivatives. The experiments described here constitute a systematic interrogation of a soil metagenome for gene clusters capable of encoding naturally occurring derivatives of biomedically relevant natural products. Our results show that previously undetected biosynthetic gene clusters with potential biomedical relevance are very common in the environment. This general process should permit the routine screening of environmental samples for gene clusters capable of encoding the systematic expansion of the structural diversity seen in biomedically relevant families of natural products.

  2. A two-host fosmid system for functional screening of (meta)genomic libraries from extreme thermophiles.

    Science.gov (United States)

    Angelov, Angel; Mientus, Markus; Liebl, Susanne; Liebl, Wolfgang

    2009-05-01

    A new cloning system is described, which allows the construction of large-insert fosmid libraries in Escherichia coli and the transfer of the recombinant libraries to the extreme thermophile Thermus thermophilus via natural transformation. Libraries are established in the thermophilic host by site-specific chromosomal insertion of the recombinant fosmids via single crossover or double crossover recombination at the T. thermophilus pyr locus. Comparative screening of a fosmid library constructed from genomic DNA from the thermophilic spirochaete, Spirochaeta thermophila, for clones expressing thermoactive xylanase activity revealed that 50% of the fosmids that conferred xylanase activity upon the corresponding T. thermophilus transformants did not give rise to xylanase-positive E. coli clones, indicating that significantly more S. thermophila genes are functionally expressed in T. thermophilus than in E. coli. The novel T. thermophilus host/vector system may be of value for the construction and functional screening of recombinant DNA libraries from individual thermophilic or extremely thermophilic organisms as well as from complex metagenomes isolated from thermophilic microbial communities.

  3. Application of 16S rRNA metagenomics to analyze bacterial communities at a respiratory care centre in Taiwan.

    Science.gov (United States)

    Tang, Chuan Yi; Yiu, Siu-Ming; Kuo, Han-Yueh; Tan, Te-Sheng; Liao, Ki-Hok; Liu, Chih-Chin; Hon, Wing-Kai; Liou, Ming-Li

    2015-03-01

    In this study, we applied a 16S ribosomal RNA (rRNA) metagenomics approach to survey inanimate hospital environments (IHEs) in a respiratory care center (RCC). A total of 16 samples, including 9 from medical devices and 7 from workstations, were analyzed. Besides, clinical isolates were retrospectively analyzed during the sampling period in the RCC. A high amount of microbial diversity was detected, with an average of 1,836 phylotypes per sample. In addition to Acinetobacter, more than 60 % of the bacterial communities present among the top 25 abundant genera were dominated by skin-associated bacteria. Differences in bacterial profiles were restricted to individual samples. Furthermore, compliance with hand hygiene guidelines may be unsatisfactory among hospital staff according to a principal coordinate analysis that indicated clustering of bacterial communities between devices and workstations for most of the sampling sites. Compared to the high incidence of clinical isolates in the RCC, only Staphylococcus and Acinetobacter were highly abundant in the IHEs. Despite Acinetobacter was the most abundant genus present in IHEs of the RCC, potential pathogens, e.g., Acinetobacter baumannii, might remain susceptible to carbapenem. This study is the first in Taiwan to demonstrate a high diversity of human-associated bacteria in the RCC via 16S rRNA metagenomics, which allows for new assessment of potential health risks in RCCs, aids in the evaluation of existing sanitation protocols, and furthers our understanding of the development of healthcare-associated infections.

  4. Identification of nitrogen-fixing genes and gene clusters from metagenomic library of acid mine drainage.

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    Zhimin Dai

    Full Text Available Biological nitrogen fixation is an essential function of acid mine drainage (AMD microbial communities. However, most acidophiles in AMD environments are uncultured microorganisms and little is known about the diversity of nitrogen-fixing genes and structure of nif gene cluster in AMD microbial communities. In this study, we used metagenomic sequencing to isolate nif genes in the AMD microbial community from Dexing Copper Mine, China. Meanwhile, a metagenome microarray containing 7,776 large-insertion fosmids was constructed to screen novel nif gene clusters. Metagenomic analyses revealed that 742 sequences were identified as nif genes including structural subunit genes nifH, nifD, nifK and various additional genes. The AMD community is massively dominated by the genus Acidithiobacillus. However, the phylogenetic diversity of nitrogen-fixing microorganisms is much higher than previously thought in the AMD community. Furthermore, a 32.5-kb genomic sequence harboring nif, fix and associated genes was screened by metagenome microarray. Comparative genome analysis indicated that most nif genes in this cluster are most similar to those of Herbaspirillum seropedicae, but the organization of the nif gene cluster had significant differences from H. seropedicae. Sequence analysis and reverse transcription PCR also suggested that distinct transcription units of nif genes exist in this gene cluster. nifQ gene falls into the same transcription unit with fixABCX genes, which have not been reported in other diazotrophs before. All of these results indicated that more novel diazotrophs survive in the AMD community.

  5. Discovery and characterization of thermophilic limonene-1,2-epoxide hydrolases from hot spring metagenomic libraries

    DEFF Research Database (Denmark)

    Ferrandi, Erica Elisa; Sayer, Christopher; Isupov, Michail N.;

    2015-01-01

    The epoxide hydrolases (EHs) represent an attractive option for the synthesis of chiral epoxides and 1,2-diols which are valuable building blocks for the synthesis of several pharmaceutical compounds. A metagenomic approach has been used to identify two new members of the atypical EH limonene-1,2...

  6. Identification of nitrogen-fixing genes and gene clusters from metagenomic library of acid mine drainage.

    Science.gov (United States)

    Dai, Zhimin; Guo, Xue; Yin, Huaqun; Liang, Yili; Cong, Jing; Liu, Xueduan

    2014-01-01

    Biological nitrogen fixation is an essential function of acid mine drainage (AMD) microbial communities. However, most acidophiles in AMD environments are uncultured microorganisms and little is known about the diversity of nitrogen-fixing genes and structure of nif gene cluster in AMD microbial communities. In this study, we used metagenomic sequencing to isolate nif genes in the AMD microbial community from Dexing Copper Mine, China. Meanwhile, a metagenome microarray containing 7,776 large-insertion fosmids was constructed to screen novel nif gene clusters. Metagenomic analyses revealed that 742 sequences were identified as nif genes including structural subunit genes nifH, nifD, nifK and various additional genes. The AMD community is massively dominated by the genus Acidithiobacillus. However, the phylogenetic diversity of nitrogen-fixing microorganisms is much higher than previously thought in the AMD community. Furthermore, a 32.5-kb genomic sequence harboring nif, fix and associated genes was screened by metagenome microarray. Comparative genome analysis indicated that most nif genes in this cluster are most similar to those of Herbaspirillum seropedicae, but the organization of the nif gene cluster had significant differences from H. seropedicae. Sequence analysis and reverse transcription PCR also suggested that distinct transcription units of nif genes exist in this gene cluster. nifQ gene falls into the same transcription unit with fixABCX genes, which have not been reported in other diazotrophs before. All of these results indicated that more novel diazotrophs survive in the AMD community.

  7. Cloning and functional characterization of endo-β-1,4-glucanase gene from metagenomic library of vermicompost.

    Science.gov (United States)

    Yasir, Muhammad; Khan, Haji; Azam, Syed Sikander; Telke, Amar; Kim, Seon Won; Chung, Young Ryun

    2013-06-01

    In the vermicomposting of paper mill sludge, the activity of earthworms is very dependent on dietetic polysaccharides including cellulose as energy sources. Most of these polymers are degraded by the host microbiota and considered potentially important source for cellulolytic enzymes. In the present study, a metagenomic library was constructed from vermicompost (VC) prepared with paper mill sludge and dairy sludge (fresh sludge, FS) and functionally screened for cellulolytic activities. Eighteen cellulase expressing clones were isolated from about 89,000 fosmid clones libraries. A short fragment library was constructed from the most active positive clone (cMGL504) and one open reading frame (ORF) of 1,092 bp encoding an endo-β-1,4-glucanase was indentified which showed 88% similarity with Cellvibrio mixtus cellulase A gene. The endo-β-1,4-glucanase cmgl504 gene was overexpressed in Escherichia coli. The purified recombinant cmgl504 cellulase displayed activities at a broad range of temperature (25-55°C) and pH (5.5-8.5). The enzyme degraded carboxymethyl cellulose (CMC) with 15.4 U, while having low activity against avicel. No detectable activity was found for xylan and laminarin. The enzyme activity was stimulated by potassium chloride. The deduced protein and three-dimensional structure of metagenome-derived cellulase cmgl504 possessed all features, including general architecture, signature motifs, and N-terminal signal peptide, followed by the catalytic domain of cellulase belonging to glycosyl hydrolase family 5 (GHF5). The cellulases cloned in this work may play important roles in the degradation of celluloses in vermicomposting process and could be exploited for industrial application in future.

  8. Identification of genes and pathways related to phenol degradation in metagenomic libraries from petroleum refinery wastewater.

    Directory of Open Access Journals (Sweden)

    Cynthia C Silva

    Full Text Available Two fosmid libraries, totaling 13,200 clones, were obtained from bioreactor sludge of petroleum refinery wastewater treatment system. The library screening based on PCR and biological activity assays revealed more than 400 positive clones for phenol degradation. From these, 100 clones were randomly selected for pyrosequencing in order to evaluate the genetic potential of the microorganisms present in wastewater treatment plant for biodegradation, focusing mainly on novel genes and pathways of phenol and aromatic compound degradation. The sequence analysis of selected clones yielded 129,635 reads at an estimated 17-fold coverage. The phylogenetic analysis showed Burkholderiales and Rhodocyclales as the most abundant orders among the selected fosmid clones. The MG-RAST analysis revealed a broad metabolic profile with important functions for wastewater treatment, including metabolism of aromatic compounds, nitrogen, sulphur and phosphorus. The predicted 2,276 proteins included phenol hydroxylases and cathecol 2,3- dioxygenases, involved in the catabolism of aromatic compounds, such as phenol, byphenol, benzoate and phenylpropanoid. The sequencing of one fosmid insert of 33 kb unraveled the gene that permitted the host, Escherichia coli EPI300, to grow in the presence of aromatic compounds. Additionally, the comparison of the whole fosmid sequence against bacterial genomes deposited in GenBank showed that about 90% of sequence showed no identity to known sequences of Proteobacteria deposited in the NCBI database. This study surveyed the functional potential of fosmid clones for aromatic compound degradation and contributed to our knowledge of the biodegradative capacity and pathways of microbial assemblages present in refinery wastewater treatment system.

  9. Identification of genes and pathways related to phenol degradation in metagenomic libraries from petroleum refinery wastewater.

    Science.gov (United States)

    Silva, Cynthia C; Hayden, Helen; Sawbridge, Tim; Mele, Pauline; De Paula, Sérgio O; Silva, Lívia C F; Vidigal, Pedro M P; Vicentini, Renato; Sousa, Maíra P; Torres, Ana Paula R; Santiago, Vânia M J; Oliveira, Valéria M

    2013-01-01

    Two fosmid libraries, totaling 13,200 clones, were obtained from bioreactor sludge of petroleum refinery wastewater treatment system. The library screening based on PCR and biological activity assays revealed more than 400 positive clones for phenol degradation. From these, 100 clones were randomly selected for pyrosequencing in order to evaluate the genetic potential of the microorganisms present in wastewater treatment plant for biodegradation, focusing mainly on novel genes and pathways of phenol and aromatic compound degradation. The sequence analysis of selected clones yielded 129,635 reads at an estimated 17-fold coverage. The phylogenetic analysis showed Burkholderiales and Rhodocyclales as the most abundant orders among the selected fosmid clones. The MG-RAST analysis revealed a broad metabolic profile with important functions for wastewater treatment, including metabolism of aromatic compounds, nitrogen, sulphur and phosphorus. The predicted 2,276 proteins included phenol hydroxylases and cathecol 2,3- dioxygenases, involved in the catabolism of aromatic compounds, such as phenol, byphenol, benzoate and phenylpropanoid. The sequencing of one fosmid insert of 33 kb unraveled the gene that permitted the host, Escherichia coli EPI300, to grow in the presence of aromatic compounds. Additionally, the comparison of the whole fosmid sequence against bacterial genomes deposited in GenBank showed that about 90% of sequence showed no identity to known sequences of Proteobacteria deposited in the NCBI database. This study surveyed the functional potential of fosmid clones for aromatic compound degradation and contributed to our knowledge of the biodegradative capacity and pathways of microbial assemblages present in refinery wastewater treatment system.

  10. Phylogenetic analysis of a spontaneous cocoa bean fermentation metagenome reveals new insights into its bacterial and fungal community diversity.

    Directory of Open Access Journals (Sweden)

    Koen Illeghems

    Full Text Available This is the first report on the phylogenetic analysis of the community diversity of a single spontaneous cocoa bean box fermentation sample through a metagenomic approach involving 454 pyrosequencing. Several sequence-based and composition-based taxonomic profiling tools were used and evaluated to avoid software-dependent results and their outcome was validated by comparison with previously obtained culture-dependent and culture-independent data. Overall, this approach revealed a wider bacterial (mainly γ-Proteobacteria and fungal diversity than previously found. Further, the use of a combination of different classification methods, in a software-independent way, helped to understand the actual composition of the microbial ecosystem under study. In addition, bacteriophage-related sequences were found. The bacterial diversity depended partially on the methods used, as composition-based methods predicted a wider diversity than sequence-based methods, and as classification methods based solely on phylogenetic marker genes predicted a more restricted diversity compared with methods that took all reads into account. The metagenomic sequencing analysis identified Hanseniaspora uvarum, Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, and Acetobacter pasteurianus as the prevailing species. Also, the presence of occasional members of the cocoa bean fermentation process was revealed (such as Erwinia tasmaniensis, Lactobacillus brevis, Lactobacillus casei, Lactobacillus rhamnosus, Lactococcus lactis, Leuconostoc mesenteroides, and Oenococcus oeni. Furthermore, the sequence reads associated with viral communities were of a restricted diversity, dominated by Myoviridae and Siphoviridae, and reflecting Lactobacillus as the dominant host. To conclude, an accurate overview of all members of a cocoa bean fermentation process sample was revealed, indicating the superiority of metagenomic sequencing over previously used techniques.

  11. Challenges of studying viral aerosol metagenomics and communities in comparison with bacterial and fungal aerosols.

    Science.gov (United States)

    Prussin, Aaron J; Marr, Linsey C; Bibby, Kyle J

    2014-08-01

    Despite the obvious importance of viral transmission and ecology to medicine, epidemiology, ecology, agriculture, and microbiology, the study of viral bioaerosols and community structure has remained a vastly underexplored area, due to both unresolved technical challenges and unrecognized importance. High-throughput, culture-independent techniques such as viral metagenomics are beginning to revolutionize the study of viral ecology. With recent developments in viral metagenomics, characterization of viral bioaerosol communities provides an opportunity for high-impact future research. However, there remain significant challenges for the study of viral bioaerosols compared with viruses in other matrices, such as water, the human gut, and soil. Collecting enough biomass is essential for successful metagenomic analysis, but this is a challenge with viral bioaerosols. Herein, we provide a perspective on the importance of studying viral bioaerosols, the challenges of studying viral community structure, and the potential opportunities for improvements in methods to study viruses in indoor and outdoor air.

  12. Applying meta-pathway analyses through metagenomics to identify the functional properties of the major bacterial communities of a single spontaneous cocoa bean fermentation process sample.

    Science.gov (United States)

    Illeghems, Koen; Weckx, Stefan; De Vuyst, Luc

    2015-09-01

    A high-resolution functional metagenomic analysis of a representative single sample of a Brazilian spontaneous cocoa bean fermentation process was carried out to gain insight into its bacterial community functioning. By reconstruction of microbial meta-pathways based on metagenomic data, the current knowledge about the metabolic capabilities of bacterial members involved in the cocoa bean fermentation ecosystem was extended. Functional meta-pathway analysis revealed the distribution of the metabolic pathways between the bacterial members involved. The metabolic capabilities of the lactic acid bacteria present were most associated with the heterolactic fermentation and citrate assimilation pathways. The role of Enterobacteriaceae in the conversion of substrates was shown through the use of the mixed-acid fermentation and methylglyoxal detoxification pathways. Furthermore, several other potential functional roles for Enterobacteriaceae were indicated, such as pectinolysis and citrate assimilation. Concerning acetic acid bacteria, metabolic pathways were partially reconstructed, in particular those related to responses toward stress, explaining their metabolic activities during cocoa bean fermentation processes. Further, the in-depth metagenomic analysis unveiled functionalities involved in bacterial competitiveness, such as the occurrence of CRISPRs and potential bacteriocin production. Finally, comparative analysis of the metagenomic data with bacterial genomes of cocoa bean fermentation isolates revealed the applicability of the selected strains as functional starter cultures.

  13. Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench).

    Science.gov (United States)

    Maropola, Mapula Kgomotso Annah; Ramond, Jean-Baptiste; Trindade, Marla

    2015-05-01

    Culture-independent studies rely on the quantity and quality of the extracted environmental metagenomic DNA (mDNA). To fully access the plant tissue microbiome, the extracted plant mDNA should allow optimal PCR applications and the genetic content must be representative of the total microbial diversity. In this study, we evaluated the endophytic bacterial diversity retrieved using different mDNA extraction procedures. Metagenomic DNA from sorghum (Sorghum bicolor L. Moench) stem and root tissues were extracted using two classical DNA extraction protocols (CTAB- and SDS-based) and five commercial kits. The mDNA yields and quality as well as the reproducibility were compared. 16S rRNA gene terminal restriction fragment length polymorphism (t-RFLP) was used to assess the impact on endophytic bacterial community structures observed. Generally, the classical protocols obtained high mDNA yields from sorghum tissues; however, they were less reproducible than the commercial kits. Commercial kits retrieved higher quality mDNA, but with lower endophytic bacterial diversities compared to classical protocols. The SDS-based protocol enabled access to the highest sorghum endophytic diversities. Therefore, "SDS-extracted" sorghum root and stem microbiome diversities were analysed via 454 pyrosequencing, and this revealed that the two tissues harbour significantly different endophytic communities. Nevertheless, both communities are dominated by agriculturally important genera such as Microbacterium, Agrobacterium, Sphingobacterium, Herbaspirillum, Erwinia, Pseudomonas and Stenotrophomonas; which have previously been shown to play a role in plant growth promotion. This study shows that DNA extraction protocols introduce biases in culture-independent studies of environmental microbial communities by influencing the mDNA quality, which impacts the microbial diversity analyses and evaluation. Using the broad-spectrum SDS-based DNA extraction protocol allows the recovery of the most

  14. Insights into bacterial cellulose biosynthesis by functional metagenomics on Antarctic soil samples.

    OpenAIRE

    Berlemont, Renaud; Delsaute, Maud; Pipers, Delphine; D'Amico, Salvino; Feller, Georges; Galleni, Moreno; Power, Pablo

    2009-01-01

    In this study, the mining of an Antarctic soil sample by functional metagenomics allowed the isolation of a cold-adapted protein (RBcel1) that hydrolyzes only carboxymethyl cellulose. The new enzyme is related to family 5 of the glycosyl hydrolase (GH5) protein from Pseudomonas stutzeri (Pst_2494) and does not possess a carbohydrate-binding domain. The protein was produced and purified to homogeneity. RBcel1 displayed an endoglucanase activity, producing cellobiose and cellotriose, using carb...

  15. The hunt for original microbial enzymes: an initiatory review on the construction and functional screening of (metagenomic libraries

    Directory of Open Access Journals (Sweden)

    Martin, M.

    2016-01-01

    Full Text Available Introduction. Discovering novel enzymes is of interest in both applied and basic science. Microbial enzymes, which are incredibly diverse and easy to produce, are increasingly sought by diverse approaches. Literature. This review first distinguishes culture-based from culture-independent methods, detailing within each group the advantages and drawbacks of sequence- and function-based methods. It then discusses the main factors affecting the success of endeavors to identify novel enzymes through construction and functional screening of genomic or metagenomic libraries: the sampled environment, how DNA is extracted and processed, the vector used (plasmid, cosmid, fosmid, BAC, or shuttle vector, the host cell chosen from the available prokaryotic and eukaryotic ones and the main screening steps. Conclusions. Library construction and screening can be tricky and requires expertise. Combining different strategies, such as working with cultivable and non-cultivable organisms, using sequence- and function-based approaches, or performing multihost screenings, is probably the best way to identify novel and diverse enzymes from an environmental sample.

  16. Comparative Metagenomic Profiling of Symbiotic Bacterial Communities Associated with Ixodes persulcatus, Ixodes pavlovskyi and Dermacentor reticulatus Ticks.

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    Alexander Kurilshikov

    Full Text Available Ixodes persulcatus, Ixodes pavlovskyi, and Dermacentor reticulatus ticks inhabiting Western Siberia are responsible for the transmission of a number of etiological agents that cause human and animal tick-borne diseases. Because these ticks are abundant in the suburbs of large cities, agricultural areas, and popular tourist sites and frequently attack people and livestock, data regarding the microbiomes of these organisms are required. Using metagenomic 16S profiling, we evaluate bacterial communities associated with I. persulcatus, I. pavlovskyi, and D. reticulatus ticks collected from the Novosibirsk region of Russia. A total of 1214 ticks were used for this study. DNA extracted from the ticks was pooled according to tick species and sex. Sequencing of the V3-V5 domains of 16S rRNA genes was performed using the Illumina Miseq platform. The following bacterial genera were prevalent in the examined communities: Acinetobacter (all three tick species, Rickettsia (I. persulcatus and D. reticulatus and Francisella (D. reticulatus. B. burgdorferi sensu lato and B. miyamotoi sequences were detected in I. persulcatus and I. pavlovskyi but not in D. reticulatus ticks. The pooled samples of all tick species studied contained bacteria from the Anaplasmataceae family, although their occurrence was low. DNA from A. phagocytophilum and Candidatus Neoehrlichia mikurensis was first observed in I. pavlovskyi ticks. Significant inter-species differences in the number of bacterial taxa as well as intra-species diversity related to tick sex were observed. The bacterial communities associated with the I. pavlovskyi ticks displayed a higher biodiversity compared with those of the I. persulcatus and D. reticulatus ticks. Bacterial community structure was also diverse across the studied tick species, as shown by permutational analysis of variance using the Bray-Curtis dissimilarity metric (p = 0.002. Between-sex variation was confirmed by PERMANOVA testing in I

  17. Characterization of a Novel Alkaline Family VIII Esterase with S-Enantiomer Preference from a Compost Metagenomic Library.

    Science.gov (United States)

    Lee, Hyun Woo; Jung, Won Kyeong; Kim, Yong Ho; Ryu, Bum Han; Kim, T Doohun; Kim, Jungho; Kim, Hoon

    2016-02-01

    A novel esterase gene, est7K, was isolated from a compost metagenomic library. The gene encoded a protein of 411 amino acids and the molecular mass of the Est7K was estimated to be 44,969 Da with no signal peptide. Est7K showed the highest identity of 57% to EstA3, which is an esterase from a drinking water metagenome, when compared with the enzymes with reported properties. Est7K had three motifs, SMTK, YSV, and WGG, which correspond to the typical motifs of family VIII esterases, SxxK, Yxx, and WGG, respectively. Est7K did not have the GxSxG motif in most lipolytic enzymes. Three additional motifs, LxxxPGxxW, PLGMxDTxF, and GGxG, were found to be conserved in family VIII enzymes. The results of the phylogenetic analysis and the alignment study suggest that family VIII enzymes could be classified into two subfamilies, VIII.1 and VIII.2. The purified Est7K was optimally active at 40°C and pH 10.0. It was activated to exhibit a 2.1-fold higher activity by the presence of 30% methanol. It preferred short-length p-nitrophenyl esters, particularly p-nitrophenyl butyrate, and efficiently hydrolyzed glyceryl tributyrate. It did not hydrolyze β-lactamase substrates, tertiary alcohol esters, glyceryl trioleate, fish oil, and olive oil. Est7K preferred an Senantiomer, such as (S)-methyl-3-hydroxy-2-methylpropionate, as the substrate. The tolerance to methanol and the substrate specificity may provide potential advantage in the use of the enzyme in pharmaceutical and other biotechnological processes.

  18. Metagenome survey of a multispecies and alga-associated biofilm revealed key elements of bacterial-algal interactions in photobioreactors.

    Science.gov (United States)

    Krohn-Molt, Ines; Wemheuer, Bernd; Alawi, Malik; Poehlein, Anja; Güllert, Simon; Schmeisser, Christel; Pommerening-Röser, Andreas; Grundhoff, Adam; Daniel, Rolf; Hanelt, Dieter; Streit, Wolfgang R

    2013-10-01

    Photobioreactors (PBRs) are very attractive for sunlight-driven production of biofuels and capturing of anthropogenic CO2. One major problem associated with PBRs however, is that the bacteria usually associated with microalgae in nonaxenic cultures can lead to biofouling and thereby affect algal productivity. Here, we report on a phylogenetic, metagenome, and functional analysis of a mixed-species bacterial biofilm associated with the microalgae Chlorella vulgaris and Scenedesmus obliquus in a PBR. The biofilm diversity and population dynamics were examined through 16S rRNA phylogeny. Overall, the diversity was rather limited, with approximately 30 bacterial species associated with the algae. The majority of the observed microorganisms were affiliated with Alphaproteobacteria, Betaproteobacteria, and Bacteroidetes. A combined approach of sequencing via GS FLX Titanium from Roche and HiSeq 2000 from Illumina resulted in the overall production of 350 Mbp of sequenced DNA, 165 Mbp of which was assembled in larger contigs with a maximum size of 0.2 Mbp. A KEGG pathway analysis suggested high metabolic diversity with respect to the use of polymers and aromatic and nonaromatic compounds. Genes associated with the biosynthesis of essential B vitamins were highly redundant and functional. Moreover, a relatively high number of predicted and functional lipase and esterase genes indicated that the alga-associated bacteria are possibly a major sink for lipids and fatty acids produced by the microalgae. This is the first metagenome study of microalga- and PBR-associated biofilm bacteria, and it gives new clues for improved biofuel production in PBRs.

  19. Identification and characterization of a cold-active phthalate esters hydrolase by screening a metagenomic library derived from biofilms of a wastewater treatment plant.

    Directory of Open Access Journals (Sweden)

    Yiying Jiao

    Full Text Available A cold-active phthalate esters hydrolase gene (designated dphB was identified through functional screening of a metagenomic library derived from biofilms of a wastewater treatment plant. The enzyme specifically catalyzed the hydrolysis of dipropyl phthalate, dibutyl phthalate, and dipentyl phthalate to the corresponding monoalkyl phthalate esters at low temperatures. The catalytic triad residues of DphB were proposed to be Ser159, Asp251, and His281.

  20. Identification and Characterization of a Cold-Active Phthalate Esters Hydrolase by Screening a Metagenomic Library Derived from Biofilms of a Wastewater Treatment Plant

    OpenAIRE

    Jiao, Yiying; CHEN Xu; Wang, Xin; Liao, Xuewei; Xiao, Lin; Miao, Aijun; Wu, Jun; Yang, Liuyan

    2013-01-01

    A cold-active phthalate esters hydrolase gene (designated dphB) was identified through functional screening of a metagenomic library derived from biofilms of a wastewater treatment plant. The enzyme specifically catalyzed the hydrolysis of dipropyl phthalate, dibutyl phthalate, and dipentyl phthalate to the corresponding monoalkyl phthalate esters at low temperatures. The catalytic triad residues of DphB were proposed to be Ser159, Asp251, and His281.

  1. Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene

    Directory of Open Access Journals (Sweden)

    Gong Xia

    2012-10-01

    Full Text Available Abstract Background Interest in cellulose degrading enzymes has increased in recent years due to the expansion of the cellulosic biofuel industry. The rumen is a highly adapted environment for the degradation of cellulose and a promising source of enzymes for industrial use. To identify cellulase enzymes that may be of such use we have undertaken a functional metagenomic screen to identify cellulase enzymes from the bacterial community in the rumen of a grass-hay fed dairy cow. Results Twenty five clones specifying cellulose activity were identified. Subcloning and sequence analysis of a subset of these hydrolase-positive clones identified 10 endoglucanase genes. Preliminary characterization of the encoded cellulases was carried out using crude extracts of each of the subclones. Zymogram analysis using carboxymethylcellulose as a substrate showed a single positive band for each subclone, confirming that only one functional cellulase gene was present in each. One cellulase gene, designated Cel14b22, was expressed at a high level in Escherichia coli and purified for further characterization. The purified recombinant enzyme showed optimal activity at pH 6.0 and 50°C. It was stable over a broad pH range, from pH 4.0 to 10.0. The activity was significantly enhanced by Mn2+ and dramatically reduced by Fe3+ or Cu2+. The enzyme hydrolyzed a wide range of beta-1,3-, and beta-1,4-linked polysaccharides, with varying activities. Activities toward microcrystalline cellulose and filter paper were relatively high, while the highest activity was toward Oat Gum. Conclusion The present study shows that a functional metagenomic approach can be used to isolate previously uncharacterized cellulases from the rumen environment.

  2. Screening and characterization of a novel ruminal cellulase gene (Umcel-1) from a metagenomic library of gayal (Bos frontalis)

    Institute of Scientific and Technical Information of China (English)

    LI Bi-feng; MAO Hua-ming; YANG Shu-Li; ZHU Ya-xin; GU Zhao-bing; CHEN Yuan; LENG Jing; GOU Xiao; FENG Li; LI Qing; XI Dong-mei

    2016-01-01

    Gayal is a rare semi-wild bovine species found in the Indo-China. They can graze grasses, including bamboo leaves, as wel as reeds and other plant species, and grow to higher mature live weights than Yunnan Yelow cattle maintained in similar harsh environments. The aim of this study was to identify speciifc celulase in the gayal rumen. A metagenomic fosmid library was constructed using genomic DNA isolated from the ruminal contents of four adult gayals. This library contained 38400 clones with an average insert size of 35.5 kb. TheUmcel-1 gene was isolated from this library. Investigation of the celulase activity of 24 random clones led to the identiifcation of theUmcel-1 gene, which exhibited the most potent celulase activity. Sequencing theUmcel-1 gene revealed that it contained an open reading frame of 942 base pairs that encoded a product of 313 amino acids. The putativegene Umcel-1 product belonged to the glycosyl hydrolase family 5 and showed the highest homology to the celulase (GenBank accession no. YP_004310852.1) fromClostridium lentocelum DSM 5427, with 44% identity and 62% similarity. TheUmcel-1 gene was heterologously expressed inEscherichia coli BL21, and recombinant Umcel-1 was puriifed. The activity of puriifed recombinant Umcel-1 was assessed, and the results revealed that it hydrolyzed carboxymethyl celulose with optimal activity at pH 5.5 and 45°C. To our knowledge, this study provides the ifrst evidence for a celulase produced by bacteria in gayal rumen.

  3. Metagenomic analysis of soil microbial communities

    Directory of Open Access Journals (Sweden)

    Đokić Lidija

    2010-01-01

    Full Text Available Ramonda serbica and Ramonda nathaliae, rare resurrection plants growing in the Balkan Peninsula, produce a high amount of phenolic compounds as a response to stress. The composition and size of bacterial communities in two rhizosphere soil samples of these plants were analyzed using a metagenomic approach. Fluorescent in situ hybridization (FISH experiments together with DAPI staining showed that the metabolically active bacteria represent only a small fraction, approximately 5%, of total soil bacteria. Using universal bacteria - specific primers 16S rDNA genes were amplified directly from metagenomic DNAs and two libraries were constructed. The Restriction Fragment Length Polymorphism (RLFP method was used in library screening. Amongst 192 clones, 35 unique operational taxonomic units (OTUs were determined from the rhizosphere of R. nathaliae, and 13 OTUs out of 80 clones in total from the library of R. serbica. Representative clones from each OTU were sequenced. The majority of sequences from metagenomes showed very little similarity to any cultured bacteria. In conclusion, the bacterial communities in the studied soil samples showed quite poor diversity. .

  4. Functional-based screening methods for lipases, esterases, and phospholipases in metagenomic libraries.

    Science.gov (United States)

    Reyes-Duarte, Dolores; Ferrer, Manuel; García-Arellano, Humberto

    2012-01-01

    The use of metagenomic techniques for enzyme discovery constitutes a powerful approach. Functional screens, in contrast to sequence homology search, enable us to select enzymes based on their activity. It is noteworthy that they additionally guarantee the identification of genes coding for enzymes that exhibited no sequence similarity to known counterparts from public databases and that even do not match any putative catalytic residues, involved in the selected catalytic function. Therefore, this strategy not only provides new enzymes for new biotechnological applications, but also allows functional assignment of many proteins, found in abundance in the databases, currently designated as "hypothetical" or "conserved hypothetical" proteins. In the past decade, there has been an exponential increase in the design of functional screening programmes, the majority of them established for hydrolases and oxidoreductases. Here, functional screening methods that guarantee the greatest enzyme diversity, for mining esterases and lipases, are described.

  5. Anthropogenic N deposition slows decay by favoring bacterial metabolism: Insights from metagenomic analyses

    Directory of Open Access Journals (Sweden)

    Zachary B. Freedman

    2016-03-01

    Full Text Available Litter decomposition is an enzymatically-complex process that is mediated by a diverse assemblage of saprophytic microorganisms. It is a globally important biogeochemical process that can be suppressed by anthropogenic N deposition. In a northern hardwood forest ecosystem located in Michigan, USA, 20 years of experimentally increased atmospheric N deposition has reduced forest floor decay and increased soil C storage. Here, we paired extracellular enzyme assays with shotgun metagenomics to assess if anthropogenic N deposition has altered the functional potential of microbial communities inhabiting decaying forest floor. Experimental N deposition significantly reduced the activity of extracellular enzymes mediating plant cell wall decay, which occurred concurrently with changes in the relative abundance of metagenomic functional gene pathways mediating the metabolism of carbohydrates, aromatic compounds, as well as microbial respiration. Moreover, experimental N deposition increased the relative abundance of 50 of the 60 gene pathways, the majority of which were associated with saprotrophic bacteria. Conversely, the relative abundance and composition of fungal genes mediating the metabolism of plant litter was not affected by experimental N deposition. Future rates of atmospheric N deposition have favored saprotrophic soil bacteria, whereas the metabolic potential of saprotrophic fungi appears resilient to this agent of environmental change. Results presented here provide evidence that changes in the functional capacity of saprotrophic soil microorganisms mediate how anthropogenic N deposition increases C storage in soil.

  6. Metagenomics reveals pervasive bacterial populations and reduced community diversity across the Alaska tundra ecosystem

    Directory of Open Access Journals (Sweden)

    Eric Robert Johnston

    2016-04-01

    Full Text Available How soil microbial communities contrast with respect to taxonomic and functional composition within and between ecosystems remains an unresolved question that is central to predicting how global anthropogenic change will affect soil functioning and services. In particular, it remains unclear how small-scale observations of soil communities based on the typical volume sampled (1-2 grams are generalizable to ecosystem-scale responses and processes. This is especially relevant for remote, northern latitude soils, which are challenging to sample and are also thought to be more vulnerable to climate change compared to temperate soils. Here, we employed well-replicated shotgun metagenome and 16S rRNA gene amplicon sequencing to characterize community composition and metabolic potential in Alaskan tundra soils, combining our own datasets with those publically available from distant tundra and temperate grassland and agriculture habitats. We found that the abundance of many taxa and metabolic functions differed substantially between tundra soil metagenomes relative to those from temperate soils, and that a high degree of OTU-sharing exists between tundra locations. Tundra soils were an order of magnitude less complex than their temperate counterparts, allowing for near-complete coverage of microbial community richness (~92% breadth by sequencing, and the recovery of twenty-seven high-quality, almost complete (>80% completeness population bins. These population bins, collectively, made up to ~10% of the metagenomic datasets, and represented diverse taxonomic groups and metabolic lifestyles tuned toward sulfur cycling, hydrogen metabolism, methanotrophy, and organic matter oxidation. Several population bins, including members of Acidobacteria, Actinobacteria, and Proteobacteria, were also present in geographically distant (~100-530 km apart tundra habitats (full genome representation and up to 99.6% genome-derived average nucleotide identity. Collectively

  7. Bacterial pathogens and community composition in advanced sewage treatment systems revealed by metagenomics analysis based on high-throughput sequencing.

    Science.gov (United States)

    Lu, Xin; Zhang, Xu-Xiang; Wang, Zhu; Huang, Kailong; Wang, Yuan; Liang, Weigang; Tan, Yunfei; Liu, Bo; Tang, Junying

    2015-01-01

    This study used 454 pyrosequencing, Illumina high-throughput sequencing and metagenomic analysis to investigate bacterial pathogens and their potential virulence in a sewage treatment plant (STP) applying both conventional and advanced treatment processes. Pyrosequencing and Illumina sequencing consistently demonstrated that Arcobacter genus occupied over 43.42% of total abundance of potential pathogens in the STP. At species level, potential pathogens Arcobacter butzleri, Aeromonas hydrophila and Klebsiella pneumonia dominated in raw sewage, which was also confirmed by quantitative real time PCR. Illumina sequencing also revealed prevalence of various types of pathogenicity islands and virulence proteins in the STP. Most of the potential pathogens and virulence factors were eliminated in the STP, and the removal efficiency mainly depended on oxidation ditch. Compared with sand filtration, magnetic resin seemed to have higher removals in most of the potential pathogens and virulence factors. However, presence of the residual A. butzleri in the final effluent still deserves more concerns. The findings indicate that sewage acts as an important source of environmental pathogens, but STPs can effectively control their spread in the environment. Joint use of the high-throughput sequencing technologies is considered a reliable method for deep and comprehensive overview of environmental bacterial virulence.

  8. Bacterial pathogens and community composition in advanced sewage treatment systems revealed by metagenomics analysis based on high-throughput sequencing.

    Directory of Open Access Journals (Sweden)

    Xin Lu

    Full Text Available This study used 454 pyrosequencing, Illumina high-throughput sequencing and metagenomic analysis to investigate bacterial pathogens and their potential virulence in a sewage treatment plant (STP applying both conventional and advanced treatment processes. Pyrosequencing and Illumina sequencing consistently demonstrated that Arcobacter genus occupied over 43.42% of total abundance of potential pathogens in the STP. At species level, potential pathogens Arcobacter butzleri, Aeromonas hydrophila and Klebsiella pneumonia dominated in raw sewage, which was also confirmed by quantitative real time PCR. Illumina sequencing also revealed prevalence of various types of pathogenicity islands and virulence proteins in the STP. Most of the potential pathogens and virulence factors were eliminated in the STP, and the removal efficiency mainly depended on oxidation ditch. Compared with sand filtration, magnetic resin seemed to have higher removals in most of the potential pathogens and virulence factors. However, presence of the residual A. butzleri in the final effluent still deserves more concerns. The findings indicate that sewage acts as an important source of environmental pathogens, but STPs can effectively control their spread in the environment. Joint use of the high-throughput sequencing technologies is considered a reliable method for deep and comprehensive overview of environmental bacterial virulence.

  9. Isolation and Characterization of a Glycosyl Hydrolase Family 16 β-Agarase from a Mangrove Soil Metagenomic Library

    Science.gov (United States)

    Mai, Zhimao; Su, Hongfei; Zhang, Si

    2016-01-01

    A mangrove soil metagenomic library was constructed and a β-agarase gene designated as AgaML was isolated by functional screening. The gene encoded for a 659-amino-acids polypeptide with an estimated molecular mass of 71.6 kDa. The deduced polypeptide sequences of AgaML showed the highest identity of 73% with the glycoside hydrolase family 16 β-agarase from Microbulbifer agarilyticus in the GenBank database. AgaML was cloned and highly expressed in Escherichia coli BL21(DE3). The purified recombinant protein, AgaML, showed optimal activity at 50 °C and pH 7.0. The kinetic parameters of Km and Vmax values toward agarose were 4.6 mg·mL−1 and 967.5 μM·min−1·mg−1, respectively. AgaML hydrolyzed the β-1,4-glycosidic linkages of agar to generate neoagarotetraose (NA4) and neoagarohexaose (NA6) as the main products. These characteristics suggest that AgaML has potential application in cosmetic, pharmaceuticals and food industries. PMID:27548158

  10. Immobilization and characterization of tannase from a metagenomic library and its use for removal of tannins from green tea infusion.

    Science.gov (United States)

    Yao, Jian; Chen, Qinglong; Zhong, Guoxiang; Cao, Wen; Yu, An; Liu, Yuhuan

    2014-01-01

    Tannase (Tan410) from a soil metagenomic library was immobilized on different supports, including mesoporous silica SBA-15, chitosan, calcium alginate, and amberlite IRC 50. Entrapment in calcium alginate beads was comparatively found to be the best method and was further characterized. The optimum pH of the immobilized Tan410 was shifted toward neutrality compared with the free enzyme (from pH 6.4 to pH 7.0). The optimum temperature was determined to be 45°C for the immobilized enzyme and 30°C for the free enzyme, respectively. The immobilized enzyme had no loss of activity after 10 cycles, and retained more than 90% of its original activity after storage for 30 days. After immobilization, the enzyme activity was only slightly affected by Hg(2+), which completely inhibited the activity of the free enzyme. The immobilized tannase was used to remove 80% of tannins from a green tea infusion on the first treatment. The beads were used for six successive runs resulting in overall hydrolysis of 56% of the tannins.

  11. Using metagenomics and metatranscriptomics to study specific bacterial species involved in biological phosphorus removal from wastewater

    DEFF Research Database (Denmark)

    Albertsen, Mads; McIlroy, Simon Jon; Stokholm-Bjerregaard, Mikkel

    profiles by metatranscriptomics. To demonstrate this we revisited the bacteria involved in enhanced biological phosphorus removal (EBPR) from wastewater treatment plants. The EBPR process is used all over the world, has a large body of information regarding the underlying microbiology, and is often studied...... to enrich for bacteria contributing to phosphorus removal and their normal competitors. To extract complete genomes we generated two metagenomes from each reactor, taken approximately 1 month apart, using the Illumina HiSeq2000 platform. Due to low micro-diversity in the reactors (2-15 dominating species...... phosphorus removal process in the treatment plant, sampling every 20 min during the 9 hour experiment. Metratranscriptome data was generated from selected samples using the stranded RNAseq Illumina protocol. We were able to extract genomes from the model polyphosphate accumulating organism (PAO) Ca...

  12. Discovery and characterization of thermophilic limonene-1,2-epoxide hydrolases from hot spring metagenomic libraries.

    Science.gov (United States)

    Ferrandi, Erica Elisa; Sayer, Christopher; Isupov, Michail N; Annovazzi, Celeste; Marchesi, Carlotta; Iacobone, Gianluca; Peng, Xu; Bonch-Osmolovskaya, Elizaveta; Wohlgemuth, Roland; Littlechild, Jennifer A; Monti, Daniela

    2015-08-01

    The epoxide hydrolases (EHs) represent an attractive option for the synthesis of chiral epoxides and 1,2-diols which are valuable building blocks for the synthesis of several pharmaceutical compounds. A metagenomic approach has been used to identify two new members of the atypical EH limonene-1,2-epoxide hydrolase (LEH) family of enzymes. These two LEHs (Tomsk-LEH and CH55-LEH) show EH activities towards different epoxide substrates, differing in most cases from those previously identified for Rhodococcus erythropolis (Re-LEH) in terms of stereoselectivity. Tomsk-LEH and CH55-LEH, both from thermophilic sources, have higher optimal temperatures and apparent melting temperatures than Re-LEH. The new LEH enzymes have been crystallized and their structures solved to high resolution in the native form and in complex with the inhibitor valpromide for Tomsk-LEH and poly(ethylene glycol) for CH55-LEH. The structural analysis has provided insights into the LEH mechanism, substrate specificity and stereoselectivity of these new LEH enzymes, which has been supported by mutagenesis studies.

  13. Bacterial bile metabolising gene abundance in Crohn's, ulcerative colitis and type 2 diabetes metagenomes.

    Directory of Open Access Journals (Sweden)

    Alain Labbé

    Full Text Available We performed an analysis to determine the importance of bile acid modification genes in the gut microbiome of inflammatory bowel disease and type 2 diabetic patients. We used publicly available metagenomic datasets from the Human Microbiome Project and the MetaHIT consortium, and determined the abundance of bile salt hydrolase gene (bsh, 7 alpha-dehydroxylase gene (adh and 7-alpha hydroxysteroid dehydrogenase gene (hsdh in fecal bacteria in diseased populations of Crohn's disease (CD, Ulcerative Colitis (UC and Type 2 diabetes mellitus (T2DM. Phylum level abundance analysis showed a significant reduction in Firmicute-derived bsh in UC and T2DM patients but not in CD patients, relative to healthy controls. Reduction of adh and hsdh genes was also seen in UC and T2DM patients, while an increase was observed in the CD population as compared to healthy controls. A further analysis of the bsh genes showed significant differences in the correlations of certain Firmicutes families with disease or healthy populations. From this observation we proceeded to analyse BSH protein sequences and identified BSH proteins clusters representing the most abundant strains in our analysis of Firmicute bsh genes. The abundance of the bsh genes corresponding to one of these protein clusters was significantly reduced in all disease states relative to healthy controls. This cluster includes bsh genes derived from Lachospiraceae, Clostridiaceae, Erysipelotrichaceae and Ruminococcaceae families. This metagenomic analysis provides evidence of the importance of bile acid modifying enzymes in health and disease. It further highlights the importance of identifying gene and protein clusters, as the same gene may be associated with health or disease, depending on the strains expressing the enzyme, and differences in the enzymes themselves.

  14. Functional environmental screening of a metagenomic library identifies stlA; a unique salt tolerance locus from the human gut microbiome.

    Directory of Open Access Journals (Sweden)

    Eamonn P Culligan

    Full Text Available Functional environmental screening of metagenomic libraries is a powerful means to identify and assign function to novel genes and their encoded proteins without any prior sequence knowledge. In the current study we describe the identification and subsequent analysis of a salt-tolerant clone from a human gut metagenomic library. Following transposon mutagenesis we identified an unknown gene (stlA, for "salt tolerance locus A" with no current known homologues in the databases. Subsequent cloning and expression in Escherichia coli MKH13 revealed that stlA confers a salt tolerance phenotype in its surrogate host. Furthermore, a detailed in silico analysis was also conducted to gain additional information on the properties of the encoded StlA protein. The stlA gene is rare when searched against human metagenome datasets such as MetaHit and the Human Microbiome Project and represents a novel and unique salt tolerance determinant which appears to be found exclusively in the human gut environment.

  15. Metagenomic analysis of Streptomyces lividans reveals host-dependent functional expression.

    Science.gov (United States)

    McMahon, Matthew D; Guan, Changhui; Handelsman, Jo; Thomas, Michael G

    2012-05-01

    Most functional metagenomic studies have been limited by the poor expression of many genes derived from metagenomic DNA in Escherichia coli, which has been the predominant surrogate host to date. To expand the range of expressed genes, we developed tools for construction and functional screening of metagenomic libraries in Streptomyces lividans. We expanded on previously published protocols by constructing a system that enables retrieval and characterization of the metagenomic DNA from biologically active clones. To test the functionality of these methods, we constructed and screened two metagenomic libraries in S. lividans. One was constructed with pooled DNA from 14 bacterial isolates cultured from Alaskan soil and the second with DNA directly extracted from the same soil. Functional screening of these libraries identified numerous clones with hemolytic activity, one clone that produces melanin by a previously unknown mechanism, and one that induces the overproduction of a secondary metabolite native to S. lividans. All bioactive clones were functional in S. lividans but not in E. coli, demonstrating the advantages of screening metagenomic libraries in more than one host.

  16. Insights into bacterial cellulose biosynthesis by functional metagenomics on Antarctic soil samples.

    Science.gov (United States)

    Berlemont, Renaud; Delsaute, Maud; Pipers, Delphine; D'Amico, Salvino; Feller, Georges; Galleni, Moreno; Power, Pablo

    2009-09-01

    In this study, the mining of an Antarctic soil sample by functional metagenomics allowed the isolation of a cold-adapted protein (RBcel1) that hydrolyzes only carboxymethyl cellulose. The new enzyme is related to family 5 of the glycosyl hydrolase (GH5) protein from Pseudomonas stutzeri (Pst_2494) and does not possess a carbohydrate-binding domain. The protein was produced and purified to homogeneity. RBcel1 displayed an endoglucanase activity, producing cellobiose and cellotriose, using carboxymethyl cellulose as a substrate. Moreover, the study of pH and the thermal dependence of the hydrolytic activity shows that RBcel1 was active from pH 6 to pH 9 and remained significantly active when temperature decreased (18% of activity at 10 degrees C). It is interesting that RBcel1 was able to synthetize non-reticulated cellulose using cellobiose as a substrate. Moreover, by a combination of bioinformatics and enzyme analysis, the physiological relevance of the RBcel1 protein and its mesophilic homologous Pst_2494 protein from P. stutzeri, A1501, was established as the key enzymes involved in the production of cellulose by bacteria. In addition, RBcel1 and Pst_2494 are the two primary enzymes belonging to the GH5 family involved in this process.

  17. Metagenomics and antibiotics.

    Science.gov (United States)

    Garmendia, L; Hernandez, A; Sanchez, M B; Martinez, J L

    2012-07-01

    Most of the bacterial species that form part of the biosphere have never been cultivated. In this situation, a comprehensive study of bacterial communities requires the utilization of non-culture-based methods, which have been named metagenomics. In this paper we review the use of different metagenomic techniques for understanding the effect of antibiotics on microbial communities, to synthesize new antimicrobial compounds and to analyse the distribution of antibiotic resistance genes in different ecosystems. These techniques include functional metagenomics, which serves to find new antibiotics or new antibiotic resistance genes, and descriptive metagenomics, which serves to analyse changes in the composition of the microbiota and to track the presence and abundance of already known antibiotic resistance genes in different ecosystems.

  18. Identification and characterization of new LuxR/LuxI-type quorum sensing systems from metagenomic libraries.

    Science.gov (United States)

    Hao, Youai; Winans, Stephen C; Glick, Bernard R; Charles, Trevor C

    2010-01-01

    Quorum sensing (QS) cell-cell communication systems are utilized by bacteria to coordinate their behaviour according to cell density. Several different types of QS signal molecules have been identified, among which acyl-homoserine lactones (AHLs) produced by Proteobacteria have been studied to the greatest extent. Although QS has been studied extensively in cultured microorganisms, little is known about the QS systems of uncultured microorganisms and the roles of these systems in microbial communities. To extend our knowledge of QS systems and to better understand the signalling that takes place in the natural environment, metagenomic libraries constructed using DNA from activated sludge and soil were screened, using an Agrobacterium biosensor strain, for novel QS synthase genes. Three cosmids (QS6-1, QS10-1 and QS10-2) that encode the production of QS signals were identified and DNA sequence analysis revealed that all three clones encode a novel luxI family AHL synthase and a luxR family transcriptional regulator. Thin layer chromatography revealed that these LuxI homologue proteins are able to synthesize multiple AHL signals. Tandem mass spectrometry analysis revealed that LuxI(QS6-1) directs the synthesis of at least three AHLs, 3-O-C14:1 HSL, 3-O-C16:1 HSL and 3-O-C14 HSL; LuxI(QS10-1) directs the synthesis of at least 3-O-C12 HSL and 3-O-C14 HSL; while LuxI(QS10-2) directs the synthesis of at least C8 HSL and C10 HSL. Two possible new AHLs, C14:3 HSL and (?)-hydroxymethyl-3-O-C14 HSL, were also found to be synthesized by LuxI(QS6-1).

  19. Biocatalysts and their small molecule products from metagenomic studies

    OpenAIRE

    2012-01-01

    The vast majority of bacteria present in environmental samples have never been cultured and therefore they have not been available to exploit their ability to produce useful biocatalysts or collections of biocatalysts that can biosynthesize interesting small molecules. Metagenomic libraries constructed using DNA extracted directly from natural bacterial communities offer access to the genetic information present in the genomes of these as yet uncultured bacteria. This review highlights recent...

  20. Identification and in silico characterization of two novel genes encoding peptidases S8 found by functional screening in a metagenomic library of Yucatán underground water.

    Science.gov (United States)

    Apolinar-Hernández, Max M; Peña-Ramírez, Yuri J; Pérez-Rueda, Ernesto; Canto-Canché, Blondy B; De Los Santos-Briones, César; O'Connor-Sánchez, Aileen

    2016-11-15

    Metagenomics is a culture-independent technology that allows access to novel and potentially useful genetic resources from a wide range of unknown microorganisms. In this study, a fosmid metagenomic library of tropical underground water was constructed, and clones were functionally screened for extracellular proteolytic activity. One of the positive clones, containing a 41,614-bp insert, had two genes with 60% and 68% identity respectively with a peptidase S8 of Chitinimonas koreensis. When these genes were individually sub-cloned, in both cases their sub-clones showed proteolytic phenotype, confirming that they both encode functional proteases. These genes -named PrAY5 and PrAY6- are next to each other. They are similar in size (1845bp and 1824bp respectively) and share 66.5% identity. An extensive in silico characterization showed that their ORFs encode complex zymogens having a signal peptide at their 5' end, followed by a pro-peptide, a catalytic region, and a PPC domain at their 3' end. Their translated sequences were classified as peptidases S8A by sequence comparisons against the non-redundant database and corroborated by Pfam and MEROPS. Phylogenetic analysis of the catalytic region showed that they encode novel proteases that clustered with the sub-family S8_13, which according to the CDD database at NCBI, is an uncharacterized subfamily. They clustered in a clade different from the other three proteases S8 found so far by functional metagenomics, and also different from proteases S8 found in sequenced environmental samples, thereby expanding the range of potentially useful proteases that have been identified by metagenomics. I-TASSER modeling corroborated that they may be subtilases, thus possibly they participate in the hydrolysis of proteins with broad specificity for peptide bonds, and have a preference for a large uncharged residue in P1.

  1. Preparation of high molecular weight gDNA and bacterial artificial chromosome (BAC) libraries in plants.

    Science.gov (United States)

    Biradar, Siddanagouda S; Nie, Xiaojun; Feng, Kewei; Weining, Song

    2014-01-01

    Bacterial artificial chromosome (BAC) libraries are extremely valuable large-insert DNA libraries for physical mapping, positional cloning, comparative genomic analysis, complete genome sequencing, and evolutionary studies. Due to their stability and relative simplicity BAC libraries are most preferred over other approaches for cloning large genomic DNA fragments for large-insert libraries. Isolation of intact high molecular weight (HMW) DNA is a critical step underlying the success of large-insert genomic DNA library construction. It requires the isolation of purified nuclei, embedding them into LMP agarose plugs, restriction digestion of the plugs, and quite often size selection using PFGE and electro-elution of insert DNA. The construction of BAC libraries is complex and challenging for most molecular laboratories. To facilitate the construction of BAC libraries, we present a step-by-step protocol for isolation of HMW DNA and construction of plant BAC libraries.

  2. Cloning the Soil Metagenome: a Strategy for Accessing the Genetic and Functional Diversity of Uncultured Microorganisms

    OpenAIRE

    2000-01-01

    Recent progress in molecular microbial ecology has revealed that traditional culturing methods fail to represent the scope of microbial diversity in nature, since only a small proportion of viable microorganisms in a sample are recovered by culturing techniques. To develop methods to investigate the full extent of microbial diversity, we used a bacterial artificial chromosome (BAC) vector to construct libraries of genomic DNA isolated directly from soil (termed metagenomic libraries). To date...

  3. Construction of Metagenomic Fosmid Library from Activated Sludge%活性污泥宏基因组Fosmid文库的构建

    Institute of Scientific and Technical Information of China (English)

    苟敏; 曲媛媛; 周集体; 许炳雯; 曹湘禹

    2012-01-01

    The construction of large-fragment metagenomic library helps to discover large-fragment functional genes and analyze their organization and function. In this paper, first, three methods, namely the CTAB method, the kit and the agarose-embedding method, were used to extract metagenomic DNA from activated sludge, and DNA fragments larger than 23 kbp were extracted by the agarose-embedding method. Then, the metagenomic DNA was liga-ted into pCClFOS vector to construct a Fosmid library consisting of 5280 clones with an average insert fragment size of 35~40kbp, which covered about 200Mbp of metagenomic DNA. Finally, one clone exhibiting amylase activity was rapidly screened from 200 randomly-picked clones via the activity-based screening. It is thus concluded that the Fosmid library constructed from activated sludge is feasible in the screening of novel functional genes by activity-based methods.%大插入片段宏基因组文库的构建是开发大片段目的基因及分析其结构与功能的基础.文中分别采用十六烷基三甲基溴化铵( CTAB)法、试剂盒及琼脂糖包埋法提取活性污泥宏基因组DNA,其中琼脂糖包埋法获得的DNA片段大于23 kbp,利用此DNA成功构建了以pCC1FOS为载体的Fosmid文库,该文库含有5280个克隆,平均插入片段长度为35~40kbp,共包含约200Mbp的宏基因组DNA.从此文库中随机挑选200个克隆,利用活性筛选方法快速筛选到了1个含有淀粉酶的阳性克隆,表明活性污泥Fosmid文库可用于功能基因的活性筛选,具有开发新基因的潜力.

  4. Identification of novel toluene monooxygenase genes in a hydrocarbon-polluted sediment using sequence- and function-based screening of metagenomic libraries.

    Science.gov (United States)

    Bouhajja, E; McGuire, M; Liles, M R; Bataille, G; Agathos, S N; George, I F

    2017-01-01

    The microbial potential for toluene degradation within sediments from a tar oil-contaminated site in Flingern, Germany, was assessed using a metagenomic approach. High molecular weight environmental DNA from contaminated sediments was extracted, purified, and cloned into fosmid and BAC vectors and transformed into Escherichia coli. The fosmid library was screened by hybridization with a PCR amplicon of the α-subunit of the toluene 4-monooxygenase gene to identify genes and pathways encoding toluene degradation. Fourteen clones were recovered from the fosmid library, among which 13 were highly divergent from known tmoA genes and several had the closest relatives among Acinetobacter species. The BAC library was transferred to the heterologous hosts Cupriavidus metallidurans (phylum Proteobacteria) and Edaphobacter aggregans (phylum Acidobacteria). The resulting libraries were screened for expression of toluene degradation in the non-degradative hosts. From expression in C. metallidurans, three novel toluene monooxygenase-encoding operons were identified that were located on IncP1 plasmids. The E. aggregans-hosted BAC library led to the isolation of a cloned genetic locus putatively derived from an Acidobacteria taxon that contained genes involved in aerobic and anaerobic toluene degradation. These data suggest the important role of plasmids in the spread of toluene degradative capacity and indicate putative novel tmoA genes present in this hydrocarbon-polluted environment.

  5. Current and future resources for functional metagenomics.

    Science.gov (United States)

    Lam, Kathy N; Cheng, Jiujun; Engel, Katja; Neufeld, Josh D; Charles, Trevor C

    2015-01-01

    Functional metagenomics is a powerful experimental approach for studying gene function, starting from the extracted DNA of mixed microbial populations. A functional approach relies on the construction and screening of metagenomic libraries-physical libraries that contain DNA cloned from environmental metagenomes. The information obtained from functional metagenomics can help in future annotations of gene function and serve as a complement to sequence-based metagenomics. In this Perspective, we begin by summarizing the technical challenges of constructing metagenomic libraries and emphasize their value as resources. We then discuss libraries constructed using the popular cloning vector, pCC1FOS, and highlight the strengths and shortcomings of this system, alongside possible strategies to maximize existing pCC1FOS-based libraries by screening in diverse hosts. Finally, we discuss the known bias of libraries constructed from human gut and marine water samples, present results that suggest bias may also occur for soil libraries, and consider factors that bias metagenomic libraries in general. We anticipate that discussion of current resources and limitations will advance tools and technologies for functional metagenomics research.

  6. Current and future resources for functional metagenomics

    Directory of Open Access Journals (Sweden)

    Kathy Nguyen Lam

    2015-10-01

    Full Text Available Functional metagenomics is a powerful experimental approach for studying gene function, starting from the extracted DNA of mixed microbial populations. A functional approach relies on the construction and screening of metagenomic libraries – physical libraries that contain DNA cloned from environmental metagenomes. The information obtained from functional metagenomics can help in future annotations of gene function and serve as a complement to sequence-based metagenomics. In this Perspective, we begin by summarizing the technical challenges of constructing metagenomic libraries and emphasize their value as resources. We then discuss libraries constructed using the popular cloning vector, pCC1FOS, and highlight the strengths and shortcomings of this system, alongside possible strategies to maximize existing pCC1FOS-based libraries by screening in diverse hosts. Finally, we discuss the known bias of libraries constructed from human gut and marine water samples, present results that suggest bias may also occur for soil libraries, and consider factors that bias metagenomic libraries in general. We anticipate that discussion of current resources and limitations will advance tools and technologies for functional metagenomics research.

  7. Intracellular screen to identify metagenomic clones that induce or inhibit a quorum-sensing biosensor.

    Science.gov (United States)

    Williamson, Lynn L; Borlee, Bradley R; Schloss, Patrick D; Guan, Changhui; Allen, Heather K; Handelsman, Jo

    2005-10-01

    The goal of this study was to design and evaluate a rapid screen to identify metagenomic clones that produce biologically active small molecules. We built metagenomic libraries with DNA from soil on the floodplain of the Tanana River in Alaska. We extracted DNA directly from the soil and cloned it into fosmid and bacterial artificial chromosome vectors, constructing eight metagenomic libraries that contain 53,000 clones with inserts ranging from 1 to 190 kb. To identify clones of interest, we designed a high throughput "intracellular" screen, designated METREX, in which metagenomic DNA is in a host cell containing a biosensor for compounds that induce bacterial quorum sensing. If the metagenomic clone produces a quorum-sensing inducer, the cell produces green fluorescent protein (GFP) and can be identified by fluorescence microscopy or captured by fluorescence-activated cell sorting. Our initial screen identified 11 clones that induce and two that inhibit expression of GFP. The intracellular screen detected quorum-sensing inducers among metagenomic clones that a traditional overlay screen would not. One inducing clone carries a LuxI homologue that directs the synthesis of an N-acyl homoserine lactone quorum-sensing signal molecule. The LuxI homologue has 62% amino acid sequence identity to its closest match in GenBank, AmfI from Pseudomonas fluorescens, and is on a 78-kb insert that contains 67 open reading frames. Another inducing clone carries a gene with homology to homocitrate synthase. Our results demonstrate the power of an intracellular screen to identify functionally active clones and biologically active small molecules in metagenomic libraries.

  8. Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics.

    Science.gov (United States)

    Colin, Pierre-Yves; Kintses, Balint; Gielen, Fabrice; Miton, Charlotte M; Fischer, Gerhard; Mohamed, Mark F; Hyvönen, Marko; Morgavi, Diego P; Janssen, Dick B; Hollfelder, Florian

    2015-12-07

    Unculturable bacterial communities provide a rich source of biocatalysts, but their experimental discovery by functional metagenomics is difficult, because the odds are stacked against the experimentor. Here we demonstrate functional screening of a million-membered metagenomic library in microfluidic picolitre droplet compartments. Using bait substrates, new hydrolases for sulfate monoesters and phosphotriesters were identified, mostly based on promiscuous activities presumed not to be under selection pressure. Spanning three protein superfamilies, these break new ground in sequence space: promiscuity now connects enzymes with only distantly related sequences. Most hits could not have been predicted by sequence analysis, because the desired activities have never been ascribed to similar sequences, showing how this approach complements bioinformatic harvesting of metagenomic sequencing data. Functional screening of a library of unprecedented size with excellent assay sensitivity has been instrumental in identifying rare genes constituting catalytically versatile hubs in sequence space as potential starting points for the acquisition of new functions.

  9. Multiplexed metagenome mining using short DNA sequence tags facilitates targeted discovery of epoxyketone proteasome inhibitors.

    Science.gov (United States)

    Owen, Jeremy G; Charlop-Powers, Zachary; Smith, Alexandra G; Ternei, Melinda A; Calle, Paula Y; Reddy, Boojala Vijay B; Montiel, Daniel; Brady, Sean F

    2015-04-07

    In molecular evolutionary analyses, short DNA sequences are used to infer phylogenetic relationships among species. Here we apply this principle to the study of bacterial biosynthesis, enabling the targeted isolation of previously unidentified natural products directly from complex metagenomes. Our approach uses short natural product sequence tags derived from conserved biosynthetic motifs to profile biosynthetic diversity in the environment and then guide the recovery of gene clusters from metagenomic libraries. The methodology is conceptually simple, requires only a small investment in sequencing, and is not computationally demanding. To demonstrate the power of this approach to natural product discovery we conducted a computational search for epoxyketone proteasome inhibitors within 185 globally distributed soil metagenomes. This led to the identification of 99 unique epoxyketone sequence tags, falling into 6 phylogenetically distinct clades. Complete gene clusters associated with nine unique tags were recovered from four saturating soil metagenomic libraries. Using heterologous expression methodologies, seven potent epoxyketone proteasome inhibitors (clarepoxcins A-E and landepoxcins A and B) were produced from these pathways, including compounds with different warhead structures and a naturally occurring halohydrin prodrug. This study provides a template for the targeted expansion of bacterially derived natural products using the global metagenome.

  10. Phylogenetic and functional diversity of metagenomic libraries of phenol degrading sludge from petroleum refinery wastewater treatment system.

    Science.gov (United States)

    Silva, Cynthia C; Hayden, Helen; Sawbridge, Tim; Mele, Pauline; Kruger, Ricardo H; Rodrigues, Marili Vn; Costa, Gustavo Gl; Vidal, Ramon O; Sousa, Maíra P; Torres, Ana Paula R; Santiago, Vânia Mj; Oliveira, Valéria M

    2012-03-27

    In petrochemical refinery wastewater treatment plants (WWTP), different concentrations of pollutant compounds are received daily in the influent stream, including significant amounts of phenolic compounds, creating propitious conditions for the development of particular microorganisms that can rapidly adapt to such environment. In the present work, the microbial sludge from a refinery WWTP was enriched for phenol, cloned into fosmid vectors and pyrosequenced. The fosmid libraries yielded 13,200 clones and a comprehensive bioinformatic analysis of the sequence data set revealed a complex and diverse bacterial community in the phenol degrading sludge. The phylogenetic analyses using MEGAN in combination with RDP classifier showed a massive predominance of Proteobacteria, represented mostly by the genera Diaphorobacter, Pseudomonas, Thauera and Comamonas. The functional classification of phenol degrading sludge sequence data set generated by MG-RAST showed the wide metabolic diversity of the microbial sludge, with a high percentage of genes involved in the aerobic and anaerobic degradation of phenol and derivatives. In addition, genes related to the metabolism of many other organic and xenobiotic compounds, such as toluene, biphenyl, naphthalene and benzoate, were found. Results gathered herein demonstrated that the phenol degrading sludge has complex phylogenetic and functional diversities, showing the potential of such community to degrade several pollutant compounds. This microbiota is likely to represent a rich resource of versatile and unknown enzymes which may be exploited for biotechnological processes such as bioremediation.

  11. The Application of BAC Library in Metagenomics Research of Rumen Microbe%瘤胃元基因组BAC文库研究

    Institute of Scientific and Technical Information of China (English)

    朱雅新; 东秀珠; 黄力; 董志扬

    2007-01-01

    目前,传统的分离培养技术在瘤胃微生物的研究中仍存在一些无法逾越的障碍.元基因组文库(metagenomic libraries)及相关技术的发展,为探讨环境中未培养微生物以及复杂的微生物间关系创立了一个新的平台.将该技术应用于瘤胃微生物研究有着广阔的前景.本文综述了元基因组文库技术在瘤胃微生物研究中的应用潜力,同时介绍了初步应用BAC文库研究瘤胃微生物工作的一些进展.

  12. Identification of novel lipolytic genes and gene families by screening of metagenomic libraries derived from soil samples of the German Biodiversity Exploratories.

    Science.gov (United States)

    Nacke, Heiko; Will, Christiane; Herzog, Sarah; Nowka, Boris; Engelhaupt, Martin; Daniel, Rolf

    2011-10-01

    Microbial metagenomes derived from soils are rich sources for the discovery of novel genes and biocatalysts. Fourteen environmental plasmid and seven fosmid libraries obtained from 10 German forest soils (A horizons) and six grassland soils (A and B horizons) were screened for genes conferring lipolytic activity. The libraries comprised approximately 29.3 Gb of cloned soil DNA. Partial activity-based screening of the constructed libraries resulted in the identification of 37 unique lipolytic clones. The amino acid sequences of the 37 corresponding lipolytic gene products shared 29-90% identity to other lipolytic enzymes, which were mainly uncharacterized or derived from uncultured microorganisms. Multiple sequence alignments and phylogenetic tree analysis revealed that 35 of the predicted proteins were new members of known families of lipolytic enzymes. The remaining two gene products represent two putatively new families. In addition, sequence analysis indicated that two genes encode true lipases, whereas the other genes encode esterases. The determination of substrate specificity and chain-length selectivity using different triacylglycerides and p-nitrophenyl esters of fatty acids as substrates supported the classification of the esterases.

  13. Construction of bacterial artificial chromosome libraries for Zhikong Scallop Chlamys farreri

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yang; ZHANG Xiaojun; Chantel F.SCHEURING; ZHANG Hongbin; LI Fuhua; XIANG Jianhai

    2008-01-01

    Two Large-insert genomic bacterial artificial chromosome (BAC) libraries of Zhikong scallop Chlamys farreri were constructed to promote our genetic and genomic research.High-quality megabase-sized DNA was isolated from the adductor muscle of the scallop and partially digested by BamH I and Mbo I,respectively.The BamH I library consisted of 53760 clones while the Mbo I library consisted of 7680 clones.Approximately 96% of the clones in BamH I library contained nuclear DNA inserts in average size of 100 kb,providing a coverage of 5.3 haploid genome equivalents.Similarly,the Mbo I library with an average insert of 145 kb and no insert-empty clones,thus providing a genome coverage of 1.1 haploid genome equivalents.

  14. Construction and characterization of a Schistosoma mansoni bacterial artificial chromosome library.

    Science.gov (United States)

    Le Paslier, M C; Pierce, R J; Merlin, F; Hirai, H; Wu, W; Williams, D L; Johnston, D; LoVerde, P T; Le Paslier, D

    2000-04-15

    A bacterial artificial chromosome (BAC) library has been established from genomic DNA isolated from the trematode parasite of human, Schistosoma mansoni. This library consists of more than 21,000 recombinant clones carrying inserts in the pBeloBAC11 vector. The mean insert size was 100 kb, representing an approximate 7.95-fold genome coverage. Library screening with eight chromosome-specific or single-copy gene probes yielded between 1 and 9 positive clones, and none of those tested was absent from the library. End sequences were obtained for 93 randomly selected clones, and 37 showed sequence identity to S. mansoni sequences (ESTs, genes, or repetitive sequences). A preliminary analysis by fluorescence in situ hybridization localized 8 clones on schistosome chromosomes 1 (2 clones), 2, 3, 5, Z, and W (3 clones). This library provides a new resource for the physical mapping and sequencing of the genome of this important human pathogen.

  15. 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.

  16. Metagenomic analysis of microbial communities and beyond

    DEFF Research Database (Denmark)

    Schreiber, Lars

    2014-01-01

    From small clone libraries to large next-generation sequencing datasets – the field of community genomics or metagenomics has developed tremendously within the last years. This chapter will summarize some of these developments and will also highlight pitfalls of current metagenomic analyses....... It will illustrate the general workflow of a metagenomic study and introduce the three different metagenomic approaches: (1) the random shotgun approach that focuses on the metagenome as a whole, (2) the targeted approach that focuses on metagenomic amplicon sequences, and (3) the function-driven approach that uses...... heterologous expression of metagenomic DNA fragments to discover novel metabolic functions. Lastly, the chapter will shortly discuss the meta-analysis of gene expression of microbial communities, more precisely metatranscriptomics and metaproteomics....

  17. Combined whole-cell high-throughput functional screening for identification of new nicotinamidases/pyrazinamidases in metagenomic/polygenomic libraries

    Directory of Open Access Journals (Sweden)

    Rubén Zapata-Pérez

    2016-11-01

    Full Text Available Nicotinamidases catalyze the hydrolysis of the amide bond in nicotinamide to produce ammonia and nicotinic acid. These enzymes are an essential component of the NAD+ salvage pathway and are implicated in the viability of several pathogenic organisms. Its absence in humans makes them a promising drug target. In addition, although they are key analytical biocatalysts for screening modulators in relevant biomedical enzymes, such as sirtuins and poly-ADP-ribosyltransferases, no commercial sources are available. Surprisingly, the finding of an affordable source of nicotinamidase from metagenomic libraries is hindered by the absence of a suitable and fast screening method. In this manuscript, we describe the development of two new whole-cell methods using the chemical property of one of the products formed in the enzymatic reaction (pyrazinoic or nicotinic acid to form colored complexes with stable iron salts, such as ammonium ferrous sulfate or sodium nitroprusside. After optimization of the assay conditions, a fosmid polygenomic expression library obtained from deep-sea mesophilic bacteria was screened, discovering several positive clones with the ammonium ferrous sulfate method. Their quantitative rescreening with the sodium nitroprusside method allowed the finding of the first nicotinamidase with balanced catalytic efficiency towards nicotinamide (nicotinamidase activity and pyrazinamide (pyrazinamidase activity. Its biochemical characterization has also made possible the development of the first high-throughput whole-cell method for prescreening of new nicotinamidase inhibitors by the naked eye, saving time and costs in the design of future antimicrobial and antiparasitic agents.

  18. [Advances of metagenomics in discovering novel biocatalysts].

    Science.gov (United States)

    Wang, Kui; Wang, Sidi; Huang, Rui; Liu, Yuhuan

    2012-04-01

    Microorganisms contain a large number of biocatalysts, which are of great potential in industrial applications. However, the traditional cultural approaches can obtain only less than 1% of microorganisms. As a culture-independent method, metagenomics is an advanced solution by means of extracting all microbial genomic DNAs in certain environmental habitat, constructing and screening metagenomic libraries to seek novel functional genes. It serves as an effective tool for studying these uncultured microorganisms. Therefore, mining novel biocatalysts from metagenome has drawn the attention of researchers in the world. In this paper, environment sample category, genomic DNA extraction, library construction and screening strategies were reviewed. Recent examples of isolated biocatalysts from metagenomic libraries were presented. Future research directions of metagenomics were also discussed.

  19. Construction of a llama bacterial artificial chromosome library with approximately 9-fold genome equivalent coverage.

    Science.gov (United States)

    Airmet, K W; Hinckley, J D; Tree, L T; Moss, M; Blumell, S; Ulicny, K; Gustafson, A K; Weed, M; Theodosis, R; Lehnardt, M; Genho, J; Stevens, M R; Kooyman, D L

    2012-01-01

    The Ilama is an important agricultural livestock in much of South America. The llama is increasing in popularity in the United States as a companion animal. Little work has been done to improve llama production using modern technology. A paucity of information is available regarding the llama genome. We report the construction of a llama bacterial artificial chromosome (BAC) library of about 196,224 clones in the vector pECBAC1. Using flow cytometry and bovine, human, mouse, and chicken as controls, we determined the llama genome size to be 2.4 × 10⁹ bp. The average insert size of the library is 137.8 kb corresponding to approximately 9-fold genome coverage. Further studies are needed to further characterize the library and llama genome. We anticipate that this new library will help facilitate future genomic studies in the llama.

  20. Comparative analysis of acidobacterial genomic fragments from terrestrial and aquatic metagenomic libraries, with emphasis on acidobacteria subdivision 6

    NARCIS (Netherlands)

    Kielak, A.M.; Van Veen, J.A.; Kowalchuk, G.A.

    2010-01-01

    The bacterial phylum Acidobacteria has a widespread distribution and is one of the most common and diverse phyla in soil habitats. However, members of this phylum have often been recalcitrant to cultivation methods, hampering the study of this presumably important bacterial group. In this study, we

  1. Complete metagenome sequencing based bacterial diversity and functional insights from basaltic hot spring of Unkeshwar, Maharashtra, India

    Directory of Open Access Journals (Sweden)

    Gajanan T. Mehetre

    2016-03-01

    Full Text Available Unkeshwar hot springs are located at geographical South East Deccan Continental basalt of India. Here, we report the microbial community analysis of this hot spring using whole metagenome shotgun sequencing approach. The analysis revealed a total of 848,096 reads with 212.87 Mbps with 50.87% G + C content. Metagenomic sequences were deposited in SRA database with accession number (SUB1242219. Community analysis revealed 99.98% sequences belonging to bacteria and 0.01% to archaea and 0.01% to Viruses. The data obtained revealed 41 phyla including bacteria and Archaea and including 719 different species. In taxonomic analysis, the dominant phyla were found as, Actinobacteria (56%, Verrucomicrobia (24%, Bacteriodes (13%, Deinococcus-Thermus (3% and firmicutes (2% and Viruses (2%. Furthermore, functional annotation using pathway information revealed dynamic potential of hot spring community in terms of metabolism, environmental information processing, cellular processes and other important aspects. Kyoto Encyclopedia of Genes and Genomes (KEGG pathway analysis of each contig sequence by assigning KEGG Orthology (KO numbers revealed contig sequences that were assigned to metabolism, organismal system, Environmental Information Processing, cellular processes and human diseases with some unclassified sequences. The Unkeshwar hot springs offer rich phylogenetic diversity and metabolic potential for biotechnological applications.

  2. Molecular cloning and characterization of a novel β-glucosidase with high hydrolyzing ability for soybean isoflavone glycosides and glucose-tolerance from soil metagenomic library.

    Science.gov (United States)

    Li, Gang; Jiang, Yang; Fan, Xin-Jiong; Liu, Yu-Huan

    2012-11-01

    A novel β-glucosidase (Bgl1269) was identified from a metagenomic library of mangrove soil by activity-based functional screening. Sequence analysis revealed that Bgl1269 encodes a protein of 422 amino acids. After being overexpressed in Escherichia coli and purified, the enzymatic properties of Bgl1269 were investigated. The recombinant enzyme displayed a pH optimum of 6.0 and a temperature optimum of 40°C, and the addition of most common metal ions (1 or 10mM) increased the enzymatic activity evidently. In addition, the enzyme showed high hydrolyzing ability for soybean isoflavone glycosides, and 0.8unit of enzyme could completely converted daidzin and genistin (0.5mg/mL) to daidzein and genistein at 40°C for 0.5h. Interestingly, Bgl1269 also exhibited a very high glucose-tolerance, with the highest inhibition constant K(i) (4.28M) among β-glucosidases reported so far. These properties make it a good candidate in the production of soybean isoflavone aglycones after further study.

  3. Molecular Cloning and Characterization of a New Cold-active Extradiol Dioxygenase from a Metagenomic Library Derived from Polychlorinated Biphenyl-contaminated Soil

    Institute of Scientific and Technical Information of China (English)

    REN He-jun; LU Yang; ZHOU Rui; DAI Chun-yan; WANG Yan; ZHANG Lan-ying

    2012-01-01

    To find new extradiol dioxygenases(EDOs,EC 1.13.11.2),a metagenomics library was constructed from polychlorinated biphenyl-contaminated soil and was screened for some dioxygenase with aromatic ring cleavage activity.A novel EDO,designated as BphC_A,was identified and heterologously expressed in Escherichia coli.The deduced amino acid sequence of BphC_A exhibited a homology of less than 60% with other known EDOs.Phylogenetic analysis of BphC_A suggests that the protein is a novel member of the EDO family.The enzyme exhibits higher substrate affinity and catalytic efficiency toward 3-methylcatechol than toward 2,3-dihydroxybiphenyl or catechol,the preferred substrate of other known EDOs.The optimum activity of purified BphC_A occurred at pH=8.5 and 35 ℃,and BphC_A showed more than 40% of its initial activity at 5 ℃.The activity of purified BphC_A was significantly induced by Mn2+ and slightly reduced bv Al3+,Cu2+ and Zn2+.

  4. Screening a novel Na+/H+ antiporter gene from a metagenomic library of halophiles colonizing in the Dagong Ancient Brine Well in China.

    Science.gov (United States)

    Xiang, Wenliang; Zhang, Jie; Li, Lin; Liang, Huazhong; Luo, Hai; Zhao, Jian; Yang, Zhirong; Sun, Qun

    2010-05-01

    Metagenomic DNA libraries constructed from the Dagong Ancient Brine Well were screened for genes with Na(+)/H(+) antiporter activity on the antiporter-deficient Escherichia coli KNabc strain. One clone with a stable Na(+)-resistant phenotype was obtained and its Na(+)/H(+) antiporter gene was sequenced and designated as m-nha. The deduced amino acid sequence of M-Nha protein consists of 523 residues with a calculated molecular weight of 58 147 Da and a pI of 5.50, which is homologous with NhaH from Halobacillus dabanensis D-8(T) (92%) and Halobacillus aidingensis AD-6(T) (86%), and with Nhe2 from Bacillus sp. NRRL B-14911 (64%). It had a hydropathy profile with 10 putative transmembrane domains and a long carboxyl terminal hydrophilic tail of 140 amino acid residues, similar to Nhap from Synechocystis sp. and Aphanothece halophytica, as well as NhaG from Bacillus subtilis. The m-nha gene in the antiporter-negative mutant E. coli KNabc conferred resistance to Na(+) and the ability to grow under alkaline conditions. The difference in amino acid sequence and the putative secondary structure suggested that the m-nha isolated from the Dagong Ancient Brine Well in this study was a novel Na(+)/H(+) antiporter gene.

  5. An exceptionally cold-adapted alpha-amylase from a metagenomic library of a cold and alkaline environment

    DEFF Research Database (Denmark)

    Vester, Jan Kjølhede; Glaring, Mikkel Andreas; Stougaard, Peter

    2015-01-01

    A cold-active α-amylase, AmyI3C6, identified by a functional metagenomics approach was expressed in Escherichia coli and purified to homogeneity. Sequence analysis showed that the AmyI3C6 amylase was similar to α-amylases from the class Clostridia and revealed classical characteristics of cold......-adapted enzymes, as did comparison of the kinetic parameters Km and kcat to a mesophilic α-amylase. AmyI3C6 was shown to be heat-labile. Temperature optimum was at 10-15 °C, and more than 70 % of the relative activity was retained at 1 °C. The pH optimum of AmyI3C6 was at pH 8-9, and the enzyme displayed activity...... in two commercial detergents tested, suggesting that the AmyI3C6 α-amylase may be useful as a detergent enzyme in environmentally friendly, low-temperature laundry processes. © 2014 Springer-Verlag Berlin Heidelberg....

  6. A study on the interaction of rhodamine B with methylthioadenosine phosphorylase protein sourced from an Antarctic soil metagenomic library.

    Science.gov (United States)

    Bartasun, Paulina; Cieśliński, Hubert; Bujacz, Anna; Wierzbicka-Woś, Anna; Kur, Józef

    2013-01-01

    The presented study examines the phenomenon of the fluorescence under UV light excitation (312 nm) of E. coli cells expressing a novel metagenomic-derived putative methylthioadenosine phosphorylase gene, called rsfp, grown on LB agar supplemented with a fluorescent dye rhodamine B. For this purpose, an rsfp gene was cloned and expressed in an LMG194 E. coli strain using an arabinose promoter. The resulting RSFP protein was purified and its UV-VIS absorbance spectrum and emission spectrum were assayed. Simultaneously, the same spectroscopic studies were carried out for rhodamine B in the absence or presence of RSFP protein or native E. coli proteins, respectively. The results of the spectroscopic studies suggested that the fluorescence of E. coli cells expressing rsfp gene under UV illumination is due to the interaction of rhodamine B molecules with the RSFP protein. Finally, this interaction was proved by a crystallographic study and then by site-directed mutagenesis of rsfp gene sequence. The crystal structures of RSFP apo form (1.98 Å) and complex RSFP/RB (1.90 Å) show a trimer of RSFP molecules located on the crystallographic six fold screw axis. The RSFP complex with rhodamine B revealed the binding site for RB, in the pocket located on the interface between symmetry related monomers.

  7. A study on the interaction of rhodamine B with methylthioadenosine phosphorylase protein sourced from an Antarctic soil metagenomic library.

    Directory of Open Access Journals (Sweden)

    Paulina Bartasun

    Full Text Available The presented study examines the phenomenon of the fluorescence under UV light excitation (312 nm of E. coli cells expressing a novel metagenomic-derived putative methylthioadenosine phosphorylase gene, called rsfp, grown on LB agar supplemented with a fluorescent dye rhodamine B. For this purpose, an rsfp gene was cloned and expressed in an LMG194 E. coli strain using an arabinose promoter. The resulting RSFP protein was purified and its UV-VIS absorbance spectrum and emission spectrum were assayed. Simultaneously, the same spectroscopic studies were carried out for rhodamine B in the absence or presence of RSFP protein or native E. coli proteins, respectively. The results of the spectroscopic studies suggested that the fluorescence of E. coli cells expressing rsfp gene under UV illumination is due to the interaction of rhodamine B molecules with the RSFP protein. Finally, this interaction was proved by a crystallographic study and then by site-directed mutagenesis of rsfp gene sequence. The crystal structures of RSFP apo form (1.98 Å and complex RSFP/RB (1.90 Å show a trimer of RSFP molecules located on the crystallographic six fold screw axis. The RSFP complex with rhodamine B revealed the binding site for RB, in the pocket located on the interface between symmetry related monomers.

  8. Metagenomic analysis of two important, but difficult to culture soil borne bacterial phyla, the Acidobacteria and the Verrucomicrobia

    NARCIS (Netherlands)

    Kielak, A.M.

    2010-01-01

    Based on phylogenetic marker genes, such as 16S rRNA genes, it is clear that numerous bacterial lineages exist that appear to be quite common in the environment, yet poorly characterized and underrepresented in culture. Two of the most common bacterial phyla in soils that fall into this category are

  9. Metagenomic analysis of bacterial community composition and antibiotic resistance genes in a wastewater treatment plant and its receiving surface water.

    Science.gov (United States)

    Tang, Junying; Bu, Yuanqing; Zhang, Xu-Xiang; Huang, Kailong; He, Xiwei; Ye, Lin; Shan, Zhengjun; Ren, Hongqiang

    2016-10-01

    The presence of pathogenic bacteria and the dissemination of antibiotic resistance genes (ARGs) may pose big risks to the rivers that receive the effluent from municipal wastewater treatment plants (WWTPs). In this study, we investigated the changes of bacterial community and ARGs along treatment processes of one WWTP, and examined the effects of the effluent discharge on the bacterial community and ARGs in the receiving river. Pyrosequencing was applied to reveal bacterial community composition including potential bacterial pathogen, and Illumina high-throughput sequencing was used for profiling ARGs. The results showed that the WWTP had good removal efficiency on potential pathogenic bacteria (especially Arcobacter butzleri) and ARGs. Moreover, the bacterial communities of downstream and upstream of the river showed no significant difference. However, the increase in the abundance of potential pathogens and ARGs at effluent outfall was observed, indicating that WWTP effluent might contribute to the dissemination of potential pathogenic bacteria and ARGs in the receiving river.

  10. Improving microbial fitness in the mammalian gut by in vivo temporal functional metagenomics.

    Science.gov (United States)

    Yaung, Stephanie J; Deng, Luxue; Li, Ning; Braff, Jonathan L; Church, George M; Bry, Lynn; Wang, Harris H; Gerber, Georg K

    2015-03-11

    Elucidating functions of commensal microbial genes in the mammalian gut is challenging because many commensals are recalcitrant to laboratory cultivation and genetic manipulation. We present Temporal FUnctional Metagenomics sequencing (TFUMseq), a platform to functionally mine bacterial genomes for genes that contribute to fitness of commensal bacteria in vivo. Our approach uses metagenomic DNA to construct large-scale heterologous expression libraries that are tracked over time in vivo by deep sequencing and computational methods. To demonstrate our approach, we built a TFUMseq plasmid library using the gut commensal Bacteroides thetaiotaomicron (Bt) and introduced Escherichia coli carrying this library into germfree mice. Population dynamics of library clones revealed Bt genes conferring significant fitness advantages in E. coli over time, including carbohydrate utilization genes, with a Bt galactokinase central to early colonization, and subsequent dominance by a Bt glycoside hydrolase enabling sucrose metabolism coupled with co-evolution of the plasmid library and E. coli genome driving increased galactose utilization. Our findings highlight the utility of functional metagenomics for engineering commensal bacteria with improved properties, including expanded colonization capabilities in vivo.

  11. Improving microbial fitness in the mammalian gut by in vivo temporal functional metagenomics.

    Science.gov (United States)

    Yaung, Stephanie J; Deng, Luxue; Li, Ning; Braff, Jonathan L; Church, George M; Bry, Lynn; Wang, Harris H; Gerber, Georg K

    2015-03-01

    Elucidating functions of commensal microbial genes in the mammalian gut is challenging because many commensals are recalcitrant to laboratory cultivation and genetic manipulation. We present Temporal FUnctional Metagenomics sequencing (TFUMseq), a platform to functionally mine bacterial genomes for genes that contribute to fitness of commensal bacteria in vivo. Our approach uses metagenomic DNA to construct large-scale heterologous expression libraries that are tracked over time in vivo by deep sequencing and computational methods. To demonstrate our approach, we built a TFUMseq plasmid library using the gut commensal Bacteroides thetaiotaomicron (Bt) and introduced Escherichia coli carrying this library into germfree mice. Population dynamics of library clones revealed Bt genes conferring significant fitness advantages in E. coli over time, including carbohydrate utilization genes, with a Bt galactokinase central to early colonization, and subsequent dominance by a Bt glycoside hydrolase enabling sucrose metabolism coupled with co-evolution of the plasmid library and E. coli genome driving increased galactose utilization. Our findings highlight the utility of functional metagenomics for engineering commensal bacteria with improved properties, including expanded colonization capabilities in vivo.

  12. Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms.

    Science.gov (United States)

    Rondon, M R; August, P R; Bettermann, A D; Brady, S F; Grossman, T H; Liles, M R; Loiacono, K A; Lynch, B A; MacNeil, I A; Minor, C; Tiong, C L; Gilman, M; Osburne, M S; Clardy, J; Handelsman, J; Goodman, R M

    2000-06-01

    Recent progress in molecular microbial ecology has revealed that traditional culturing methods fail to represent the scope of microbial diversity in nature, since only a small proportion of viable microorganisms in a sample are recovered by culturing techniques. To develop methods to investigate the full extent of microbial diversity, we used a bacterial artificial chromosome (BAC) vector to construct libraries of genomic DNA isolated directly from soil (termed metagenomic libraries). To date, we have constructed two such libraries, which contain more than 1 Gbp of DNA. Phylogenetic analysis of 16S rRNA gene sequences recovered from one of the libraries indicates that the BAC libraries contain DNA from a wide diversity of microbial phyla, including sequences from diverse taxa such as the low-G+C, gram-positive Acidobacterium, Cytophagales, and Proteobacteria. Initial screening of the libraries in Escherichia coli identified several clones that express heterologous genes from the inserts, confirming that the BAC vector can be used to maintain, express, and analyze environmental DNA. The phenotypes expressed by these clones include antibacterial, lipase, amylase, nuclease, and hemolytic activities. Metagenomic libraries are a powerful tool for exploring soil microbial diversity, providing access to the genetic information of uncultured soil microorganisms. Such libraries will be the basis of new initiatives to conduct genomic studies that link phylogenetic and functional information about the microbiota of environments dominated by microorganisms that are refractory to cultivation.

  13. Quality control of the sheep bacterial artificial chromosome library, CHORI-243

    Directory of Open Access Journals (Sweden)

    Kirkness Ewen F

    2010-12-01

    Full Text Available Abstract Background The sheep CHORI-243 bacterial artificial chromosome (BAC library is being used in the construction of the virtual sheep genome, the sequencing and construction of the actual sheep genome assembly and as a source of DNA for regions of the genome of biological interest. The objective of our study is to assess the integrity of the clones and plates which make up the CHORI-243 library using the virtual sheep genome. Findings A series of analyses were undertaken based on the mapping the sheep BAC-end sequences (BESs to the virtual sheep genome. Overall, very few plate specific biases were identified, with only three of the 528 plates in the library significantly affected. The analysis of the number of tail-to-tail (concordant BACs on the plates identified a number of plates with lower than average numbers of such BACs. For plates 198 and 213 a partial swap of the BESs determined with one of the two primers appear to have occurred. A third plate, 341, also with a significant deficit in tail-to-tail BACs, appeared to contain a substantial number of sequences determined from contaminating eubacterial 16 S rRNA DNA. Additionally a small number of eubacterial 16 S rRNA DNA sequences were present on two other plates, 111 and 338, in the library. Conclusions The comparative genomic approach can be used to assess BAC library integrity in the absence of fingerprinting. The sequences of the sheep CHORI-243 library BACs have high integrity, especially with the corrections detailed above. The library represents a high quality resource for use by the sheep genomics community.

  14. Hot Spring Metagenomics

    Directory of Open Access Journals (Sweden)

    Olalla López-López

    2013-04-01

    Full Text Available Hot springs have been investigated since the XIX century, but isolation and examination of their thermophilic microbial inhabitants did not start until the 1950s. Many thermophilic microorganisms and their viruses have since been discovered, although the real complexity of thermal communities was envisaged when research based on PCR amplification of the 16S rRNA genes arose. Thereafter, the possibility of cloning and sequencing the total environmental DNA, defined as metagenome, and the study of the genes rescued in the metagenomic libraries and assemblies made it possible to gain a more comprehensive understanding of microbial communities—their diversity, structure, the interactions existing between their components, and the factors shaping the nature of these communities. In the last decade, hot springs have been a source of thermophilic enzymes of industrial interest, encouraging further study of the poorly understood diversity of microbial life in these habitats.

  15. Construction and screening of metagenomic libraries derived from enrichment cultures: generation of a gene bank for genes conferring alcohol oxidoreductase activity on Escherichia coli.

    Science.gov (United States)

    Knietsch, Anja; Waschkowitz, Tanja; Bowien, Susanne; Henne, Anke; Daniel, Rolf

    2003-03-01

    Enrichment of microorganisms with special traits and the construction of metagenomic libraries by direct cloning of environmental DNA have great potential for identifying genes and gene products for biotechnological purposes. We have combined these techniques to isolate novel genes conferring oxidation of short-chain (C(2) to C(4)) polyols or reduction of the corresponding carbonyls. In order to favor the growth of microorganisms containing the targeted genes, samples collected from four different environments were incubated in the presence of glycerol and 1,2-propanediol. Subsequently, the DNA was extracted from the four samples and used to construct complex plasmid libraries. Approximately 100,000 Escherichia coli strains of each library per test substrate were screened for the production of carbonyls from polyols on indicator agar. Twenty-four positive E. coli clones were obtained during the initial screen. Sixteen of them contained a plasmid (pAK101 to pAK116) which conferred a stable carbonyl-forming phenotype. Eight of the positive clones exhibited NAD(H)-dependent alcohol oxidoreductase activity with polyols or carbonyls as the substrates in crude extracts. Sequencing revealed that the inserts of pAK101 to pAK116 encoded 36 complete and 17 incomplete presumptive protein-encoding genes. Fifty of these genes showed similarity to sequenced genes from a broad collection of different microorganisms. The genes responsible for the carbonyl formation of E. coli were identified for nine of the plasmids (pAK101, pAK102, pAK105, pAK107 to pAK110, pAK115, and pAK116). Analyses of the amino acid sequences deduced from these genes revealed that three (orf12, orf14, and orf22) encoded novel alcohol dehydrogenases of different types, four (orf5, sucB, fdhD, and yabF) encoded novel putative oxidoreductases belonging to groups distinct from alcohol dehydrogenases, one (glpK) encoded a putative glycerol kinase, and one (orf1) encoded a protein which showed no similarity to any

  16. Feasibility of physical map construction from fingerprinted bacterial artificial chromosome libraries of polyploid plant species

    Directory of Open Access Journals (Sweden)

    Doležel Jaroslav

    2010-02-01

    Full Text Available Abstract Background The presence of closely related genomes in polyploid species makes the assembly of total genomic sequence from shotgun sequence reads produced by the current sequencing platforms exceedingly difficult, if not impossible. Genomes of polyploid species could be sequenced following the ordered-clone sequencing approach employing contigs of bacterial artificial chromosome (BAC clones and BAC-based physical maps. Although BAC contigs can currently be constructed for virtually any diploid organism with the SNaPshot high-information-content-fingerprinting (HICF technology, it is currently unknown if this is also true for polyploid species. It is possible that BAC clones from orthologous regions of homoeologous chromosomes would share numerous restriction fragments and be therefore included into common contigs. Because of this and other concerns, physical mapping utilizing the SNaPshot HICF of BAC libraries of polyploid species has not been pursued and the possibility of doing so has not been assessed. The sole exception has been in common wheat, an allohexaploid in which it is possible to construct single-chromosome or single-chromosome-arm BAC libraries from DNA of flow-sorted chromosomes and bypass the obstacles created by polyploidy. Results The potential of the SNaPshot HICF technology for physical mapping of polyploid plants utilizing global BAC libraries was evaluated by assembling contigs of fingerprinted clones in an in silico merged BAC library composed of single-chromosome libraries of two wheat homoeologous chromosome arms, 3AS and 3DS, and complete chromosome 3B. Because the chromosome arm origin of each clone was known, it was possible to estimate the fidelity of contig assembly. On average 97.78% or more clones, depending on the library, were from a single chromosome arm. A large portion of the remaining clones was shown to be library contamination from other chromosomes, a feature that is unavoidable during the

  17. A bacterial artificial chromosome library for Biomphalaria glabrata, intermediate snail host of Schistosoma mansoni

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    Coen M Adema

    2006-10-01

    Full Text Available To provide a novel resource for analysis of the genome of Biomphalaria glabrata, members of the international Biomphalaria glabrata Genome Initiative (biology.unm.edu/biomphalaria-genome.html, working with the Arizona Genomics Institute (AGI and supported by the National Human Genome Research Institute (NHGRI, produced a high quality bacterial artificial chromosome (BAC library. The BB02 strain B. glabrata, a field isolate (Belo Horizonte, Minas Gerais, Brasil that is susceptible to several strains of Schistosoma mansoni, was selfed for two generations to reduce haplotype diversity in the offspring. High molecular weight DNA was isolated from ovotestes of 40 snails, partially digested with HindIII, and ligated into pAGIBAC1 vector. The resulting B. glabrata BAC library (BG_BBa consists of 61824 clones (136.3 kb average insert size and provides 9.05 × coverage of the 931 Mb genome. Probing with single/low copy number genes from B. glabrata and fingerprinting of selected BAC clones indicated that the BAC library sufficiently represents the gene complement. BAC end sequence data (514 reads, 299860 nt indicated that the genome of B. glabrata contains ~ 63% AT, and disclosed several novel genes, transposable elements, and groups of high frequency sequence elements. This BG_BBa BAC library, available from AGI at cost to the research community, gains in relevance because BB02 strain B. glabrata is targeted whole genome sequencing by NHGRI.

  18. Comparison of soil bacterial communities of Pinus patula of Nilgiris, western ghats with other biogeographically distant pine forest clone libraries.

    Science.gov (United States)

    Rohini-Kumar, M; Osborne, Jabez W; Saravanan, V S

    2013-07-01

    The bacterial community structure of the rhizosphere and non-rhizosphere soil of Pinus patula, found in the Nilgiris region of Western Ghats, was studied by constructing 16S rRNA gene clone libraries. In the rhizosphere and non-rhizosphere soil clone libraries constructed, 13 and 15 bacterial phyla were identified, respectively. The clone libraries showed the predominance of members of culturally underrepresented phyla like Acidobacteria and Verrucomicrobia. The Alphaproteobacteria and Acidobacteria clones were predominant in rhizosphere and non-rhizosphere soil samples, respectively. In rhizosphere, amongst Alphaproteobacteria members, Bradyrhizobium formed the significant proportion, whereas in non-rhizosphere, members of subdivision-6 of phylum Acidobacteria were abundant. The diversity analysis of P. patula soil libraries showed that the phylotypes (16S rRNA gene similarity cutoff, ≥97 %) of Acidobacteria and Bacteroidetes were relatively predominant and diverse followed by Alphaproteobacteria and Verrucomicrobia. The diversity indices estimated higher richness and abundance of bacteria in P. patula soil clone libraries than the pine forest clone libraries retrieved from previous studies. The tools like principal co-ordinate analysis and Jackknife cluster analysis, which were under UniFrac analysis indicated that variations in soil bacterial communities were attributed to their respective geographical locations due to the phylogenetic divergence amongst the clone libraries. Overall, the P. patula rhizosphere and non-rhizosphere clone libraries were found significantly unique in composition, evenly distributed and highly rich in phylotypes, amongst the biogeographically distant clone libraries. It was finally hypothesised that the phylogenetic divergence amongst the bacterial phylotypes and natural selection plays a pivotal role in the variations of bacterial communities across the geographical distance.

  19. Bacterial communities in semen from men of infertile couples: metagenomic sequencing reveals relationships of seminal microbiota to semen quality.

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    Shun-Long Weng

    Full Text Available Some previous studies have identified bacteria in semen as being a potential factor in male infertility. However, only few types of bacteria were taken into consideration while using PCR-based or culturing methods. Here we present an analysis approach using next-generation sequencing technology and bioinformatics analysis to investigate the associations between bacterial communities and semen quality. Ninety-six semen samples collected were examined for bacterial communities, measuring seven clinical criteria for semen quality (semen volume, sperm concentration, motility, Kruger's strict morphology, antisperm antibody (IgA, Atypical, and leukocytes. Computer-assisted semen analysis (CASA was also performed. Results showed that the most abundant genera among all samples were Lactobacillus (19.9%, Pseudomonas (9.85%, Prevotella (8.51% and Gardnerella (4.21%. The proportion of Lactobacillus and Gardnerella was significantly higher in the normal samples, while that of Prevotella was significantly higher in the low quality samples. Unsupervised clustering analysis demonstrated that the seminal bacterial communities were clustered into three main groups: Lactobacillus, Pseudomonas, and Prevotella predominant group. Remarkably, most normal samples (80.6% were clustered in Lactobacillus predominant group. The analysis results showed seminal bacteria community types were highly associated with semen health. Lactobacillus might not only be a potential probiotic for semen quality maintenance, but also might be helpful in countering the negative influence of Prevotella and Pseudomonas. In this study, we investigated whole seminal bacterial communities and provided the most comprehensive analysis of the association between bacterial community and semen quality. The study significantly contributes to the current understanding of the etiology of male fertility.

  20. Integrated Metagenomic and Metatranscriptomic Analyses of Microbial Communities in the Meso- and Bathypelagic Realm of North Pacific Ocean

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    Deirdre R. Meldrum

    2013-10-01

    Full Text Available Although emerging evidence indicates that deep-sea water contains an untapped reservoir of high metabolic and genetic diversity, this realm has not been studied well compared with surface sea water. The study provided the first integrated meta-genomic and -transcriptomic analysis of the microbial communities in deep-sea water of North Pacific Ocean. DNA/RNA amplifications and simultaneous metagenomic and metatranscriptomic analyses were employed to discover information concerning deep-sea microbial communities from four different deep-sea sites ranging from the mesopelagic to pelagic ocean. Within the prokaryotic community, bacteria is absolutely dominant (~90% over archaea in both metagenomic and metatranscriptomic data pools. The emergence of archaeal phyla Crenarchaeota, Euryarchaeota, Thaumarchaeota, bacterial phyla Actinobacteria, Firmicutes, sub-phyla Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria, and the decrease of bacterial phyla Bacteroidetes and Alphaproteobacteria are the main composition changes of prokaryotic communities in the deep-sea water, when compared with the reference Global Ocean Sampling Expedition (GOS surface water. Photosynthetic Cyanobacteria exist in all four metagenomic libraries and two metatranscriptomic libraries. In Eukaryota community, decreased abundance of fungi and algae in deep sea was observed. RNA/DNA ratio was employed as an index to show metabolic activity strength of microbes in deep sea. Functional analysis indicated that deep-sea microbes are leading a defensive lifestyle.

  1. Construction and Analysis of Siberian Tiger Bacterial Artificial Chromosome Library with Approximately 6.5-Fold Genome Equivalent Coverage

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    Changqing Liu

    2014-03-01

    Full Text Available Bacterial artificial chromosome (BAC libraries are extremely valuable for the genome-wide genetic dissection of complex organisms. The Siberian tiger, one of the most well-known wild primitive carnivores in China, is an endangered animal. In order to promote research on its genome, a high-redundancy BAC library of the Siberian tiger was constructed and characterized. The library is divided into two sub-libraries prepared from blood cells and two sub-libraries prepared from fibroblasts. This BAC library contains 153,600 individually archived clones; for PCR-based screening of the library, BACs were placed into 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 116.5 kb, representing approximately 6.46 genome equivalents of the haploid genome and affording a 98.86% statistical probability of obtaining at least one clone containing a unique DNA sequence. Screening the library with 19 microsatellite markers and a SRY sequence revealed that each of these markers were present in the library; the average number of positive clones per marker was 6.74 (range 2 to 12, consistent with 6.46 coverage of the tiger genome. Additionally, we identified 72 microsatellite markers that could potentially be used as genetic markers. This BAC library will serve as a valuable resource for physical mapping, comparative genomic study and large-scale genome sequencing in the tiger.

  2. Construction and analysis of Siberian tiger bacterial artificial chromosome library with approximately 6.5-fold genome equivalent coverage.

    Science.gov (United States)

    Liu, Changqing; Bai, Chunyu; Guo, Yu; Liu, Dan; Lu, Taofeng; Li, Xiangchen; Ma, Jianzhang; Ma, Yuehui; Guan, Weijun

    2014-03-07

    Bacterial artificial chromosome (BAC) libraries are extremely valuable for the genome-wide genetic dissection of complex organisms. The Siberian tiger, one of the most well-known wild primitive carnivores in China, is an endangered animal. In order to promote research on its genome, a high-redundancy BAC library of the Siberian tiger was constructed and characterized. The library is divided into two sub-libraries prepared from blood cells and two sub-libraries prepared from fibroblasts. This BAC library contains 153,600 individually archived clones; for PCR-based screening of the library, BACs were placed into 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 116.5 kb, representing approximately 6.46 genome equivalents of the haploid genome and affording a 98.86% statistical probability of obtaining at least one clone containing a unique DNA sequence. Screening the library with 19 microsatellite markers and a SRY sequence revealed that each of these markers were present in the library; the average number of positive clones per marker was 6.74 (range 2 to 12), consistent with 6.46 coverage of the tiger genome. Additionally, we identified 72 microsatellite markers that could potentially be used as genetic markers. This BAC library will serve as a valuable resource for physical mapping, comparative genomic study and large-scale genome sequencing in the tiger.

  3. A diverse bacterial community in an anoxic quinoline-degrading bioreactor determined by using pyrosequencing and clone library analysis.

    Science.gov (United States)

    Zhang, Xiaojun; Yue, Siqing; Zhong, Huihui; Hua, Weiying; Chen, Ruijia; Cao, Youfang; Zhao, Liping

    2011-07-01

    There is a concern of whether the structure and diversity of a microbial community can be effectively revealed by short-length pyrosequencing reads. In this study, we performed a microbial community analysis on a sample from a high-efficiency denitrifying quinoline-degrading bioreactor and compared the results generated by pyrosequencing with those generated by clone library technology. By both technologies, 16S rRNA gene analysis indicated that the bacteria in the sample were closely related to, for example, Proteobacteria, Actinobacteria, and Bacteroidetes. The sequences belonging to Rhodococcus were the most predominant, and Pseudomonas, Sphingomonas, Acidovorax, and Zoogloea were also abundant. Both methods revealed a similar overall bacterial community structure. However, the 622 pyrosequencing reads of the hypervariable V3 region of the 16S rRNA gene revealed much higher bacterial diversity than the 130 sequences from the full-length 16S rRNA gene clone library. The 92 operational taxonomic unit (OTUs) detected using pyrosequencing belonged to 45 families, whereas the 37 OTUs found in the clone library belonged to 25 families. Most sequences obtained from the clone library had equivalents in the pyrosequencing reads. However, 64 OTUs detected by pyrosequencing were not represented in the clone library. Our results demonstrate that pyrosequencing of the V3 region of the 16S rRNA gene is not only a powerful tool for discovering low-abundance bacterial populations but is also reliable for dissecting the bacterial community structure in a wastewater environment.

  4. Identification of Bacterial Surface Antigens by Screening Peptide Phage Libraries Using Whole Bacteria Cell-Purified Antisera

    Science.gov (United States)

    Hu, Yun-Fei; Zhao, Dun; Yu, Xing-Long; Hu, Yu-Li; Li, Run-Cheng; Ge, Meng; Xu, Tian-Qi; Liu, Xiao-Bo; Liao, Hua-Yuan

    2017-01-01

    Bacterial surface proteins can be good vaccine candidates. In the present study, we used polyclonal antibodies purified with intact Erysipelothrix rhusiopthiae to screen phage-displayed random dodecapeptide and loop-constrained heptapeptide libraries, which led to the identification of mimotopes. Homology search of the mimotope sequences against E. rhusiopthiae-encoded ORF sequences revealed 14 new antigens that may localize on the surface of E. rhusiopthiae. When these putative surface proteins were used to immunize mice, 9/11 antigens induced protective immunity. Thus, we have demonstrated that a combination of using the whole bacterial cells to purify antibodies and using the phage-displayed peptide libraries to determine the antigen specificities of the antibodies can lead to the discovery of novel bacterial surface antigens. This can be a general approach for identifying surface antigens for other bacterial species. PMID:28184219

  5. Screening and expression of genes from metagenomes.

    Science.gov (United States)

    Leis, Benedikt; Angelov, Angel; Liebl, Wolfgang

    2013-01-01

    Microorganisms are the most abundant and widely spread organisms on earth. They colonize a huge variety of natural and anthropogenic environments, including very specialized ecological niches and even extreme habitats, which are made possible by the immense metabolic diversity and genetic adaptability of microbes. As most of the organisms from environmental samples defy cultivation, cultivation-independent metagenomics approaches have been applied since more than one decade to access and characterize the phylogenetic diversity in microbial communities as well as their metabolic potential and ecological functions. Thereby, metagenomics has fully emerged as an own scientific field for mining new biocatalysts for many industrially relevant processes in biotechnology and pharmaceutics. This review summarizes common metagenomic approaches ranging from sampling, isolation of nucleic acids, construction of metagenomic libraries and their evaluation. Sequence-based screenings implement next-generation sequencing platforms, microarrays or PCR-based methods, while function-based analysis covers heterologous expression of metagenomic libraries in diverse screening setups. Major constraints and advantages of each strategy are described. The importance of alternative host-vector systems is discussed, and in order to underline the role of phylogenetic and physiological distance from the gene donor and the expression host employed, a case study is presented that describes the screening of a genomic library from an extreme thermophilic bacterium in both Escherichia coli and Thermus thermophilus. Metatranscriptomics, metaproteomics and single-cell-based methods are expected to complement metagenomic screening efforts to identify novel biocatalysts from environmental samples.

  6. 牦牛瘤胃元基因组文库中木聚糖酶基因的分析%Analysis of xylanases derived from the metagenomic BAC clone library of yak rumen

    Institute of Scientific and Technical Information of China (English)

    王敏; 陈富荣; 张山; 朱雅新; 东秀珠; 黄力; 田瑞华; 董志扬; 戴欣

    2011-01-01

    [ Objective ] The diversity of rumen microbial xylanases and their degradation characteristics were studied and the resources of new xylanases genes and enzymes were provided. [ Methods ] According to the result of the high-throughput sequencing of the rumen microbial metagenome bacterial artificial chromosome ( B AC ) clones library, the diversity of xylanase genes was analyzed and screened. Then one xylanase and its downstream xylosidase gene screened was cloned and expressed in Escherichia coli. The enzyme characterization of the recombinant xylanase and xylosidase and their synergistic effect were studied. [Results] All 14 xylanases screened from the library belong to GH10 family proteins. These xylanases shared the amino acid sequence similarity between 20. 5% and 91. 3% . Intriguingly, 7 xylanase genes in different contigs were found to be followed by xylosidase gene. The specific enzyme activity of the xylanase (Xyn32) was 1.98 U/mg and no ferulic acid esterase activity was detected. The specific enzyme activity of the coupled xylosidase (Xyl33) was 0.07 IU/mg, and xylosidase ( Xyl33 ) also displayed the activity of arabinofuranosidase. In addition, the in vitro experiment confirmed the synergistic effect between the coupled xylanase and xylosidase.%[目的]了解牦牛瘤胃微生物木聚糖酶多样性及其降解特征,为木聚糖降解提供新的基因资源.[方法]根据对已构建的瘤胃微生物元基因组细菌人工染色体( BAC)克隆文库高通量测序结果的注释,筛选其中编码木聚糖酶的基因并进行多样性分析;对其中一个木聚糖酶基因及其连锁的木糖苷酶基因进行克隆表达和酶学性质表征,分析其协同作用.[结果]共筛选到14个木聚糖酶基因,均编码GH1O家族木聚糖酶,其氨基酸序列之间的相似性为20.5% -91.3%;其中7个木聚糖酶基因所在的不同的DNA片段(contig)上存在木糖苷酶基因,编码的木糖苷酶属于GH43或GH3糖苷水解酶家

  7. Functional metagenomics: a high throughput screening method to decipher microbiota-driven NF-κB modulation in the human gut.

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    Omar Lakhdari

    Full Text Available BACKGROUND/AIM: The human intestinal microbiota plays an important role in modulation of mucosal immune responses. To study interactions between intestinal epithelial cells (IECs and commensal bacteria, a functional metagenomic approach was developed. One interest of metagenomics is to provide access to genomes of uncultured microbes. We aimed at identifying bacterial genes involved in regulation of NF-κB signaling in IECs. A high throughput cell-based screening assay allowing rapid detection of NF-κB modulation in IECs was established using the reporter-gene strategy to screen metagenomic libraries issued from the human intestinal microbiota. METHODS: A plasmid containing the secreted alkaline phosphatase (SEAP gene under the control of NF-κB binding elements was stably transfected in HT-29 cells. The reporter clone HT-29/kb-seap-25 was selected and characterized. Then, a first screening of a metagenomic library from Crohn's disease patients was performed to identify NF-κB modulating clones. Furthermore, genes potentially involved in the effect of one stimulatory metagenomic clone were determined by sequence analysis associated to mutagenesis by transposition. RESULTS: The two proinflammatory cytokines, TNF-α and IL-1β, were able to activate the reporter system, translating the activation of the NF-κB signaling pathway and NF-κB inhibitors, BAY 11-7082, caffeic acid phenethyl ester and MG132 were efficient. A screening of 2640 metagenomic clones led to the identification of 171 modulating clones. Among them, one stimulatory metagenomic clone, 52B7, was further characterized. Sequence analysis revealed that its metagenomic DNA insert might belong to a new Bacteroides strain and we identified 2 loci encoding an ABC transport system and a putative lipoprotein potentially involved in 52B7 effect on NF-κB. CONCLUSIONS: We have established a robust high throughput screening assay for metagenomic libraries derived from the human intestinal

  8. Construction of bacterial artificial chromosome libraries for the Lake Malawi cichlid (Metriaclima zebra), and the blind cavefish (Astyanax mexicanus).

    Science.gov (United States)

    Di Palma, Federica; Kidd, Celeste; Borowsky, Richard; Kocher, Thomas D

    2007-01-01

    Teleost fishes have become important models for studying the evolution of the genetic mechanisms of development. A key resource for comparative genomics and positional cloning are large-insert libraries constructed in bacterial artificial chromosomes. We have constructed bacterial artificial chromosome libraries for two species of teleost fish that are important models for the study of developmental evolution. Metriaclima zebra is one of several hundred closely related, morphologically diverse, haplochromine cichlids which have evolved over the last one million years in Lake Malawi, East Africa. The Mexican tetra, Astyanax mexicanus, is well known for adaptations related to the recent evolution of blind cave-dwelling forms. Clones and high-density filters for each library are available to the scientific community through the Hubbard Center for Genome Studies.

  9. Current and future trends in metagenomics : Development of knowledge bases

    Science.gov (United States)

    Mori, Hiroshi; Yamada, Takuji; Kurokawa, Ken

    Microbes are essential for every part of life on Earth. Numerous microbes inhabit the biosphere, many of which are uncharacterized or uncultivable. They form a complex microbial community that deeply affects against surrounding environments. Metagenome analysis provides a radically new way of examining such complex microbial community without isolation or cultivation of individual bacterial community members. In this article, we present a brief discussion about a metagenomics and the development of knowledge bases, and also discuss about the future trends in metagenomics.

  10. Construction and Preliminary Characterization Analysis of Wuzhishan Miniature Pig Bacterial Artificial Chromosome Library with Approximately 8-Fold Genome Equivalent Coverage

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    Changqing Liu

    2013-01-01

    Full Text Available Bacterial artificial chromosome (BAC libraries have been invaluable tools for the genome-wide genetic dissection of complex organisms. Here, we report the construction and characterization of a high-redundancy BAC library from a very valuable pig breed in China, Wuzhishan miniature pig (Sus scrofa, using its blood cells and fibroblasts, respectively. The library contains approximately 153,600 clones ordered in 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 152.3 kb, representing approximately 7.68 genome equivalents of the porcine haploid genome and a 99.93% statistical probability of obtaining at least one clone containing a unique DNA sequence in the library. 19 pairs of microsatellite marker primers covering porcine chromosomes were used for screening the BAC library, which showed that each of these markers was positive in the library; the positive clone number was 2 to 9, and the average number was 7.89, which was consistent with 7.68-fold coverage of the porcine genome. And there were no significant differences of genomic BAC library from blood cells and fibroblast cells. Therefore, we identified 19 microsatellite markers that could potentially be used as genetic markers. As a result, this BAC library will serve as a valuable resource for gene identification, physical mapping, and comparative genomics and large-scale genome sequencing in the porcine.

  11. Functional metagenomics reveals novel pathways of prebiotic breakdown by human gut bacteria.

    Science.gov (United States)

    Cecchini, Davide A; Laville, Elisabeth; Laguerre, Sandrine; Robe, Patrick; Leclerc, Marion; Doré, Joël; Henrissat, Bernard; Remaud-Siméon, Magali; Monsan, Pierre; Potocki-Véronèse, Gabrielle

    2013-01-01

    The human intestine hosts a complex bacterial community that plays a major role in nutrition and in maintaining human health. A functional metagenomic approach was used to explore the prebiotic breakdown potential of human gut bacteria, including non-cultivated ones. Two metagenomic libraries, constructed from ileum mucosa and fecal microbiota, were screened for hydrolytic activities on the prebiotic carbohydrates inulin, fructo-oligosaccharides, xylo-oligosaccharides, galacto-oligosaccharides and lactulose. The DNA inserts of 17 clones, selected from the 167 hits that were identified, were pyrosequenced in-depth, yielding in total 407, 420 bp of metagenomic DNA. From these sequences, we discovered novel prebiotic degradation pathways containing carbohydrate transporters and hydrolysing enzymes, for which we provided the first experimental proof of function. Twenty of these proteins are encoded by genes that are also present in the gut metagenome of at least 100 subjects, whatever are their ages or their geographical origin. The sequence taxonomic assignment indicated that still unknown bacteria, for which neither culture conditions nor genome sequence are available, possess the enzymatic machinery to hydrolyse the prebiotic carbohydrates tested. The results expand the vision on how prebiotics are metabolized along the intestine, and open new perspectives for the design of functional foods.

  12. Functional metagenomics reveals novel pathways of prebiotic breakdown by human gut bacteria.

    Directory of Open Access Journals (Sweden)

    Davide A Cecchini

    Full Text Available The human intestine hosts a complex bacterial community that plays a major role in nutrition and in maintaining human health. A functional metagenomic approach was used to explore the prebiotic breakdown potential of human gut bacteria, including non-cultivated ones. Two metagenomic libraries, constructed from ileum mucosa and fecal microbiota, were screened for hydrolytic activities on the prebiotic carbohydrates inulin, fructo-oligosaccharides, xylo-oligosaccharides, galacto-oligosaccharides and lactulose. The DNA inserts of 17 clones, selected from the 167 hits that were identified, were pyrosequenced in-depth, yielding in total 407, 420 bp of metagenomic DNA. From these sequences, we discovered novel prebiotic degradation pathways containing carbohydrate transporters and hydrolysing enzymes, for which we provided the first experimental proof of function. Twenty of these proteins are encoded by genes that are also present in the gut metagenome of at least 100 subjects, whatever are their ages or their geographical origin. The sequence taxonomic assignment indicated that still unknown bacteria, for which neither culture conditions nor genome sequence are available, possess the enzymatic machinery to hydrolyse the prebiotic carbohydrates tested. The results expand the vision on how prebiotics are metabolized along the intestine, and open new perspectives for the design of functional foods.

  13. Under-detection of endospore-forming Firmicutes in metagenomic data.

    Science.gov (United States)

    Filippidou, Sevasti; Junier, Thomas; Wunderlin, Tina; Lo, Chien-Chi; Li, Po-E; Chain, Patrick S; Junier, Pilar

    2015-01-01

    Microbial diversity studies based on metagenomic sequencing have greatly enhanced our knowledge of the microbial world. However, one caveat is the fact that not all microorganisms are equally well detected, questioning the universality of this approach. Firmicutes are known to be a dominant bacterial group. Several Firmicutes species are endospore formers and this property makes them hardy in potentially harsh conditions, and thus likely to be present in a wide variety of environments, even as residents and not functional players. While metagenomic libraries can be expected to contain endospore formers, endospores are known to be resilient to many traditional methods of DNA isolation and thus potentially undetectable. In this study we evaluated the representation of endospore-forming Firmicutes in 73 published metagenomic datasets using two molecular markers unique to this bacterial group (spo0A and gpr). Both markers were notably absent in well-known habitats of Firmicutes such as soil, with spo0A found only in three mammalian gut microbiomes. A tailored DNA extraction method resulted in the detection of a large diversity of endospore-formers in amplicon sequencing of the 16S rRNA and spo0A genes. However, shotgun classification was still poor with only a minor fraction of the community assigned to Firmicutes. Thus, removing a specific bias in a molecular workflow improves detection in amplicon sequencing, but it was insufficient to overcome the limitations for detecting endospore-forming Firmicutes in whole-genome metagenomics. In conclusion, this study highlights the importance of understanding the specific methodological biases that can contribute to improve the universality of metagenomic approaches.

  14. Under-detection of endospore-forming Firmicutes in metagenomic data

    Directory of Open Access Journals (Sweden)

    Sevasti Filippidou

    2015-01-01

    Full Text Available Microbial diversity studies based on metagenomic sequencing have greatly enhanced our knowledge of the microbial world. However, one caveat is the fact that not all microorganisms are equally well detected, questioning the universality of this approach. Firmicutes are known to be a dominant bacterial group. Several Firmicutes species are endospore formers and this property makes them hardy in potentially harsh conditions, and thus likely to be present in a wide variety of environments, even as residents and not functional players. While metagenomic libraries can be expected to contain endospore formers, endospores are known to be resilient to many traditional methods of DNA isolation and thus potentially undetectable. In this study we evaluated the representation of endospore-forming Firmicutes in 73 published metagenomic datasets using two molecular markers unique to this bacterial group (spo0A and gpr. Both markers were notably absent in well-known habitats of Firmicutes such as soil, with spo0A found only in three mammalian gut microbiomes. A tailored DNA extraction method resulted in the detection of a large diversity of endospore-formers in amplicon sequencing of the 16S rRNA and spo0A genes. However, shotgun classification was still poor with only a minor fraction of the community assigned to Firmicutes. Thus, removing a specific bias in a molecular workflow improves detection in amplicon sequencing, but it was insufficient to overcome the limitations for detecting endospore-forming Firmicutes in whole-genome metagenomics. In conclusion, this study highlights the importance of understanding the specific methodological biases that can contribute to improve the universality of metagenomic approaches.

  15. Bioactivity Screening of Fermentation Crude Extracts from Deep Sea Metagenomic Library Clones%深海宏基因组文库克隆子发酵产物的生物活性筛选

    Institute of Scientific and Technical Information of China (English)

    汤熙翔; 易志伟; 李宁; 马群; 李慧; 秦丹; 肖湘

    2011-01-01

    目的:对前期构建的深海宏基因组文库克隆子发酵产物进行生物活性筛选.方法:利用CCK8法及琼脂扩散法和双层琼脂法进行了细胞毒活性及抗菌活性筛选,利用荧光定量PCR法进行了抗乙肝病毒活性筛选.结果:筛选发现3个具有细胞毒活性的混合克隆子.HPLC指纹图谱分析表明这3个混合克隆子具有与大肠杆菌宿主对照不同的指纹图谱,其活性与出现的指纹峰成分可能相关.结论:研究结果提示对深海宏基因组文库克隆子发酵产物进行化学筛选为常规的生物活性功能筛选之外的有意义途径.%Objective: Detect bioactivities of fermentation crude extracts originated from previously constructed deep sea metagenomic libraries. Methods : Functional screening of antitumor( cytotoxic ) ,antibacterial and anti HBV activity from fermentation crude extracts was performed by CCK-8 method, agar diffusion method,double agar method and real time PCR method. Results: No antibacterial and anti HBV clones were identified and three mixed clones showed good cytotoxic activity. HPLC analysis of these three mixed clones fingerprints indicated they possess obviously different fingerprints with E. coli host which could be useful to next isolation of cytotoxic compounds from deep sea metagenomic library. Conclusion: These results indicated that chemical screening could be potential way for screening bioactivity substance from deep sea metagenomic libraries besides functional screening and homology screening.

  16. Metagenomic Analysis of Streptomyces lividans Reveals Host-Dependent Functional Expression

    OpenAIRE

    2012-01-01

    Most functional metagenomic studies have been limited by the poor expression of many genes derived from metagenomic DNA in Escherichia coli, which has been the predominant surrogate host to date. To expand the range of expressed genes, we developed tools for construction and functional screening of metagenomic libraries in Streptomyces lividans. We expanded on previously published protocols by constructing a system that enables retrieval and characterization of the metagenomic DNA from biolog...

  17. Construction of an Americn mink Bacterial Artificial Chromosome (BAC) library and sequencing candidate genes important for the fur industry

    DEFF Research Database (Denmark)

    Anistoroaei, Razvan Marian; Hallers, Boudewijn ten; Nefedov, Michael

    2011-01-01

    consisting of 18,432 clones spotted in duplicate, have been produced for hybridization screening and are publicly available. Overgo probes derived from expressed sequence tags (ESTs), representing 21 candidate genes for traits important for the mink industry, were used to screen the BAC library......BACKGROUND: Bacterial artificial chromosome (BAC) libraries continue to be invaluable tools for the genomic analysis of complex organisms. Complemented by the newly and fast growing deep sequencing technologies, they provide an excellent source of information in genomics projects. RESULTS: Here, we...... report the construction and characterization of the CHORI-231 BAC library constructed from a Danish-farmed, male American mink (Neovison vison). The library contains approximately 165,888 clones with an average insert size of 170 kb, representing approximately 10-fold coverage. High-density filters, each...

  18. Identification of a new lipase family in the Brazilian Atlantic Forest soil metagenome.

    Science.gov (United States)

    Faoro, Helisson; Glogauer, Arnaldo; Souza, Emanuel M; Rigo, Liu U; Cruz, Leonardo M; Monteiro, Rose A; Pedrosa, Fábio O

    2011-12-01

    Lipases are the most investigated class of enzymes in metagenomics. Phylogenetic classification of bacterial lipases comprises eight families. Here we describe the construction and screening of three metagenomic libraries from Brazilian Atlantic Forest soil and identification of a new lipase family. The metagenomic libraries, MAF1, MAF2 and MAF3, contained 34 560, 29 280 and 36 288 clones respectively. Lipase screening on triolein-rhodamine B plates resulted in one positive clone, Lip018. The DNA insert of Lip018 was fully sequenced and 20 ORFs were identified by comparison against the GenBank. Transposon mutagenesis revealed that ORF15, similar to serine peptidases, and ORF16, a hypothetical protein, were both required for lipase activity. ORF16 has a typical lipase conserved pentapeptide G-X-S-X-G and the comparison against the Pfam database showed that ORF16 belongs to family 5 of αβ-hydrolase. Phylogenetic analyses indicated that ORF16, together with other related proteins, may be a member of a new lipase family, named LipAP, activated by a putative serine protease. Partial characterization of ORF16 lipase showed that the enzyme has activity against a broad range of p-nitrophenyl esters, but only after activation by the predicted peptidase ORF15.

  19. 土壤宏基因组文库的构建及格尔德霉素类PKS基因的初步筛选%Construction of a soil metagenomic library and preliminary screening of geldanamycin-like type-I polyketide synthase gene

    Institute of Scientific and Technical Information of China (English)

    林灵; 陈菲菲; 王以光; 赫卫清; 王相晶

    2011-01-01

    目的 构建土壤宏基因组文库并对格尔德霉素类I型聚酮合系(polyketide synthase,PKS)基因进行初步筛选研究.方法 直接提取土壤样品中宏基因组DNA,以Fosmid为载体,构建宏基因组文库.根据格尔德霉素的I型PKS基因的保守序列设计引物,使用菌落PCR方法直接筛选所获得的宏基因组文库.结果 成功构建了土壤宏基因组文库,获得约6800个克隆子,其平均插入片段在25kb以上,覆盖至少170Mb的基因组信息.通过PKS基因筛选,获得了新的PKS基因片段.结论 本文报道了土壤宏基因组文库的成功构建,并为利用宏基因组技术寻找新的聚酮类次级代谢产物的生物合成基因,以便于其异源表达或组合生物合成新的聚酮类次级代谢产物奠定基础.%Objective Construction of a soil metagenomic library and preliminary screening of geldanamycinlike type-Ⅰ polyketide synthase genes.Methods A metagenomic library was constructed by inserting the metagenomic DNA directly extracted from soil into Fosmid vector, and then transduced into E.coli EPI300.The primers for screening polyketide synthase (PKS) genes were designed according to the conserved region of geldanamycin type Ⅰ PKS genes.Colony-PCR was performed for screening the metagenomic library.Results A soil metagenomic library was constructed successfully, which was consisted of about 6800 clones containing over 25kb inserted exogenous DNA on average, and covered almost 170Mb genomic information.And a new PKS gene fragment was obtained by screening from metagenomic library.Conclusion The strategy for finding novel PKS gene using metagenomic-technology was reported in this study and could facilitate to heterologous expressing or combinatorial biosynthesis of novel polyketide secondary metabolites.

  20. Taxonomic and functional assignment of cloned sequences from high Andean forest soil metagenome

    NARCIS (Netherlands)

    Montaña, José Salvador; Jiménez Avella, Diego; Angel, Tatiana; Hernández, Mónica; Baena, Sandra

    2012-01-01

    Total metagenomic DNA was isolated from high Andean forest soil and subjected to taxonomical and functional composition analyses by means of clone library generation and sequencing. The obtained yield of 1.7 μg of DNA/g of soil was used to construct a metagenomic library of approximately 20,000 clon

  1. A novel cold active esterase derived from Colombian high Andean forest soil metagenome

    NARCIS (Netherlands)

    Jiménez, Diego Javier; Montaña, José Salvador; Alvarez, Diana; Baena, Sandra

    2012-01-01

    In order to search new lipolytic enzymes and conduct bioprospecting of microbial communities from high Andean forest soil, a metagenomic library of approximately 20,000 clones was constructed in Escherichia coli using plasmid p-Bluescript II SK+. The library covered 80 Mb of the metagenomic DNA main

  2. Swine Fecal Metagenomics

    Science.gov (United States)

    Metagenomic approaches are providing rapid and more robust means to investigate the composition and functional genetic potential of complex microbial communities. In this study, we utilized a metagenomic approach to further understand the functional diversity of the swine gut. To...

  3. 16S ribosomal DNA clone libraries to reveal bacterial diversity in anaerobic reactor-degraded tetrabromobisphenol A.

    Science.gov (United States)

    Peng, Xingxing; Zhang, Zaili; Zhao, Ziling; Jia, Xiaoshan

    2012-05-01

    Microorganisms able to rapidly degrade tetrabromobisphenol A (TBBPA) were domesticated in an anaerobic reactor and added to gradually increased concentrations of TBBPA. After 240 days of domestication, the degradation rate reached 96.0% in cultivated batch experiments lasting 20 days. The optimum cultivating temperature and pH were 30°C and 7.0. The bacterial community's composition and diversity in the reactor was studied by comparative analysis with 16S ribosomal DNA clone libraries. Amplified rDNA restriction analysis of 200 clones from the library indicate that the rDNA richness was high (Coverage C 99.5%) and that evenness was not high (Shannon-Weaver index 2.42). Phylogenetic analysis of 63 bacterial sequences from the reactor libraries demonstrated the presence of Betaproteobacteria (33.1%), Gammaproteobacteria (18.7%), Bacteroidetes (13.9%), Firmicutes (11.4%), Chloroflexi (3.6%), Actinobacteria (0.6%), the candidate division TM7 (4.2%) and other unknown, uncultured bacterial groups (14.5%). Comamonas, Achromobacter, Pseudomonas and Flavobacterium were the dominant types.

  4. A bacterial artificial chromosome library for soybean PI 437654 and identification of clones associated with cyst nematode resistance.

    Science.gov (United States)

    Tomkins, J P; Mahalingam, R; Smith, H; Goicoechea, J L; Knap, H T; Wing, R A

    1999-09-01

    We have constructed a soybean bacterial artificial chromosome (BAC) library using the plant introduction (PI) 437654. The library contains 73 728 clones stored in 192 384-well microtiter plates. A random sampling of 230 BACs indicated an average insert size of 136 kb with a range of 20 to 325 kb, and less than 4% of the clones do not contain inserts. Ninety percent of BAC clones in the library have an average insert size greater than 100 kb. Based on a genome size of 1115 Mb, library coverage is 9 haploid genome equivalents. Screening the BAC library colony filters with cpDNA sequences showed that contamination of the genomic library with chloroplast clones was low (1.85%). Library screening with three genomic RFLP probes linked to soybean cyst nematode (SCN) resistance genes resulted in an average of 18 hits per probe (range 7 to 30). Two separate pools of forward and reverse suppression subtractive cDNAs obtained from SCN-infected and uninfected roots of PI437654 were hybridized to the BAC library filters. The 488 BACs identified from positive signals were fingerprinted and analyzed using FPC software (version 4.0) resulting in 85 different contigs. Contigs were grouped and analyzed in three categories: (1) contigs of BAC clones which hybridized to forward subtracted cDNAs, (2) contigs of BAC clones which hybridized to reverse subtracted cDNAs, and (3) contigs of BAC clones which hybridized to both forward and reverse subtracted cDNAs. This protocol provides an estimate of the number of genomic regions involved in early resistance response to a pathogenic attack.

  5. Construction and Identification of Bacterial Artificial Chromosome Library for 0-613-2R in Upland Cotton

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    A bacterial artificial chromosome (BAC) library containing a large genomic DNA insert is an important tool for genome physical mapping, map-based cloning, and genome sequencing. To isolate genes via a map-based cloning strategy and to perform physical mapping of the cotton genome, a high-quality BAC library containing large cotton DNA inserts is needed. We have developed a BAC library of the restoring line 0-613-2R for isolating the fertility restorer (Rf1) gene and genomic research in cotton (Gossypium hirsutum L.). The BAC library contains 97 825 clones stored in 255 pieces of a 384-well microtiter plate. Random samples of BACs digested with the Notl enzyme indicated that the average insert size is approximately 130 kb, with a range of 80-275 kb,and 95.7% of the BAC clones in the library have an average insert size larger than 100 kb. Based on a cotton genome size of 2 250 Mb, library coverage is 5.7 x haploid genome equivalents. Four clones were selected randomly from the library to determine the stability of the BAC clones. There were no different fingerprints for 0 and 100 generations of each clone digested with Notl and Hindlll enzymes. Thus, the stability of a single BAC clone can be sustained at least for 100 generations. Eight simple sequence repeat (SSR) markers flanking the Rf1 gene were chosen to screen the BAC library by pool using PCR method and 25 positive clones were identified with 3.1 positive clones per SSR marker.

  6. 肠道微生物宏基因组文库构建及酯解酶基因筛选%Screening of lipolytic genes from the metagenomic library of human intestine microbes

    Institute of Scientific and Technical Information of China (English)

    段嘉; 程功; 胡永飞; 李晶; 律娜; 朱宝利

    2013-01-01

    利用宏基因组学方法,以人肠道微生物样品为原材料,构建了1个约30 000个克隆的fosmid文库.以三丁酸甘油酯为底物,通过功能筛选,获得1个酯解酶阳性克隆.对该阳性克隆构建亚克隆,挑选具有酯解酶活性的阳性亚克隆进行测序分析,最终获得1个肠道微生物来源的酯解酶基因(GenBank登录号:JQ972699).结果表明,文库克隆的平均插入片段约为40 kb,没有重复插入片段克隆.获得的酯解酶基因推演蛋白与Pyramidobacter piscolens W5455的patatin样磷脂酶同源性最高,氨基酸一致性为95%.生物信息学分析结果表明该基因可能通过Vd型分泌方式进行分泌并发挥功能.本研究是通过构建人肠道微生物宏基因组大片段文库并结合重组子功能筛选获得酯解酶的首次报道,可为食品工业提供新的酯解酶来源和筛选方法.%Metagenomic DNA was extracted from a human fecal sample and DNA fragments around 40 kb was isolated and purified. A fosmid library containing approximately 30 000 clones was created. Restriction analysis by EcoR I revealed a high diversity of the cloned DNA fragments and the estimated average insert size of the library was 40 kb. Tributyrin was used as substrate for activity-based screening of the library and one lipolytic enzyme positive fosmid clone was obtained. The lipolytic gene sequence was obtained after subcloning and sequencing, which showed high homology to the patatin-like phospholipase from Pyramidobacter piscolens W5455 with an amino acid identity of 95% . This is the first report of function-based metagenomics on screening of lipolytic genes from human gut microbiota. It also provides food industry with a new source of lipolytic genes and screening method.

  7. Metagenomics as a Tool for Enzyme Discovery: Hydrolytic Enzymes from Marine-Related Metagenomes.

    Science.gov (United States)

    Popovic, Ana; Tchigvintsev, Anatoly; Tran, Hai; Chernikova, Tatyana N; Golyshina, Olga V; Yakimov, Michail M; Golyshin, Peter N; Yakunin, Alexander F

    2015-01-01

    This chapter discusses metagenomics and its application for enzyme discovery, with a focus on hydrolytic enzymes from marine metagenomic libraries. With less than one percent of culturable microorganisms in the environment, metagenomics, or the collective study of community genetics, has opened up a rich pool of uncharacterized metabolic pathways, enzymes, and adaptations. This great untapped pool of genes provides the particularly exciting potential to mine for new biochemical activities or novel enzymes with activities tailored to peculiar sets of environmental conditions. Metagenomes also represent a huge reservoir of novel enzymes for applications in biocatalysis, biofuels, and bioremediation. Here we present the results of enzyme discovery for four enzyme activities, of particular industrial or environmental interest, including esterase/lipase, glycosyl hydrolase, protease and dehalogenase.

  8. Construction of a Bacterial Artificial Chromosome Library of TM-1, a Standard Line for Genetics and Genomics in Upland Cotton

    Institute of Scientific and Technical Information of China (English)

    Yan Hu; Wang-Zhen Guo; Tian-Zhen Zhang

    2009-01-01

    A bacterial artificial chromosome (BAC) library was constructed for Gossyplum hirsutum acc. TM-1, a genetic and genomic standard line for Upland cotton. The library consists of 147 456 clones with an average insert size of 122.8 kb ranging from 97 to 240 kb. About 96.0% of the clones have inserts over 100 kb. Therefore, this library represents theoretically 7.4 haploid genome equivalents based on an AD genome size of 2 425 Mb. Clones were stored in 384 384- well plates and arrayed into multiplex pools for rapid and reliable library screening. BAC screening was carded out by four-round polymerase chain reactions using 23 simple sequence repeats (SSR) markers, three sequence-related amplified polymorphism markers and one pair of pdmere for a gene associated with fiber development to test the quality of the library. Correspondingly, in total 92 positive BAC clones were Identified with an average four positive clones per SSR marker, ranging from one to eight hits. Additionally, since these SSR markers have been localized to chromosome 12 (A12) and 26 (D12) according to the genetic map, these BAC clonee are expected to serve as seeds for the physical mapping of these two homologous chromosomes, sequentially map-based cloning of quantitative trait loci or genes associated with Important agronomic traits.

  9. Construction and characterization of a bacterial artificial chromosome library of thermo-sensitive genic male-sterile rice 5460S

    Institute of Scientific and Technical Information of China (English)

    邱芳; 金德敏; 伏健民; 张超良; 谢纬武; 王斌; 杨仁崔; 张洪斌

    1999-01-01

    In order to develop a detailed physical map of the thermo-sensitive genie male-sterile (TGMS) gene-encompassing region and finally clone the TGMS gene, a high-quality rice bacterial artificial chromosome (BAC) library from TGMS rice 5460S was constructed. The method of constructing BAC library was examined and optimized. The 5460S library consists of 19 584 BAC clones with an average insert size of 110 kb, which represents about 5 times rice haploid genome equivalents. Rice inserts of up to 140 kb and 250 kb were isolated and appeared stable after 100 generations of serial growth. Hybridization of BAC clones with mitochondrial and chloroplastic genes as probes demonstrated that this library has no organellar contamination. The 5460S library was screened with 3 molecular markers linked to tmsl gene as probes and at least 1 BAC clone was identified with each probe. The insert ends of positive clones were successfully isolated using thermal asymmetric interlaced PCR (TAIL-PCR) technique.

  10. Challenges and Opportunities of Airborne Metagenomics

    KAUST Repository

    Behzad, H.

    2015-05-06

    Recent metagenomic studies of environments, such as marine and soil, have significantly enhanced our understanding of the diverse microbial communities living in these habitats and their essential roles in sustaining vast ecosystems. The increase in the number of publications related to soil and marine metagenomics is in sharp contrast to those of air, yet airborne microbes are thought to have significant impacts on many aspects of our lives from their potential roles in atmospheric events such as cloud formation, precipitation, and atmospheric chemistry to their major impact on human health. In this review, we will discuss the current progress in airborne metagenomics, with a special focus on exploring the challenges and opportunities of undertaking such studies. The main challenges of conducting metagenomic studies of airborne microbes are as follows: 1) Low density of microorganisms in the air, 2) efficient retrieval of microorganisms from the air, 3) variability in airborne microbial community composition, 4) the lack of standardized protocols and methodologies, and 5) DNA sequencing and bioinformatics-related challenges. Overcoming these challenges could provide the groundwork for comprehensive analysis of airborne microbes and their potential impact on the atmosphere, global climate, and our health. Metagenomic studies offer a unique opportunity to examine viral and bacterial diversity in the air and monitor their spread locally or across the globe, including threats from pathogenic microorganisms. Airborne metagenomic studies could also lead to discoveries of novel genes and metabolic pathways relevant to meteorological and industrial applications, environmental bioremediation, and biogeochemical cycles.

  11. Challenges and opportunities of airborne metagenomics.

    Science.gov (United States)

    Behzad, Hayedeh; Gojobori, Takashi; Mineta, Katsuhiko

    2015-05-06

    Recent metagenomic studies of environments, such as marine and soil, have significantly enhanced our understanding of the diverse microbial communities living in these habitats and their essential roles in sustaining vast ecosystems. The increase in the number of publications related to soil and marine metagenomics is in sharp contrast to those of air, yet airborne microbes are thought to have significant impacts on many aspects of our lives from their potential roles in atmospheric events such as cloud formation, precipitation, and atmospheric chemistry to their major impact on human health. In this review, we will discuss the current progress in airborne metagenomics, with a special focus on exploring the challenges and opportunities of undertaking such studies. The main challenges of conducting metagenomic studies of airborne microbes are as follows: 1) Low density of microorganisms in the air, 2) efficient retrieval of microorganisms from the air, 3) variability in airborne microbial community composition, 4) the lack of standardized protocols and methodologies, and 5) DNA sequencing and bioinformatics-related challenges. Overcoming these challenges could provide the groundwork for comprehensive analysis of airborne microbes and their potential impact on the atmosphere, global climate, and our health. Metagenomic studies offer a unique opportunity to examine viral and bacterial diversity in the air and monitor their spread locally or across the globe, including threats from pathogenic microorganisms. Airborne metagenomic studies could also lead to discoveries of novel genes and metabolic pathways relevant to meteorological and industrial applications, environmental bioremediation, and biogeochemical cycles.

  12. Metagenomic and functional analyses of the consequences of reduction of bacterial diversity on soil functions and bioremediation in diesel-contaminated microcosms.

    Science.gov (United States)

    Jung, Jaejoon; Philippot, Laurent; Park, Woojun

    2016-03-14

    The relationship between microbial biodiversity and soil function is an important issue in ecology, yet most studies have been performed in pristine ecosystems. Here, we assess the role of microbial diversity in ecological function and remediation strategies in diesel-contaminated soils. Soil microbial diversity was manipulated using a removal by dilution approach and microbial functions were determined using both metagenomic analyses and enzymatic assays. A shift from Proteobacteria- to Actinobacteria-dominant communities was observed when species diversity was reduced. Metagenomic analysis showed that a large proportion of functional gene categories were significantly altered by the reduction in biodiversity. The abundance of genes related to the nitrogen cycle was significantly reduced in the low-diversity community, impairing denitrification. In contrast, the efficiency of diesel biodegradation was increased in the low-diversity community and was further enhanced by addition of red clay as a stimulating agent. Our results suggest that the relationship between microbial diversity and ecological function involves trade-offs among ecological processes, and should not be generalized as a positive, neutral, or negative relationship.

  13. Isolation and characterization of novel lipases/esterases from a bovine rumen metagenome.

    Science.gov (United States)

    Privé, Florence; Newbold, C Jamie; Kaderbhai, Naheed N; Girdwood, Susan G; Golyshina, Olga V; Golyshin, Peter N; Scollan, Nigel D; Huws, Sharon A

    2015-07-01

    Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78% following BlastP searches. Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses.

  14. Expression cloning of different bacterial phosphatase-encoding genes by histochemical screening of genomic libraries onto an indicator medium containing phenolphthalein diphosphate and methyl green.

    Science.gov (United States)

    Riccio, M L; Rossolini, G M; Lombardi, G; Chiesurin, A; Satta, G

    1997-02-01

    A system for expression cloning of bacterial phosphatase-encoding genes has been developed, and its potential has been investigated. The system is based on histochemical screening of bacterial genomic libraries, constructed in an Escherichia coli multicopy plasmid vector, for phosphatase-producing clones using an indicator medium (named TPMG) made of Tryptose-Phosphate agar supplemented with the phosphatase substrate phenolphthalein diphosphate and the stain methyl green. To test the performance of this system, three genomic libraries were constructed from bacterial strains of different species which showed different patterns of phosphatase activity, and were screened using the TPMG medium. Following a partial screening, three different phosphatase-encoding genes (respectively encoding a class A non-specific acid phosphatase, an acid-hexose phosphatase and a non-specific alkaline phosphatase) were shotgun-cloned from the above libraries, indicating that the TPMG-based expression cloning system can be useful for rapid isolation of different bacterial phosphatase-encoding genes.

  15. Construction and Characterization of a Bacterial Artificial Chromosome Library for the A-Genome of Cotton (G. arboreum L.

    Directory of Open Access Journals (Sweden)

    Yan Hu

    2011-01-01

    Full Text Available A bacterial artificial chromosome (BAC library for the A-genome of cotton has been constructed from the leaves of G. arboreum L cv. Jianglinzhongmian. It is used as elite A-genome germplasm resources in the present cotton breeding program and has been used to build a genetic reference map of cotton. The BAC library consists of 123,648 clones stored in 322 384-well plates. Statistical analysis of a set of 103 randomly selected BAC clones indicated that each clone has an average insert length of 100.2 kb per plasmid, with a range of 30 to 190 kb. Theoretically, this represents 7.2 haploid genome equivalents based on an A-genome size of 1697 Mb. The BAC library has been arranged in column pools and superpools allowing screening with various PCR-based markers. In the future, the A-genome cotton BAC library will serve as both a giant gene resource and a valuable tool for map-based gene isolation, physical mapping and comparative genome analysis.

  16. Microbial Diversity and Biochemical Potential Encoded by Thermal Spring Metagenomes Derived from the Kamchatka Peninsula

    Directory of Open Access Journals (Sweden)

    Bernd Wemheuer

    2013-01-01

    Full Text Available Volcanic regions contain a variety of environments suitable for extremophiles. This study was focused on assessing and exploiting the prokaryotic diversity of two microbial communities derived from different Kamchatkian thermal springs by metagenomic approaches. Samples were taken from a thermoacidophilic spring near the Mutnovsky Volcano and from a thermophilic spring in the Uzon Caldera. Environmental DNA for metagenomic analysis was isolated from collected sediment samples by direct cell lysis. The prokaryotic community composition was examined by analysis of archaeal and bacterial 16S rRNA genes. A total number of 1235 16S rRNA gene sequences were obtained and used for taxonomic classification. Most abundant in the samples were members of Thaumarchaeota, Thermotogae, and Proteobacteria. The Mutnovsky hot spring was dominated by the Terrestrial Hot Spring Group, Kosmotoga, and Acidithiobacillus. The Uzon Caldera was dominated by uncultured members of the Miscellaneous Crenarchaeotic Group and Enterobacteriaceae. The remaining 16S rRNA gene sequences belonged to the Aquificae, Dictyoglomi, Euryarchaeota, Korarchaeota, Thermodesulfobacteria, Firmicutes, and some potential new phyla. In addition, the recovered DNA was used for generation of metagenomic libraries, which were subsequently mined for genes encoding lipolytic and proteolytic enzymes. Three novel genes conferring lipolytic and one gene conferring proteolytic activity were identified.

  17. 从植物共生菌宏基因组文库筛选新的生物催化酶基因%Screening of new genes with biocatalytic potential from a plant microbiota metagenomic library

    Institute of Scientific and Technical Information of China (English)

    付小莉; 王浩鑫; 陈倩倩; 赵沛基; 曾英

    2012-01-01

    [目的]通过建立宏基因组文库的高通量保存与基于探针洗脱的多次膜杂交筛选方法,从植物共生菌宏基因组文库筛选具有生物催化潜力的新酶基因.[方法]首先根据滴度将初始文库噬菌体包装颗粒感染到EPI300TM-T1R E.coli,过夜培养后对应保存于96孔板;提取粘粒进行文库的杂交筛选.[结果]描述的洗脱条件可完全去除尼龙膜上与靶DNA结合的探针,并且尼龙膜上的靶DNA至少可用于7次探针杂交,从而明显提高宏基因组文库的筛选效率.[结论]以Enoate reductase (ER)和短链脱氢酶(SDR)的同源基因片段为探针,运用该方法经两轮筛选获得候选单克隆并进行了部分粘粒的测序,发现了新的ER和SDR同源基因,并克隆到相应的全长基因序列用于后续的表达与酶化学研究.%[Objective] We aim to find the new genes with biocatalytic potential from the plant microbiota metagenomic library by the means of high-throughput screening combined with multiple hybridizations based on probe stripping. (Methods] First, the phage particles were used to infect EPI300?T1R E. coli cells according to the titer of the phage particles as a primary library. After incubation the mixture was then divided into aliquots of 96 and cultured in the medium overnight, followed by storage in 2-mL 96-well plates. The resulting fosmids were hybridized to screen the library for the new enzyme genes. [Results] We found a thoroughly removal of the probe by striping the nylon membrane as described here, and the target DNA on the nylon membrane can be used repeatedly for at least 7 times. All these resulted in a highly efficient means for storage and screening of the metagenomic library. [Conclusion] By using the enoate reductase and short-chain dehydrogenase (SDR) as probes, candidate fosmid clones were obtained after two cycles of screening. Based on fosmid sequence analyses, new homologues of enoate reductase and SDR were found and cloned for

  18. Construction of a bacterial artificial chromosome library of S-type CMS maize mitochondria

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    In order to isolate mitochondrial genes easily, we have developed a new method to construct S-type CMS maize mitochondrial gene library by means of embedding mitochondria and enzymatic digesting mitochondria in situ, preparing mtDNA by electrophoresis, digesting LMP agarose with β-agarase, using BAC vector and electroporation. About 2 500 white clones of Mo17 CMS-J mitochondrial gene library were obtained with the average size of 18.24 kb, ranging from 5 to 40 kb, 63.6% inserts came from mitochondrial genome and represented 48 ′ mitochondrial genome equivalents. All the probes had detected the positive clones in the gene library. It is helpful to elucidating the maize mitochondrial genome structure and mechanism of S-type CMS, and may give some valuable reference to the construction of other plant mitochondrial genome library.

  19. Construction of a nurse shark (Ginglymostoma cirratum bacterial artificial chromosome (BAC library and a preliminary genome survey

    Directory of Open Access Journals (Sweden)

    Inoko Hidetoshi

    2006-05-01

    Full Text Available Abstract Background Sharks are members of the taxonomic class Chondrichthyes, the oldest living jawed vertebrates. Genomic studies of this group, in comparison to representative species in other vertebrate taxa, will allow us to theorize about the fundamental genetic, developmental, and functional characteristics in the common ancestor of all jawed vertebrates. Aims In order to obtain mapping and sequencing data for comparative genomics, we constructed a bacterial artificial chromosome (BAC library for the nurse shark, Ginglymostoma cirratum. Results The BAC library consists of 313,344 clones with an average insert size of 144 kb, covering ~4.5 × 1010 bp and thus providing an 11-fold coverage of the haploid genome. BAC end sequence analyses revealed, in addition to LINEs and SINEs commonly found in other animal and plant genomes, two new groups of nurse shark-specific repetitive elements, NSRE1 and NSRE2 that seem to be major components of the nurse shark genome. Screening the library with single-copy or multi-copy gene probes showed 6–28 primary positive clones per probe of which 50–90% were true positives, demonstrating that the BAC library is representative of the different regions of the nurse shark genome. Furthermore, some BAC clones contained multiple genes, making physical mapping feasible. Conclusion We have constructed a deep-coverage, high-quality, large insert, and publicly available BAC library for a cartilaginous fish. It will be very useful to the scientific community interested in shark genomic structure, comparative genomics, and functional studies. We found two new groups of repetitive elements specific to the nurse shark genome, which may contribute to the architecture and evolution of the nurse shark genome.

  20. Development of High-Throughput Phenotyping of Metagenomic Clones from the Human Gut Microbiome for Modulation of Eukaryotic Cell Growth▿

    OpenAIRE

    2007-01-01

    Metagenomic libraries derived from human intestinal microbiota (20,725 clones) were screened for epithelial cell growth modulation. Modulatory clones belonging to the four phyla represented among the metagenomic libraries were identified (hit rate, 0.04 to 8.7% depending on the screening cutoff). Several candidate loci were identified by transposon mutagenesis and subcloning.

  1. Construction of an American mink Bacterial Artificial Chromosome (BAC library and sequencing candidate genes important for the fur industry

    Directory of Open Access Journals (Sweden)

    Christensen Knud

    2011-07-01

    Full Text Available Abstract Background Bacterial artificial chromosome (BAC libraries continue to be invaluable tools for the genomic analysis of complex organisms. Complemented by the newly and fast growing deep sequencing technologies, they provide an excellent source of information in genomics projects. Results Here, we report the construction and characterization of the CHORI-231 BAC library constructed from a Danish-farmed, male American mink (Neovison vison. The library contains approximately 165,888 clones with an average insert size of 170 kb, representing approximately 10-fold coverage. High-density filters, each consisting of 18,432 clones spotted in duplicate, have been produced for hybridization screening and are publicly available. Overgo probes derived from expressed sequence tags (ESTs, representing 21 candidate genes for traits important for the mink industry, were used to screen the BAC library. These included candidate genes for coat coloring, hair growth and length, coarseness, and some receptors potentially involved in viral diseases in mink. The extensive screening yielded positive results for 19 of these genes. Thirty-five clones corresponding to 19 genes were sequenced using 454 Roche, and large contigs (184 kb in average were assembled. Knowing the complete sequences of these candidate genes will enable confirmation of the association with a phenotype and the finding of causative mutations for the targeted phenotypes. Additionally, 1577 BAC clones were end sequenced; 2505 BAC end sequences (80% of BACs were obtained. An excess of 2 Mb has been analyzed, thus giving a snapshot of the mink genome. Conclusions The availability of the CHORI-321 American mink BAC library will aid in identification of genes and genomic regions of interest. We have demonstrated how the library can be used to identify specific genes of interest, develop genetic markers, and for BAC end sequencing and deep sequencing of selected clones. To our knowledge, this is the

  2. Expanding the Repertoire of Carbapenem-Hydrolyzing Metallo-ß-Lactamases by Functional Metagenomic Analysis of Soil Microbiota.

    Science.gov (United States)

    Gudeta, Dereje D; Bortolaia, Valeria; Pollini, Simona; Docquier, Jean-Denis; Rossolini, Gian M; Amos, Gregory C A; Wellington, Elizabeth M H; Guardabassi, Luca

    2016-01-01

    Carbapenemases are bacterial enzymes that hydrolyze carbapenems, a group of last-resort β-lactam antibiotics used for treatment of severe bacterial infections. They belong to three β-lactamase classes based amino acid sequence (A, B, and D). The aim of this study was to elucidate occurrence, diversity and functionality of carbapenemase-encoding genes in soil microbiota by functional metagenomics. Ten plasmid libraries were generated by cloning metagenomic DNA from agricultural (n = 6) and grassland (n = 4) soil into Escherichia coli. The libraries were cultured on amoxicillin-containing agar and up to 100 colonies per library were screened for carbapenemase production by CarbaNP test. Presumptive carbapenemases were characterized with regard to DNA sequence, minimum inhibitory concentration (MIC) of β-lactams, and imipenem hydrolysis. Nine distinct class B carbapenemases, also known as metallo-beta-lactamases (MBLs), were identified in six soil samples, including two subclass B1 (GRD23-1 and SPN79-1) and seven subclass B3 (CRD3-1, PEDO-1, GRD33-1, ESP-2, ALG6-1, ALG11-1, and DHT2-1). Except PEDO-1 and ESP-2, these enzymes were distantly related to any previously described MBLs (33 to 59% identity). RAIphy analysis indicated that six enzymes (CRD3-1, GRD23-1, DHT2-1, SPN79-1, ALG6-1, and ALG11-1) originated from Proteobacteria, two (PEDO-1 and ESP-2) from Bacteroidetes and one (GRD33-1) from Gemmatimonadetes. All MBLs detected in soil microbiota were functional when expressed in E. coli, resulting in detectable imipenem-hydrolyzing activity and significantly increased MICs of clinically relevant ß-lactams. Interestingly, the MBLs yielded by functional metagenomics generally differed from those detected in the same soil samples by antibiotic selective culture, showing that the two approaches targeted different subpopulations in soil microbiota.

  3. Expanding the Repertoire of Carbapenem-Hydrolyzing Metallo-ß-Lactamases by Functional Metagenomic Analysis of Soil Microbiota

    Science.gov (United States)

    Gudeta, Dereje D.; Bortolaia, Valeria; Pollini, Simona; Docquier, Jean-Denis; Rossolini, Gian M.; Amos, Gregory C. A.; Wellington, Elizabeth M. H.; Guardabassi, Luca

    2016-01-01

    Carbapenemases are bacterial enzymes that hydrolyze carbapenems, a group of last-resort β-lactam antibiotics used for treatment of severe bacterial infections. They belong to three β-lactamase classes based amino acid sequence (A, B, and D). The aim of this study was to elucidate occurrence, diversity and functionality of carbapenemase-encoding genes in soil microbiota by functional metagenomics. Ten plasmid libraries were generated by cloning metagenomic DNA from agricultural (n = 6) and grassland (n = 4) soil into Escherichia coli. The libraries were cultured on amoxicillin-containing agar and up to 100 colonies per library were screened for carbapenemase production by CarbaNP test. Presumptive carbapenemases were characterized with regard to DNA sequence, minimum inhibitory concentration (MIC) of β-lactams, and imipenem hydrolysis. Nine distinct class B carbapenemases, also known as metallo-beta-lactamases (MBLs), were identified in six soil samples, including two subclass B1 (GRD23-1 and SPN79-1) and seven subclass B3 (CRD3-1, PEDO-1, GRD33-1, ESP-2, ALG6-1, ALG11-1, and DHT2-1). Except PEDO-1 and ESP-2, these enzymes were distantly related to any previously described MBLs (33 to 59% identity). RAIphy analysis indicated that six enzymes (CRD3-1, GRD23-1, DHT2-1, SPN79-1, ALG6-1, and ALG11-1) originated from Proteobacteria, two (PEDO-1 and ESP-2) from Bacteroidetes and one (GRD33-1) from Gemmatimonadetes. All MBLs detected in soil microbiota were functional when expressed in E. coli, resulting in detectable imipenem-hydrolyzing activity and significantly increased MICs of clinically relevant ß-lactams. Interestingly, the MBLs yielded by functional metagenomics generally differed from those detected in the same soil samples by antibiotic selective culture, showing that the two approaches targeted different subpopulations in soil microbiota. PMID:28082950

  4. Livermore Metagenomics Analysis Toolkit

    Energy Technology Data Exchange (ETDEWEB)

    2012-10-01

    LMAT is designed to take as input a collection of raw metagenomic sequencer reads, and search each read against a reference genome database and assign a taxonomic label and confidence value to each read and report a summary of the predicted taxonomic contents of the metagenomic sample.

  5. A human gut microbial gene catalogue established by metagenomic sequencing

    DEFF Research Database (Denmark)

    dos Santos, Marcelo Bertalan Quintanilha; Sicheritz-Pontén, Thomas; Nielsen, Henrik Bjørn;

    2010-01-01

    To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence...... gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively....

  6. Metagenomics at Grass Roots

    Indian Academy of Sciences (India)

    Sudeshna Mazumdar-Leighton; Vivek K Choudhary

    2017-03-01

    Metagenomics is a robust, interdisciplinary approach for studyingmicrobial community composition, function, and dynamics.It typically involves a core of molecular biology, microbiology,ecology, statistics, and computational biology. Excitingoutcomes anticipated from these studies include unravelingof complex interactions that characterize the ecologicalmilieu of microbial communities. Diverse habitats fromwhich metagenomes have been reported include human guts,caterpillar guts, thermal vents in oceans, ore deposits, polarcaps, and even soils that adhere to plant roots. Knowledgegenerated from metagenomic projects has tremendous potentialto benefit human health, agriculture, and ecosystemfunctions. This article provides a brief history of technicaladvances in metagenomics, including DNA sequencing methods,and some case studies. A specific example is providedof microbial metagenomes found at the roots of native grassspecies (family Poaceae) that can grow on degraded lands undergoingrevegetation.

  7. Expression screening of bacterial libraries of recombinant alpha-1 proteinase inhibitor variants for candidates with thrombin inhibitory capacity.

    Science.gov (United States)

    Bhakta, Varsha; Gierczak, Richard F; Sheffield, William P

    2013-12-01

    Exhaustive mutagenesis studies of the reactive centre loop (RCL), a key structural component of proteins belonging to the serpin superfamily of protease inhibitors, are complicated by the size of the RCL, serpin conformational complexity, and, for most serpins, the lack of a serpin-dependent phenotype of expressing cells. Here, we describe a thrombin capture assay that distinguished thrombin-inhibitory recombinant human alpha-1 proteinase inhibitor (API M358R) from non-inhibitory API variants in Escherichia coli lysates prepared from either single clones or pools. Binding of API proteins in the lysates to thrombin immobilized on microtiter plate wells was quantified via colour generated by a peroxidase-coupled anti-API antibody. Bacterial expression plasmids encoding inhibitory API M358R were mixed 1:99 with plasmids encoding non-inhibitory API T345R/M358R and the resulting library screened in pools of 10. All above-background signals arising from pools or subsequently re-probed single clones were linked to the presence of plasmids encoding API M358R. Screening of a portion of another expression library encoding hypervariable API with all possibilities at codons 352-358 also yielded only novel, thrombin-inhibitory variants. Probing a smaller library expressing all possible codons at Ala347 yielded the wild type, 6 different functional variants, one partially active variant, and two variants with no thrombin-inhibitory activity. API antigen levels varied considerably less among Ala347 variants than activity levels, and comparison of rate constants of inhibition of purified API variants to their corresponding thrombin capture assay lysate values was used to establish the sensitivity and specificity of the assay. The results indicate that the approach is sufficiently robust to correctly identify functional versus non-functional candidates in API expression libraries, and could be of value in systematically probing structure/function relationships not only in the API

  8. Metagenomics of an alkaline hot spring in Galicia (Spain: microbial diversity analysis and screening for novel lipolytic enzymes

    Directory of Open Access Journals (Sweden)

    Olalla eLópez-López

    2015-11-01

    Full Text Available A fosmid library was constructed with the metagenomic DNA from the water of the Lobios hot spring (76°C, pH=8.2 located in Ourense (Spain. Metagenomic sequencing of the fosmid library allowed the assembly of 9,722 contigs ranging in size from 500 to 56,677 bp and spanning approximately 18 Mbp. 23,207 ORFs (Open Reading Frames were predicted from the assembly. Biodiversity was explored by taxonomic classification and it revealed that bacteria were predominant, while the archaea were less abundant. The 6 most abundant bacterial phyla were Deinococcus-Thermus, Proteobacteria, Firmicutes, Acidobacteria, Aquificae and Chloroflexi. Within the archaeal superkingdom, the phylum Thaumarchaeota was predominant with the dominant species Candidatus Caldiarchaeum subterraneum. Functional classification revealed the genes associated to one-carbon metabolism as the most abundant. Both taxonomic and functional classifications showed a mixture of different microbial metabolic patterns: aerobic and anaerobic, chemoorganotrophic and chemolithotrophic, autotrophic and heterotrophic. Remarkably, the presence of genes encoding enzymes with potential biotechnological interest, such as xylanases, galactosidases, proteases and lipases, was also revealed in the metagenomic library.Functional screening of this library was subsequently done looking for genes encoding lipolytic enzymes. Six genes conferring lipolytic activity were identified and one was cloned and characterized. This gene was named LOB4Est and it was expressed in a yeast mesophilic host. LOB4Est codes for a novel esterase of family VIII, with sequence similarity to β-lactamases, but with unusual wide substrate specificity. When the enzyme was purified from the mesophilic host it showed half-life of 1 h and 43 minutes at 50°C, and maximal activity at 40°C and pH 7.5 with p-nitrophenyl-laurate as substrate. Interestingly, the enzyme retained more than 80% of maximal activity in a broad range of pH from 6.5-8.

  9. Metagenomics of an Alkaline Hot Spring in Galicia (Spain): Microbial Diversity Analysis and Screening for Novel Lipolytic Enzymes

    Science.gov (United States)

    López-López, Olalla; Knapik, Kamila; Cerdán, Maria-Esperanza; González-Siso, María-Isabel

    2015-01-01

    A fosmid library was constructed with the metagenomic DNA from the water of the Lobios hot spring (76°C, pH = 8.2) located in Ourense (Spain). Metagenomic sequencing of the fosmid library allowed the assembly of 9722 contigs ranging in size from 500 to 56,677 bp and spanning ~18 Mbp. 23,207 ORFs (Open Reading Frames) were predicted from the assembly. Biodiversity was explored by taxonomic classification and it revealed that bacteria were predominant, while the archaea were less abundant. The six most abundant bacterial phyla were Deinococcus-Thermus, Proteobacteria, Firmicutes, Acidobacteria, Aquificae, and Chloroflexi. Within the archaeal superkingdom, the phylum Thaumarchaeota was predominant with the dominant species “Candidatus Caldiarchaeum subterraneum.” Functional classification revealed the genes associated to one-carbon metabolism as the most abundant. Both taxonomic and functional classifications showed a mixture of different microbial metabolic patterns: aerobic and anaerobic, chemoorganotrophic and chemolithotrophic, autotrophic and heterotrophic. Remarkably, the presence of genes encoding enzymes with potential biotechnological interest, such as xylanases, galactosidases, proteases, and lipases, was also revealed in the metagenomic library. Functional screening of this library was subsequently done looking for genes encoding lipolytic enzymes. Six genes conferring lipolytic activity were identified and one was cloned and characterized. This gene was named LOB4Est and it was expressed in a yeast mesophilic host. LOB4Est codes for a novel esterase of family VIII, with sequence similarity to β-lactamases, but with unusual wide substrate specificity. When the enzyme was purified from the mesophilic host it showed half-life of 1 h and 43 min at 50°C, and maximal activity at 40°C and pH 7.5 with p-nitrophenyl-laurate as substrate. Interestingly, the enzyme retained more than 80% of maximal activity in a broad range of pH from 6.5 to 8. PMID:26635759

  10. Salt resistance genes revealed by functional metagenomics from brines and moderate-salinity rhizosphere within a hypersaline environment.

    Science.gov (United States)

    Mirete, Salvador; Mora-Ruiz, Merit R; Lamprecht-Grandío, María; de Figueras, Carolina G; Rosselló-Móra, Ramon; González-Pastor, José E

    2015-01-01

    Hypersaline environments are considered one of the most extreme habitats on earth and microorganisms have developed diverse molecular mechanisms of adaptation to withstand these conditions. The present study was aimed at identifying novel genes from the microbial communities of a moderate-salinity rhizosphere and brine from the Es Trenc saltern (Mallorca, Spain), which could confer increased salt resistance to Escherichia coli. The microbial diversity assessed by pyrosequencing of 16S rRNA gene libraries revealed the presence of communities that are typical in such environments and the remarkable presence of three bacterial groups never revealed as major components of salt brines. Metagenomic libraries from brine and rhizosphere samples, were transferred to the osmosensitive strain E. coli MKH13, and screened for salt resistance. Eleven genes that conferred salt resistance were identified, some encoding for well-known proteins previously related to osmoadaptation such as a glycerol transporter and a proton pump, whereas others encoded proteins not previously related to this function in microorganisms such as DNA/RNA helicases, an endonuclease III (Nth) and hypothetical proteins of unknown function. Furthermore, four of the retrieved genes were cloned and expressed in Bacillus subtilis and they also conferred salt resistance to this bacterium, broadening the spectrum of bacterial species in which these genes can function. This is the first report of salt resistance genes recovered from metagenomes of a hypersaline environment.

  11. Comparative analysis of bacterial community-metagenomics in coastal Gulf of Mexico sediment microcosms following exposure to Macondo oil (MC252).

    Science.gov (United States)

    Koo, Hyunmin; Mojib, Nazia; Thacker, Robert W; Bej, Asim K

    2014-11-01

    The indigenous bacterial communities in sediment microcosms from Dauphin Island (DI), Petit Bois Island (PB) and Perdido Pass (PP) of the coastal Gulf of Mexico were compared following treatment with Macondo oil (MC252) using pyrosequencing and culture-based approaches. After quality-based trimming, 28,991 partial 16S rRNA sequence reads were analyzed by rarefaction, confirming that analyses of bacterial communities were saturated with respect to species diversity. Changes in the relative abundances of Proteobacteria, Bacteroidetes and Firmicutes played an important role in structuring bacterial communities in oil-treated sediments. Proteobacteria were dominant in oil-treated samples, whereas Firmicutes and Bacteroidetes were either the second or the third most abundant taxa. Tenericutes, members of which are known for oil biodegradation, were detected shortly after treatment, and continued to increase in DI and PP sediments. Multivariate statistical analyses (ADONIS) revealed significant dissimilarity of bacterial communities between oil-treated and untreated samples and among locations. In addition, a similarity percentage analysis showed the contribution of each species to the contrast between untreated and oil-treated samples. PCR amplification using DNA from pure cultures of Exiguobacterium,  Pseudoalteromonas,  Halomonas and Dyadobacter, isolated from oil-treated microcosm sediments, produced amplicons similar to polycyclic aromatic hydrocarbon-degrading genes. In the context of the 2010 Macondo blowout, the results from our study demonstrated that the indigenous bacterial communities in coastal Gulf of Mexico sediment microcosms responded to the MC252 oil with altered community structure and species composition. The rapid proliferation of hydrocarbonoclastic bacteria suggests their involvement in the degradation of the spilt oil in the Gulf of Mexico ecosystem.

  12. Comparative analysis of bacterial community-metagenomics in coastal Gulf of Mexico sediment microcosms following exposure to Macondo oil (MC252)

    KAUST Repository

    Koo, Hyunmin

    2014-09-10

    The indigenous bacterial communities in sediment microcosms from Dauphin Island (DI), Petit Bois Island (PB) and Perdido Pass (PP) of the coastal Gulf of Mexico were compared following treatment with Macondo oil (MC252) using pyrosequencing and culture-based approaches. After quality-based trimming, 28,991 partial 16S rRNA sequence reads were analyzed by rarefaction, confirming that analyses of bacterial communities were saturated with respect to species diversity. Changes in the relative abundances of Proteobacteria, Bacteroidetes and Firmicutes played an important role in structuring bacterial communities in oil-treated sediments. Proteobacteria were dominant in oil-treated samples, whereas Firmicutes and Bacteroidetes were either the second or the third most abundant taxa. Tenericutes, members of which are known for oil biodegradation, were detected shortly after treatment, and continued to increase in DI and PP sediments. Multivariate statistical analyses (ADONIS) revealed significant dissimilarity of bacterial communities between oil-treated and untreated samples and among locations. In addition, a similarity percentage analysis showed the contribution of each species to the contrast between untreated and oil-treated samples. PCR amplification using DNA from pure cultures of Exiguobacterium,  Pseudoalteromonas,  Halomonas and Dyadobacter, isolated from oil-treated microcosm sediments, produced amplicons similar to polycyclic aromatic hydrocarbon-degrading genes. In the context of the 2010 Macondo blowout, the results from our study demonstrated that the indigenous bacterial communities in coastal Gulf of Mexico sediment microcosms responded to the MC252 oil with altered community structure and species composition. The rapid proliferation of hydrocarbonoclastic bacteria suggests their involvement in the degradation of the spilt oil in the Gulf of Mexico ecosystem.

  13. METAGENassist: a comprehensive web server for comparative metagenomics.

    Science.gov (United States)

    Arndt, David; Xia, Jianguo; Liu, Yifeng; Zhou, You; Guo, An Chi; Cruz, Joseph A; Sinelnikov, Igor; Budwill, Karen; Nesbø, Camilla L; Wishart, David S

    2012-07-01

    With recent improvements in DNA sequencing and sample extraction techniques, the quantity and quality of metagenomic data are now growing exponentially. This abundance of richly annotated metagenomic data and bacterial census information has spawned a new branch of microbiology called comparative metagenomics. Comparative metagenomics involves the comparison of bacterial populations between different environmental samples, different culture conditions or different microbial hosts. However, in order to do comparative metagenomics, one typically requires a sophisticated knowledge of multivariate statistics and/or advanced software programming skills. To make comparative metagenomics more accessible to microbiologists, we have developed a freely accessible, easy-to-use web server for comparative metagenomic analysis called METAGENassist. Users can upload their bacterial census data from a wide variety of common formats, using either amplified 16S rRNA data or shotgun metagenomic data. Metadata concerning environmental, culture, or host conditions can also be uploaded. During the data upload process, METAGENassist also performs an automated taxonomic-to-phenotypic mapping. Phenotypic information covering nearly 20 functional categories such as GC content, genome size, oxygen requirements, energy sources and preferred temperature range is automatically generated from the taxonomic input data. Using this phenotypically enriched data, users can then perform a variety of multivariate and univariate data analyses including fold change analysis, t-tests, PCA, PLS-DA, clustering and classification. To facilitate data processing, users are guided through a step-by-step analysis workflow using a variety of menus, information hyperlinks and check boxes. METAGENassist also generates colorful, publication quality tables and graphs that can be downloaded and used directly in the preparation of scientific papers. METAGENassist is available at http://www.metagenassist.ca.

  14. Construction of a full bacterial artificial chromosome (BAC) library of Oryza sativa genome

    Institute of Scientific and Technical Information of China (English)

    TAOQUANZHOU; HAIYINGZHAO; 等

    1994-01-01

    We have constructed a full BAC library for the superior early indica variety of Oryza sativa,Guang Lu Ai 4.The MAX Efficiency DH10B with increased stability of inserts was used as BAC host cells.The potent pBelo BACII with double selection markers was used as cloning vector.The cloning efficiency we have reached was as high as 98%,and the transformation efficiency was raised up to 106 transformants/μg of large fragment DNA.The BAC recombinant transformants were picked at random and analyzed for the size of inserts,which turned out to be of 120 kb in length on average.We have obtained more than 20,000 such BAC clones.According to conventional probability equation,they covered the entire rice genome of 420,000 kb in length.The entire length of inserts of the library obtained has the 5-to 6-fold coverage of the genome.To our knowledge,this is the first reported full BAC library for a complex genome.

  15. Analysis of bacterial communities in soil by use of denaturing gradient gel electrophoresis and clone libraries, as influenced by different reverse primers

    NARCIS (Netherlands)

    Brons, Jolanda; van Elsas, J.D.

    2008-01-01

    To assess soil bacterial diversity, PCR systems consisting of several slightly different reverse primers together with forward primer F968-GC were used along with subsequent denaturing gradient gel electrophoresis (DGGE) or clone library analyses. In this study, a set of 13 previously used and novel

  16. Universal Probe Library based real-time PCR for rapid detection of bacterial pathogens from positive blood culture bottles.

    Science.gov (United States)

    Zhu, Lingxiang; Shen, Ding-Xia; Zhou, Qiming; Liu, Chao-Jun; Li, Zexia; Fang, Xiangdong; Li, Quan-Zhen

    2014-03-01

    A set of real-time PCR based assays using the locked nucleic acid probes from Roche Universal ProbeLibrary were developed for rapid detection of eight bacterial species from positive blood culture bottles. Four duplex real-time PCR reactions targeting to one Gram-positive bacterium and one Gram-negative bacterium were optimized for species identification according to Gram stain results. We also included mecA-specific primers and probes in the assays to indicate the presence of methicillin resistance in the bacterial species. The analytical sensitivity was in the range of 1-10 CFU per PCR reaction mixture. The specificity and cross reactivity of the assay was validated by 28 ATCC reference strains and 77 negative blood culture specimens. No cross-reactivity was observed in these samples thus demonstrating 100 % specificity. 72 previously characterized clinical isolates were tested by the real-time PCR assay and validated the accuracy and feasibility of the real-time PCR assay. Furthermore, 55 positive blood culture samples were tested using real-time PCR and 50 (90.9 %) of them were identified as the same species as judged by biochemical analysis. In total, real-time PCR showed 98.2 % consistent to that of traditional methods. Real-time PCR can be used as a supplement for early detection of the frequently-occurred pathogens from the positive blood cultures.

  17. Identification and characterization of a new true lipase isolated through metagenomic approach

    Directory of Open Access Journals (Sweden)

    de Souza Emanuel M

    2011-07-01

    Full Text Available Abstract Background Metagenomics, the application of molecular genomics to consortia of non-cultivated microbes, has the potential to have a substantial impact on the search for novel industrial enzymes such as esterases (carboxyl ester hydrolases, EC 3.1.1.1 and lipases (triacylglycerol lipases, EC 3.1.1.3. In the current work, a novel lipase gene was identified from a fosmid metagenomic library constructed with the "prokaryotic-enriched" DNA from a fat-contaminated soil collected from a wastewater treatment plant. Results In preliminary screening on agar containing 1% tributyrin, 2661 of the approximately 500,000 clones in the metagenomic library showed activity. Of these, 127 showed activity on agar containing 1% tricaprylin, while 32 were shown to be true lipase producers through screening on agar containing 1% triolein. The clone with the largest halo was further characterized. Its lipase gene showed 72% identity to a putative lipase of Yersinia enterocolitica subsp. palearctica Y11. The lipase, named LipC12, belongs to family I.1 of bacterial lipases, has a chaperone-independent folding, does not possess disulfide bridges and is calcium ion dependent. It is stable from pH 6 to 11 and has activity from pH 4.5 to 10, with higher activities at alkaline pH values. LipC12 is stable up to 3.7 M NaCl and from 20 to 50°C, with maximum activity at 30°C over a 1 h incubation. The pure enzyme has specific activities of 1722 U/mg and 1767 U/mg against olive oil and pig fat, respectively. Moreover, it is highly stable in organic solvents at 15% and 30% (v/v. Conclusions The combination of the use of a fat-contaminated soil, enrichment of prokaryotic DNA and a three-step screening strategy led to a high number of lipase-producing clones in the metagenomic library. The most notable properties of the new lipase that was isolated and characterized were a high specific activity against long chain triacylglycerols, activity and stability over a wide range

  18. Novel bacterial sulfur oxygenase reductases from bioreactors treating gold-bearing concentrates

    DEFF Research Database (Denmark)

    Chen, Z-W; Liu, Y-Y; Wu, J-F;

    2007-01-01

    The microbial community and sulfur oxygenase reductases of metagenomic DNA from bioreactors treating gold-bearing concentrates were studied by 16S rRNA library, real-time polymerase chain reaction (RT-PCR), conventional cultivation, and molecular cloning. Results indicated that major bacterial......) of bacteria and archaea were 4.59 x 10(9) and 6.68 x 10(5), respectively. Bacterial strains representing Acidithiobacillus, Leptospirillum, and Sulfobacillus were isolated from the bioreactors. To study sulfur oxidation in the reactors, pairs of new PCR primers were designed for the detection of sulfur...... oxygenase reductase (SOR) genes. Three sor-like genes, namely, sor (Fx), sor (SA), and sor (SB) were identified from metagenomic DNAs of the bioreactors. The sor (Fx) is an inactivated SOR gene and is identical to the pseudo-SOR gene of Ferroplasma acidarmanus. The sor (SA) and sor (SB) showed...

  19. Identifying contamination with advanced visualization and analysis practices: metagenomic approaches for eukaryotic genome assemblies

    Science.gov (United States)

    Delmont, Tom O.

    2016-01-01

    High-throughput sequencing provides a fast and cost-effective mean to recover genomes of organisms from all domains of life. However, adequate curation of the assembly results against potential contamination of non-target organisms requires advanced bioinformatics approaches and practices. Here, we re-analyzed the sequencing data generated for the tardigrade Hypsibius dujardini, and created a holistic display of the eukaryotic genome assembly using DNA data originating from two groups and eleven sequencing libraries. By using bacterial single-copy genes, k-mer frequencies, and coverage values of scaffolds we could identify and characterize multiple near-complete bacterial genomes from the raw assembly, and curate a 182 Mbp draft genome for H. dujardini supported by RNA-Seq data. Our results indicate that most contaminant scaffolds were assembled from Moleculo long-read libraries, and most of these contaminants have differed between library preparations. Our re-analysis shows that visualization and curation of eukaryotic genome assemblies can benefit from tools designed to address the needs of today’s microbiologists, who are constantly challenged by the difficulties associated with the identification of distinct microbial genomes in complex environmental metagenomes. PMID:27069789

  20. Identifying contamination with advanced visualization and analysis practices: metagenomic approaches for eukaryotic genome assemblies

    Directory of Open Access Journals (Sweden)

    Tom O. Delmont

    2016-03-01

    Full Text Available High-throughput sequencing provides a fast and cost-effective mean to recover genomes of organisms from all domains of life. However, adequate curation of the assembly results against potential contamination of non-target organisms requires advanced bioinformatics approaches and practices. Here, we re-analyzed the sequencing data generated for the tardigrade Hypsibius dujardini, and created a holistic display of the eukaryotic genome assembly using DNA data originating from two groups and eleven sequencing libraries. By using bacterial single-copy genes, k-mer frequencies, and coverage values of scaffolds we could identify and characterize multiple near-complete bacterial genomes from the raw assembly, and curate a 182 Mbp draft genome for H. dujardini supported by RNA-Seq data. Our results indicate that most contaminant scaffolds were assembled from Moleculo long-read libraries, and most of these contaminants have differed between library preparations. Our re-analysis shows that visualization and curation of eukaryotic genome assemblies can benefit from tools designed to address the needs of today’s microbiologists, who are constantly challenged by the difficulties associated with the identification of distinct microbial genomes in complex environmental metagenomes.

  1. Identifying contamination with advanced visualization and analysis practices: metagenomic approaches for eukaryotic genome assemblies.

    Science.gov (United States)

    Delmont, Tom O; Eren, A Murat

    2016-01-01

    High-throughput sequencing provides a fast and cost-effective mean to recover genomes of organisms from all domains of life. However, adequate curation of the assembly results against potential contamination of non-target organisms requires advanced bioinformatics approaches and practices. Here, we re-analyzed the sequencing data generated for the tardigrade Hypsibius dujardini, and created a holistic display of the eukaryotic genome assembly using DNA data originating from two groups and eleven sequencing libraries. By using bacterial single-copy genes, k-mer frequencies, and coverage values of scaffolds we could identify and characterize multiple near-complete bacterial genomes from the raw assembly, and curate a 182 Mbp draft genome for H. dujardini supported by RNA-Seq data. Our results indicate that most contaminant scaffolds were assembled from Moleculo long-read libraries, and most of these contaminants have differed between library preparations. Our re-analysis shows that visualization and curation of eukaryotic genome assemblies can benefit from tools designed to address the needs of today's microbiologists, who are constantly challenged by the difficulties associated with the identification of distinct microbial genomes in complex environmental metagenomes.

  2. 深海沉积物宏基因组文库构建及淀粉酶筛选%Construction of metagenomic DNA library of deep-sea sediments and screening of amylase

    Institute of Scientific and Technical Information of China (English)

    黄雅丽; 张炯; 曹理想; 谭红铭; 陆勇军; 周世宁

    2013-01-01

    Deep sea environment is usually characterized by extremely high salinity, high pressure, high/low temperature and darkness. The physicochemical features ensured that may exist in microorganisms unique metabolic and physiological activities, molecular and cellular structure to adapt to these extreme conditions. Therefore, the exploration of biological and function diversity of microorganisms in deep sea are expected to yield novel metabolites. To access to the microbial genetic resources of deep-sea sediment with a culture-independent approach, The sediment DNA was extracted from deep-sea sediments of the South China Sea and cloned into fosmid vector generating a library of 39 600 clones with inserts of 24 -45 kb and an average insert size of 33 kb. The metagenomic library represented about 1 320 Mbp of genomic DNA in deep-sea sediments. The library was screened for amylase activity and three recombinant clones showed amylolytic activity. The clone, amy7, with lower optimal temperature was further analyzed. Subsequent library of insert DNA of amy7 was constructed and a positive subclone, designated as amy7-4, was screened. Sequencing of the clone revealed that it contains a 3 291 bp fragment. An open reading frame of 1 920 bp was identified from the fragment and showed high identity with genes of glycosidase from various organisms.%深海环境通常具有高盐,高压,高/低温,无光照等特点,使得海洋微生物存在一套独特的生理代谢机制和分子细胞结构,然而迄今绝大部分深海微生物不能在实验室条件下被分离培养,深海微生物资源开发遇到很大挑战.本研究通过不依赖培养的方法研究海洋微生物的基因资源,构建了南海深海沉积物fosmid宏基因组文库,共获得约39 600个克隆,插入片段范围在24~45 kb之间,平均插入片段大小为33 kb,克隆片段的总库容达到1 320 Mb.通过功能筛选获得3个具有淀粉酶活性的克隆子,选取其中最适温度较低的amy7

  3. Ocean microbial metagenomics

    Science.gov (United States)

    Kerkhof, Lee J.; Goodman, Robert M.

    2009-09-01

    Technology for accessing the genomic DNA of microorganisms, directly from environmental samples without prior cultivation, has opened new vistas to understanding microbial diversity and functions. Especially as applied to soils and the oceans, environments on Earth where microbial diversity is vast, metagenomics and its emergent approaches have the power to transform rapidly our understanding of environmental microbiology. Here we explore select recent applications of the metagenomic suite to ocean microbiology.

  4. The future of skin metagenomics.

    Science.gov (United States)

    Mathieu, Alban; Vogel, Timothy M; Simonet, Pascal

    2014-01-01

    Metagenomics, the direct exploitation of environmental microbial DNA, is complementary to traditional culture-based approaches for deciphering taxonomic and functional microbial diversity in a plethora of ecosystems, including those related to the human body such as the mouth, saliva, teeth, gut or skin. DNA extracted from human skin analyzed by sequencing the PCR-amplified rrs gene has already revealed the taxonomic diversity of microbial communities colonizing the human skin ("skin microbiome"). Each individual possesses his/her own skin microbial community structure, with marked taxonomic differences between different parts of the body and temporal evolution depending on physical and chemical conditions (sweat, washing etc.). However, technical limitations due to the low bacterial density at the surface of the human skin or contamination by human DNA still has inhibited extended use of the metagenomic approach for investigating the skin microbiome at a functional level. These difficulties have been overcome in part by the new generation of sequencing platforms that now provide sequences describing the genes and functions carried out by skin bacteria. These methodological advances should help us understand the mechanisms by which these microorganisms adapt to the specific chemical composition of each skin and thereby lead to a better understanding of bacteria/human host interdependence. This knowledge will pave the way for more systemic and individualized pharmaceutical and cosmetic applications.

  5. A primer on metagenomics.

    Directory of Open Access Journals (Sweden)

    John C Wooley

    2010-02-01

    Full Text Available Metagenomics is a discipline that enables the genomic study of uncultured microorganisms. Faster, cheaper sequencing technologies and the ability to sequence uncultured microbes sampled directly from their habitats are expanding and transforming our view of the microbial world. Distilling meaningful information from the millions of new genomic sequences presents a serious challenge to bioinformaticians. In cultured microbes, the genomic data come from a single clone, making sequence assembly and annotation tractable. In metagenomics, the data come from heterogeneous microbial communities, sometimes containing more than 10,000 species, with the sequence data being noisy and partial. From sampling, to assembly, to gene calling and function prediction, bioinformatics faces new demands in interpreting voluminous, noisy, and often partial sequence data. Although metagenomics is a relative newcomer to science, the past few years have seen an explosion in computational methods applied to metagenomic-based research. It is therefore not within the scope of this article to provide an exhaustive review. Rather, we provide here a concise yet comprehensive introduction to the current computational requirements presented by metagenomics, and review the recent progress made. We also note whether there is software that implements any of the methods presented here, and briefly review its utility. Nevertheless, it would be useful if readers of this article would avail themselves of the comment section provided by this journal, and relate their own experiences. Finally, the last section of this article provides a few representative studies illustrating different facets of recent scientific discoveries made using metagenomics.

  6. A primer on metagenomics.

    Science.gov (United States)

    Wooley, John C; Godzik, Adam; Friedberg, Iddo

    2010-02-26

    Metagenomics is a discipline that enables the genomic study of uncultured microorganisms. Faster, cheaper sequencing technologies and the ability to sequence uncultured microbes sampled directly from their habitats are expanding and transforming our view of the microbial world. Distilling meaningful information from the millions of new genomic sequences presents a serious challenge to bioinformaticians. In cultured microbes, the genomic data come from a single clone, making sequence assembly and annotation tractable. In metagenomics, the data come from heterogeneous microbial communities, sometimes containing more than 10,000 species, with the sequence data being noisy and partial. From sampling, to assembly, to gene calling and function prediction, bioinformatics faces new demands in interpreting voluminous, noisy, and often partial sequence data. Although metagenomics is a relative newcomer to science, the past few years have seen an explosion in computational methods applied to metagenomic-based research. It is therefore not within the scope of this article to provide an exhaustive review. Rather, we provide here a concise yet comprehensive introduction to the current computational requirements presented by metagenomics, and review the recent progress made. We also note whether there is software that implements any of the methods presented here, and briefly review its utility. Nevertheless, it would be useful if readers of this article would avail themselves of the comment section provided by this journal, and relate their own experiences. Finally, the last section of this article provides a few representative studies illustrating different facets of recent scientific discoveries made using metagenomics.

  7. Identifying Differentially Abundant Metabolic Pathways in Metagenomic Datasets

    Science.gov (United States)

    Liu, Bo; Pop, Mihai

    Enabled by rapid advances in sequencing technology, metagenomic studies aim to characterize entire communities of microbes bypassing the need for culturing individual bacterial members. One major goal of such studies is to identify specific functional adaptations of microbial communities to their habitats. Here we describe a powerful analytical method (MetaPath) that can identify differentially abundant pathways in metagenomic data-sets, relying on a combination of metagenomic sequence data and prior metabolic pathway knowledge. We show that MetaPath outperforms other common approaches when evaluated on simulated datasets. We also demonstrate the power of our methods in analyzing two, publicly available, metagenomic datasets: a comparison of the gut microbiome of obese and lean twins; and a comparison of the gut microbiome of infant and adult subjects. We demonstrate that the subpathways identified by our method provide valuable insights into the biological activities of the microbiome.

  8. Signal processing for metagenomics: extracting information from the soup.

    Science.gov (United States)

    Rosen, Gail L; Sokhansanj, Bahrad A; Polikar, Robi; Bruns, Mary Ann; Russell, Jacob; Garbarine, Elaine; Essinger, Steve; Yok, Non

    2009-11-01

    Traditionally, studies in microbial genomics have focused on single-genomes from cultured species, thereby limiting their focus to the small percentage of species that can be cultured outside their natural environment. Fortunately, recent advances in high-throughput sequencing and computational analyses have ushered in the new field of metagenomics, which aims to decode the genomes of microbes from natural communities without the need for cultivation. Although metagenomic studies have shed a great deal of insight into bacterial diversity and coding capacity, several computational challenges remain due to the massive size and complexity of metagenomic sequence data. Current tools and techniques are reviewed in this paper which address challenges in 1) genomic fragment annotation, 2) phylogenetic reconstruction, 3) functional classification of samples, and 4) interpreting complementary metaproteomics and metametabolomics data. Also surveyed are important applications of metagenomic studies, including microbial forensics and the roles of microbial communities in shaping human health and soil ecology.

  9. Reference databases for taxonomic assignment in metagenomics.

    Science.gov (United States)

    Santamaria, Monica; Fosso, Bruno; Consiglio, Arianna; De Caro, Giorgio; Grillo, Giorgio; Licciulli, Flavio; Liuni, Sabino; Marzano, Marinella; Alonso-Alemany, Daniel; Valiente, Gabriel; Pesole, Graziano

    2012-11-01

    Metagenomics is providing an unprecedented access to the environmental microbial diversity. The amplicon-based metagenomics approach involves the PCR-targeted sequencing of a genetic locus fitting different features. Namely, it must be ubiquitous in the taxonomic range of interest, variable enough to discriminate between different species but flanked by highly conserved sequences, and of suitable size to be sequenced through next-generation platforms. The internal transcribed spacers 1 and 2 (ITS1 and ITS2) of the ribosomal DNA operon and one or more hyper-variable regions of 16S ribosomal RNA gene are typically used to identify fungal and bacterial species, respectively. In this context, reliable reference databases and taxonomies are crucial to assign amplicon sequence reads to the correct phylogenetic ranks. Several resources provide consistent phylogenetic classification of publicly available 16S ribosomal DNA sequences, whereas the state of ribosomal internal transcribed spacers reference databases is notably less advanced. In this review, we aim to give an overview of existing reference resources for both types of markers, highlighting strengths and possible shortcomings of their use for metagenomics purposes. Moreover, we present a new database, ITSoneDB, of well annotated and phylogenetically classified ITS1 sequences to be used as a reference collection in metagenomic studies of environmental fungal communities. ITSoneDB is available for download and browsing at http://itsonedb.ba.itb.cnr.it/.

  10. The effects of variable sample biomass on comparative metagenomics.

    Science.gov (United States)

    Chafee, Meghan; Maignien, Loïs; Simmons, Sheri L

    2015-07-01

    Longitudinal studies that integrate samples with variable biomass are essential to understand microbial community dynamics across space or time. Shotgun metagenomics is widely used to investigate these communities at the functional level, but little is known about the effects of combining low and high biomass samples on downstream analysis. We investigated the interacting effects of DNA input and library amplification by polymerase chain reaction on comparative metagenomic analysis using dilutions of a single complex template from an Arabidopsis thaliana-associated microbial community. We modified the Illumina Nextera kit to generate high-quality large-insert (680 bp) paired-end libraries using a range of 50 pg to 50 ng of input DNA. Using assembly-based metagenomic analysis, we demonstrate that DNA input level has a significant impact on community structure due to overrepresentation of low-GC genomic regions following library amplification. In our system, these differences were largely superseded by variations between biological replicates, but our results advocate verifying the influence of library amplification on a case-by-case basis. Overall, this study provides recommendations for quality filtering and de-replication prior to analysis, as well as a practical framework to address the issue of low biomass or biomass heterogeneity in longitudinal metagenomic surveys.

  11. Rapid editing and evolution of bacterial genomes using libraries of synthetic DNA.

    Science.gov (United States)

    Gallagher, Ryan R; Li, Zhe; Lewis, Aaron O; Isaacs, Farren J

    2014-10-01

    Multiplex automated genome engineering (MAGE) is a powerful technology for in vivo genome editing that uses synthetic single-stranded DNA (ssDNA) to introduce targeted modifications directly into the Escherichia coli chromosome. MAGE is a cyclical process that involves transformation of ssDNA (by electroporation) followed by outgrowth, during which bacteriophage homologous recombination proteins mediate annealing of ssDNAs to their genomic targets. By iteratively introducing libraries of mutagenic ssDNAs targeting multiple sites, MAGE can generate combinatorial genetic diversity in a cell population. Alternatively, MAGE can introduce precise mutant alleles at many loci for genome-wide editing or for recoding projects that are not possible with other methods. In recent technological advances, MAGE has been improved by strain modifications and selection techniques that enhance allelic replacement. This protocol describes the manual execution of MAGE wherein each cycle takes ≈ 2.5 h, which, if carried out by two people, allows ≈ 10 continuous cycles of MAGE-based mutagenesis per day.

  12. A Bioinformatician's Guide to Metagenomics

    Energy Technology Data Exchange (ETDEWEB)

    Kunin, Victor; Copeland, Alex; Lapidus, Alla; Mavromatis, Konstantinos; Hugenholtz, Philip

    2008-08-01

    As random shotgun metagenomic projects proliferate and become the dominant source of publicly available sequence data, procedures for best practices in their execution and analysis become increasingly important. Based on our experience at the Joint Genome Institute, we describe step-by-step the chain of decisions accompanying a metagenomic project from the viewpoint of a bioinformatician. We guide the reader through a standard workflow for a metagenomic project beginning with pre-sequencing considerations such as community composition and sequence data type that will greatly influence downstream analyses. We proceed with recommendations for sampling and data generation including sample and metadata collection, community profiling, construction of shotgun libraries and sequencing strategies. We then discuss the application of generic sequence processing steps (read preprocessing, assembly, and gene prediction and annotation) to metagenomic datasets by contrast to genome projects. Different types of data analyses particular to metagenomes are then presented including binning, dominant population analysis and gene-centric analysis. Finally data management systems and issues are presented and discussed. We hope that this review will assist bioinformaticians and biologists in making better-informed decisions on their journey during a metagenomic project.

  13. Insights into antibiotic resistance through metagenomic approaches.

    Science.gov (United States)

    Schmieder, Robert; Edwards, Robert

    2012-01-01

    The consequences of bacterial infections have been curtailed by the introduction of a wide range of antibiotics. However, infections continue to be a leading cause of mortality, in part due to the evolution and acquisition of antibiotic-resistance genes. Antibiotic misuse and overprescription have created a driving force influencing the selection of resistance. Despite the problem of antibiotic resistance in infectious bacteria, little is known about the diversity, distribution and origins of resistance genes, especially for the unculturable majority of environmental bacteria. Functional and sequence-based metagenomics have been used for the discovery of novel resistance determinants and the improved understanding of antibiotic-resistance mechanisms in clinical and natural environments. This review discusses recent findings and future challenges in the study of antibiotic resistance through metagenomic approaches.

  14. The YNP metagenome project

    DEFF Research Database (Denmark)

    Inskeep, William P.; Jay, Zackary J.; Tringe, Susannah G.

    2013-01-01

    , or lake habitats and therefore offer a tremendous opportunity for studying the structure and function of different model microbial communities using environmental metagenomics. One of the broader goals of this study was to establish linkages among microbial distribution, metabolic potential......, and environmental variables. Twenty geochemically distinct geothermal ecosystems representing a broad spectrum of Yellowstone hot-spring environments were used for metagenomic and geochemical analysis and included approximately equal numbers of: (1) phototrophic mats, (2) “filamentous streamer” communities, and (3......) archaeal-dominated sediments. The metagenomes were analyzed using a suite of complementary and integrative bioinformatic tools, including phylogenetic and functional analysis of both individual sequence reads and assemblies of predominant phylotypes.This volume identifies major environmental determinants...

  15. Salt resistance genes revealed by functional metagenomics from brines and moderate-salinity rhizosphere within a hypersaline environment

    Directory of Open Access Journals (Sweden)

    Salvador eMirete

    2015-10-01

    Full Text Available Hypersaline environments are considered one of the most extreme habitats on earth and microorganisms have developed diverse molecular mechanisms of adaptation to withstand these conditions. The present study was aimed at identifying novel genes involved in salt resistance from the microbial communities of brines and the rhizosphere from the Es Trenc saltern (Mallorca, Spain. The microbial diversity assessed by pyrosequencing of 16S rRNA gene libraries revealed the presence of communities that are typical in such environments. Metagenomic libraries from brine and rhizosphere samples, were transferred to the osmosensitive strain Escherichia coli MKH13, and screened for salt resistance. As a result, eleven genes that conferred salt resistance were identified, some encoding for well known proteins previously related to osmoadaptation as a glycerol and a proton pump, whereas others encoded for proteins not previously related to this function in microorganisms as DNA/RNA helicases, an endonuclease III (Nth and hypothetical proteins of unknown function. Furthermore, four of the retrieved genes were cloned and expressed in Bacillus subtilis and they also exhibited salt resistance in this bacterium, broadening the spectrum of bacterial species where these genes can operate. This is the first report of salt resistance genes recovered from metagenomes of a hypersaline environment.

  16. Metagenomics of extreme environments.

    Science.gov (United States)

    Cowan, D A; Ramond, J-B; Makhalanyane, T P; De Maayer, P

    2015-06-01

    Whether they are exposed to extremes of heat or cold, or buried deep beneath the Earth's surface, microorganisms have an uncanny ability to survive under these conditions. This ability to survive has fascinated scientists for nearly a century, but the recent development of metagenomics and 'omics' tools has allowed us to make huge leaps in understanding the remarkable complexity and versatility of extremophile communities. Here, in the context of the recently developed metagenomic tools, we discuss recent research on the community composition, adaptive strategies and biological functions of extremophiles.

  17. Highly Effective Inhibition of Biofilm Formation by the First Metagenome-Derived AI-2 Quenching Enzyme

    Science.gov (United States)

    Weiland-Bräuer, Nancy; Kisch, Martin J.; Pinnow, Nicole; Liese, Andreas; Schmitz, Ruth A.

    2016-01-01

    Bacterial cell–cell communication (quorum sensing, QS) represents a fundamental process crucial for biofilm formation, pathogenicity, and virulence allowing coordinated, concerted actions of bacteria depending on their cell density. With the widespread appearance of antibiotic-resistance of biofilms, there is an increasing need for novel strategies to control harmful biofilms. One attractive and most likely effective approach is to target bacterial communication systems for novel drug design in biotechnological and medical applications. In this study, metagenomic large-insert libraries were constructed and screened for QS interfering activities (quorum quenching, QQ) using recently established reporter strains. Overall, 142 out of 46,400 metagenomic clones were identified to interfere with acyl-homoserine lactones (AHLs), 13 with autoinducer-2 (AI-2). Five cosmid clones with highest simultaneous interfering activities were further analyzed and the respective open reading frames conferring QQ activities identified. Those showed homologies to bacterial oxidoreductases, proteases, amidases and aminotransferases. Evaluating the ability of the respective purified QQ-proteins to prevent biofilm formation of several model systems demonstrated highest inhibitory effects of QQ-2 using the crystal violet biofilm assay. This was confirmed by heterologous expression of the respective QQ proteins in Klebsiella oxytoca M5a1 and monitoring biofilm formation in a continuous flow cell system. Moreover, QQ-2 chemically immobilized to the glass surface of the flow cell effectively inhibited biofilm formation of K. oxytoca as well as clinical K. pneumoniae isolates derived from patients with urinary tract infections. Indications were obtained by molecular and biochemical characterizations that QQ-2 represents an oxidoreductase most likely reducing the signaling molecules AHL and AI-2 to QS-inactive hydroxy-derivatives. Overall, we propose that the identified novel QQ-2 protein

  18. Highly effective inhibition of biofilm formation by the first metagenome-derived AI-2 quenching enzyme

    Directory of Open Access Journals (Sweden)

    Nancy Weiland-Bräuer

    2016-07-01

    Full Text Available Bacterial cell-cell communication (quorum sensing, QS represents a fundamental process crucial for biofilm formation, pathogenicity, and virulence allowing coordinated, concerted actions of bacteria depending on their cell density. With the widespread appearance of antibiotic-resistance of biofilms, there is an increasing need for novel strategies to control harmful biofilms. One attractive and most likely effective approach is to target bacterial communication systems for novel drug design in biotechnological and medical applications. In this study, metagenomic large-insert libraries were constructed and screened for QS interfering activities (quorum quenching, QQ using recently established reporter strains. Overall, 142 out of 46,400 metagenomic clones were identified to interfere with acyl-homoserine lactones (AHLs, 13 with autoinducer-2 (AI-2. Five cosmid clones with highest simultaneous interfering activities were further analyzed and the respective open reading frames conferring QQ activities identified. Those showed homologies to bacterial oxidoreductases, proteases, amidases and aminotransferases. Evaluating the ability of the respective purified QQ-proteins to prevent biofilm formation of several model systems demonstrated highest inhibitory effects of QQ-2 using the crystal violet biofilm assay. This was confirmed by heterologous expression of the respective QQ proteins in Klebsiella oxytoca M5a1 and monitoring biofilm formation in a continuous flow cell system. Moreover, QQ-2 chemically immobilized to the glass surface of the flow cell effectively inhibited biofilm formation of K. oxytoca as well as clinical K. pneumoniae isolates derived from patients with urinary tract infections. Indications were obtained by molecular and biochemical characterizations that QQ-2 represents an oxidoreductase most likely reducing the signaling molecules AHL and AI-2 to QS-inactive hydroxy-derivatives. Overall, we propose that the identified novel QQ-2

  19. A Metagenomic Study Highlights Phylogenetic Proximity of Quorum-Quenching and Xenobiotic-Degrading Amidases of the AS-Family

    Science.gov (United States)

    Tannières, Mélanie; Beury-Cirou, Amélie; Vigouroux, Armelle; Mondy, Samuel; Pellissier, Franck; Dessaux, Yves; Faure, Denis

    2013-01-01

    Quorum-sensing (QS) signals of the N-acylhomoserine lactone (NAHL) class are cleaved by quorum-quenching enzymes, collectively named NAHLases. Here, functional metagenomics allowed the discovery of a novel bacterial NAHLase in a rhizosphere that was treated with γ-caprolactone. As revealed by rrs-DGGE and rrs-pyrosequencing, this treatment increased the percentage of the NAHL-degrading bacteria and strongly biased the structure of the bacterial community, among which Azospirillum dominated. Among the 29 760 fosmids of the metagenomic library, a single one was detected that expressed the qsdB gene conferring NAHL-degradation upon E. coli and decreased QS-regulated virulence in Pectobacterium. Phylogenetic analysis of the 34 orfs of the fosmid suggested that it would belong to an unknown Proteobacterium - probably a γ-proteobacterium. qPCR quantification of the NAHLase-encoding genes attM, qsdA, and qsdB revealed their higher abundance in the γ-caprolactone-treated rhizosphere as compared to an untreated control. The purified QsdB enzyme exhibited amidase activity. QsdB is the first amidase signature (AS) family member exhibiting NAHLase-activity. Point mutations in the AS-family catalytic triad K-S-S abolished the NAHLase activity of QsdB. This study extends the diversity of NAHLases and highlights a common phylogenic origin of AS-family enzymes involved in the degradation of natural compounds, such as NAHLs, and xenobiotics, such as nylon and linuron. PMID:23762380

  20. Novel tools for the functional expression of metagenomic DNA.

    Science.gov (United States)

    Troeschel, Sonja Christina; Drepper, Thomas; Leggewie, Christian; Streit, Wolfgang R; Jaeger, Karl-Erich

    2010-01-01

    The functional expression of environmental genes in a particular host bacterium is hampered by various limitations including inefficient transcription of target genes as well as improper assembly of the corresponding enzymes. Therefore, the identification of novel enzymes from metagenomic libraries by activity-based screening requires efficient expression and screening systems. In the following chapter, we present two novel tools to improve the functional expression of metagenomic genes. (1) Comparative screenings of metagenomic libraries demonstrated that different enzymes were detected when phylogenetically distinct expression host strains were used. Thus, we have developed a strategy, which comprises library construction using a shuttle vector that allows comparative expression and screening of metagenomic DNA in Escherichia coli, Pseudomonas putida, and Bacillus subtilis. (2) Expression studies have revealed that functional expression of environmental genes in heterologous expression hosts is often limited by insufficient promoter recognition. Therefore, a method is described allowing to enhance the expression capacity of E. coli by using the transposon MuExpress. This recombinant transposon is able to insert randomly into environmental DNA fragments thereby facilitating gene expression from its two inducible promoters.

  1. Recent progresses in metagenomics

    Science.gov (United States)

    Metagenomics addresses the collective genetic structure and functional composition of a microbial community at its native habitat. This approach has emerged as a powerful tool to study the structure and function of the microbiota for the past few years and is revolutionizing studies of microbial ec...

  2. The Correlation between Bacterial Metagenomic DNA Concentration and the Total Number of Bacteria Colonies in Drinking Water%自来水中细菌宏基因组 DNA的浓度与细菌菌落总数的相关性分析

    Institute of Scientific and Technical Information of China (English)

    刘艳超; 郝文利; 宿庄; 高玉敏

    2015-01-01

    分析自来水中细菌宏基因组DNA浓度与细菌菌落总数之间的相关性,探索新的自来水细菌菌落总数检测方法。采用国标法检测自来水中的细菌菌落总数约为(165±5) CFU/mL,高于国标限值(100 CFU/mL)。通过计算得出,检测600 mL本实验水样中的细菌菌落总数相当于检测1000 mL(1 L)100 CFU/mL的自来水。首先将600 mL及低于和高于该体积不同体积梯度的自来水经滤膜过滤收集菌体;然后使用细菌基因组提取试剂盒提取滤膜上的细菌宏基因组DNA,电泳分析提取的宏基因组质量;最后检测各个体积自来水宏基因组DNA浓度,根据检测结果分析自来水中宏基因组DNA含量与细菌菌落总数之间的相关性。提取的自来水宏基因组DNA主条带清晰,可以进行基因组DNA含量测定分析;自来水中细菌宏基因组DNA浓度与细菌菌落总数之间呈正相关的线性关系。建立一种通过检测自来水中细菌宏基因组DNA浓度来反映细菌菌落总数的检测方法,检测灵敏度可达7 CFU/mL,且省时、重复性好。%Through analyzing the correlation between the bacterial metagenomic DNA concentration and the total count of bacterial colonies in drinking water, we explore a new method for the detection of total number of bacterial colonies in drinking water.The total number of bacteria colonies in the water from our lab was detected by a tradi-tional technology, and it was about ( 165 ±5 ) CFU/mL, which is higher than 100 CFU/mL that is the security boundary value which was made by Chinese health department in 2006.To limit the total number of bacterial colo-nies within 100 CFU/mL,the volume of the detection sample must below 600 mL.Firstly, the sample with different volumes higher or lower than 600 mL is treated with microfiltration; Secondly, extract the bacterial metagenomic DNA of filter membrane by Bacterial Genome Extraction Kit;Thirdly, analyze the DNA

  3. Identification and Preliminary Analysis of Several Centromere-associated Bacterial Artificial Chromosome Clones from a Diploid Wheat Library

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Although the centromeres of some plants have been investigated previously, our knowledge of the wheat centromere is still very limited. To understand the structure and function of the wheat centromere, we used two centromeric repeats (RCS1 and CCS1-5ab) to obtain some centromere-associated bacterial artificial chromosome (BAC) clones in 32 RCS1-related BAC clones that had been screened out from a diploid wheat (Triticum boeoticum Boiss.; 2n=2x=14) BAC library. Southern hybridization results indicated that, of the 32 candidates,there were 28 RCS1-positive clones. Based on gel blot patterns, the frequency of RCS1 was approximately one copy every 69.4 kb in these 28 RCS1-positive BAC clones. More bands were detected when the same filter was probed with CCS1-5ab. Furthermore, the CCS1 bands covered all the bands detected by RCS1, which suggests that some CCS1 repeats were distributed together with RCS1. The frequency of CCS1 families was once every 35.8 kb, nearly twice that of RCS1. Fluorescence in situ hybridization (FISH) analysis indicated that the five BAC clones containing RCS1 and CCS1 sequences all detected signals at the centromeric regions in hexaploid wheat, but the signal intensities on the A-genome chromosomes were stronger than those on the B- and/or D-genome chromosomes. The FISH analysis among nine Triticeae cereals indicated that there were A-genomespecific (or rich) sequences dispersing on chromosome arms in the BAC clone TbBAC5. In addition, at the interphase cells, the centromeres of diploid species usually clustered at one pole and formed a ring-like allocation in the period before metaphase.

  4. Cloning and characterization of a novel α-amylase from a fecal microbial metagenome.

    Science.gov (United States)

    Xu, Bo; Yang, Fuya; Xiong, Caiyun; Li, Junjun; Tang, Xianghua; Zhou, Junpei; Xie, Zhenrong; Ding, Junmei; Yang, Yunjuan; Huang, Zunxi

    2014-04-01

    To isolate novel and useful microbial enzymes from uncultured gastrointestinal microorganisms, a fecal microbial metagenomic library of the pygmy loris was constructed. The library was screened for amylolytic activity, and 8 of 50,000 recombinant clones showed amylolytic activity. Subcloning and sequence analysis of a positive clone led to the identification a novel gene (amyPL) coding for α-amylase. AmyPL was expressed in Escherichia coli BL21 (DE3) and the purified AmyPL was enzymatically characterized. This study is the first to report the molecular and biochemical characterization of a novel α-amylase from a gastrointestinal metagenomic library.

  5. Soil metagenomics and tropical soil productivity

    OpenAIRE

    Karen A Garrett

    2009-01-01

    This presentation summarizes research in the soil metagenomics cross cutting research activity. Soil metagenomics studies soil microbial communities as contributors to soil health.C CCRA-4 (Soil Metagenomics)

  6. A retrospective metagenomics approach to studying Blastocystis.

    Science.gov (United States)

    Andersen, Lee O'Brien; Bonde, Ida; Nielsen, Henrik Bjørn; Stensvold, Christen Rune

    2015-07-01

    Blastocystis is a common single-celled intestinal parasitic genus, comprising several subtypes. Here, we screened data obtained by metagenomic analysis of faecal DNA for Blastocystis by searching for subtype-specific genes in coabundance gene groups, which are groups of genes that covary across a selection of 316 human faecal samples, hence representing genes originating from a single subtype. The 316 faecal samples were from 236 healthy individuals, 13 patients with Crohn's disease (CD) and 67 patients with ulcerative colitis (UC). The prevalence of Blastocystis was 20.3% in the healthy individuals and 14.9% in patients with UC. Meanwhile, Blastocystis was absent in patients with CD. Individuals with intestinal microbiota dominated by Bacteroides were much less prone to having Blastocystis-positive stool (Matthew's correlation coefficient = -0.25, P metagenomics approach. The study serves as an example of how it is possible to retrospectively investigate microbial eukaryotic communities in the gut using metagenomic datasets targeting the bacterial component of the intestinal microbiome and the interplay between these microbial communities.

  7. Metagenomic studies of the Red Sea.

    Science.gov (United States)

    Behzad, Hayedeh; Ibarra, Martin Augusto; Mineta, Katsuhiko; Gojobori, Takashi

    2016-02-01

    Metagenomics has significantly advanced the field of marine microbial ecology, revealing the vast diversity of previously unknown microbial life forms in different marine niches. The tremendous amount of data generated has enabled identification of a large number of microbial genes (metagenomes), their community interactions, adaptation mechanisms, and their potential applications in pharmaceutical and biotechnology-based industries. Comparative metagenomics reveals that microbial diversity is a function of the local environment, meaning that unique or unusual environments typically harbor novel microbial species with unique genes and metabolic pathways. The Red Sea has an abundance of unique characteristics; however, its microbiota is one of the least studied among marine environments. The Red Sea harbors approximately 25 hot anoxic brine pools, plus a vibrant coral reef ecosystem. Physiochemical studies describe the Red Sea as an oligotrophic environment that contains one of the warmest and saltiest waters in the world with year-round high UV radiations. These characteristics are believed to have shaped the evolution of microbial communities in the Red Sea. Over-representation of genes involved in DNA repair, high-intensity light responses, and osmoregulation were found in the Red Sea metagenomic databases suggesting acquisition of specific environmental adaptation by the Red Sea microbiota. The Red Sea brine pools harbor a diverse range of halophilic and thermophilic bacterial and archaeal communities, which are potential sources of enzymes for pharmaceutical and biotechnology-based application. Understanding the mechanisms of these adaptations and their function within the larger ecosystem could also prove useful in light of predicted global warming scenarios where global ocean temperatures are expected to rise by 1-3°C in the next few decades. In this review, we provide an overview of the published metagenomic studies that were conducted in the Red Sea, and

  8. Construction and characterization of two bacterial artificial chromosome libraries of pea (Pisum sativum L.) for the isolation of economically important genes.

    Science.gov (United States)

    Coyne, C J; McClendon, M T; Walling, J G; Timmerman-Vaughan, G M; Murray, S; Meksem, K; Lightfoot, D A; Shultz, J L; Keller, K E; Martin, R R; Inglis, D A; Rajesh, P N; McPhee, K E; Weeden, N F; Grusak, M A; Li, C-M; Storlie, E W

    2007-09-01

    Pea (Pisum sativum L.) has a genome of about 4 Gb that appears to share conserved synteny with model legumes having genomes of 0.2-0.4 Gb despite extensive intergenic expansion. Pea plant inventory (PI) accession 269818 has been used to introgress genetic diversity into the cultivated germplasm pool. The aim here was to develop pea bacterial artificial chromosome (BAC) libraries that would enable the isolation of genes involved in plant disease resistance or control of economically important traits. The BAC libraries encompassed about 3.2 haploid genome equivalents consisting of partially HindIII-digested DNA fragments with a mean size of 105 kb that were inserted in 1 of 2 vectors. The low-copy oriT-based T-DNA vector (pCLD04541) library contained 55 680 clones. The single-copy oriS-based vector (pIndigoBAC-5) library contained 65 280 clones. Colony hybridization of a universal chloroplast probe indicated that about 1% of clones in the libraries were of chloroplast origin. The presence of about 0.1% empty vectors was inferred by white/blue colony plate counts. The usefulness of the libraries was tested by 2 replicated methods. First, high-density filters were probed with low copy number sequences. Second, BAC plate-pool DNA was used successfully to PCR amplify 7 of 9 published pea resistance gene analogs (RGAs) and several other low copy number pea sequences. Individual BAC clones encoding specific sequences were identified. Therefore, the HindIII BAC libraries of pea, based on germplasm accession PI 269818, will be useful for the isolation of genes underlying disease resistance and other economically important traits.

  9. Metagenomic Analysis of Koumiss in Kazakhstan

    Directory of Open Access Journals (Sweden)

    Samat Kozhakhmetov

    2014-12-01

    Full Text Available Introduction. Koumiss is a low-alcohol product made from fermented mare's milk, which is popular in Kazakhstan, Russia, and other countries of Central Asia, China, and Mongolia. Natural mare's milk is fermented in symbiosis of two types of microorganisms (lactobacteria and yeast. Koumiss’s microbial composition varies depending on the geographical, climatic, and cultural conditions. Based on a phenotypic characteristic from samples, Wu, R. and colleagues identified the following bacteria isolated in inner Mongolia, an autonomous region of China: L.casei, L.helveticus, L.plantarum, L.coryniformis subsp. coryniformis, L.paracasei, L.kefiranofaciens, L.curvatus, L.fermentum, and W.kandleri. Studies of the yeast composition in koumiss also showed significant variations. Thus, there were Saccharomyces unisporus related 48.3% of isolates, to Kluyveromyces marxianus (27.6%, Pichia membranaefaciens (15.0%, and Saccharomyces cerevisiae (9.2% from 87 isolated yeast cultures. The purpose of this study was to examine the bacterial composition in koumiss.Methods. To extract DNA, 1.8 ml of fermented milk was centrifuged to generate a pellet, which was suspended in 450 µl of lysis buffer P1 from the Powerfood Microbial DNA Isolation kit (MoBio Laboratories Inc, USA. Amplification of the microflora was used to determine the composition of a fragment of the gene 16S rRNA and ITS1. Plasmid library with target insertion was obtained on the basis of height copy plasmid vectors producing high pGem-T. The definition of direct nucleotide sequencing was performed by the method of Sanger using a set of "BigDye Terminanor v 3.1 Cycle sequencing Kit with automatic genetic analyzer ABI 3730xl  (Applied Biosystems, USA.  Informax Vector NTI Suite 9, Sequence Scanner v 1.0  software package used for the analysis.Results. Our studies showed that in the most samples of koumiss isolated from Akmola region (Central Kazakhstan prevailed the following bacteria species

  10. Screening for novel enzymes from metagenome and SIGEX, as a way to improve it

    Directory of Open Access Journals (Sweden)

    Ryu Sangryeol

    2005-03-01

    Full Text Available Abstract Metagenomics has been successfully applied to isolate novel biocatalysts from the uncultured microbiota in the environment. Two types of screening have been used to identify clones carrying desired traits from metagenomic libraries: function-based screening, and sequence-based screening. Both function- and sequence- based screening have individual advantages and disadvantages, and they have been applied successfully to discover biocatalysts from metagenome. However, both strategies are laborious and tedious because of the low frequency of screening hits. A recent paper introduced a high throughput screening strategy, termed substrate-induced gene-expression screening (SIGEX. SIGEX is designed to select the clones harboring catabolic genes induced by various substrates in concert with fluorescence activated cell sorting (FACS. This method was applied successfully to isolate aromatic hydrocarbon-induced genes from a metagenomic library. Although SIGEX has many limitations, it is expected to provide economic advantages, especially to industry.

  11. Environmental Metagenomics: The Data Assembly and Data Analysis Perspectives

    Science.gov (United States)

    Kumar, Vinay; Maitra, S. S.; Shukla, Rohit Nandan

    2015-01-01

    Novel gene finding is one of the emerging fields in the environmental research. In the past decades the research was focused mainly on the discovery of microorganisms which were capable of degrading a particular compound. A lot of methods are available in literature about the cultivation and screening of these novel microorganisms. All of these methods are efficient for screening of microbes which can be cultivated in the laboratory. Microorganisms which live in extreme conditions like hot springs, frozen glaciers, acid mine drainage, etc. cannot be cultivated in the laboratory, this is because of incomplete knowledge about their growth requirements like temperature, nutrients and their mutual dependence on each other. The microbes that can be cultivated correspond only to less than 1 % of the total microbes which are present in the earth. Rest of the 99 % of uncultivated majority remains inaccessible. Metagenomics transcends the culture requirements of microbes. In metagenomics DNA is directly extracted from the environmental samples such as soil, seawater, acid mine drainage etc., followed by construction and screening of metagenomic library. With the ongoing research, a huge amount of metagenomic data is accumulating. Understanding this data is an essential step to extract novel genes of industrial importance. Various bioinformatics tools have been designed to analyze and annotate the data produced from the metagenome. The Bio-informatic requirements of metagenomics data analysis are different in theory and practice. This paper reviews the tools that are available for metagenomic data analysis and the capability such tools—what they can do and their web availability.

  12. Soil-specific limitations for access and analysis of soil microbial communities by metagenomics.

    Science.gov (United States)

    Lombard, Nathalie; Prestat, Emmanuel; van Elsas, Jan Dirk; Simonet, Pascal

    2011-10-01

    Metagenomics approaches represent an important way to acquire information on the microbial communities present in complex environments like soil. However, to what extent do these approaches provide us with a true picture of soil microbial diversity? Soil is a challenging environment to work with. Its physicochemical properties affect microbial distributions inside the soil matrix, metagenome extraction and its subsequent analyses. To better understand the bias inherent to soil metagenome 'processing', we focus on soil physicochemical properties and their effects on the perceived bacterial distribution. In the light of this information, each step of soil metagenome processing is then discussed, with an emphasis on strategies for optimal soil sampling. Then, the interaction of cells and DNA with the soil matrix and the consequences for microbial DNA extraction are examined. Soil DNA extraction methods are compared and the veracity of the microbial profiles obtained is discussed. Finally, soil metagenomic sequence analysis and exploitation methods are reviewed.

  13. Genome signature analysis of thermal virus metagenomes reveals Archaea and thermophilic signatures

    Directory of Open Access Journals (Sweden)

    Pride David T

    2008-09-01

    Full Text Available Abstract Background Metagenomic analysis provides a rich source of biological information for otherwise intractable viral communities. However, study of viral metagenomes has been hampered by its nearly complete reliance on BLAST algorithms for identification of DNA sequences. We sought to develop algorithms for examination of viral metagenomes to identify the origin of sequences independent of BLAST algorithms. We chose viral metagenomes obtained from two hot springs, Bear Paw and Octopus, in Yellowstone National Park, as they represent simple microbial populations where comparatively large contigs were obtained. Thermal spring metagenomes have high proportions of sequences without significant Genbank homology, which has hampered identification of viruses and their linkage with hosts. To analyze each metagenome, we developed a method to classify DNA fragments using genome signature-based phylogenetic classification (GSPC, where metagenomic fragments are compared to a database of oligonucleotide signatures for all previously sequenced Bacteria, Archaea, and viruses. Results From both Bear Paw and Octopus hot springs, each assembled contig had more similarity to other metagenome contigs than to any sequenced microbial genome based on GSPC analysis, suggesting a genome signature common to each of these extreme environments. While viral metagenomes from Bear Paw and Octopus share some similarity, the genome signatures from each locale are largely unique. GSPC using a microbial database predicts most of the Octopus metagenome has archaeal signatures, while bacterial signatures predominate in Bear Paw; a finding consistent with those of Genbank BLAST. When using a viral database, the majority of the Octopus metagenome is predicted to belong to archaeal virus Families Globuloviridae and Fuselloviridae, while none of the Bear Paw metagenome is predicted to belong to archaeal viruses. As expected, when microbial and viral databases are combined, each of

  14. Exploring Antibiotic Resistance Genes and Metal Resistance Genes in Plasmid Metagenomes from Wastewater Treatment Plants

    Directory of Open Access Journals (Sweden)

    An-Dong eLi

    2015-09-01

    Full Text Available Plasmids operate as independent genetic elements in microorganism communities. Through horizontal gene transfer, they can provide their host microorganisms with important functions such as antibiotic resistance and heavy metal resistance. In this study, six metagenomic libraries were constructed with plasmid DNA extracted from influent, activated sludge and digested sludge of two wastewater treatment plants. Compared with the metagenomes of the total DNA extracted from the same sectors of the wastewater treatment plant, the plasmid metagenomes had significantly higher annotation rates, indicating that the functional genes on plasmids are commonly shared by those studied microorganisms. Meanwhile, the plasmid metagenomes also encoded many more genes related to defense mechanisms, including ARGs. Searching against an antibiotic resistance genes (ARGs database and a metal resistance genes (MRGs database revealed a broad-spectrum of antibiotic (323 out of a total 618 subtypes and metal resistance genes (23 out of a total 23 types on these plasmid metagenomes. The influent plasmid metagenomes contained many more resistance genes (both ARGs and MRGs than the activated sludge and the digested sludge metagenomes. Sixteen novel plasmids with a complete circular structure that carried these resistance genes were assembled from the plasmid metagenomes. The results of this study demonstrated that the plasmids in wastewater treatment plants could be important reservoirs for resistance genes, and may play a significant role in the horizontal transfer of these genes.

  15. Exploring antibiotic resistance genes and metal resistance genes in plasmid metagenomes from wastewater treatment plants.

    Science.gov (United States)

    Li, An-Dong; Li, Li-Guan; Zhang, Tong

    2015-01-01

    Plasmids operate as independent genetic elements in microorganism communities. Through horizontal gene transfer (HGT), they can provide their host microorganisms with important functions such as antibiotic resistance and heavy metal resistance. In this study, six metagenomic libraries were constructed with plasmid DNA extracted from influent, activated sludge (AS) and digested sludge (DS) of two wastewater treatment plants (WWTPs). Compared with the metagenomes of the total DNA extracted from the same sectors of the wastewater treatment plant, the plasmid metagenomes had significantly higher annotation rates, indicating that the functional genes on plasmids are commonly shared by those studied microorganisms. Meanwhile, the plasmid metagenomes also encoded many more genes related to defense mechanisms, including ARGs. Searching against an antibiotic resistance genes (ARGs) database and a metal resistance genes (MRGs) database revealed a broad-spectrum of antibiotic (323 out of a total 618 subtypes) and MRGs (23 out of a total 23 types) on these plasmid metagenomes. The influent plasmid metagenomes contained many more resistance genes (both ARGs and MRGs) than the AS and the DS metagenomes. Sixteen novel plasmids with a complete circular structure that carried these resistance genes were assembled from the plasmid metagenomes. The results of this study demonstrated that the plasmids in WWTPs could be important reservoirs for resistance genes, and may play a significant role in the horizontal transfer of these genes.

  16. The great screen anomaly--a new frontier in product discovery through functional metagenomics.

    Science.gov (United States)

    Ekkers, David Matthias; Cretoiu, Mariana Silvia; Kielak, Anna Maria; Elsas, Jan Dirk van

    2012-02-01

    Functional metagenomics, the study of the collective genome of a microbial community by expressing it in a foreign host, is an emerging field in biotechnology. Over the past years, the possibility of novel product discovery through metagenomics has developed rapidly. Thus, metagenomics has been heralded as a promising mining strategy of resources for the biotechnological and pharmaceutical industry. However, in spite of innovative work in the field of functional genomics in recent years, yields from function-based metagenomics studies still fall short of producing significant amounts of new products that are valuable for biotechnological processes. Thus, a new set of strategies is required with respect to fostering gene expression in comparison to the traditional work. These new strategies should address a major issue, that is, how to successfully express a set of unknown genes of unknown origin in a foreign host in high throughput. This article is an opinionating review of functional metagenomic screening of natural microbial communities, with a focus on the optimization of new product discovery. It first summarizes current major bottlenecks in functional metagenomics and then provides an overview of the general metagenomic assessment strategies, with a focus on the challenges that are met in the screening for, and selection of, target genes in metagenomic libraries. To identify possible screening limitations, strategies to achieve optimal gene expression are reviewed, examining the molecular events all the way from the transcription level through to the secretion of the target gene product.

  17. Application of metagenomics in understanding oral health and disease.

    Science.gov (United States)

    Xu, Ping; Gunsolley, John

    2014-04-01

    Oral diseases including periodontal disease and caries are some of the most prevalent infectious diseases in humans. Different microbial species cohabitate and form a polymicrobial biofilm called dental plaque in the oral cavity. Metagenomics using next generation sequencing technologies has produced bacterial profiles and genomic profiles to study the relationships between microbial diversity, genetic variation, and oral diseases. Several oral metagenomic studies have examined the oral microbiome of periodontal disease and caries. Gene annotations in these studies support the association of specific genes or metabolic pathways with oral health and with specific diseases. The roles of pathogenic species and functions of specific genes in oral disease development have been recognized by metagenomic analysis. A model is proposed in which three levels of interactions occur in the oral microbiome that determines oral health or disease.

  18. The YNP metagenome project

    DEFF Research Database (Denmark)

    Inskeep, William P.; Jay, Zackary J.; Tringe, Susannah G.;

    2013-01-01

    The Yellowstone geothermal complex contains over 10,000 diverse geothermal features that host numerous phylogenetically deeply rooted and poorly understood archaea, bacteria, and viruses. Microbial communities in high-temperature environments are generally less diverse than soil, marine, sediment......, and environmental variables. Twenty geochemically distinct geothermal ecosystems representing a broad spectrum of Yellowstone hot-spring environments were used for metagenomic and geochemical analysis and included approximately equal numbers of: (1) phototrophic mats, (2) “filamentous streamer” communities, and (3...

  19. 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...

  20. Databases of the marine metagenomics

    KAUST Repository

    Mineta, Katsuhiko

    2015-10-28

    The metagenomic data obtained from marine environments is significantly useful for understanding marine microbial communities. In comparison with the conventional amplicon-based approach of metagenomics, the recent shotgun sequencing-based approach has become a powerful tool that provides an efficient way of grasping a diversity of the entire microbial community at a sampling point in the sea. However, this approach accelerates accumulation of the metagenome data as well as increase of data complexity. Moreover, when metagenomic approach is used for monitoring a time change of marine environments at multiple locations of the seawater, accumulation of metagenomics data will become tremendous with an enormous speed. Because this kind of situation has started becoming of reality at many marine research institutions and stations all over the world, it looks obvious that the data management and analysis will be confronted by the so-called Big Data issues such as how the database can be constructed in an efficient way and how useful knowledge should be extracted from a vast amount of the data. In this review, we summarize the outline of all the major databases of marine metagenome that are currently publically available, noting that database exclusively on marine metagenome is none but the number of metagenome databases including marine metagenome data are six, unexpectedly still small. We also extend our explanation to the databases, as reference database we call, that will be useful for constructing a marine metagenome database as well as complementing important information with the database. Then, we would point out a number of challenges to be conquered in constructing the marine metagenome database.

  1. Databases of the marine metagenomics.

    Science.gov (United States)

    Mineta, Katsuhiko; Gojobori, Takashi

    2016-02-01

    The metagenomic data obtained from marine environments is significantly useful for understanding marine microbial communities. In comparison with the conventional amplicon-based approach of metagenomics, the recent shotgun sequencing-based approach has become a powerful tool that provides an efficient way of grasping a diversity of the entire microbial community at a sampling point in the sea. However, this approach accelerates accumulation of the metagenome data as well as increase of data complexity. Moreover, when metagenomic approach is used for monitoring a time change of marine environments at multiple locations of the seawater, accumulation of metagenomics data will become tremendous with an enormous speed. Because this kind of situation has started becoming of reality at many marine research institutions and stations all over the world, it looks obvious that the data management and analysis will be confronted by the so-called Big Data issues such as how the database can be constructed in an efficient way and how useful knowledge should be extracted from a vast amount of the data. In this review, we summarize the outline of all the major databases of marine metagenome that are currently publically available, noting that database exclusively on marine metagenome is none but the number of metagenome databases including marine metagenome data are six, unexpectedly still small. We also extend our explanation to the databases, as reference database we call, that will be useful for constructing a marine metagenome database as well as complementing important information with the database. Then, we would point out a number of challenges to be conquered in constructing the marine metagenome database.

  2. Comparison of mammalian and bacterial expression library screening to detect recombinant alpha-1 proteinase inhibitor variants with enhanced thrombin inhibitory capacity.

    Science.gov (United States)

    Gierczak, Richard F; Bhakta, Varsha; Xie, Michael; Sheffield, William P

    2015-08-20

    Serpins are a widely distributed family of serine proteases. A key determinant of their specificity is the reactive centre loop (RCL), a surface motif of ∼20 amino acids in length. Expression libraries of variant serpins could be rapidly probed with proteases to develop novel inhibitors if optimal systems were available. The serpin variant alpha-1 proteinase inhibitor M358R (API M358R) inhibits the coagulation protease thrombin, but at sub-maximal rates compared to other serpins. Here we compared two approaches to isolate functional API variants from serpin expression libraries, using the same small library of API randomized at residue 358 (M358X): flow cytometry of transfected HEK 293 cells expressing membrane-displayed API; and a thrombin capture assay (TCA) performed on pools of bacterial lysates expressing soluble API. No enrichment for specific P1 residues was observed when the RCL codons of the 1% of sorted transfected 293 cells with the highest fluorescent thrombin-binding signals were subcloned and sequenced. In contrast, screening of 16 pools of bacterial API-expressing transformants led to the facile identification of API M358R and M358K as functional variants. Kinetic characterization showed that API M358R inhibited thrombin 17-fold more rapidly than API M358K. Reducing the incubation time with immobilized thrombin improved the sensitivity of TCA to detect supra-active API M358R variants and was used to screen a hypervariable library of API variants expressing 16 different amino acids at residues 352-357. The most active variant isolated, with TLSATP substituted for FLEAI, inhibited thrombin 2.9-fold more rapidly than API M358R. Our results indicate that flow cytometric approaches used in protein engineering of antibodies are not appropriate for serpins, and highlight the utility of the optimized TCA for serpin protein engineering.

  3. Microbial metagenomics: beyond the genome.

    Science.gov (United States)

    Gilbert, Jack A; Dupont, Christopher L

    2011-01-01

    Metagenomics literally means "beyond the genome." Marine microbial metagenomic databases presently comprise approximately 400 billion base pairs of DNA, only approximately 3% of that found in 1 ml of seawater. Very soon a trillion-base-pair sequence run will be feasible, so it is time to reflect on what we have learned from metagenomics. We review the impact of metagenomics on our understanding of marine microbial communities. We consider the studies facilitated by data generated through the Global Ocean Sampling expedition, as well as the revolution wrought at the individual laboratory level through next generation sequencing technologies. We review recent studies and discoveries since 2008, provide a discussion of bioinformatic analyses, including conceptual pipelines and sequence annotation and predict the future of metagenomics, with suggestions of collaborative community studies tailored toward answering some of the fundamental questions in marine microbial ecology.

  4. Microbial Metagenomics: Beyond the Genome

    Science.gov (United States)

    Gilbert, Jack A.; Dupont, Christopher L.

    2011-01-01

    Metagenomics literally means “beyond the genome.” Marine microbial metagenomic databases presently comprise ˜400 billion base pairs of DNA, only ˜3% of that found in 1 ml of seawater. Very soon a trillion-base-pair sequence run will be feasible, so it is time to reflect on what we have learned from metagenomics. We review the impact of metagenomics on our understanding of marine microbial communities. We consider the studies facilitated by data generated through the Global Ocean Sampling expedition, as well as the revolution wrought at the individual laboratory level through next generation sequencing technologies. We review recent studies and discoveries since 2008, provide a discussion of bioinformatic analyses, including conceptual pipelines and sequence annotation and predict the future of metagenomics, with suggestions of collaborative community studies tailored toward answering some of the fundamental questions in marine microbial ecology.

  5. Captured metagenomics: large-scale targeting of genes based on 'sequence capture' reveals functional diversity in soils.

    Science.gov (United States)

    Manoharan, Lokeshwaran; Kushwaha, Sandeep K; Hedlund, Katarina; Ahrén, Dag

    2015-12-01

    Microbial enzyme diversity is a key to understand many ecosystem processes. Whole metagenome sequencing (WMG) obtains information on functional genes, but it is costly and inefficient due to large amount of sequencing that is required. In this study, we have applied a captured metagenomics technique for functional genes in soil microorganisms, as an alternative to WMG. Large-scale targeting of functional genes, coding for enzymes related to organic matter degradation, was applied to two agricultural soil communities through captured metagenomics. Captured metagenomics uses custom-designed, hybridization-based oligonucleotide probes that enrich functional genes of interest in metagenomic libraries where only probe-bound DNA fragments are sequenced. The captured metagenomes were highly enriched with targeted genes while maintaining their target diversity and their taxonomic distribution correlated well with the traditional ribosomal sequencing. The captured metagenomes were highly enriched with genes related to organic matter degradation; at least five times more than similar, publicly available soil WMG projects. This target enrichment technique also preserves the functional representation of the soils, thereby facilitating comparative metagenomics projects. Here, we present the first study that applies the captured metagenomics approach in large scale, and this novel method allows deep investigations of central ecosystem processes by studying functional gene abundances.

  6. Evaluating the Quantitative Capabilities of Metagenomic Analysis Software.

    Science.gov (United States)

    Kerepesi, Csaba; Grolmusz, Vince

    2016-05-01

    DNA sequencing technologies are applied widely and frequently today to describe metagenomes, i.e., microbial communities in environmental or clinical samples, without the need for culturing them. These technologies usually return short (100-300 base-pairs long) DNA reads, and these reads are processed by metagenomic analysis software that assign phylogenetic composition-information to the dataset. Here we evaluate three metagenomic analysis software (AmphoraNet--a webserver implementation of AMPHORA2--, MG-RAST, and MEGAN5) for their capabilities of assigning quantitative phylogenetic information for the data, describing the frequency of appearance of the microorganisms of the same taxa in the sample. The difficulties of the task arise from the fact that longer genomes produce more reads from the same organism than shorter genomes, and some software assign higher frequencies to species with longer genomes than to those with shorter ones. This phenomenon is called the "genome length bias." Dozens of complex artificial metagenome benchmarks can be found in the literature. Because of the complexity of those benchmarks, it is usually difficult to judge the resistance of a metagenomic software to this "genome length bias." Therefore, we have made a simple benchmark for the evaluation of the "taxon-counting" in a metagenomic sample: we have taken the same number of copies of three full bacterial genomes of different lengths, break them up randomly to short reads of average length of 150 bp, and mixed the reads, creating our simple benchmark. Because of its simplicity, the benchmark is not supposed to serve as a mock metagenome, but if a software fails on that simple task, it will surely fail on most real metagenomes. We applied three software for the benchmark. The ideal quantitative solution would assign the same proportion to the three bacterial taxa. We have found that AMPHORA2/AmphoraNet gave the most accurate results and the other two software were under

  7. The metagenome-derived enzymes LipS and LipT increase the diversity of known lipases.

    Directory of Open Access Journals (Sweden)

    Jennifer Chow

    Full Text Available Triacylglycerol lipases (EC 3.1.1.3 catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75 °C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70 °C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70 °C. LipS had an optimum temperature at 70 °C and LipT at 75 °C. Both enzymes catalyzed hydrolysis of long-chain (C(12 and C(14 fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R-ibuprofen-phenyl ester with an enantiomeric excess (ee of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70 °C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 Å revealing an unusually compact lid structure.

  8. A five-fold pig bacterial artificial chromosome library:a resource for positional cloning and physical mapping

    Institute of Scientific and Technical Information of China (English)

    LIU Wei; LI Ning; ZHANG Ying; LIU Zhaoliang; GUO Li; WANG Xiaobo; FEI Jing; FENG Jidong; ZHAO Rui; HU Xiaoxiang

    2006-01-01

    A pig BAC library was constructed with genomic DNA from a male Erhualian pig. After partial digestion with Hind Ⅲ or BamH I the fragments obtained were cloned into the pBeloBAC11 vector. The library consists of 184320 clones which stored in 480pieces 384-well plates (20 plates per superpool). A two-step 4-dimension PCR screening system was established to screen the positive clones. An average insert size of 128 kb was estimated from 105 randomly isolated clones, which indicates that the library is more than five times of genomic coverage. For the demonstration of the probability to pick out any unique genes or DNA markers from the library, 10single-copy genes were screened out and the positive clones were yielded between 1 and 8 with an average of 3.6. Positive superpools were obtained for 32 microsatellite markers selected from different regions of pig genome. The number of positive superpools for each marker varies from 1 to 9 with an average of 4.78. This BAC library provides an additional resource for pig physical mapping and gene identification.

  9. An enrichment of CRISPR and other defense-related features in marine sponge-associated microbial metagenomes

    Directory of Open Access Journals (Sweden)

    Hannes Horn

    2016-11-01

    Full Text Available Many marine sponges are populated by dense and taxonomically diverse microbial consortia. We employed a metagenomics approach to unravel the differences in the functional gene repertoire among three Mediterranean sponge species, Petrosia ficiformis, Sarcotragus foetidus, Aplysina aerophoba and seawater. Different signatures were observed between sponge and seawater metagenomes with regard to microbial community composition, GC content, and estimated bacterial genome size. Our analysis showed further a pronounced repertoire for defense systems in sponge metagenomes. Specifically, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR, restriction modification, DNA phosphorothioation and phage growth limitation systems were enriched in sponge metagenomes. These data suggest that defense is an important functional trait for an existence within sponges that requires mechanisms to defend against foreign DNA from microorganisms and viruses. This study contributes to an understanding of the evolutionary arms race between viruses/phages and bacterial genomes and it sheds light on the bacterial defenses that have evolved in the context of the sponge holobiont.

  10. Megraft: a software package to graft ribosomal small subunit (16S/18S) fragments onto full-length sequences for accurate species richness and sequencing depth analysis in pyrosequencing-length metagenomes and similar environmental datasets.

    Science.gov (United States)

    Bengtsson, Johan; Hartmann, Martin; Unterseher, Martin; Vaishampayan, Parag; Abarenkov, Kessy; Durso, Lisa; Bik, Elisabeth M; Garey, James R; Eriksson, K Martin; Nilsson, R Henrik

    2012-07-01

    Metagenomic libraries represent subsamples of the total DNA found at a study site and offer unprecedented opportunities to study ecological and functional aspects of microbial communities. To examine the depth of a community sequencing effort, rarefaction analysis of the ribosomal small subunit (SSU/16S/18S) gene in the metagenome is usually performed. The fragmentary, non-overlapping nature of SSU sequences in metagenomic libraries poses a problem for this analysis, however. We introduce a software package - Megraft - that grafts SSU fragments onto full-length SSU sequences, accounting for observed and unobserved variability, for accurate assessment of species richness and sequencing depth in metagenomics endeavors.

  11. A retrospective metagenomics approach to studying Blastocystis

    DEFF Research Database (Denmark)

    Andersen, Lee O'Brien; Bonde, Ida; Nielsen, Henrik Bjørn

    2015-01-01

    a selection of 316 human faecal samples, hence representing genes originating from a single subtype. The 316 faecal samples were from 236 healthy individuals, 13 patients with Crohn's disease (CD) and 67 patients with ulcerative colitis (UC). The prevalence of Blastocystis was 20.3% in the healthy individuals...... metagenomic datasets targeting the bacterial component of the intestinal microbiome and the interplay between these microbial communities....

  12. Assembly of a marine viral metagenome after physical fractionation.

    Directory of Open Access Journals (Sweden)

    Jennifer R Brum

    Full Text Available Metagenomic analyses of marine viruses generate an overview of viral genes present in a sample, but the percentage of the resulting sequence fragments that can be reassembled is low and the phenotype of the virus from which a given sequence derives is usually unknown. In this study, we employed physical fractionation to characterize the morphological and genomic traits of a subset of uncultivated viruses from a natural marine assemblage. Viruses from Kāne'ohe Bay, Hawai'i were fractionated by equilibrium buoyant density centrifugation in a cesium chloride (CsCl gradient, and one fraction from the CsCl gradient was then further fractionated by strong anion-exchange chromatography. One of the fractions resulting from this two-dimensional separation appeared to be dominated by only a few virus types based on genome sizes and morphology. Sequences generated from a shotgun clone library of the viruses in this fraction were assembled into significantly more numerous contigs than have been generated with previous metagenomic investigations of whole DNA viral assemblages with comparable sequencing effort. Analysis of the longer contigs (up to 6.5 kb assembled from our metagenome allowed us to assess gene arrangement in this subset of marine viruses. Our results demonstrate the potential for physical fractionation to facilitate sequence assembly from viral metagenomes and permit linking of morphological and genomic data for uncultivated viruses.

  13. Metagenomics of the deep Mediterranean, a warm bathypelagic habitat.

    Directory of Open Access Journals (Sweden)

    Ana-Belen Martín-Cuadrado

    Full Text Available BACKGROUND: Metagenomics is emerging as a powerful method to study the function and physiology of the unexplored microbial biosphere, and is causing us to re-evaluate basic precepts of microbial ecology and evolution. Most marine metagenomic analyses have been nearly exclusively devoted to photic waters. METHODOLOGY/PRINCIPAL FINDINGS: We constructed a metagenomic fosmid library from 3,000 m-deep Mediterranean plankton, which is much warmer (approximately 14 degrees C than waters of similar depth in open oceans (approximately 2 degrees C. We analyzed the library both by phylogenetic screening based on 16S rRNA gene amplification from clone pools and by sequencing both insert extremities of ca. 5,000 fosmids. Genome recruitment strategies showed that the majority of high scoring pairs corresponded to genomes from Rhizobiales within the Alphaproteobacteria, Cenarchaeum symbiosum, Planctomycetes, Acidobacteria, Chloroflexi and Gammaproteobacteria. We have found a community structure similar to that found in the aphotic zone of the Pacific. However, the similarities were significantly higher to the mesopelagic (500-700 m deep in the Pacific than to the single 4000 m deep sample studied at this location. Metabolic genes were mostly related to catabolism, transport and degradation of complex organic molecules, in agreement with a prevalent heterotrophic lifestyle for deep-sea microbes. However, we observed a high percentage of genes encoding dehydrogenases and, among them, cox genes, suggesting that aerobic carbon monoxide oxidation may be important in the deep ocean as an additional energy source. CONCLUSIONS/SIGNIFICANCE: The comparison of metagenomic libraries from the deep Mediterranean and the Pacific ALOHA water column showed that bathypelagic Mediterranean communities resemble more mesopelagic communities in the Pacific, and suggests that, in the absence of light, temperature is a major stratifying factor in the oceanic water column, overriding

  14. MALINA: a web service for visual analytics of human gut microbiota whole-genome metagenomic reads.

    Science.gov (United States)

    Tyakht, Alexander V; Popenko, Anna S; Belenikin, Maxim S; Altukhov, Ilya A; Pavlenko, Alexander V; Kostryukova, Elena S; Selezneva, Oksana V; Larin, Andrei K; Karpova, Irina Y; Alexeev, Dmitry G

    2012-12-07

    MALINA is a web service for bioinformatic analysis of whole-genome metagenomic data obtained from human gut microbiota sequencing. As input data, it accepts metagenomic reads of various sequencing technologies, including long reads (such as Sanger and 454 sequencing) and next-generation (including SOLiD and Illumina). It is the first metagenomic web service that is capable of processing SOLiD color-space reads, to authors' knowledge. The web service allows phylogenetic and functional profiling of metagenomic samples using coverage depth resulting from the alignment of the reads to the catalogue of reference sequences which are built into the pipeline and contain prevalent microbial genomes and genes of human gut microbiota. The obtained metagenomic composition vectors are processed by the statistical analysis and visualization module containing methods for clustering, dimension reduction and group comparison. Additionally, the MALINA database includes vectors of bacterial and functional composition for human gut microbiota samples from a large number of existing studies allowing their comparative analysis together with user samples, namely datasets from Russian Metagenome project, MetaHIT and Human Microbiome Project (downloaded from http://hmpdacc.org). MALINA is made freely available on the web at http://malina.metagenome.ru. The website is implemented in JavaScript (using Ext JS), Microsoft .NET Framework, MS SQL, Python, with all major browsers supported.

  15. Construction of an Americn mink Bacterial Artificial Chromosome (BAC) library and sequencing candidate genes important for the fur industry

    DEFF Research Database (Denmark)

    Anistoroaei, Razvan Marian; Hallers, Boudewijn ten; Nefedov, Michael;

    2011-01-01

    consisting of 18,432 clones spotted in duplicate, have been produced for hybridization screening and are publicly available. Overgo probes derived from expressed sequence tags (ESTs), representing 21 candidate genes for traits important for the mink industry, were used to screen the BAC library...

  16. The cystic fibrosis lower airways microbial metagenome

    Science.gov (United States)

    Moran Losada, Patricia; Chouvarine, Philippe; Dorda, Marie; Hedtfeld, Silke; Mielke, Samira; Schulz, Angela; Wiehlmann, Lutz

    2016-01-01

    Chronic airway infections determine most morbidity in people with cystic fibrosis (CF). Herein, we present unbiased quantitative data about the frequency and abundance of DNA viruses, archaea, bacteria, moulds and fungi in CF lower airways. Induced sputa were collected on several occasions from children, adolescents and adults with CF. Deep sputum metagenome sequencing identified, on average, approximately 10 DNA viruses or fungi and several hundred bacterial taxa. The metagenome of a CF patient was typically found to be made up of an individual signature of multiple, lowly abundant species superimposed by few disease-associated pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, as major components. The host-associated signatures ranged from inconspicuous polymicrobial communities in healthy subjects to low-complexity microbiomes dominated by the typical CF pathogens in patients with advanced lung disease. The DNA virus community in CF lungs mainly consisted of phages and occasionally of human pathogens, such as adeno- and herpesviruses. The S. aureus and P. aeruginosa populations were composed of one major and numerous minor clone types. The rare clones constitute a low copy genetic resource that could rapidly expand as a response to habitat alterations, such as antimicrobial chemotherapy or invasion of novel microbes. PMID:27730195

  17. A Cell-Based Approach for the Biosynthesis/Screening of Cyclic Peptide Libraries against Bacterial Toxins

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A; Kimura, R; Woo, Y; Cantor, J; Steenblock, E

    2007-10-24

    Available methods for developing and screening small drug-like molecules able to knockout toxins or pathogenic microorganisms have some limitations. In order to be useful, these new methods must provide high-throughput analysis and identify specific binders in a short period of time. To meet this need, we are developing an approach that uses living cells to generate libraries of small biomolecules, which are then screened inside the cell for activity. Our group is using this new, combined approach to find highly specific ligands capable of disabling anthrax Lethal Factor (LF) as proof of principle. Key to our approach is the development of a method for the biosynthesis of libraries of cyclic peptides, and an efficient screening process that can be carried out inside the cell.

  18. Study of anaerobic ammonium oxidation bacterial community in the aged refuse bioreactor with 16S rRNA gene library technique.

    Science.gov (United States)

    Wang, Chao; Xie, Bing; Han, Lu; Xu, Xiaofan

    2013-10-01

    In order to investigate the anaerobic ammonium-oxidation (Anammox) nitrogen removal pathway of the aged refuse bioreactor treating landfill leachate, a lab-scale bioreactor was established and run for 35 weeks, the performance of the bioreactor and its bacterial community structure of Planctomycetes were analyzed. The results showed that the average TN removal rate of landfill leachate could be reached to 89%. 16S rRNA gene library of Planctomycetes revealed that Anammox sequences accounted for 28.3% of the total Planctomycetes sequences in the bioreactor, and previously recognized Anammox bacterium Candidatus Kuenenia stuttgartiensis was the only detected Anammox species in the reactor. It was also found that Anammox bacteria distributed at different sites of the bioreactor while mostly concentrated in the middle and low-middle part. Results above confirmed that Anammox process could happen in aged refuse bioreactor treating landfill leachate and provided an alternative nitrogen removal pathway in practical landfills.

  19. A high-throughput strategy for screening of bacterial artificial chromosome libraries and anchoring of clones on a genetic map constructed with single nucleotide polymorphisms

    Directory of Open Access Journals (Sweden)

    Deal Karin R

    2009-01-01

    Full Text Available Abstract Background Current techniques of screening bacterial artificial chromosome (BAC libraries for molecular markers during the construction of physical maps are slow, laborious and often assign multiple BAC contigs to a single locus on a genetic map. These limitations are the principal impediment in the construction of physical maps of large eukaryotic genomes. It is hypothesized that this impediment can be overcome by screening multidimensional pools of BAC clones using the highly parallel Illumina GoldenGate™ assay. Results To test the efficacy of the Golden Gate assay in BAC library screening, multidimensional pools involving 302976 Aegilops tauschii BAC clones were genotyped for the presence/absence of specific gene sequences with multiplexed Illumina GoldenGate oligonucleotide assays previously used to place single nucleotide polymorphisms on an Ae. tauschii genetic map. Of 1384 allele-informative oligonucleotide assays, 87.6% successfully clustered BAC pools into those positive for a BAC clone harboring a specific gene locus and those negative for it. The location of the positive BAC clones within contigs assembled from 199190 fingerprinted Ae. tauschii BAC clones was used to evaluate the precision of anchoring of BAC clones and contigs on the Ae. tauschii genetic map. For 41 (95% assays, positive BAC clones were neighbors in single contigs. Those contigs could be unequivocally assigned to loci on the genetic map. For two (5% assays, positive clones were in two different contigs and the relationships of these contigs to loci on the Ae. tauschii genetic map were equivocal. Screening of BAC libraries with a simple five-dimensional BAC pooling strategy was evaluated and shown to allow direct detection of positive BAC clones without the need for manual deconvolution of BAC clone pools. Conclusion The highly parallel Illumina oligonucleotide assay is shown here to be an efficient tool for screening BAC libraries and a strategy for high

  20. Screening and identification of a novel target specific for hepatoma cell line HepG2 from the FliTrx bacterial peptide library

    Institute of Scientific and Technical Information of China (English)

    Wenhan Li; Ping Lei; Bing Yu; Sha Wu; Jilin Peng; Xiaoping Zhao; Huffen Zhu; Michael Kirschfink; Guanxin Shen

    2008-01-01

    To explore new targets for hepatoma research, we used a surface display library to screen novel tumor cell-specific peptides. The bacterial FliTrx system was screened with living normal liver cell line L02 and hepatoma cell line HepG2 successively to search for hepatoma-specific peptides. Three clones (Hep1, Hep2, and Hep3) were identified to be specific to HepG2 compared with L02 and other cancer cell lines.Three-dimensional structural prediction proved that peptides inserted into the active site of Escherichia coli thioredoxin (TrxA) formed certain loop structures protruding out of the surface. Western blot analysis showed that FliC/TrxA-pepfide fusion proteins could be directly used to detect HepG2 cells.Three different FliC/TrxA-peptide fusion proteins targeted the same molecule, at approximately 140 kDa, on HepG2 cells.This work presented for the first time the application of the FliTrx library in screening living cells. Three peptides were obtained that could be potential candidates for targeted liver cancer therapy.

  1. A metagenomic study highlights phylogenetic proximity of quorum-quenching and xenobiotic-degrading amidases of the AS-family.

    Directory of Open Access Journals (Sweden)

    Mélanie Tannières

    Full Text Available Quorum-sensing (QS signals of the N-acylhomoserine lactone (NAHL class are cleaved by quorum-quenching enzymes, collectively named NAHLases. Here, functional metagenomics allowed the discovery of a novel bacterial NAHLase in a rhizosphere that was treated with γ-caprolactone. As revealed by rrs-DGGE and rrs-pyrosequencing, this treatment increased the percentage of the NAHL-degrading bacteria and strongly biased the structure of the bacterial community, among which Azospirillum dominated. Among the 29 760 fosmids of the metagenomic library, a single one was detected that expressed the qsdB gene conferring NAHL-degradation upon E. coli and decreased QS-regulated virulence in Pectobacterium. Phylogenetic analysis of the 34 orfs of the fosmid suggested that it would belong to an unknown Proteobacterium - probably a γ-proteobacterium. qPCR quantification of the NAHLase-encoding genes attM, qsdA, and qsdB revealed their higher abundance in the γ-caprolactone-treated rhizosphere as compared to an untreated control. The purified QsdB enzyme exhibited amidase activity. QsdB is the first amidase signature (AS family member exhibiting NAHLase-activity. Point mutations in the AS-family catalytic triad K-S-S abolished the NAHLase activity of QsdB. This study extends the diversity of NAHLases and highlights a common phylogenic origin of AS-family enzymes involved in the degradation of natural compounds, such as NAHLs, and xenobiotics, such as nylon and linuron.

  2. Repetitive genome elements in a European corn borer, Ostrinia nubilalis, bacterial artificial chromosome library were indicated by bacterial artificial chromosome end sequencing and development of sequence tag site markers: implications for lepidopteran genomic research.

    Science.gov (United States)

    Coates, Brad S; Sumerford, Douglas V; Hellmich, Richard L; Lewis, Leslie C

    2009-01-01

    The European corn borer, Ostrinia nubilalis, is a serious pest of food, fiber, and biofuel crops in Europe, North America, and Asia and a model system for insect olfaction and speciation. A bacterial artificial chromosome library constructed for O. nubilalis contains 36 864 clones with an estimated average insert size of >or=120 kb and genome coverage of 8.8-fold. Screening OnB1 clones comprising approximately 2.76 genome equivalents determined the physical position of 24 sequence tag site markers, including markers linked to ecologically important and Bacillus thuringiensis toxin resistance traits. OnB1 bacterial artificial chromosome end sequence reads (GenBank dbGSS accessions ET217010 to ET217273) showed homology to annotated genes or expressed sequence tags and identified repetitive genome elements, O. nubilalis miniature subterminal inverted repeat transposable elements (OnMITE01 and OnMITE02), and ezi-like long interspersed nuclear elements. Mobility of OnMITE01 was demonstrated by the presence or absence in O. nubilalis of introns at two different loci. A (GTCT)n tetranucleotide repeat at the 5' ends of OnMITE01 and OnMITE02 are evidence for transposon-mediated movement of lepidopteran microsatellite loci. The number of repetitive elements in lepidopteran genomes will affect genome assembly and marker development. Single-locus sequence tag site markers described here have downstream application for integration within linkage maps and comparative genomic studies.

  3. Metagenome Analyses of Corroded Concrete Wastewater Pipe Biofilms Reveals a Complex Microbial System

    Science.gov (United States)

    Analysis of whole-metagenome pyrosequencing data and 16S rRNA gene clone libraries was used to determine microbial composition and functional genes associated with biomass harvested from crown (top) and invert (bottom) sections of a corroded wastewater pipe. Taxonomic and functio...

  4. Culture-independent discovery of natural products from soil metagenomes.

    Science.gov (United States)

    Katz, Micah; Hover, Bradley M; Brady, Sean F

    2016-03-01

    Bacterial natural products have proven to be invaluable starting points in the development of many currently used therapeutic agents. Unfortunately, traditional culture-based methods for natural product discovery have been deemphasized by pharmaceutical companies due in large part to high rediscovery rates. Culture-independent, or "metagenomic," methods, which rely on the heterologous expression of DNA extracted directly from environmental samples (eDNA), have the potential to provide access to metabolites encoded by a large fraction of the earth's microbial biosynthetic diversity. As soil is both ubiquitous and rich in bacterial diversity, it is an appealing starting point for culture-independent natural product discovery efforts. This review provides an overview of the history of soil metagenome-driven natural product discovery studies and elaborates on the recent development of new tools for sequence-based, high-throughput profiling of environmental samples used in discovering novel natural product biosynthetic gene clusters. We conclude with several examples of these new tools being employed to facilitate the recovery of novel secondary metabolite encoding gene clusters from soil metagenomes and the subsequent heterologous expression of these clusters to produce bioactive small molecules.

  5. Metagenome Skimming of Insect Specimen Pools: Potential for Comparative Genomics.

    Science.gov (United States)

    Linard, Benjamin; Crampton-Platt, Alex; Gillett, Conrad P D T; Timmermans, Martijn J T N; Vogler, Alfried P

    2015-05-14

    Metagenomic analyses are challenging in metazoans, but high-copy number and repeat regions can be assembled from low-coverage sequencing by "genome skimming," which is applied here as a new way of characterizing metagenomes obtained in an ecological or taxonomic context. Illumina shotgun sequencing on two pools of Coleoptera (beetles) of approximately 200 species each were assembled into tens of thousands of scaffolds. Repeated low-coverage sequencing recovered similar scaffold sets consistently, although approximately 70% of scaffolds could not be identified against existing genome databases. Identifiable scaffolds included mitochondrial DNA, conserved sequences with hits to expressed sequence tag and protein databases, and known repeat elements of high and low complexity, including numerous copies of rRNA and histone genes. Assemblies of histones captured a diversity of gene order and primary sequence in Coleoptera. Scaffolds with similarity to multiple sites in available coleopteran genome sequences for Dendroctonus and Tribolium revealed high specificity of scaffolds to either of these genomes, in particular for high-copy number repeats. Numerous "clusters" of scaffolds mapped to the same genomic site revealed intra- and/or intergenomic variation within a metagenome pool. In addition to effect of taxonomic composition of the metagenomes, the number of mapped scaffolds also revealed structural differences between the two reference genomes, although the significance of this striking finding remains unclear. Finally, apparently exogenous sequences were recovered, including potential food plants, fungal pathogens, and bacterial symbionts. The "metagenome skimming" approach is useful for capturing the genomic diversity of poorly studied, species-rich lineages and opens new prospects in environmental genomics.

  6. Functional metagenomics to decipher food-microbe-host crosstalk.

    Science.gov (United States)

    Larraufie, Pierre; de Wouters, Tomas; Potocki-Veronese, Gabrielle; Blottière, Hervé M; Doré, Joël

    2015-02-01

    The recent developments of metagenomics permit an extremely high-resolution molecular scan of the intestinal microbiota giving new insights and opening perspectives for clinical applications. Beyond the unprecedented vision of the intestinal microbiota given by large-scale quantitative metagenomics studies, such as the EU MetaHIT project, functional metagenomics tools allow the exploration of fine interactions between food constituents, microbiota and host, leading to the identification of signals and intimate mechanisms of crosstalk, especially between bacteria and human cells. Cloning of large genome fragments, either from complex intestinal communities or from selected bacteria, allows the screening of these biological resources for bioactivity towards complex plant polymers or functional food such as prebiotics. This permitted identification of novel carbohydrate-active enzyme families involved in dietary fibre and host glycan breakdown, and highlighted unsuspected bacterial players at the top of the intestinal microbial food chain. Similarly, exposure of fractions from genomic and metagenomic clones onto human cells engineered with reporter systems to track modulation of immune response, cell proliferation or cell metabolism has allowed the identification of bioactive clones modulating key cell signalling pathways or the induction of specific genes. This opens the possibility to decipher mechanisms by which commensal bacteria or candidate probiotics can modulate the activity of cells in the intestinal epithelium or even in distal organs such as the liver, adipose tissue or the brain. Hence, in spite of our inability to culture many of the dominant microbes of the human intestine, functional metagenomics open a new window for the exploration of food-microbe-host crosstalk.

  7. Web Resources for Metagenomics Studies

    Institute of Scientific and Technical Information of China (English)

    Pravin Dudhagara; Sunil Bhavsar; Chintan Bhagat; Anjana Ghelani; Shreyas Bhatt; Rajesh Patel

    2015-01-01

    The development of next-generation sequencing (NGS) platforms spawned an enormous volume of data. This explosion in data has unearthed new scalability challenges for existing bioinformatics tools. The analysis of metagenomic sequences using bioinformatics pipelines is complicated by the substantial complexity of these data. In this article, we review several commonly-used online tools for metagenomics data analysis with respect to their quality and detail of analysis using simulated metagenomics data. There are at least a dozen such software tools presently available in the public domain. Among them, MGRAST, IMG/M, and METAVIR are the most well-known tools according to the number of citations by peer-reviewed scientific media up to mid-2015. Here, we describe 12 online tools with respect to their web link, annotation pipelines, clustering methods, online user support, and availability of data storage. We have also done the rating for each tool to screen more potential and preferential tools and evaluated five best tools using synthetic metagenome. The article comprehensively deals with the contemporary problems and the prospects of metagenomics from a bioinformatics viewpoint.

  8. Web Resources for Metagenomics Studies

    Directory of Open Access Journals (Sweden)

    Pravin Dudhagara

    2015-10-01

    Full Text Available The development of next-generation sequencing (NGS platforms spawned an enormous volume of data. This explosion in data has unearthed new scalability challenges for existing bioinformatics tools. The analysis of metagenomic sequences using bioinformatics pipelines is complicated by the substantial complexity of these data. In this article, we review several commonly-used online tools for metagenomics data analysis with respect to their quality and detail of analysis using simulated metagenomics data. There are at least a dozen such software tools presently available in the public domain. Among them, MGRAST, IMG/M, and METAVIR are the most well-known tools according to the number of citations by peer-reviewed scientific media up to mid-2015. Here, we describe 12 online tools with respect to their web link, annotation pipelines, clustering methods, online user support, and availability of data storage. We have also done the rating for each tool to screen more potential and preferential tools and evaluated five best tools using synthetic metagenome. The article comprehensively deals with the contemporary problems and the prospects of metagenomics from a bioinformatics viewpoint.

  9. Web Resources for Metagenomics Studies.

    Science.gov (United States)

    Dudhagara, Pravin; Bhavsar, Sunil; Bhagat, Chintan; Ghelani, Anjana; Bhatt, Shreyas; Patel, Rajesh

    2015-10-01

    The development of next-generation sequencing (NGS) platforms spawned an enormous volume of data. This explosion in data has unearthed new scalability challenges for existing bioinformatics tools. The analysis of metagenomic sequences using bioinformatics pipelines is complicated by the substantial complexity of these data. In this article, we review several commonly-used online tools for metagenomics data analysis with respect to their quality and detail of analysis using simulated metagenomics data. There are at least a dozen such software tools presently available in the public domain. Among them, MGRAST, IMG/M, and METAVIR are the most well-known tools according to the number of citations by peer-reviewed scientific media up to mid-2015. Here, we describe 12 online tools with respect to their web link, annotation pipelines, clustering methods, online user support, and availability of data storage. We have also done the rating for each tool to screen more potential and preferential tools and evaluated five best tools using synthetic metagenome. The article comprehensively deals with the contemporary problems and the prospects of metagenomics from a bioinformatics viewpoint.

  10. Identification of a Potent Allosteric Inhibitor of Human Protein Kinase CK2 by Bacterial Surface Display Library Screening.

    Science.gov (United States)

    Nienberg, Christian; Garmann, Claudia; Gratz, Andreas; Bollacke, Andre; Götz, Claudia; Jose, Joachim

    2017-01-05

    Human protein kinase CK2 has emerged as promising target for the treatment of neoplastic diseases. The vast majority of kinase inhibitors known today target the ATP binding site, which is highly conserved among kinases and hence leads to limited selectivity. In order to identify non-ATP competitive inhibitors, a 12-mer peptide library of 6 × 10⁵ variants was displayed on the surface of E. coli by autodisplay. Screening of this peptide library on variants with affinity to CK2 was performed by fluorophore-conjugated CK2 and subsequent flow cytometry. Single cell sorting of CK2-bound E. coli yielded new peptide variants, which were tested on inhibition of CK2 by a CE-based assay. Peptide B2 (DCRGLIVMIKLH) was the most potent inhibitor of both, CK2 holoenzyme and the catalytic CK2α subunit (IC50 = 0.8 µM). Using different ATP concentrations and different substrate concentrations for IC50 determination, B2 was shown to be neither ATP- nor substrate competitive. By microscale thermophoresis (MST) the KD value of B2 with CK2α was determined to be 2.16 µM, whereas no binding of B2 to CK2β-subunit was detectable. To our surprise, besides inhibition of enzymatic activity, B2 also disturbed the interaction of CK2α with CK2β at higher concentrations (≥25 µM).

  11. Identification of a Potent Allosteric Inhibitor of Human Protein Kinase CK2 by Bacterial Surface Display Library Screening

    Directory of Open Access Journals (Sweden)

    Christian Nienberg

    2017-01-01

    Full Text Available Human protein kinase CK2 has emerged as promising target for the treatment of neoplastic diseases. The vast majority of kinase inhibitors known today target the ATP binding site, which is highly conserved among kinases and hence leads to limited selectivity. In order to identify non-ATP competitive inhibitors, a 12-mer peptide library of 6 × 105 variants was displayed on the surface of E. coli by autodisplay. Screening of this peptide library on variants with affinity to CK2 was performed by fluorophore-conjugated CK2 and subsequent flow cytometry. Single cell sorting of CK2-bound E. coli yielded new peptide variants, which were tested on inhibition of CK2 by a CE-based assay. Peptide B2 (DCRGLIVMIKLH was the most potent inhibitor of both, CK2 holoenzyme and the catalytic CK2α subunit (IC50 = 0.8 µM. Using different ATP concentrations and different substrate concentrations for IC50 determination, B2 was shown to be neither ATP- nor substrate competitive. By microscale thermophoresis (MST the KD value of B2 with CK2α was determined to be 2.16 µM, whereas no binding of B2 to CK2β-subunit was detectable. To our surprise, besides inhibition of enzymatic activity, B2 also disturbed the interaction of CK2α with CK2β at higher concentrations (≥25 µM.

  12. Genome sequences of rare, uncultured bacteria obtained by differential coverage binning of multiple metagenomes

    DEFF Research Database (Denmark)

    Albertsen, Mads; Hugenholtz, Philip; Skarshewski, Adam;

    2013-01-01

    Reference genomes are required to understand the diverse roles of microorganisms in ecology, evolution, human and animal health, but most species remain uncultured. Here we present a sequence composition–independent approach to recover high-quality microbial genomes from deeply sequenced...... metagenomes. Multiple metagenomes of the same community, which differ in relative population abundances, were used to assemble 31 bacterial genomes, including rare (genomes were assembled into complete or near-complete chromosomes....... Four belong to the candidate bacterial phylum TM7 and represent the most complete genomes for this phylum to date (relative abundances, 0.06–1.58%). Reanalysis of published metagenomes reveals that differential coverage binning facilitates recovery of more complete and higher fidelity genome bins than...

  13. Precision Metagenomics: Rapid Metagenomic Analyses for Infectious Disease Diagnostics and Public Health Surveillance

    Science.gov (United States)

    Afshinnekoo, Ebrahim; Chou, Chou; Alexander, Noah; Ahsanuddin, Sofia; Schuetz, Audrey N.; Mason, Christopher E.

    2017-01-01

    Next-generation sequencing (NGS) technologies have ushered in the era of precision medicine, transforming the way we treat cancer patients and diagnose disease. Concomitantly, the advent of these technologies has created a surge of microbiome and metagenomic studies over the last decade, many of which are focused on investigating the host-gene-microbial interactions responsible for the development and spread of infectious diseases, as well as delineating their key role in maintaining health. As we continue to discover more information about the etiology of infectious diseases, the translational potential of metagenomic NGS methods for treatment and rapid diagnosis is becoming abundantly clear. Here, we present a robust protocol for the implementation and application of “precision metagenomics” across various sequencing platforms for clinical samples. Such a pipeline integrates DNA/RNA extraction, library preparation, sequencing, and bioinformatics analyses for taxonomic classification, antimicrobial resistance (AMR) marker screening, and functional analysis (biochemical and metabolic pathway abundance). Moreover, the pipeline has 3 tracks: STAT for results within 24 h; Comprehensive that affords a more in-depth analysis and takes between 5 and 7 d, but offers antimicrobial resistance information; and Targeted, which also requires 5–7 d, but with more sensitive analysis for specific pathogens. Finally, we discuss the challenges that need to be addressed before full integration in the clinical setting.

  14. Identification of the Bacterial Cellular Lipid Fraction by Using Fast GC × GC-MS and Innovative MS Libraries

    Science.gov (United States)

    Mondello, Luigi; Tranchida, Peter Quinto; Purcaro, Giorgia; Fanali, Chiara; Dugo, Paola; La Camera, Erminia; Bisignano, Carlo

    The bacteria fatty acid (FA) profile has been extensively studied for taxonomic classification purposes, since bacteria, in general, contain particular and rare fatty acids, compared to animal and plant tissues. In the last few years, the concern about pathogenic microorganisms used as bioterrorist agents has increased; therefore, rapid methods for the characterization of bacteria are necessary. In the present research, a half-an-hour procedure, to analyze bacteria, was developed: a 2-min one-step sample preparation step, was followed by a relatively fast comprehensive 2D GC-MS separation (25 min). Furthermore, dedicated mass spectrometry libraries were constructed for bacteria and FA identification. Finally, data-processing was carried out with the support of novel comprehensive 2D GC software.

  15. Microbial community profiling of human saliva using shotgun metagenomic sequencing.

    Directory of Open Access Journals (Sweden)

    Nur A Hasan

    Full Text Available Human saliva is clinically informative of both oral and general health. Since next generation shotgun sequencing (NGS is now widely used to identify and quantify bacteria, we investigated the bacterial flora of saliva microbiomes of two healthy volunteers and five datasets from the Human Microbiome Project, along with a control dataset containing short NGS reads from bacterial species representative of the bacterial flora of human saliva. GENIUS, a system designed to identify and quantify bacterial species using unassembled short NGS reads was used to identify the bacterial species comprising the microbiomes of the saliva samples and datasets. Results, achieved within minutes and at greater than 90% accuracy, showed more than 175 bacterial species comprised the bacterial flora of human saliva, including bacteria known to be commensal human flora but also Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, and Gamma proteobacteria. Basic Local Alignment Search Tool (BLASTn analysis in parallel, reported ca. five times more species than those actually comprising the in silico sample. Both GENIUS and BLAST analyses of saliva samples identified major genera comprising the bacterial flora of saliva, but GENIUS provided a more precise description of species composition, identifying to strain in most cases and delivered results at least 10,000 times faster. Therefore, GENIUS offers a facile and accurate system for identification and quantification of bacterial species and/or strains in metagenomic samples.

  16. Screening and Cloning of IMP Cyclohydrolase from a Metagenomic Cosmid Library of a Deep-sea Sediment Sample%深海沉积物微生物宏基因组文库中IMP环水解酶的筛选及基因克隆

    Institute of Scientific and Technical Information of China (English)

    陈兴麟; 赵晶; 徐宪忠; 曾润颖

    2011-01-01

    为了解嘌呤合成途径的关键酶——IMP环水解酶的多样性,从深海沉积物样品中提取总DNA构建Cosmid宏基因组文库,从中筛选出一个具有IMP环水解酶活性的克隆(CAIMP1).通过基因步移的方法从CAIMP1的约35 kb的插入片段中扩增出长度为l 599 bp的完整的IMP环水解酶基因,该基因包含MGS和AICARFT_IMPCHas两个保守结构域.活性检测结果表明CAIMP1克隆的发酵液上清具有较强的IMP环水解酶活性.该酶与数据库中收录的IMP环水解酶的氨基酸序列同源性较低,系统发育分析也表明该酶与其它参照序列亲缘关系较远,表明该序列可能为一个新的IMP环水解酶基因.%IMP cyclohydrolase is a key enzyme in the purine synthesis pathway. A clone (CAIMP1) producing IMP cyclohydrolase activity was isolated from a metagenomic library which was constructed from metagenomic DNA of a deep-sea sediment sample. The gene coding for the IMP cyclohydrolase with the size of 1 599 bp was subcloned from the 35 kb inserted fragment of CAIMP1 by gene walking. Two conserved domains, MGS and AICARFT_IMPCHas, was found in the gene. The CAIMP1 exhibited high activity towards 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR). The alignment and phylogenetic analysis of amino acid sequence of the CAIMP1 IMP cyclohydrolase showed that it had low homology with other referenced sequences in GenBank, indicating that it might be a new sequence. Fig 5, Tab 3, Ref 17

  17. 红树林土壤微生物宏基因组文库的构建及两种新颖的β-葡萄糖苷酶基因的鉴定%Construction of a Mangrove Soil Metagenome Library and Identification of Two Novelβ-Glucosidase Genes

    Institute of Scientific and Technical Information of China (English)

    麦志茂; 苏宏飞; 李丽珍; 张偲

    2014-01-01

    宏基因组技术是挖掘微生物新酶的重要途径。通过构建红树林土壤微生物Fosmid文库,共获得了约100000个阳性克隆,该文库外源插入片段平均长度约为30 kb,文库容量达3 Gb。通过对文库中10000个克隆进行功能筛选,获得了17个具有β-葡萄糖苷酶活性的克隆。对其中2个表达β-葡萄糖苷酶的克隆进行亚克隆,获得2个新颖的β-葡萄糖苷酶的基因,分别命名MhGH3和bgl66。生物信息学分析表明,MhGH3基因由1992个碱基对组成,bgl66基因由2025个碱基对组成。在氨基酸水平上, MhGH3和bgl66编码的蛋白质与GenBank数据库中已知β-葡萄糖苷酶的一致性分别为64%和55%。%Metagenomics provides a means of exploring new enzymes in microorganisms. A mangrove soil metagenome library with 100 000 clones was constructed. The average length of inserted DNA fragment is about 30 kb, and the total capacity of inserted DNA reached about 3 Gb. Seventeen clones withβ-glucosidase activity were detected by screening 10 000 fosmid clones. The subcloning and sequencing revealed two novelβ-glucosidase genes(MhGH3 and bgl66). Bioinformatics analysis indicated that MhGH3 and bgl66 composed of 1 992 bp and 2 025 bp in length, respectively. The deduced amino acid sequence of MhGH3 and bgl66 showed the highest sequence identity of 64%and 55%with the knownβ-glucosidase in GenBank datebase, respectively.

  18. An integrated metagenome and -proteome analysis of the microbial community residing in a biogas production plant.

    Science.gov (United States)

    Ortseifen, Vera; Stolze, Yvonne; Maus, Irena; Sczyrba, Alexander; Bremges, Andreas; Albaum, Stefan P; Jaenicke, Sebastian; Fracowiak, Jochen; Pühler, Alfred; Schlüter, Andreas

    2016-08-10

    To study the metaproteome of a biogas-producing microbial community, fermentation samples were taken from an agricultural biogas plant for microbial cell and protein extraction and corresponding metagenome analyses. Based on metagenome sequence data, taxonomic community profiling was performed to elucidate the composition of bacterial and archaeal sub-communities. The community's cytosolic metaproteome was represented in a 2D-PAGE approach. Metaproteome databases for protein identification were compiled based on the assembled metagenome sequence dataset for the biogas plant analyzed and non-corresponding biogas metagenomes. Protein identification results revealed that the corresponding biogas protein database facilitated the highest identification rate followed by other biogas-specific databases, whereas common public databases yielded insufficient identification rates. Proteins of the biogas microbiome identified as highly abundant were assigned to the pathways involved in methanogenesis, transport and carbon metabolism. Moreover, the integrated metagenome/-proteome approach enabled the examination of genetic-context information for genes encoding identified proteins by studying neighboring genes on the corresponding contig. Exemplarily, this approach led to the identification of a Methanoculleus sp. contig encoding 16 methanogenesis-related gene products, three of which were also detected as abundant proteins within the community's metaproteome. Thus, metagenome contigs provide additional information on the genetic environment of identified abundant proteins.

  19. Metagenomic analysis of uncultured Cytophaga and beta-1,4 glycanases in marine consortia

    Energy Technology Data Exchange (ETDEWEB)

    David Kirchman

    2005-12-15

    Culture-independent studies have shown that microbial consortia in natural environments are incredibly diverse and are dominated by bacteria and archaea substantially different from microbes maintained in pure laboratory cultures. Recent studies indicate, however, that previous culture-independent studies using PCR-based methods have largely overlook an important group of uncultured bacteria, the Cytophagales. These bacteria appear to be abundant in the oceans and probably other oxic environments. Although well known to be active in degradation of structural glycans such as cellulose and chitin, no cellulase or chitinase gene has been sequenced from a Cytophaga, except those recently found by whole genome sequencing of Cytophaga hutchinsonii by the DOE Joint Genome Institute (JGI). We hypothesize that the key to understanding consortia and their function in organic matter mineralization in oxic environments is to focus on uncultured Cytophagales, genes encoding endoglycanases, and other functional genes. The ''metagenomic'' approach used by this project consisted of constructing large insert libraries with DNA directly (no PCR) from uncultured microbial consortia found in the Arctic Ocean and Delaware Estuary. Our results provide insights into new types of bacterial metabolisms which have not considered adequately before, but which may change our views of the global carbon cycle.

  20. Reconciling Phylogeny and Function During Plant Litter Decomposition by High-Throughput Functional Metagenomics

    Science.gov (United States)

    Nyyssonen, M.; Weihe, C.; Goulden, M.; Treseder, K. K.; Martiny, J.; Martiny, A.; Allison, S. D.; Brodie, E. L.

    2012-12-01

    Integrating information on microbial diversity and functionality with ecosystem processes may be critical to predicting how ecosystems respond to environmental change. While theoretical models can be used to link microbial processes to environmental responses and rates, accurate predictions of ecosystem functioning would benefit from detailed information on microbial community composition and function. In this study, our aim was to identify functional traits involved in plant litter decomposition, a model process for carbon cycling, from decomposing plant litter. The overall goal is then to link these traits with individual microbial taxa and use this information to build predictive trait-based models of ecosystem responses to global change. In order to identify activities involved in plant litter decomposition we used automated high-throughput assays for functional screening of metagenomic fosmid libraries prepared from decomposing plant litter. Litter was collected over 15 month period from a global change field experiment undergoing rainfall and nitrogen manipulations. We identified over 600 cellulose, hemicellulose, chitin and starch hydrolyzing clones following screening of over 300,000 clones. The frequency of positive clones was ten times lower during dry season but no significant differences in hit rates were observed between different treatments. The positive clones were shotgun sequenced on the Illumina sequencing platform and the identified hydrolytic genes were shown to represent variety bacterial taxonomic groups including Proteobacteria and Bacteroidetes.

  1. Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes

    DEFF Research Database (Denmark)

    Nielsen, Henrik Bjørn; Almeida, Mathieu; Juncker, Agnieszka

    2014-01-01

    , such as particular bacterial strains or viruses, remains a largely unsolved problem. Here we present a method, based on binning co-abundant genes across a series of metagenomic samples, that enables comprehensive discovery of new microbial organisms, viruses and co-inherited genetic entities and aids assembly...

  2. Comparative analysis of tertiary alcohol esterase activity in bacterial strains isolated from enrichment cultures and from screening strain libraries.

    Science.gov (United States)

    Herter, Susanne; Nguyen, Giang-Son; Thompson, Mark L; Steffen-Munsberg, Fabian; Schauer, Frieder; Bornscheuer, Uwe T; Kourist, Robert

    2011-05-01

    The preparation of enantiopure tertiary alcohols is of great contemporary interest due to the application of these versatile building blocks in organic synthesis and as precursors towards high value pharmaceutical compounds. Herein, we describe two approaches taken towards the discovery of novel biocatalysts for the synthesis of these valuable compounds. The first approach was initiated with screening of 47 bacterial strains for hydrolytic activity towards the simple tertiary alcohol ester tert-butyl acetate. In conjunction, a second method focussed on the isolation of strains competent for growth on tert-butyl acetate as the sole source of carbon and energy. From functional screening, 10 Gram-positive Actinomycetes showed hydrolytic activity, whilst enrichment selection resulted in the identification of 14 active strains, of which five belong to the Gram-negative cell-wall type. Bacterial strains obtained from both approaches were viable for enantioselective hydrolysis of pyridine substituted tertiary alcohol esters in addition to bulky aliphatic and keto-derived substrates from the same class. Activity towards each of the test substrates was uncovered, with promising enantioselectivities of up to E = 71 in the hydrolysis of a para-substituted pyridine tertiary alcohol ester using a strain of Rhodococcus ruber. Interestingly strains of Microbacterium and Alcaligenes sp. gave opposite enantiopreference in the hydrolysis of a meta-substituted pyridine tertiary alcohol ester with E values of 17 and 54. These approaches show that via both possibilities, screening established strain collections and performing enrichment selection, it is possible to identify novel species which show activity towards sterically challenging substrates.

  3. Production of indole antibiotics induced by exogenous gene derived from sponge metagenomes.

    Science.gov (United States)

    Takeshige, Yuya; Egami, Yoko; Wakimoto, Toshiyuki; Abe, Ikuro

    2015-05-01

    Sponge metagenomes are accessible genetic sources containing genes and gene clusters responsible for the biosynthesis of sponge-derived bioactive natural products. In this study, we obtained the clone pDC112, producing turbomycin A and 2,2-di(3-indolyl)-3-indolone, based on the functional screening of the metagenome library derived from the marine sponge Discodermia calyx. The subcloning experiment identified ORF 25, which is homologous to inosine 5'-monophosphate dehydrogenase and required for the production of 2,2-di(3-indolyl)-3-indolone in Escherichia coli.

  4. 白眉长臂猿基因组BAC文库的构建%Construction of Genome Bacterial Artificial Chromosome Library of Hylobates Hoolock

    Institute of Scientific and Technical Information of China (English)

    王起明; 孙烨超; 厉申捷; 叶建平

    2015-01-01

    High quality genomic DNA of Hylobates hoolock was obtained by gentle physical homogenization. The DNA was partially digested with EcoRⅠand EcoRⅠmethylase, and cloned to pCC1BAC vector. The positive clones were stored in 384-well plates. The constructed BAC library consists of 85800 clones. DNA from randomly selected 250 BAC clones was restricted with Not I restriction enzyme and fragments were separated by pulsed field gel electrophoresis. The result shows that the average insert size is estimated as approximately 110 kb, and the ratio of non-recombinant clones is 10. 0%. If the genome size of Hylobates hoolock is 3 ×106 kilo-base, the library could cover 3 times the number of genome.%通过温和的物理方法获得白眉长臂猿高质量的基因组DNA,EcoRⅠ和EcoRⅠ甲基化酶部分酶切后经回收、连接、转化、阳性克隆的保存,构建了含有85800个克隆的全基因组BAC( Bacterial artificial chromosome)文库.随机选取250个BAC克隆进行Not I酶切及脉冲场电泳分析,结果表明该文库的平均插入片段大小为110 kb,非重组克隆(无插入片段)的比率为10.0%.假定白眉长臂猿的基因组大小为3×106 kb,根据文库的平均插入片段大小,则该文库具有3倍的基因组覆盖率.

  5. Shotgun metagenomic sequencing based microbial diversity assessment of Lasundra hot spring, India

    Directory of Open Access Journals (Sweden)

    Amit V. Mangrola

    2015-06-01

    Full Text Available This is the first report on the metagenomic approach for unveiling the microbial diversity of Lasundra hot spring, Gujarat State, India. High-throughput sequencing of community DNA was performed on an Ion Torrent PGM platform. Metagenome consisted of 606,867 sequences represent 98,567,305 bps size with an average length of 162 bps and 46% G + C content. Metagenome sequence information is available at EBI under EBI Metagenomic database with accession no. ERP009313. MG-RAST assisted community analysis revealed that 99.21% sequences were bacterial origin, 0.43% was fit to eukaryotes and 0.11% belongs to archaea. A total of 29 bacterial, 20 eukaryotic and 4 archaeal phyla were detected. Abundant genera were Bacillus (86.7%, Geobacillus (2.4%, Paenibacillus (1.0%, Clostridium (0.7% and Listeria (0.5%, that represent 91.52% in metagenome. In functional analysis, Cluster of Orthologous Group (COG based annotation revealed that 45.4% was metabolism connected and 19.6% falls in poorly characterized group. Subsystem based annotation approach suggests that the 14.0% was carbohydrates, 7.0% was protein metabolism and 3.0% genes for various stress responses together with the versatile presence of commercially useful traits.

  6. Metagenomic Classification and Characterization Marine Actinobacteria from the Gulf of Maine without Representative Genomes

    Science.gov (United States)

    Sachdeva, R.; Heidelberg, J.

    2012-12-01

    Actinobacteria represent one of the largest and most diverse bacterial phyla and unlike most marine prokaryotes are gram-positive. This phylum encompasses a broad range of physiologies, morphologies, and metabolic properties with a broad array of lifestyles. The marine actinobacterial assemblage is dominated by the orders Actinomycetales and Acidimicrobiales (also known as the marine Actinobacteria clade). The Acidimicrobiales bacteria typically outnumber the Actinomycetales bacteria and are mostly represented by the OCS155 group. Although bacteria of the order Acidimicrobiales make up ~7.6% of the 16S matches from the Global Ocean Survey shotgun metagenomic libraries; very little is known about their potential function and role in biogeochemical cycling. Samples were collected from surface seawater samples in the Gulf of Maine (GOM) from the summer and winter of 2006. Sanger sequences were generated from the 0.1-0.8 μm fractions using paired-end medium insert shotgun libraries. The resulting 2.2 Gb were assembled using the Celera Assembler package into 280 Mb of non-redundant scaffolds. Putative actinobacterial assemblies were identified using (1) ribosomal RNA genes (16S and 23S), (2) phylogenetically informative non-ribosomal core genes thought to be resistant to horizontal gene transfer (e.g. RecA and RpoB) and (3) compositional binning using oligonucleotide frequency pattern based hierarchical clustering. Binning resulted in 3.6 Mb (4.2X coverage) of actinobacterial scaffolds that were comprised of 15.1 Mb of unassembled reads. Putative actinobacterial assemblies included both summer and winter reads demonstrating that the Actinobacteria are abundant year round. Classification reveals that all of the sampled Actinobacteria are from the orders Acidimicrobiales and Actinomycetales and are similar to those found in the global ocean. The GOM Actinobacteria show a broad range of G+C % content (32-66%) indicating a high level of genomic diversity. Those assemblies

  7. Functional Intestinal Metagenomics (Chapter 18)

    NARCIS (Netherlands)

    Bogert, van den B.; Leimena, M.M.; Vos, de W.M.; Zoetendal, E.G.; Kleerebezem, M.

    2011-01-01

    The premiere two-volume reference on revelations from studying complex microbial communities in many distinct habitats Metagenomics is an emerging field that has changed the way microbiologists study microorganisms. It involves the genomic analysis of microorganisms by extraction and cloning of DNA

  8. Multivariate analysis of functional metagenomes

    Directory of Open Access Journals (Sweden)

    Elizabeth A. Dinsdale

    2013-04-01

    Full Text Available Metagenomics is a primary tool for the description of microbial and viral communities. The sheer magnitude of the data generated in each metagenome makes identifying key differences in the function and taxonomy between communities difficult to elucidate. Here we discuss the application of seven different data mining and statistical analyses by comparing and contrasting the metabolic functions of 212 microbial metagenomes within and between 10 environments. Not all approaches are appropriate for all questions, and researchers should decide which approach addresses their questions. This work demonstrated the use of each approach: for example, random forests provided a robust and enlightening description of both the clustering of metagenomes and the metabolic processes that were important in separating microbial communities from different environments. All analyses identified that the presence of phage genes within the microbial community was a predictor of whether the microbial community was host associated or free living. Several analyses identified the subtle differences that occur with environments, such as those seen in different regions of the marine environment.

  9. Estimating richness from phage metagenomes

    Science.gov (United States)

    Bacteriophages are important drivers of ecosystem functions, yet little is known about the vast majority of phages. Phage metagenomics, or the study of the collective genome of an assemblage of phages, enables the investigation of broad ecological questions in phage communities. One ecological cha...

  10. 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.

  11. Bioprospecting metagenomics of decaying wood: mining for new glycoside hydrolases

    Energy Technology Data Exchange (ETDEWEB)

    Li L. L.; van der Lelie D.; Taghavi, S.; McCorkle, S. M.; Zhang, Y.-B.; Blewitt, M. G.; Brunecky, R.; Adney, W. S.; Himmel, M. E.; Brumm, P.; Drinkwater, C.; Mead, D. A.; Tringe, S. G.

    2011-08-01

    To efficiently deconstruct recalcitrant plant biomass to fermentable sugars in industrial processes, biocatalysts of higher performance and lower cost are required. The genetic diversity found in the metagenomes of natural microbial biomass decay communities may harbor such enzymes. Our goal was to discover and characterize new glycoside hydrolases (GHases) from microbial biomass decay communities, especially those from unknown or never previously cultivated microorganisms. From the metagenome sequences of an anaerobic microbial community actively decaying poplar biomass, we identified approximately 4,000 GHase homologs. Based on homology to GHase families/activities of interest and the quality of the sequences, candidates were selected for full-length cloning and subsequent expression. As an alternative strategy, a metagenome expression library was constructed and screened for GHase activities. These combined efforts resulted in the cloning of four novel GHases that could be successfully expressed in Escherichia coli. Further characterization showed that two enzymes showed significant activity on p-nitrophenyl-{alpha}-L-arabinofuranoside, one enzyme had significant activity against p-nitrophenyl-{beta}-D-glucopyranoside, and one enzyme showed significant activity against p-nitrophenyl-{beta}-D-xylopyranoside. Enzymes were also tested in the presence of ionic liquids. Metagenomics provides a good resource for mining novel biomass degrading enzymes and for screening of cellulolytic enzyme activities. The four GHases that were cloned may have potential application for deconstruction of biomass pretreated with ionic liquids, as they remain active in the presence of up to 20% ionic liquid (except for 1-ethyl-3-methylimidazolium diethyl phosphate). Alternatively, ionic liquids might be used to immobilize or stabilize these enzymes for minimal solvent processing of biomass.

  12. Bioprospecting metagenomics of decaying wood: mining for new glycoside hydrolases

    Directory of Open Access Journals (Sweden)

    Li Luen-Luen

    2011-08-01

    Full Text Available Abstract Background To efficiently deconstruct recalcitrant plant biomass to fermentable sugars in industrial processes, biocatalysts of higher performance and lower cost are required. The genetic diversity found in the metagenomes of natural microbial biomass decay communities may harbor such enzymes. Our goal was to discover and characterize new glycoside hydrolases (GHases from microbial biomass decay communities, especially those from unknown or never previously cultivated microorganisms. Results From the metagenome sequences of an anaerobic microbial community actively decaying poplar biomass, we identified approximately 4,000 GHase homologs. Based on homology to GHase families/activities of interest and the quality of the sequences, candidates were selected for full-length cloning and subsequent expression. As an alternative strategy, a metagenome expression library was constructed and screened for GHase activities. These combined efforts resulted in the cloning of four novel GHases that could be successfully expressed in Escherichia coli. Further characterization showed that two enzymes showed significant activity on p-nitrophenyl-α-L-arabinofuranoside, one enzyme had significant activity against p-nitrophenyl-β-D-glucopyranoside, and one enzyme showed significant activity against p-nitrophenyl-β-D-xylopyranoside. Enzymes were also tested in the presence of ionic liquids. Conclusions Metagenomics provides a good resource for mining novel biomass degrading enzymes and for screening of cellulolytic enzyme activities. The four GHases that were cloned may have potential application for deconstruction of biomass pretreated with ionic liquids, as they remain active in the presence of up to 20% ionic liquid (except for 1-ethyl-3-methylimidazolium diethyl phosphate. Alternatively, ionic liquids might be used to immobilize or stabilize these enzymes for minimal solvent processing of biomass.

  13. PhylOTU: a high-throughput procedure quantifies microbial community diversity and resolves novel taxa from metagenomic data.

    Directory of Open Access Journals (Sweden)

    Thomas J Sharpton

    Full Text Available Microbial diversity is typically characterized by clustering ribosomal RNA (SSU-rRNA sequences into operational taxonomic units (OTUs. Targeted sequencing of environmental SSU-rRNA markers via PCR may fail to detect OTUs due to biases in priming and amplification. Analysis of shotgun sequenced environmental DNA, known as metagenomics, avoids amplification bias but generates fragmentary, non-overlapping sequence reads that cannot be clustered by existing OTU-finding methods. To circumvent these limitations, we developed PhylOTU, a computational workflow that identifies OTUs from metagenomic SSU-rRNA sequence data through the use of phylogenetic principles and probabilistic sequence profiles. Using simulated metagenomic data, we quantified the accuracy with which PhylOTU clusters reads into OTUs. Comparisons of PCR and shotgun sequenced SSU-rRNA markers derived from the global open ocean revealed that while PCR libraries identify more OTUs per sequenced residue, metagenomic libraries recover a greater taxonomic diversity of OTUs. In addition, we discover novel species, genera and families in the metagenomic libraries, including OTUs from phyla missed by analysis of PCR sequences. Taken together, these results suggest that PhylOTU enables characterization of part of the biosphere currently hidden from PCR-based surveys of diversity?

  14. PhylOTU: a high-throughput procedure quantifies microbial community diversity and resolves novel taxa from metagenomic data.

    Science.gov (United States)

    Sharpton, Thomas J; Riesenfeld, Samantha J; Kembel, Steven W; Ladau, Joshua; O'Dwyer, James P; Green, Jessica L; Eisen, Jonathan A; Pollard, Katherine S

    2011-01-20

    Microbial diversity is typically characterized by clustering ribosomal RNA (SSU-rRNA) sequences into operational taxonomic units (OTUs). Targeted sequencing of environmental SSU-rRNA markers via PCR may fail to detect OTUs due to biases in priming and amplification. Analysis of shotgun sequenced environmental DNA, known as metagenomics, avoids amplification bias but generates fragmentary, non-overlapping sequence reads that cannot be clustered by existing OTU-finding methods. To circumvent these limitations, we developed PhylOTU, a computational workflow that identifies OTUs from metagenomic SSU-rRNA sequence data through the use of phylogenetic principles and probabilistic sequence profiles. Using simulated metagenomic data, we quantified the accuracy with which PhylOTU clusters reads into OTUs. Comparisons of PCR and shotgun sequenced SSU-rRNA markers derived from the global open ocean revealed that while PCR libraries identify more OTUs per sequenced residue, metagenomic libraries recover a greater taxonomic diversity of OTUs. In addition, we discover novel species, genera and families in the metagenomic libraries, including OTUs from phyla missed by analysis of PCR sequences. Taken together, these results suggest that PhylOTU enables characterization of part of the biosphere currently hidden from PCR-based surveys of diversity?

  15. Marine metagenomics: strategies for the discovery of novel enzymes with biotechnological applications from marine environments

    Directory of Open Access Journals (Sweden)

    Dobson Alan DW

    2008-08-01

    Full Text Available Abstract Metagenomic based strategies have previously been successfully employed as powerful tools to isolate and identify enzymes with novel biocatalytic activities from the unculturable component of microbial communities from various terrestrial environmental niches. Both sequence based and function based screening approaches have been employed to identify genes encoding novel biocatalytic activities and metabolic pathways from metagenomic libraries. While much of the focus to date has centred on terrestrial based microbial ecosystems, it is clear that the marine environment has enormous microbial biodiversity that remains largely unstudied. Marine microbes are both extremely abundant and diverse; the environments they occupy likewise consist of very diverse niches. As culture-dependent methods have thus far resulted in the isolation of only a tiny percentage of the marine microbiota the application of metagenomic strategies holds great potential to study and exploit the enormous microbial biodiversity which is present within these marine environments.

  16. Challenges of the Unknown: Clinical Application of Microbial Metagenomics

    Directory of Open Access Journals (Sweden)

    Graham Rose

    2015-01-01

    Full Text Available Availability of fast, high throughput and low cost whole genome sequencing holds great promise within public health microbiology, with applications ranging from outbreak detection and tracking transmission events to understanding the role played by microbial communities in health and disease. Within clinical metagenomics, identifying microorganisms from a complex and host enriched background remains a central computational challenge. As proof of principle, we sequenced two metagenomic samples, a known viral mixture of 25 human pathogens and an unknown complex biological model using benchtop technology. The datasets were then analysed using a bioinformatic pipeline developed around recent fast classification methods. A targeted approach was able to detect 20 of the viruses against a background of host contamination from multiple sources and bacterial contamination. An alternative untargeted identification method was highly correlated with these classifications, and over 1,600 species were identified when applied to the complex biological model, including several species captured at over 50% genome coverage. In summary, this study demonstrates the great potential of applying metagenomics within the clinical laboratory setting and that this can be achieved using infrastructure available to nondedicated sequencing centres.

  17. [Mini review] metagenomic studies of the Red Sea

    KAUST Repository

    Behzad, Hayedeh

    2015-10-23

    Metagenomics has significantly advanced the field of marine microbial ecology, revealing the vast diversity of previously unknown microbial life forms in different marine niches. The tremendous amount of data generated has enabled identification of a large number of microbial genes (metagenomes), their community interactions, adaptation mechanisms, and their potential applications in pharmaceutical and biotechnology-based industries. Comparative metagenomics reveals that microbial diversity is a function of the local environment, meaning that unique or unusual environments typically harbor novel microbial species with unique genes and metabolic pathways. The Red Sea has an abundance of unique characteristics; however, its microbiota is one of the least studied amongst marine environments. The Red Sea harbors approximately 25 hot anoxic brine pools, plus a vibrant coral reef ecosystem. Physiochemical studies describe the Red Sea as an oligotrophic environment that contains one of the warmest and saltiest waters in the world with year-round high UV radiations. These characteristics are believed to have shaped the evolution of microbial communities in the Red Sea. Over-representation of genes involved in DNA repair, high-intensity light responses, and osmolyte C1 oxidation were found in the Red Sea metagenomic databases suggesting acquisition of specific environmental adaptation by the Red Sea microbiota. The Red Sea brine pools harbor a diverse range of halophilic and thermophilic bacterial and archaeal communities, which are potential sources of enzymes for pharmaceutical and biotechnology-based application. Understanding the mechanisms of these adaptations and their function within the larger ecosystem could also prove useful in light of predicted global warming scenarios where global ocean temperatures are expected to rise by 1–3 °C in the next few decades. In this review, we provide an overview of the published metagenomic studies that were conducted in the

  18. An application of statistics to comparative metagenomics

    Directory of Open Access Journals (Sweden)

    Rohwer Forest

    2006-03-01

    Full Text Available Abstract Background Metagenomics, sequence analyses of genomic DNA isolated directly from the environments, can be used to identify organisms and model community dynamics of a particular ecosystem. Metagenomics also has the potential to identify significantly different metabolic potential in different environments. Results Here we use a statistical method to compare curated subsystems, to predict the physiology, metabolism, and ecology from metagenomes. This approach can be used to identify those subsystems that are significantly different between metagenome sequences. Subsystems that were overrepresented in the Sargasso Sea and Acid Mine Drainage metagenome when compared to non-redundant databases were identified. Conclusion The methodology described herein applies statistics to the comparisons of metabolic potential in metagenomes. This analysis reveals those subsystems that are more, or less, represented in the different environments that are compared. These differences in metabolic potential lead to several testable hypotheses about physiology and metabolism of microbes from these ecosystems.

  19. Biofilm-growing bacteria involved in the corrosion of concrete wastewater pipes: protocols for comparative metagenomic analyses.

    Science.gov (United States)

    Gomez-Alvarez, Vicente

    2014-01-01

    Advances in high-throughput next-generation sequencing (NGS) technology for direct sequencing of environmental DNA (i.e., shotgun metagenomics) are transforming the field of microbiology. NGS technologies are now regularly being applied in comparative metagenomic studies, which provide the data for functional annotations, taxonomic comparisons, community profile, and metabolic reconstructions. For example, comparative metagenomic analysis of corroded pipes unveiled novel insights on the bacterial populations associated with the sulfur and nitrogen cycle, which may be directly or indirectly implicated in concrete wastewater pipe corrosion. The objective of this chapter is to describe the steps involved in the taxonomic and functional analysis of metagenome datasets from biofilm involved in microbial-induced concrete corrosion (MICC).

  20. Metagenomic Sequencing for Surveillance of Food- and Waterborne Viral Diseases

    Science.gov (United States)

    Nieuwenhuijse, David F.; Koopmans, Marion P. G.

    2017-01-01

    A plethora of viruses can be transmitted by the food- and waterborne route. However, their recognition is challenging because of the variety of viruses, heterogeneity of symptoms, the lack of awareness of clinicians, and limited surveillance efforts. Classical food- and waterborne viral disease outbreaks are mainly caused by caliciviruses, but the source of the virus is often not known and the foodborne mode of transmission is difficult to discriminate from human-to-human transmission. Atypical food- and waterborne viral disease can be caused by viruses such as hepatitis A and hepatitis E. In addition, a source of novel emerging viruses with a potential to spread via the food- and waterborne route is the repeated interaction of humans with wildlife. Wildlife-to-human adaptation may give rise to self- limiting outbreaks in some cases, but when fully adjusted to the human host can be devastating. Metagenomic sequencing has been investigated as a promising solution for surveillance purposes as it detects all viruses in a single protocol, delivers additional genomic information for outbreak tracing, and detects novel unknown viruses. Nevertheless, several issues must be addressed to apply metagenomic sequencing in surveillance. First, sample preparation is difficult since the genomic material of viruses is generally overshadowed by host- and bacterial genomes. Second, several data analysis issues hamper the efficient, robust, and automated processing of metagenomic data. Third, interpretation of metagenomic data is hard, because of the lack of general knowledge of the virome in the food chain and the environment. Further developments in virus-specific nucleic acid extraction methods, bioinformatic data processing applications, and unifying data visualization tools are needed to gain insightful surveillance knowledge from suspect food samples. PMID:28261185

  1. [Meta-Mesh: metagenomic data analysis system].

    Science.gov (United States)

    Su, Xiaoquan; Song, Baoxing; Wang, Xuetao; Ma, Xinle; Xu, Jian; Ning, Kang

    2014-01-01

    With the current accumulation of metagenome data, it is possible to build an integrated platform for processing of rigorously selected metagenomic samples (also referred as "metagenomic communities" here) of interests. Any metagenomic samples could then be searched against this database to find the most similar sample(s). However, on one hand, current databases with a large number of metagenomic samples mostly serve as data repositories but not well annotated database, and only offer few functions for analysis. On the other hand, the few available methods to measure the similarity of metagenomic data could only compare a few pre-defined set of metagenome. It has long been intriguing scientists to effectively calculate similarities between microbial communities in a large repository, to examine how similar these samples are and to find the correlation of the meta-information of these samples. In this work we propose a novel system, Meta-Mesh, which includes a metagenomic database and its companion analysis platform that could systematically and efficiently analyze, compare and search similar metagenomic samples. In the database part, we have collected more than 7 000 high quality and well annotated metagenomic samples from the public domain and in-house facilities. The analysis platform supplies a list of online tools which could accept metagenomic samples, build taxonomical annotations, compare sample in multiple angle, and then search for similar samples against its database by a fast indexing strategy and scoring function. We also used case studies of "database search for identification" and "samples clustering based on similarity matrix" using human-associated habitat samples to demonstrate the performance of Meta-Mesh in metagenomic analysis. Therefore, Meta-Mesh would serve as a database and data analysis system to quickly parse and identify similar

  2. Genomic libraries: I. Construction and screening of fosmid genomic libraries.

    Science.gov (United States)

    Quail, Mike A; Matthews, Lucy; Sims, Sarah; Lloyd, Christine; Beasley, Helen; Baxter, Simon W

    2011-01-01

    Large insert genome libraries have been a core resource required to sequence genomes, analyze haplotypes, and aid gene discovery. While next generation sequencing technologies are revolutionizing the field of genomics, traditional genome libraries will still be required for accurate genome assembly. Their utility is also being extended to functional studies for understanding DNA regulatory elements. Here, we present a detailed method for constructing genomic fosmid libraries, testing for common contaminants, gridding the library to nylon membranes, then hybridizing the library membranes with a radiolabeled probe to identify corresponding genomic clones. While this chapter focuses on fosmid libraries, many of these steps can also be applied to bacterial artificial chromosome libraries.

  3. Whole-Genome Enrichment Provides Deep Insights into Vibrio cholerae Metagenome from an African River.

    Science.gov (United States)

    Vezzulli, L; Grande, C; Tassistro, G; Brettar, I; Höfle, M G; Pereira, R P A; Mushi, D; Pallavicini, A; Vassallo, P; Pruzzo, C

    2016-11-25

    The detection and typing of Vibrio cholerae in natural aquatic environments encounter major methodological challenges related to the fact that the bacterium is often present in environmental matrices at very low abundance in nonculturable state. This study applied, for the first time to our knowledge, a whole-genome enrichment (WGE) and next-generation sequencing (NGS) approach for direct genotyping and metagenomic analysis of low abundant V. cholerae DNA (V. cholerae metagenomic DNA via hybridization. An enriched V. cholerae metagenome library was generated and sequenced on an Illumina MiSeq platform. Up to 1.8 × 10(7) bp (4.5× mean read depth) were found to map against V. cholerae reference genome sequences representing an increase of about 2500 times in target DNA coverage compared to theoretical calculations of performance for shotgun metagenomics. Analysis of metagenomic data revealed the presence of several V. cholerae virulence and virulence associated genes in river water including major virulence regions (e.g. CTX prophage and Vibrio pathogenicity island-1) and genetic markers of epidemic strains (e.g. O1-antigen biosynthesis gene cluster) that were not detectable by standard culture and molecular techniques. Overall, besides providing a powerful tool for direct genotyping of V. cholerae in complex environmental matrices, this study provides a 'proof of concept' on the methodological gap that might currently preclude a more comprehensive understanding of toxigenic V. cholerae emergence from natural aquatic environments.

  4. Expanding the Repertoire of Carbapenem-Hydrolyzing Metallo-ß-Lactamases by Functional Metagenomic Analysis of Soil Microbiota

    DEFF Research Database (Denmark)

    Gudeta, Dereje D.; Bortolaia, Valeria; Pollini, Simona;

    2016-01-01

    , resulting in detectable imipenem-hydrolyzing activity and significantly increased MICs of clinically relevant ss-lactams. Interestingly, the MBLs yielded by functional metagenomics generally differed from those detected in the same soil samples by antibiotic selective culture, showing that the two......, diversity and functionality of carbapenemase-encoding genes in soil microbiota by functional metagenomics. Ten plasmid libraries were generated by cloning metagenomic DNA from agricultural (n = 6) and grassland (n = 4) soil into Escherichia coli. The libraries were cultured on amoxicillin-containing agar...... as metallo-beta-lactamases (MBLs), were identified in six soil samples, including two subclass B1 (GRD23-1 and SPN79-1) and seven subclass B3 (CRD3-1, PEDO-1, GRD33-1, ESP-2, ALG6-1, ALG11-1, and DHT2-1). Except PEDO-1 and ESP-2, these enzymes were distantly related to any previously described MBLs (33 to 59...

  5. Marine metagenomics as a source for bioprospecting

    KAUST Repository

    Kodzius, Rimantas

    2015-08-12

    This review summarizes usage of genome-editing technologies for metagenomic studies; these studies are used to retrieve and modify valuable microorganisms for production, particularly in marine metagenomics. Organisms may be cultivable or uncultivable. Metagenomics is providing especially valuable information for uncultivable samples. The novel genes, pathways and genomes can be deducted. Therefore, metagenomics, particularly genome engineering and system biology, allows for the enhancement of biological and chemical producers and the creation of novel bioresources. With natural resources rapidly depleting, genomics may be an effective way to efficiently produce quantities of known and novel foods, livestock feed, fuels, pharmaceuticals and fine or bulk chemicals.

  6. Marine metagenomics as a source for bioprospecting.

    Science.gov (United States)

    Kodzius, Rimantas; Gojobori, Takashi

    2015-12-01

    This review summarizes usage of genome-editing technologies for metagenomic studies; these studies are used to retrieve and modify valuable microorganisms for production, particularly in marine metagenomics. Organisms may be cultivable or uncultivable. Metagenomics is providing especially valuable information for uncultivable samples. The novel genes, pathways and genomes can be deducted. Therefore, metagenomics, particularly genome engineering and system biology, allows for the enhancement of biological and chemical producers and the creation of novel bioresources. With natural resources rapidly depleting, genomics may be an effective way to efficiently produce quantities of known and novel foods, livestock feed, fuels, pharmaceuticals and fine or bulk chemicals.

  7. Exploration of noncoding sequences in metagenomes.

    Directory of Open Access Journals (Sweden)

    Fabián Tobar-Tosse

    Full Text Available Environment-dependent genomic features have been defined for different metagenomes, whose genes and their associated processes are related to specific environments. Identification of ORFs and their functional categories are the most common methods for association between functional and environmental features. However, this analysis based on finding ORFs misses noncoding sequences and, therefore, some metagenome regulatory or structural information could be discarded. In this work we analyzed 23 whole metagenomes, including coding and noncoding sequences using the following sequence patterns: (G+C content, Codon Usage (Cd, Trinucleotide Usage (Tn, and functional assignments for ORF prediction. Herein, we present evidence of a high proportion of noncoding sequences discarded in common similarity-based methods in metagenomics, and the kind of relevant information present in those. We found a high density of trinucleotide repeat sequences (TRS in noncoding sequences, with a regulatory and adaptive function for metagenome communities. We present associations between trinucleotide values and gene function, where metagenome clustering correlate with microorganism adaptations and kinds of metagenomes. We propose here that noncoding sequences have relevant information to describe metagenomes that could be considered in a whole metagenome analysis in order to improve their organization, classification protocols, and their relation with the environment.

  8. Metagenomic Assembly: Overview, Challenges and Applications

    Science.gov (United States)

    Ghurye, Jay S.; Cepeda-Espinoza, Victoria; Pop, Mihai

    2016-01-01

    Advances in sequencing technologies have led to the increased use of high throughput sequencing in characterizing the microbial communities associated with our bodies and our environment. Critical to the analysis of the resulting data are sequence assembly algorithms able to reconstruct genes and organisms from complex mixtures. Metagenomic assembly involves new computational challenges due to the specific characteristics of the metagenomic data. In this survey, we focus on major algorithmic approaches for genome and metagenome assembly, and discuss the new challenges and opportunities afforded by this new field. We also review several applications of metagenome assembly in addressing interesting biological problems. PMID:27698619

  9. Metagenomic approaches for exploiting uncultivated bacteria as a resource for novel biosynthetic enzymology.

    Science.gov (United States)

    Wilson, Micheal C; Piel, Jörn

    2013-05-23

    Most biologically active microbial natural products are known from strains that can be isolated and cultivated in the laboratory. However, the genomics era has revealed that cultured bacteria represent a mere fraction of total estimated bacterial biodiversity. With the development of community genomics, termed metagenomics, the uncultivated majority became accessible for functional analysis. Through metagenomic studies, novel biocatalysts and biosynthetic pathways are being discovered at a pace previously not possible using traditional molecular biology techniques. Additionally, the study of uncultivated bacteria has provided valuable insights into previously overlooked biocatalysts from cultured strains. This perspective highlights recent discoveries from metagenomics of uncultivated bacteria and discusses the impact of those findings on the field of natural products.

  10. Metagenomics as a tool to obtain full genomes of process-critical bacteria in engineered systems

    DEFF Research Database (Denmark)

    Albertsen, Mads; Hugenholtz, Philip; Tyson, Gene W.;

    Bacteria play a pivotal role in engineered systems such as wastewater treatment plants. Obtaining genomes of the bacteria provides the genetic potential of the system and also allows studies of in situ functions through transcriptomics and proteomics. Hence, it enables correlations of operational...... parameters with functions of specific bacteria within the ecosystems in order to decipher principles that might be used to control and predict ecosystem performance. The main bottleneck in obtaining genomes from the environment is that the vast majority of bacteria are not readily cultured. Metagenomics......, the sequencing of bulk genomic DNA from environmental samples, has the potential to provide genomes of this uncultured majority. However, so far only few bacterial genomes have been obtained from metagenomic data. In this study we present a new approach to obtain individual genomes from metagenomes. We deeply...

  11. Functional metagenomics of extreme environments.

    Science.gov (United States)

    Mirete, Salvador; Morgante, Verónica; González-Pastor, José Eduardo

    2016-04-01

    The bioprospecting of enzymes that operate under extreme conditions is of particular interest for many biotechnological and industrial processes. Nevertheless, there is a considerable limitation to retrieve novel enzymes as only a small fraction of microorganisms derived from extreme environments can be cultured under standard laboratory conditions. Functional metagenomics has the advantage of not requiring the cultivation of microorganisms or previous sequence information to known genes, thus representing a valuable approach for mining enzymes with new features. In this review, we summarize studies showing how functional metagenomics was employed to retrieve genes encoding for proteins involved not only in molecular adaptation and resistance to extreme environmental conditions but also in other enzymatic activities of biotechnological interest.

  12. Growth dynamics of gut microbiota in health and disease inferred from single metagenomic samples

    Science.gov (United States)

    Avnit-Sagi, Tali; Pompan-Lotan, Maya; Matot, Elad; Jona, Ghil; Harmelin, Alon; Cohen, Nadav; Sirota-Madi, Alexandra; Thaiss, Christoph A.; Pevsner-Fischer, Meirav; Sorek, Rotem; Xavier, Ramnik; Elinav, Eran; Segal, Eran

    2016-01-01

    Metagenomic sequencing increased our understanding of the role of the microbiome in health and disease, yet it only provides a snapshot of a highly dynamic ecosystem. Here, we show that the pattern of metagenomic sequencing read coverage for different microbial genomes contains a single trough and a single peak, the latter coinciding with the bacterial origin of replication. Furthermore, the ratio of sequencing coverage between the peak and trough provides a quantitative measure of a species' growth rate. We demonstrate this in vitro and in vivo, under different growth conditions, and in complex bacterial communities. For several bacterial species, peak to trough coverage ratios, but not relative abundances, correlated with the manifestation of inflammatory bowel disease and type II diabetes. PMID:26229116

  13. Filtration and Normalization of Sequencing Read Data in Whole-Metagenome Shotgun Samples

    Science.gov (United States)

    Chouvarine, Philippe; Wiehlmann, Lutz; Moran Losada, Patricia; DeLuca, David S.; Tümmler, Burkhard

    2016-01-01

    Ever-increasing affordability of next-generation sequencing makes whole-metagenome sequencing an attractive alternative to traditional 16S rDNA, RFLP, or culturing approaches for the analysis of microbiome samples. The advantage of whole-metagenome sequencing is that it allows direct inference of the metabolic capacity and physiological features of the studied metagenome without reliance on the knowledge of genotypes and phenotypes of the members of the bacterial community. It also makes it possible to overcome problems of 16S rDNA sequencing, such as unknown copy number of the 16S gene and lack of sufficient sequence similarity of the “universal” 16S primers to some of the target 16S genes. On the other hand, next-generation sequencing suffers from biases resulting in non-uniform coverage of the sequenced genomes. To overcome this difficulty, we present a model of GC-bias in sequencing metagenomic samples as well as filtration and normalization techniques necessary for accurate quantification of microbial organisms. While there has been substantial research in normalization and filtration of read-count data in such techniques as RNA-seq or Chip-seq, to our knowledge, this has not been the case for the field of whole-metagenome shotgun sequencing. The presented methods assume that complete genome references are available for most microorganisms of interest present in metagenomic samples. This is often a valid assumption in such fields as medical diagnostics of patient microbiota. Testing the model on two validation datasets showed four-fold reduction in root-mean-square error compared to non-normalized data in both cases. The presented methods can be applied to any pipeline for whole metagenome sequencing analysis relying on complete microbial genome references. We demonstrate that such pre-processing reduces the number of false positive hits and increases accuracy of abundance estimates. PMID:27760173

  14. Metagenomic Analysis of Kimchi, a Traditional Korean Fermented Food ▿ †

    Science.gov (United States)

    Jung, Ji Young; Lee, Se Hee; Kim, Jeong Myeong; Park, Moon Su; Bae, Jin-Woo; Hahn, Yoonsoo; Madsen, Eugene L.; Jeon, Che Ok

    2011-01-01

    Kimchi, a traditional food in the Korean culture, is made from vegetables by fermentation. In this study, metagenomic approaches were used to monitor changes in bacterial populations, metabolic potential, and overall genetic features of the microbial community during the 29-day fermentation process. Metagenomic DNA was extracted from kimchi samples obtained periodically and was sequenced using a 454 GS FLX Titanium system, which yielded a total of 701,556 reads, with an average read length of 438 bp. Phylogenetic analysis based on 16S rRNA genes from the metagenome indicated that the kimchi microbiome was dominated by members of three genera: Leuconostoc, Lactobacillus, and Weissella. Assignment of metagenomic sequences to SEED categories of the Metagenome Rapid Annotation using Subsystem Technology (MG-RAST) server revealed a genetic profile characteristic of heterotrophic lactic acid fermentation of carbohydrates, which was supported by the detection of mannitol, lactate, acetate, and ethanol as fermentation products. When the metagenomic reads were mapped onto the database of completed genomes, the Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 and Lactobacillus sakei subsp. sakei 23K genomes were highly represented. These same two genera were confirmed to be important in kimchi fermentation when the majority of kimchi metagenomic sequences showed very high identity to Leuconostoc mesenteroides and Lactobacillus genes. Besides microbial genome sequences, a surprisingly large number of phage DNA sequences were identified from the cellular fractions, possibly indicating that a high proportion of cells were infected by bacteriophages during fermentation. Overall, these results provide insights into the kimchi microbial community and also shed light on fermentation processes carried out broadly by complex microbial communities. PMID:21317261

  15. Subtractive assembly for comparative metagenomics, and its application to type 2 diabetes metagenomes.

    Science.gov (United States)

    Wang, Mingjie; Doak, Thomas G; Ye, Yuzhen

    2015-11-02

    Comparative metagenomics remains challenging due to the size and complexity of metagenomic datasets. Here we introduce subtractive assembly, a de novo assembly approach for comparative metagenomics that directly assembles only the differential reads that distinguish between two groups of metagenomes. Using simulated datasets, we show it improves both the efficiency of the assembly and the assembly quality of the differential genomes and genes. Further, its application to type 2 diabetes (T2D) metagenomic datasets reveals clear signatures of the T2D gut microbiome, revealing new phylogenetic and functional features of the gut microbial communities associated with T2D.

  16. Metagenomic analysis reveals presence of Treponema denticola in a tissue biopsy of the Iceman.

    Directory of Open Access Journals (Sweden)

    Frank Maixner

    Full Text Available Ancient hominoid genome studies can be regarded by definition as metagenomic analyses since they represent a mixture of both hominoid and microbial sequences in an environment. Here, we report the molecular detection of the oral spirochete Treponema denticola in ancient human tissue biopsies of the Iceman, a 5,300-year-old Copper Age natural ice mummy. Initially, the metagenomic data of the Iceman's genomic survey was screened for bacterial ribosomal RNA (rRNA specific reads. Through ranking the reads by abundance a relatively high number of rRNA reads most similar to T. denticola was detected. Mapping of the metagenome sequences against the T. denticola genome revealed additional reads most similar to this opportunistic pathogen. The DNA damage pattern of specifically mapped reads suggests an ancient origin of these sequences. The haematogenous spread of bacteria of the oral microbiome often reported in the recent literature could already explain the presence of metagenomic reads specific for T. denticola in the Iceman's bone biopsy. We extended, however, our survey to an Iceman gingival tissue sample and a mouth swab sample and could thereby detect T. denticola and Porphyrimonas gingivalis, another important member of the human commensal oral microflora. Taken together, this study clearly underlines the opportunity to detect disease-associated microorganisms when applying metagenomics-enabled approaches on datasets of ancient human remains.

  17. Exploring neighborhoods in the metagenome universe.

    Science.gov (United States)

    Aßhauer, Kathrin P; Klingenberg, Heiner; Lingner, Thomas; Meinicke, Peter

    2014-07-14

    The variety of metagenomes in current databases provides a rapidly growing source of information for comparative studies. However, the quantity and quality of supplementary metadata is still lagging behind. It is therefore important to be able to identify related metagenomes by means of the available sequence data alone. We have studied efficient sequence-based methods for large-scale identification of similar metagenomes within a database retrieval context. In a broad comparison of different profiling methods we found that vector-based distance measures are well-suitable for the detection of metagenomic neighbors. Our evaluation on more than 1700 publicly available metagenomes indicates that for a query metagenome from a particular habitat on average nine out of ten nearest neighbors represent the same habitat category independent of the utilized profiling method or distance measure. While for well-defined labels a neighborhood accuracy of 100% can be achieved, in general the neighbor detection is severely affected by a natural overlap of manually annotated categories. In addition, we present results of a novel visualization method that is able to reflect the similarity of metagenomes in a 2D scatter plot. The visualization method shows a similarly high accuracy in the reduced space as compared with the high-dimensional profile space. Our study suggests that for inspection of metagenome neighborhoods the profiling methods and distance measures can be chosen to provide a convenient interpretation of results in terms of the underlying features. Furthermore, supplementary metadata of metagenome samples in the future needs to comply with readily available ontologies for fine-grained and standardized annotation. To make profile-based k-nearest-neighbor search and the 2D-visualization of the metagenome universe available to the research community, we included the proposed methods in our CoMet-Universe server for comparative metagenome analysis.

  18. Metagenomic data analysis : computational methods and applications

    NARCIS (Netherlands)

    Gori, F.

    2013-01-01

    Metagenomics is the study of the genomic content of microbial communities, acquired through DNA sequencing technology. The main advantage of metagenomics is that it can overcome the limitations of individual genome sequencing, that can work only on the few culturable microbes. Unfortunately, the an

  19. Metagenomic Detection Methods in Biopreparedness Outbreak Scenarios

    DEFF Research Database (Denmark)

    Karlsson, Oskar Erik; Hansen, Trine; Knutsson, Rickard

    2013-01-01

    of a clinical sample, creating a metagenome, in a single week of laboratory work. As new technologies emerge, their dissemination and capacity building must be facilitated, and criteria for use, as well as guidelines on how to report results, must be established. This article focuses on the use of metagenomics...

  20. Exploring the viral world through metagenomics.

    Science.gov (United States)

    Rosario, Karyna; Breitbart, Mya

    2011-10-01

    Viral metagenomics, or shotgun sequencing of purified viral particles, has revolutionized the field of environmental virology by allowing the exploration of viral communities in a variety of sample types throughout the biosphere. The introduction of viral metagenomics has demonstrated that dominant viruses in environmental communities are not well-represented by the cultured viruses in existing sequence databases. Viral metagenomic studies have provided insights into viral ecology by elucidating the genetic potential, community structure, and biogeography of environmental viruses. In addition, viral metagenomics has expanded current knowledge of virus-host interactions by uncovering genes that may allow viruses to manipulate their hosts in unexpected ways. The intrinsic potential for virus discovery through viral metagenomics can help advance a wide array of disciplines including evolutionary biology, pathogen surveillance, and biotechnology.

  1. Metagenomics using next-generation sequencing.

    Science.gov (United States)

    Bragg, Lauren; Tyson, Gene W

    2014-01-01

    Traditionally, microbial genome sequencing has been restricted to the small number of species that can be grown in pure culture. The progressive development of culture-independent methods over the last 15 years now allows researchers to sequence microbial communities directly from environmental samples. This approach is commonly referred to as "metagenomics" or "community genomics". However, the term metagenomics is applied liberally in the literature to describe any culture-independent analysis of microbial communities. Here, we define metagenomics as shotgun ("random") sequencing of the genomic DNA of a sample taken directly from the environment. The metagenome can be thought of as a sampling of the collective genome of the microbial community. We outline the considerations and analyses that should be undertaken to ensure the success of a metagenomic sequencing project, including the choice of sequencing platform and methods for assembly, binning, annotation, and comparative analysis.

  2. Isolation and Biochemical Characterization of a Glucose Dehydrogenase from a Hay Infusion Metagenome

    OpenAIRE

    Alexander Basner; Garabed Antranikian

    2014-01-01

    Glucose hydrolyzing enzymes are essential to determine blood glucose level. A high-throughput screening approach was established to identify NAD(P)-dependent glucose dehydrogenases for the application in test stripes and the respective blood glucose meters. In the current report a glucose hydrolyzing enzyme, derived from a metagenomic library by expressing recombinant DNA fragments isolated from hay infusion, was characterized. The recombinant clone showing activity on glucose as substrate ex...

  3. Targeted metagenomics: finding rare tryptophan dimer natural products in the environment.

    Science.gov (United States)

    Chang, Fang-Yuan; Ternei, Melinda A; Calle, Paula Y; Brady, Sean F

    2015-05-13

    Natural product discovery from environmental genomes (metagenomics) has largely been limited to the screening of existing environmental DNA (eDNA) libraries. Here, we have coupled a chemical-biogeographic survey of chromopyrrolic acid synthase (CPAS) gene diversity with targeted eDNA library production to more efficiently access rare tryptophan dimer (TD) biosynthetic gene clusters. A combination of traditional and synthetic biology-based heterologous expression efforts using eDNA-derived gene clusters led to the production of hydroxysporine (1) and reductasporine (2), two bioactive TDs. As suggested by our phylogenetic analysis of CPAS genes, identified in our survey of crude eDNA extracts, reductasporine (2) contains an unprecedented TD core structure: a pyrrolinium indolocarbazole core that is likely key to its unusual bioactivity profile. This work demonstrates the potential for the discovery of structurally rare and biologically interesting natural products using targeted metagenomics, where environmental samples are prescreened to identify the most phylogenetically unique gene sequences and molecules associated with these genes are accessed through targeted metagenomic library construction and heterologous expression.

  4. Comparing viral metagenomics methods using a highly multiplexed human viral pathogens reagent.

    Science.gov (United States)

    Li, Linlin; Deng, Xutao; Mee, Edward T; Collot-Teixeira, Sophie; Anderson, Rob; Schepelmann, Silke; Minor, Philip D; Delwart, Eric

    2015-03-01

    Unbiased metagenomic sequencing holds significant potential as a diagnostic tool for the simultaneous detection of any previously genetically described viral nucleic acids in clinical samples. Viral genome sequences can also inform on likely phenotypes including drug susceptibility or neutralization serotypes. In this study, different variables of the laboratory methods often used to generate viral metagenomics libraries were compared for their abilities to detect multiple viruses and generate full genome coverage. A biological reagent consisting of 25 different human RNA and DNA viral pathogens was used to estimate the effect of filtration and nuclease digestion, DNA/RNA extraction methods, pre-amplification and the use of different library preparation kits on the detection of viral nucleic acids. Filtration and nuclease treatment led to slight decreases in the percentage of viral sequence reads and number of viruses detected. For nucleic acid extractions silica spin columns improved viral sequence recovery relative to magnetic beads and Trizol extraction. Pre-amplification using random RT-PCR while generating more viral sequence reads resulted in detection of fewer viruses, more overlapping sequences, and lower genome coverage. The ScriptSeq library preparation method retrieved more viruses and a greater fraction of their genomes than the TruSeq and Nextera methods. Viral metagenomics sequencing was able to simultaneously detect up to 22 different viruses in the biological reagent analyzed including all those detected by qPCR. Further optimization will be required for the detection of viruses in biologically more complex samples such as tissues, blood, or feces.

  5. Bacterial diversity analysis of Huanglongbing pathogen-infected citrus, using PhyloChip and 16S rRNA gene clone library sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Shankar Sagaram, U.; DeAngelis, K.M.; Trivedi, P.; Andersen, G.L.; Lu, S.-E.; Wang, N.

    2009-03-01

    The bacterial diversity associated with citrus leaf midribs was characterized 1 from citrus groves that contained the Huanglongbing (HLB) pathogen, which has yet to be cultivated in vitro. We employed a combination of high-density phylogenetic 16S rDNA microarray and 16S rDNA clone library sequencing to determine the microbial community composition of symptomatic and asymptomatic citrus midribs. Our results revealed that citrus leaf midribs can support a diversity of microbes. PhyloChip analysis indicated that 47 orders of bacteria from 15 phyla were present in the citrus leaf midribs while 20 orders from phyla were observed with the cloning and sequencing method. PhyloChip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs compared to asymptomatic midribs. Candidatus Liberibacter asiaticus (Las) was detected at a very low level in asymptomatic plants, but was over 200 times more abundant in symptomatic plants. The PhyloChip analysis was further verified by sequencing 16S rDNA clone libraries, which indicated the dominance of Las in symptomatic leaves. These data implicate Las as the pathogen responsible for HLB disease. Citrus is the most important commercial fruit crop in Florida. In recent years, citrus Huanglongbing (HLB), also called citrus greening, has severely affected Florida's citrus production and hence has drawn an enormous amount of attention. HLB is one of the most devastating diseases of citrus (6,13), characterized by blotchy mottling with green islands on leaves, as well as stunting, fruit decline, and small, lopsided fruits with poor coloration. The disease tends to be associated with a phloem-limited fastidious {alpha}-proteobacterium given a provisional Candidatus status (Candidatus Liberobacter spp. later changed to Candidatus Liberibacter spp.) in nomenclature (18,25,34). Previous studies indicate that HLB infection causes disorder in the phloem and severely impairs the translocation of assimilates in

  6. Exploration of soil metagenome diversity for prospection of enzymes involved in lignocellulosic biomass conversion

    Energy Technology Data Exchange (ETDEWEB)

    Alvarez, T.M.; Squina, F.M. [Laboratorio Nacional de Luz Sincrotron (LNLS), Campinas, SP (Brazil); Paixao, D.A.A.; Franco Cairo, J.P.L.; Buchli, F.; Ruller, R. [Laboratorio Nacional de Ciencia e Tecnologia do Bioetanol (CTBE), Campinas, SP (Brazil); Prade, R. [Oklahoma State University, Sillwater, OK (United States)

    2012-07-01

    Full text: Metagenomics allows access to genetic information encoded in DNA of microorganisms recalcitrant to cultivation. They represent a reservoir of novel biocatalyst with potential application in environmental friendly techniques aiming to overcome the dependence on fossil fuels and also to diminish air and water pollution. The focus of our work is the generation of a tool kit of lignocellulolytic enzymes from soil metagenome, which could be used for second generation ethanol production. Environmental samples were collected at a sugarcane field after harvesting, where it is expected that the microbial population involved on lignocellulose degradation was enriched due to the presence of straws covering the soil. Sugarcane Bagasse-Degrading-Soil (SBDS) metagenome was massively-parallel-454-Roche-sequenced. We identified a full repertoire of genes with significant match to glycosyl hydrolases catalytic domain and carbohydrate-binding modules. Soil metagenomics libraries cloned into pUC19 were screened through functional assays. CMC-agar screening resulted in positive clones, revealing new cellulases coding genes. Through a CMC-zymogram it was possible to observe that one of these genes, nominated as E-1, corresponds to an enzyme that is secreted to the extracellular medium, suggesting that the cloned gene carried the original signal peptide. Enzymatic assays and analysis through capillary electrophoresis showed that E-1 was able to cleave internal glycosidic bonds of cellulose. New rounds of functional screenings through chromogenic substrates are being conducted aiming the generation of a library of lignocellulolytic enzymes derived from soil metagenome, which may become key component for development of second generation biofuels. (author)

  7. Changes in the Composition of Drinking Water Bacterial Clone Libraries Introduced by Using Two Different 16S rRNA Gene PCR Primers

    Science.gov (United States)

    Sequence analysis of 16S rRNA gene clone libraries is a popular tool used to describe the composition of natural microbial communities. Commonly, clone libraries are developed by direct cloning of 16S rRNA gene PCR products. Different primers are often employed in the initial amp...

  8. Diel Metagenomics and Metatranscriptomics of Elkhorn Slough Hypersaline Microbial Mat

    Science.gov (United States)

    Lee, J.; Detweiler, A. M.; Everroad, R. C.; Bebout, L. E.; Weber, P. K.; Pett-Ridge, J.; Bebout, B.

    2014-12-01

    To understand the variation in gene expression associated with the daytime oxygenic phototrophic and nighttime fermentation regimes seen in hypersaline microbial mats, a contiguous mat piece was subjected to sampling at regular intervals over a 24-hour diel period. Additionally, to understand the impact of sulfate reduction on biohydrogen consumption, molybdate was added to a parallel experiment in the same run. 4 metagenome and 12 metatranscriptome Illumina HiSeq lanes were completed over day / night, and control / molybdate experiments. Preliminary comparative examination of noon and midnight metatranscriptomic samples mapped using bowtie2 to reference genomes has revealed several notable results about the dominant mat-building cyanobacterium Microcoleus chthonoplastes PCC 7420. Dominant cyanobacterium M. chthonoplastes PCC 7420 shows expression in several pathways for nitrogen scavenging, including nitrogen fixation. Reads mapped to M. chthonoplastes PCC 7420 shows expression of two starch storage and utilization pathways, one as a starch-trehalose-maltose-glucose pathway, another through UDP-glucose-cellulose-β-1,4 glucan-glucose pathway. The overall trend of gene expression was primarily light driven up-regulation followed by down-regulation in dark, while much of the remaining expression profile appears to be constitutive. Co-assembly of quality-controlled reads from 4 metagenomes was performed using Ray Meta with progressively smaller K-mer sizes, with bins identified and filtered using principal component analysis of coverages from all libraries and a %GC filter, followed by reassembly of the remaining co-assembly reads and binned reads. Despite having relatively similar abundance profiles in each metagenome, this binning approach was able to distinctly resolve bins from dominant taxa, but also sulfate reducing bacteria that are desired for understanding molybdate inhibition. Bins generated from this iterative assembly process will be used for downstream

  9. Bioprospecting metagenomes: Glycosyl hydrolases for converting biomass

    Energy Technology Data Exchange (ETDEWEB)

    Li, L.; van der Lelie, D.; McCorkle, S. R.; Monchy, S.; Taghavi, S.

    2009-05-18

    Throughout immeasurable time, microorganisms evolved and accumulated remarkable physiological and functional heterogeneity, and now constitute the major reserve for genetic diversity on earth. Using metagenomics, namely genetic material recovered directly from environmental samples, this biogenetic diversification can be accessed without the need to cultivate cells. Accordingly, microbial communities and their metagenomes, isolated from biotopes with high turnover rates of recalcitrant biomass, such as lignocellulosic plant cell walls, have become a major resource for bioprospecting; furthermore, this material is a major asset in the search for new biocatalytics (enzymes) for various industrial processes, including the production of biofuels from plant feedstocks. However, despite the contributions from metagenomics technologies consequent upon the discovery of novel enzymes, this relatively new enterprise requires major improvements. In this review, we compare function-based metagenome screening and sequence-based metagenome data mining, discussing the advantages and limitations of both methods. We also describe the unusual enzymes discovered via metagenomics approaches, and discuss the future prospects for metagenome technologies.

  10. 环境细菌宏基因组研究及海洋细菌生物活性物质BAC文库筛选%Exploration of Environmental Bacterial Metagenome and Library Screening of Bioactive Materials of Oceanic Bacteria

    Institute of Scientific and Technical Information of China (English)

    杨官品; 茅云翔

    2001-01-01

    宏基因组是特定环境全部生物遗传物质总和,决定生物群体生命现象.特定生物种基因组研究使人们的认识单元实现了从单一基因到基因集合的转变,宏基因组研究将使人们摆脱物种界限,揭示更高更复杂层次上的生命运动规律.在目前的基因结构功能认识和基因操作技术背景下,环境细菌宏基因组成为研究和开发的主要对象.环境细菌宏基因组细菌人工染色体文库筛选和基因系统学分析使研究者能更有效地开发细菌基因资源,更深入地洞察环境细菌多样性.

  11. 瘤胃微生物基因组文库中BAC末端序列分析%Analysis of bacterial artificial chromosome (BAC) end sequences from microbial metagenomic library of rumen

    Institute of Scientific and Technical Information of China (English)

    赵圣国; 王加启; 李发弟; 刘开朗; 卜登攀; 李旦; 于萍

    2010-01-01

    基于前期从瘤胃微生物基因组文库中筛选的功能酶克隆菌,利用T7和pIBRP引物,测定功能酶克隆菌的BAC末端序列,并利用NCBI Blast系统对BAC末端序列进行分析.结果表明:共得到26条BAC末端序列,经Blast比对分析,与已知编码基因的匹配度低,而大部分BAC末端序列与阪崎肠杆菌和环境中的微生物匹配度高,说明瘤胃中的微生物与其它环境中的微生物具有一定的相似性,进一步丰富了对瘤胃微生物的认识.

  12. Metagenomics and future perspectives in virus discovery.

    Science.gov (United States)

    Mokili, John L; Rohwer, Forest; Dutilh, Bas E

    2012-02-01

    Monitoring the emergence and re-emergence of viral diseases with the goal of containing the spread of viral agents requires both adequate preparedness and quick response. Identifying the causative agent of a new epidemic is one of the most important steps for effective response to disease outbreaks. Traditionally, virus discovery required propagation of the virus in cell culture, a proven technique responsible for the identification of the vast majority of viruses known to date. However, many viruses cannot be easily propagated in cell culture, thus limiting our knowledge of viruses. Viral metagenomic analyses of environmental samples suggest that the field of virology has explored less than 1% of the extant viral diversity. In the last decade, the culture-independent and sequence-independent metagenomic approach has permitted the discovery of many viruses in a wide range of samples. Phylogenetically, some of these viruses are distantly related to previously discovered viruses. In addition, 60-99% of the sequences generated in different viral metagenomic studies are not homologous to known viruses. In this review, we discuss the advances in the area of viral metagenomics during the last decade and their relevance to virus discovery, clinical microbiology and public health. We discuss the potential of metagenomics for characterization of the normal viral population in a healthy community and identification of viruses that could pose a threat to humans through zoonosis. In addition, we propose a new model of the Koch's postulates named the 'Metagenomic Koch's Postulates'. Unlike the original Koch's postulates and the Molecular Koch's postulates as formulated by Falkow, the metagenomic Koch's postulates focus on the identification of metagenomic traits in disease cases. The metagenomic traits that can be traced after healthy individuals have been exposed to the source of the suspected pathogen.

  13. Analysis of bacterial community diversity in anaerobic fluidized bed bioreactors treating 2,4-dinitroanisole (DNAN) and n-methyl-4-nitroaniline (MNA) using 16S rRNA gene clone libraries.

    Science.gov (United States)

    Arnett, Clint M; Rodriguez, Giselle; Maloney, Stephen W

    2009-01-01

    Clone libraries were used to evaluate the effects of 2,4-dinitroanisole (DNAN) and n-methyl-4-nitroaniline (MNA) on bacterial populations within three anaerobic bioreactors. Prior to the addition of DNAN and MNA greater than 69% of the clones in each reactor were identified as a single Desulfuromonales species. However, after 60 days of treatment the Desulfuromonales distribution decreased to less than 13% of the distribution and a clone identified as a Levilinea sp. became the dominant organism at greater than 27% of the clone distribution in each reactor suggesting the species may play an important roll in the reduction of DNAN and MNA.

  14. EBI metagenomics--a new resource for the analysis and archiving of metagenomic data.

    Science.gov (United States)

    Hunter, Sarah; Corbett, Matthew; Denise, Hubert; Fraser, Matthew; Gonzalez-Beltran, Alejandra; Hunter, Christopher; Jones, Philip; Leinonen, Rasko; McAnulla, Craig; Maguire, Eamonn; Maslen, John; Mitchell, Alex; Nuka, Gift; Oisel, Arnaud; Pesseat, Sebastien; Radhakrishnan, Rajesh; Rocca-Serra, Philippe; Scheremetjew, Maxim; Sterk, Peter; Vaughan, Daniel; Cochrane, Guy; Field, Dawn; Sansone, Susanna-Assunta

    2014-01-01

    Metagenomics is a relatively recently established but rapidly expanding field that uses high-throughput next-generation sequencing technologies to characterize the microbial communities inhabiting different ecosystems (including oceans, lakes, soil, tundra, plants and body sites). Metagenomics brings with it a number of challenges, including the management, analysis, storage and sharing of data. In response to these challenges, we have developed a new metagenomics resource (http://www.ebi.ac.uk/metagenomics/) that allows users to easily submit raw nucleotide reads for functional and taxonomic analysis by a state-of-the-art pipeline, and have them automatically stored (together with descriptive, standards-compliant metadata) in the European Nucleotide Archive.

  15. Bacterial diversity analysis of Huanglongbing pathogen-infected citrus, using PhyloChip and 16S rRNA gene clone library sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Shankar Sagaram, U.; DeAngelis, K.M.; Trivedi, P.; Andersen, G.L.; Lu, S.-E.; Wang, N.

    2009-03-01

    The bacterial diversity associated with citrus leaf midribs was characterized 1 from citrus groves that contained the Huanglongbing (HLB) pathogen, which has yet to be cultivated in vitro. We employed a combination of high-density phylogenetic 16S rDNA microarray and 16S rDNA clone library sequencing to determine the microbial community composition of symptomatic and asymptomatic citrus midribs. Our results revealed that citrus leaf midribs can support a diversity of microbes. PhyloChip analysis indicated that 47 orders of bacteria from 15 phyla were present in the citrus leaf midribs while 20 orders from phyla were observed with the cloning and sequencing method. PhyloChip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs compared to asymptomatic midribs. Candidatus Liberibacter asiaticus (Las) was detected at a very low level in asymptomatic plants, but was over 200 times more abundant in symptomatic plants. The PhyloChip analysis was further verified by sequencing 16S rDNA clone libraries, which indicated the dominance of Las in symptomatic leaves. These data implicate Las as the pathogen responsible for HLB disease. Citrus is the most important commercial fruit crop in Florida. In recent years, citrus Huanglongbing (HLB), also called citrus greening, has severely affected Florida's citrus production and hence has drawn an enormous amount of attention. HLB is one of the most devastating diseases of citrus (6,13), characterized by blotchy mottling with green islands on leaves, as well as stunting, fruit decline, and small, lopsided fruits with poor coloration. The disease tends to be associated with a phloem-limited fastidious {alpha}-proteobacterium given a provisional Candidatus status (Candidatus Liberobacter spp. later changed to Candidatus Liberibacter spp.) in nomenclature (18,25,34). Previous studies indicate that HLB infection causes disorder in the phloem and severely impairs the translocation of assimilates in

  16. Metagenomic Sequencing Indicates That the Oropharyngeal Phageome of Individuals With Schizophrenia Differs From That of Controls

    OpenAIRE

    2015-01-01

    Mucosal sites such as the oropharynx contain a wide range of microorganisms, collectively designated as the microbiome. The microbiome can affect behavior through a number of neurobiological and immunological mechanisms. Most previous studies have focused on the bacterial components of the microbiome. However, the microbiome also includes viruses such as bacteriophages, which are viruses that infect bacteria and alter their metabolism and replication. We employed metagenomic analysis to chara...

  17. Functional Metagenomics of the Bronchial Microbiome in COPD.

    Directory of Open Access Journals (Sweden)

    Laura Millares

    Full Text Available The course of chronic obstructive pulmonary disease (COPD is frequently aggravated by exacerbations, and changes in the composition and activity of the microbiome may be implicated in their appearance. The aim of this study was to analyse the composition and the gene content of the microbial community in bronchial secretions of COPD patients in both stability and exacerbation. Taxonomic data were obtained by 16S rRNA gene amplification and pyrosequencing, and metabolic information through shotgun metagenomics, using the Metagenomics RAST server (MG-RAST, and the PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States programme, which predict metagenomes from 16S data. Eight severe COPD patients provided good quality sputum samples, and no significant differences in the relative abundance of any phyla and genera were found between stability and exacerbation. Bacterial biodiversity (Chao1 and Shannon indexes did not show statistical differences and beta-diversity analysis (Bray-Curtis dissimilarity index showed a similar microbial composition in the two clinical situations. Four functional categories showed statistically significant differences with MG-RAST at KEGG level 2: in exacerbation, Cell growth and Death and Transport and Catabolism decreased in abundance [1.6 (0.2-2.3 vs 3.6 (3.3-6.9, p = 0.012; and 1.8 (0-3.3 vs 3.6 (1.8-5.1, p = 0.025 respectively], while Cancer and Carbohydrate Metabolism increased [0.8 (0-1.5 vs 0 (0-0.5, p = 0.043; and 7 (6.4-9 vs 5.9 (6.3-6.1, p = 0.012 respectively]. In conclusion, the bronchial microbiome as a whole is not significantly modified when exacerbation symptoms appear in severe COPD patients, but its functional metabolic capabilities show significant changes in several pathways.

  18. Functional Metagenomics of the Bronchial Microbiome in COPD.

    Science.gov (United States)

    Millares, Laura; Pérez-Brocal, Vicente; Ferrari, Rafaela; Gallego, Miguel; Pomares, Xavier; García-Núñez, Marian; Montón, Concepción; Capilla, Silvia; Monsó, Eduard; Moya, Andrés

    2015-01-01

    The course of chronic obstructive pulmonary disease (COPD) is frequently aggravated by exacerbations, and changes in the composition and activity of the microbiome may be implicated in their appearance. The aim of this study was to analyse the composition and the gene content of the microbial community in bronchial secretions of COPD patients in both stability and exacerbation. Taxonomic data were obtained by 16S rRNA gene amplification and pyrosequencing, and metabolic information through shotgun metagenomics, using the Metagenomics RAST server (MG-RAST), and the PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) programme, which predict metagenomes from 16S data. Eight severe COPD patients provided good quality sputum samples, and no significant differences in the relative abundance of any phyla and genera were found between stability and exacerbation. Bacterial biodiversity (Chao1 and Shannon indexes) did not show statistical differences and beta-diversity analysis (Bray-Curtis dissimilarity index) showed a similar microbial composition in the two clinical situations. Four functional categories showed statistically significant differences with MG-RAST at KEGG level 2: in exacerbation, Cell growth and Death and Transport and Catabolism decreased in abundance [1.6 (0.2-2.3) vs 3.6 (3.3-6.9), p = 0.012; and 1.8 (0-3.3) vs 3.6 (1.8-5.1), p = 0.025 respectively], while Cancer and Carbohydrate Metabolism increased [0.8 (0-1.5) vs 0 (0-0.5), p = 0.043; and 7 (6.4-9) vs 5.9 (6.3-6.1), p = 0.012 respectively]. In conclusion, the bronchial microbiome as a whole is not significantly modified when exacerbation symptoms appear in severe COPD patients, but its functional metabolic capabilities show significant changes in several pathways.

  19. Metagenomics and its Applications in the Field of Marine Microorganisms%宏基因组学及其在海洋微生物领域的应用∗

    Institute of Scientific and Technical Information of China (English)

    刘如运; 黄毅梅

    2013-01-01

      Metagenomics is a new field of science applied to study gene function and interaction ecotype communi-ties. This review is analyzed the strategies of metagenomics on extraction of metagenome DNA and screening of met-agenome library, and discussed the applications of metagenomics in the field of marine microorganisms.%  宏基因组学是研究生态群体基因功能和相互作用的新科学领域。文章从宏基因组DNA的提取和宏基因文库筛选两方面分析了宏基因组学的研究策略,并综述了宏基因组学在海洋微生物领域的应用。

  20. Bacterial communities associated with the rhizosphere of pioneer plants (Bahia xylopoda and Viguiera linearis) growing on heavy metals-contaminated soils.

    Science.gov (United States)

    Navarro-Noya, Yendi E; Jan-Roblero, Janet; González-Chávez, Maria del Carmen; Hernández-Gama, Regina; Hernández-Rodríguez, César

    2010-05-01

    In this study, the bacterial communities associated with the rhizospheres of pioneer plants Bahia xylopoda and Viguiera linearis were explored. These plants grow on silver mine tailings with high concentration of heavy metals in Zacatecas, Mexico. Metagenomic DNAs from rhizosphere and bulk soil were extracted to perform a denaturing gradient gel electrophoresis analysis (DGGE) and to construct 16S rRNA gene libraries. A moderate bacterial diversity and twelve major phylogenetic groups including Proteobacteria, Acidobacteria, Bacteroidetes, Gemmatimonadetes, Chloroflexi, Firmicutes, Verrucomicrobia, Nitrospirae and Actinobacteria phyla, and divisions TM7, OP10 and OD1 were recognized in the rhizospheres. Only 25.5% from the phylotypes were common in the rhizosphere libraries and the most abundant groups were members of the phyla Acidobacteria and Betaproteobacteria (Thiobacillus spp., Nitrosomonadaceae). The most abundant groups in bulk soil library were Acidobacteria and Actinobacteria, and no common phylotypes were shared with the rhizosphere libraries. Many of the clones detected were related with chemolithotrophic and sulfur-oxidizing bacteria, characteristic of an environment with a high concentration of heavy metal-sulfur complexes, and lacking carbon and organic energy sources.

  1. Comparative Metagenomic Analysis Of Microbial Communities From Active Layer And Permafrost After Short-Term Thaw

    Science.gov (United States)

    Vishnivetskaya, T. A.; Chauhan, A.; Saarunya, G.; Murphy, J.; Williams, D.; Layton, A. C.; Pfiffner, S. M.; Stackhouse, B. T.; Sanders, R.; Lau, C. M.; myneni, S.; Phelps, T. J.; Fountain, A. G.; Onstott, T. C.

    2012-12-01

    decreased with depth from 7x108 to 1x108 copies /g of soil. Characterization of the metagenomic sequences derived from the samples before thawing elucidated differences between the permafrost and active-layer with Acidobacteria and Alpha-Proteobacteria are being significantly higher in active layer than in permafrost, on the contrary permafrost had higher abundance of Actinobacteria and Bacteroidetes than active layer. Currently additional metagenomic DNA illumina libraries for 20 samples after 1 week thawing are in preparation and will be analyzed to determine whether variations in the metagenomic sequences are correlated to the geochemical data.

  2. Comparative metagenomics of the Red Sea

    KAUST Repository

    Mineta, Katsuhiko

    2016-01-26

    Metagenome produces a tremendous amount of data that comes from the organisms living in the environments. This big data enables us to examine not only microbial genes but also the community structure, interaction and adaptation mechanisms at the specific location and condition. The Red Sea has several unique characteristics such as high salinity, high temperature and low nutrition. These features must contribute to form the unique microbial community during the evolutionary process. Since 2014, we started monthly samplings of the metagenomes in the Red Sea under KAUST-CCF project. In collaboration with Kitasato University, we also collected the metagenome data from the ocean in Japan, which shows contrasting features to the Red Sea. Therefore, the comparative metagenomics of those data provides a comprehensive view of the Red Sea microbes, leading to identify key microbes, genes and networks related to those environmental differences.

  3. Toward Accurate and Quantitative Comparative Metagenomics

    Science.gov (United States)

    Nayfach, Stephen; Pollard, Katherine S.

    2016-01-01

    Shotgun metagenomics and computational analysis are used to compare the taxonomic and functional profiles of microbial communities. Leveraging this approach to understand roles of microbes in human biology and other environments requires quantitative data summaries whose values are comparable across samples and studies. Comparability is currently hampered by the use of abundance statistics that do not estimate a meaningful parameter of the microbial community and biases introduced by experimental protocols and data-cleaning approaches. Addressing these challenges, along with improving study design, data access, metadata standardization, and analysis tools, will enable accurate comparative metagenomics. We envision a future in which microbiome studies are replicable and new metagenomes are easily and rapidly integrated with existing data. Only then can the potential of metagenomics for predictive ecological modeling, well-powered association studies, and effective microbiome medicine be fully realized. PMID:27565341

  4. [Pathology and viral metagenomics, a recent history].

    Science.gov (United States)

    Bernardo, Pauline; Albina, Emmanuel; Eloit, Marc; Roumagnac, Philippe

    2013-05-01

    Human, animal and plant viral diseases have greatly benefited from recent metagenomics developments. Viral metagenomics is a culture-independent approach used to investigate the complete viral genetic populations of a sample. During the last decade, metagenomics concepts and techniques that were first used by ecologists progressively spread into the scientific field of viral pathology. The sample, which was first for ecologists a fraction of ecosystem, became for pathologists an organism that hosts millions of microbes and viruses. This new approach, providing without a priori high resolution qualitative and quantitative data on the viral diversity, is now revolutionizing the way pathologists decipher viral diseases. This review describes the very last improvements of the high throughput next generation sequencing methods and discusses the applications of viral metagenomics in viral pathology, including discovery of novel viruses, viral surveillance and diagnostic, large-scale molecular epidemiology, and viral evolution.

  5. Diverse Bacterial Groups Contribute to the Alkane Degradation Potential of Chronically Polluted Subantarctic Coastal Sediments.

    Science.gov (United States)

    Guibert, Lilian M; Loviso, Claudia L; Borglin, Sharon; Jansson, Janet K; Dionisi, Hebe M; Lozada, Mariana

    2016-01-01

    We aimed to gain insight into the alkane degradation potential of microbial communities from chronically polluted sediments of a subantarctic coastal environment using a combination of metagenomic approaches. A total of 6178 sequences annotated as alkane-1-monooxygenases (EC 1.14.15.3) were retrieved from a shotgun metagenomic dataset that included two sites analyzed in triplicate. The majority of the sequences binned with AlkB described in Bacteroidetes (32 ± 13 %) or Proteobacteria (29 ± 7 %), although a large proportion remained unclassified at the phylum level. Operational taxonomic unit (OTU)-based analyses showed small differences in AlkB distribution among samples that could be correlated with alkane concentrations, as well as with site-specific variations in pH and salinity. A number of low-abundance OTUs, mostly affiliated with Actinobacterial sequences, were found to be only present in the most contaminated samples. On the other hand, the molecular screening of a large-insert metagenomic library of intertidal sediments from one of the sampling sites identified two genomic fragments containing novel alkB gene sequences, as well as various contiguous genes related to lipid metabolism. Both genomic fragments were affiliated with the phylum Planctomycetes, and one could be further assigned to the genus Rhodopirellula due to the presence of a partial sequence of the 23S ribosomal RNA (rRNA) gene. This work highlights the diversity of bacterial groups contributing to the alkane degradation potential and reveals patterns of functional diversity in relation with environmental stressors in a chronically polluted, high-latitude coastal environment. In addition, alkane biodegradation genes are described for the first time in members of Planctomycetes.

  6. Diverse Bacterial Groups Contribute to the Alkane Degradation Potential of Chronically Polluted Subantarctic Coastal Sediments

    Energy Technology Data Exchange (ETDEWEB)

    Guibert, Lilian M.; Loviso, Claudia L.; Borglin, Sharon; Jansson, Janet K.; Dionisi, Hebe M.; Lozada, Mariana

    2015-11-07

    We aimed to gain insight into the alkane degradation potential of microbial communities from chronically polluted sediments of a subantarctic coastal environment using a combination of metagenomic approaches. A total of 6178 sequences annotated as alkane-1-monooxygenases (EC 1.14.15.3) were retrieved from a shotgun metagenomic dataset that included two sites analyzed in triplicate. The majority of the sequences binned with AlkB described in Bacteroidetes (32 ± 13 %) or Proteobacteria (29 ± 7 %), although a large proportion remained unclassified at the phylum level. Operational taxonomic unit (OTU)-based analyses showed small differences in AlkB distribution among samples that could be correlated with alkane concentrations, as well as with site-specific variations in pH and salinity. A number of low-abundance OTUs, mostly affiliated with Actinobacterial sequences, were found to be only present in the most contaminated samples. On the other hand, the molecular screening of a large-insert metagenomic library of intertidal sediments from one of the sampling sites identified two genomic fragments containing novel alkB gene sequences, as well as various contiguous genes related to lipid metabolism. Both genomic fragments were affiliated with the phylum Planctomycetes, and one could be further assigned to the genus Rhodopirellula due to the presence of a partial sequence of the 23S ribosomal RNA (rRNA) gene. This work highlights the diversity of bacterial groups contributing to the alkane degradation potential and reveals patterns of functional diversity in relation with environmental stressors in a chronically polluted, high-latitude coastal environment. In addition, alkane biodegradation genes are described for the first time in members of Planctomycetes.

  7. Metagenome Fragment Classification Using -Mer Frequency Profiles

    OpenAIRE

    Gail Rosen; Elaine Garbarine; Diamantino Caseiro; Robi Polikar; Bahrad Sokhansanj

    2008-01-01

    A vast amount of microbial sequencing data is being generated through large-scale projects in ecology, agriculture, and human health. Efficient high-throughput methods are needed to analyze the mass amounts of metagenomic data, all DNA present in an environmental sample. A major obstacle in metagenomics is the inability to obtain accuracy using technology that yields short reads. We construct the unique -mer frequency profiles of 635 microbial genomes publicly available as of February 2008....

  8. Snowball: Strain aware gene assembly of Metagenomes

    OpenAIRE

    Gregor, I.; Schönhuth, A.; McHardy, A. C.

    2015-01-01

    Gene assembly is an important step in functional analysis of shotgun metagenomic data. Nonetheless, strain aware assembly remains a challenging task, as current assembly tools often fail to distinguish among strain variants or require closely related reference genomes of the studied species to be available. We have developed Snowball, a novel strain aware and reference-free gene assembler for shotgun metagenomic data. It uses profile hidden Markov models (HMMs) of gene domains of interest to ...

  9. Metagenomic analysis of a Southern Maritime Antarctic soil

    Directory of Open Access Journals (Sweden)

    David Anthony Pearce

    2012-12-01

    Full Text Available Our current understanding of Antarctic soils is derived from direct culture on selective media, biodiversity studies based on clone library construction and analysis, quantitative PCR amplification of specific gene sequences and the application of generic microarrays for microbial community analysis. Here, we investigated the biodiversity and functional potential of a soil community at Mars Oasis on Alexander Island in the southern Maritime Antarctic, by applying 454 pyrosequencing technology to a metagenomic library constructed from soil genomic DNA. The results suggest that the commonly cited range of phylotypes used in clone library construction and analysis of 78-730 OTUs (de-replicated to 30-140 provides low coverage of the major groups present (~5%. The vast majority of functional genes (>77% were for structure, carbohydrate metabolism and DNA/RNA processing and modification. This study suggests that prokaryotic diversity in Antarctic terrestrial environments appears to be limited at the generic level, with Proteobacteria, Actinobacteria being common. Cyanobacteria were surprisingly under-represented at 2.6% of sequences, although ~1% of the genes identified were involved in CO2 fixation. At the sequence level there appeared to be much greater heterogeneity, and this might be due to high divergence within the relatively restricted lineages which have successfully colonized Antarctic terrestrial environments.

  10. Screening for cellulase-encoding clones in metagenomic libraries.

    Science.gov (United States)

    Ilmberger, Nele; Streit, Wolfgang R

    2010-01-01

    Modern biotechnology has the steady need to continuously identify novel enzymes for use in biotechnological applications. In industrial applications, however, enzymes often have to function under extreme and nonnatural conditions (i.e., in the presence of solvents, high temperature and/or at extreme pH values). Cellulases have many industrial applications from the generation of bioethanol, a realistic long-term energy source, to the finishing of textiles. These industrial processes require cellulolytic activity under a range of pH, temperature, and ionic conditions, and they are usually carried out by mixtures of cellulases. Investigation of the broad diversity of cellulolytic enzymes involved in the natural degradation of cellulose is necessary for optimization of these processes.

  11. Eu-Detect: An algorithm for detecting eukaryotic sequences in metagenomic data sets

    Indian Academy of Sciences (India)

    Monzoorul Haque Mohammed; Sudha Chadaram Dinakar; Dinakar Komanduri; Tarini Shankar Ghosh; Sharmila S Mande

    2011-09-01

    Physical partitioning techniques are routinely employed (during sample preparation stage) for segregating the prokaryotic and eukaryotic fractions of metagenomic samples. In spite of these efforts, several metagenomic studies focusing on bacterial and archaeal populations have reported the presence of contaminating eukaryotic sequences inmetagenomic data sets. Contaminating sequences originate not only from genomes of micro-eukaryotic species but also from genomes of (higher) eukaryotic host cells. The latter scenario usually occurs in the case of host-associatedmetagenomes. Identification and removal of contaminating sequences is important, since these sequences not only impact estimates of microbial diversity but also affect the accuracy of several downstream analyses. Currently, the computational techniques used for identifying contaminating eukaryotic sequences, being alignment based, are slow, inefficient, and require huge computing resources. In this article, we present Eu-Detect, an alignment-free algorithm that can rapidly identify eukaryotic sequences contaminating metagenomic data sets. Validation results indicate that on a desktop with modest hardware specifications, the Eu-Detect algorithm is able to rapidly segregate DNA sequence fragments of prokaryotic and eukaryotic origin, with high sensitivity. A Web server for the Eu-Detect algorithm is available at http://metagenomics.atc.tcs.com/Eu-Detect/.

  12. Use of Metagenomics and Isolation of Actinobacteria in Brazil's Atlantic Rainforest Soil for Antimicrobial Prospecting

    Science.gov (United States)

    Assis, Danyelle Alves Martins; Rezende, Rachel Passos; Dias, João Carlos Teixeira

    2014-01-01

    Modern techniques involving molecular biology, such as metagenomics, have the advantage of exploiting a higher number of microorganisms; however, classic isolation and culture methods used to obtain antimicrobials continue to be promising, especially in the isolation of Actinobacteria, which are responsible for the production of many of these compounds. In this work, two methodologies were used to search for antimicrobial substances—isolation of Actinobacteria and metagenomics of the Atlantic Rainforest soil and of the cultivation of cocoa intercropped with acai berry in the Atlantic Rainforest. The metagenomic libraries were constructed with the CopyControl Fosmid Library kit EPICENTRE, resulting in a total of 2688 clones, 1344 of each soil sample. None of the clones presented antimicrobial activity against the microorganisms tested: S. aureus, Bacillus subtilis, and Salmonella choleraesuis. A total of 46 isolates were obtained from the isolation of soil Actinobacteria: 24 isolates from Atlantic Rainforest soil and 22 isolates from the intercrop cultivation soil. Of these, two Atlantic Rainforest soil isolates inhibited the growth of S. aureus including a clinical isolate of S. aureus MRSA—a promising result, since it is an important multidrug-resistant human pathogen. PMID:25937991

  13. Metagenome mining reveals polytheonamides as posttranslationally modified ribosomal peptides.

    Science.gov (United States)

    Freeman, Michael F; Gurgui, Cristian; Helf, Maximilian J; Morinaka, Brandon I; Uria, Agustinus R; Oldham, Neil J; Sahl, Hans-Georg; Matsunaga, Shigeki; Piel, Jörn

    2012-10-19

    It is held as a paradigm that ribosomally synthesized peptides and proteins contain only l-amino acids. We demonstrate a ribosomal origin of the marine sponge-derived polytheonamides, exceptionally potent, giant natural-product toxins. Isolation of the biosynthetic genes from the sponge metagenome revealed a bacterial gene architecture. Only six candidate enzymes were identified for 48 posttranslational modifications, including 18 epimerizations and 17 methylations of nonactivated carbon centers. Three enzymes were functionally validated, which showed that a radical S-adenosylmethionine enzyme is responsible for the unidirectional epimerization of multiple and different amino acids. Collectively, these complex alterations create toxins that function as unimolecular minimalistic ion channels with near-femtomolar activity. This study broadens the biosynthetic scope of ribosomal systems and creates new opportunities for peptide and protein bioengineering.

  14. Current Advances on Virus Discovery and Diagnostic Role of Viral Metagenomics in Aquatic Organisms

    Science.gov (United States)

    Munang'andu, Hetron M.; Mugimba, Kizito K.; Byarugaba, Denis K.; Mutoloki, Stephen; Evensen, Øystein

    2017-01-01

    The global expansion of the aquaculture industry has brought with it a corresponding increase of novel viruses infecting different aquatic organisms. These emerging viral pathogens have proved to be a challenge to the use of traditional cell-cultures and immunoassays for identification of new viruses especially in situations where the novel viruses are unculturable and no antibodies exist for their identification. Viral metagenomics has the potential to identify novel viruses without prior knowledge of their genomic sequence data and may provide a solution for the study of unculturable viruses. This review provides a synopsis on the contribution of viral metagenomics to the discovery of viruses infecting different aquatic organisms as well as its potential role in viral diagnostics. High throughput Next Generation sequencing (NGS) and library construction used in metagenomic projects have simplified the task of generating complete viral genomes unlike the challenge faced in traditional methods that use multiple primers targeted at different segments and VPs to generate the entire genome of a novel virus. In terms of diagnostics, studies carried out this far show that viral metagenomics has the potential to serve as a multifaceted tool able to study and identify etiological agents of single infections, co-infections, tissue tropism, profiling viral infections of different aquatic organisms, epidemiological monitoring of disease prevalence, evolutionary phylogenetic analyses, and the study of genomic diversity in quasispecies viruses. With sequencing technologies and bioinformatics analytical tools becoming cheaper and easier, we anticipate that metagenomics will soon become a routine tool for the discovery, study, and identification of novel pathogens including viruses to enable timely disease control for emerging diseases in aquaculture.

  15. Potential and pitfalls of eukaryotic metagenome skimming: a test case for lichens.

    Science.gov (United States)

    Greshake, Bastian; Zehr, Simonida; Dal Grande, Francesco; Meiser, Anjuli; Schmitt, Imke; Ebersberger, Ingo

    2016-03-01

    Whole-genome shotgun sequencing of multispecies communities using only a single library layout is commonly used to assess taxonomic and functional diversity of microbial assemblages. Here, we investigate to what extent such metagenome skimming approaches are applicable for in-depth genomic characterizations of eukaryotic communities, for example lichens. We address how to best assemble a particular eukaryotic metagenome skimming data, what pitfalls can occur, and what genome quality can be expected from these data. To facilitate a project-specific benchmarking, we introduce the concept of twin sets, simulated data resembling the outcome of a particular metagenome sequencing study. We show that the quality of genome reconstructions depends essentially on assembler choice. Individual tools, including the metagenome assemblers Omega and MetaVelvet, are surprisingly sensitive to low and uneven coverages. In combination with the routine of assembly parameter choice to optimize the assembly N50 size, these tools can preclude an entire genome from the assembly. In contrast, MIRA, an all-purpose overlap assembler, and SPAdes, a multisized de Bruijn graph assembler, facilitate a comprehensive view on the individual genomes across a wide range of coverage ratios. Testing assemblers on a real-world metagenome skimming data from the lichen Lasallia pustulata demonstrates the applicability of twin sets for guiding method selection. Furthermore, it reveals that the assembly outcome for the photobiont Trebouxia sp. falls behind the a priori expectation given the simulations. Although the underlying reasons remain still unclear, this highlights that further studies on this organism require special attention during sequence data generation and downstream analysis.

  16. The metagenomics RAST server – a public resource for the automatic phylogenetic and functional analysis of metagenomes

    Directory of Open Access Journals (Sweden)

    Stevens R

    2008-09-01

    Full Text Available Abstract Background Random community genomes (metagenomes are now commonly used to study microbes in different environments. Over the past few years, the major challenge associated with metagenomics shifted from generating to analyzing sequences. High-throughput, low-cost next-generation sequencing has provided access to metagenomics to a wide range of researchers. Results A high-throughput pipeline has been constructed to provide high-performance computing to all researchers interested in using metagenomics. The pipeline produces automated functional assignments of sequences in the metagenome by comparing both protein and nucleotide databases. Phylogenetic and functional summaries of the metagenomes are generated, and tools for comparative metagenomics are incorporated into the standard views. User access is controlled to ensure data privacy, but the collaborative environment underpinning the service provides a framework for sharing datasets between multiple users. In the metagenomics RAST, all users retain full control of their data, and everything is available for download in a variety of formats. Conclusion The open-source metagenomics RAST service provides a new paradigm for the annotation and analysis of metagenomes. With built-in support for multiple data sources and a back end that houses abstract data types, the metagenomics RAST is stable, extensible, and freely available to all researchers. This service has removed one of the primary bottlenecks in metagenome sequence analysis – the availability of high-performance computing for annotating the data. http://metagenomics.nmpdr.org

  17. Interactive metagenomic visualization in a Web browser

    Directory of Open Access Journals (Sweden)

    Phillippy Adam M

    2011-09-01

    Full Text Available Abstract Background A critical output of metagenomic studies is the estimation of abundances of taxonomical or functional groups. The inherent uncertainty in assignments to these groups makes it important to consider both their hierarchical contexts and their prediction confidence. The current tools for visualizing metagenomic data, however, omit or distort quantitative hierarchical relationships and lack the facility for displaying secondary variables. Results Here we present Krona, a new visualization tool that allows intuitive exploration of relative abundances and confidences within the complex hierarchies of metagenomic classifications. Krona combines a variant of radial, space-filling displays with parametric coloring and interactive polar-coordinate zooming. The HTML5 and JavaScript implementation enables fully interactive charts that can be explored with any modern Web browser, without the need for installed software or plug-ins. This Web-based architecture also allows each chart to be an independent document, making them easy to share via e-mail or post to a standard Web server. To illustrate Krona's utility, we describe its application to various metagenomic data sets and its compatibility with popular metagenomic analysis tools. Conclusions Krona is both a powerful metagenomic visualization tool and a demonstration of the potential of HTML5 for highly accessible bioinformatic visualizations. Its rich and interactive displays facilitate more informed interpretations of metagenomic analyses, while its implementation as a browser-based application makes it extremely portable and easily adopted into existing analysis packages. Both the Krona rendering code and conversion tools are freely available under a BSD open-source license, and available from: http://krona.sourceforge.net.

  18. Metagenomic Analysis of Water Distribution System Bacterial Communities

    Science.gov (United States)

    The microbial quality of drinking water is assessed using culture-based methods that are highly selective and that tend to underestimate the densities and diversity of microbial populations inhabiting distribution systems. In order to better understand the effect of different dis...

  19. Bacterial Communities: Interactions to Scale

    Directory of Open Access Journals (Sweden)

    Reed M. Stubbendieck

    2016-08-01

    Full Text Available In the environment, bacteria live in complex multispecies communities. These communities span in scale from small, multicellular aggregates to billions or trillions of cells within the gastrointestinal tract of animals. The dynamics of bacterial communities are determined by pairwise interactions that occur between different species in the community. Though interactions occur between a few cells at a time, the outcomes of these interchanges have ramifications that ripple through many orders of magnitude, and ultimately affect the macroscopic world including the health of host organisms. In this review we cover how bacterial competition influences the structures of bacterial communities. We also emphasize methods and insights garnered from culture-dependent pairwise interaction studies, metagenomic analyses, and modeling experiments. Finally, we argue that the integration of multiple approaches will be instrumental to future understanding of the underlying dynamics of bacterial communities.

  20. Metagenomics as a preliminary screen for antimicrobial bioprospecting.

    Science.gov (United States)

    Al-Amoudi, Soha; Razali, Rozaimi; Essack, Magbubah; Amini, Mohammad Shoaib; Bougouffa, Salim; Archer, John A C; Lafi, Feras F; Bajic, Vladimir B

    2016-12-15

    Since the composition of soil directs the diversity of the contained microbiome and its potential to produce bioactive compounds, many studies has been focused on sediment types with unique features characteristic of extreme environments. However, not much is known about the potential of microbiomes that inhabit the highly saline and hot Red Sea lagoons. This case study explores mangrove mud and the microbial mat of sediments collected from the Rabigh harbor lagoon and Al Kharrar lagoon for antimicrobial bioprospecting. Rabigh harbor lagoon appears the better location, and the best sediment type for this purpose is mangrove mud. On the other hand, Al Kharrar lagoon displayed increased anaerobic hydrocarbon degradation and an abundance of bacterial DNA associated with antibiotic resistance. Moreover, our findings show an identical shift in phyla associated with historic hydrocarbon contamination exposure reported in previous studies (that is, enrichment of Gamma- and Delta-proteobacteria), but we also report that bacterial DNA sequences associated with antibiotic synthesis enzymes are derived from Gamma-, Delta- and Alpha-proteobacteria. This suggests that selection pressure associated with hydrocarbon contamination tend to enrich the bacterial classes DNA associated with antibiotic synthesis enzymes. Although Actinobacteria tends to be the common target for research when it comes to antimicrobial bioprospecting, our study suggests that Firmicutes (Bacilli and Clostridia), Bacteroidetes, Cyanobacteria, and Proteobacteria should be antimicrobial bioprospecting targets as well. To the best of our knowledge, this is the first metagenomic study that analyzed the microbiomes in Red Sea lagoons for antimicrobial bioprospecting.

  1. Single sample resolution of rare microbial dark matter in a marine invertebrate metagenome

    Science.gov (United States)

    Miller, Ian J.; Weyna, Theodore R.; Fong, Stephen S.; Lim-Fong, Grace E.; Kwan, Jason C.

    2016-01-01

    Direct, untargeted sequencing of environmental samples (metagenomics) and de novo genome assembly enable the study of uncultured and phylogenetically divergent organisms. However, separating individual genomes from a mixed community has often relied on the differential-coverage analysis of multiple, deeply sequenced samples. In the metagenomic investigation of the marine bryozoan Bugula neritina, we uncovered seven bacterial genomes associated with a single B. neritina individual that appeared to be transient associates, two of which were unique to one individual and undetectable using certain “universal” 16S rRNA primers and probes. We recovered high quality genome assemblies for several rare instances of “microbial dark matter,” or phylogenetically divergent bacteria lacking genomes in reference databases, from a single tissue sample that was not subjected to any physical or chemical pre-treatment. One of these rare, divergent organisms has a small (593 kbp), poorly annotated genome with low GC content (20.9%) and a 16S rRNA gene with just 65% sequence similarity to the closest reference sequence. Our findings illustrate the importance of sampling strategy and de novo assembly of metagenomic reads to understand the extent and function of bacterial biodiversity. PMID:27681823

  2. Shotgun metagenomic data streams: surfing without fear

    Energy Technology Data Exchange (ETDEWEB)

    Berendzen, Joel R [Los Alamos National Laboratory

    2010-12-06

    Timely information about bio-threat prevalence, consequence, propagation, attribution, and mitigation is needed to support decision-making, both routinely and in a crisis. One DNA sequencer can stream 25 Gbp of information per day, but sampling strategies and analysis techniques are needed to turn raw sequencing power into actionable knowledge. Shotgun metagenomics can enable biosurveillance at the level of a single city, hospital, or airplane. Metagenomics characterizes viruses and bacteria from complex environments such as soil, air filters, or sewage. Unlike targeted-primer-based sequencing, shotgun methods are not blind to sequences that are truly novel, and they can measure absolute prevalence. Shotgun metagenomic sampling can be non-invasive, efficient, and inexpensive while being informative. We have developed analysis techniques for shotgun metagenomic sequencing that rely upon phylogenetic signature patterns. They work by indexing local sequence patterns in a manner similar to web search engines. Our methods are laptop-fast and favorable scaling properties ensure they will be sustainable as sequencing methods grow. We show examples of application to soil metagenomic samples.

  3. Preliminary High-Throughput Metagenome Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Dusheyko, Serge; Furman, Craig; Pangilinan, Jasmyn; Shapiro, Harris; Tu, Hank

    2007-03-26

    Metagenome data sets present a qualitatively different assembly problem than traditional single-organism whole-genome shotgun (WGS) assembly. The unique aspects of such projects include the presence of a potentially large number of distinct organisms and their representation in the data set at widely different fractions. In addition, multiple closely related strains could be present, which would be difficult to assemble separately. Failure to take these issues into account can result in poor assemblies that either jumble together different strains or which fail to yield useful results. The DOE Joint Genome Institute has sequenced a number of metagenomic projects and plans to considerably increase this number in the coming year. As a result, the JGI has a need for high-throughput tools and techniques for handling metagenome projects. We present the techniques developed to handle metagenome assemblies in a high-throughput environment. This includes a streamlined assembly wrapper, based on the JGI?s in-house WGS assembler, Jazz. It also includes the selection of sensible defaults targeted for metagenome data sets, as well as quality control automation for cleaning up the raw results. While analysis is ongoing, we will discuss preliminary assessments of the quality of the assembly results (http://fames.jgi-psf.org).

  4. Viral Metagenomics: MetaView Software

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, C; Smith, J

    2007-10-22

    The purpose of this report is to design and develop a tool for analysis of raw sequence read data from viral metagenomics experiments. The tool should compare read sequences of known viral nucleic acid sequence data and enable a user to attempt to determine, with some degree of confidence, what virus groups may be present in the sample. This project was conducted in two phases. In phase 1 we surveyed the literature and examined existing metagenomics tools to educate ourselves and to more precisely define the problem of analyzing raw read data from viral metagenomic experiments. In phase 2 we devised an approach and built a prototype code and database. This code takes viral metagenomic read data in fasta format as input and accesses all complete viral genomes from Kpath for sequence comparison. The system executes at the UNIX command line, producing output that is stored in an Oracle relational database. We provide here a description of the approach we came up with for handling un-assembled, short read data sets from viral metagenomics experiments. We include a discussion of the current MetaView code capabilities and additional functionality that we believe should be added, should additional funding be acquired to continue the work.

  5. Metagenomic Analysis of Chicken Gut Microbiota for Improving Metabolism and Health of Chickens - A Review.

    Science.gov (United States)

    Choi, Ki Young; Lee, Tae Kwon; Sul, Woo Jun

    2015-09-01

    Chicken is a major food source for humans, hence it is important to understand the mechanisms involved in nutrient absorption in chicken. In the gastrointestinal tract (GIT), the microbiota plays a central role in enhancing nutrient absorption and strengthening the immune system, thereby affecting both growth and health of chicken. There is little information on the diversity and functions of chicken GIT microbiota, its impact on the host, and the interactions between the microbiota and host. Here, we review the recent metagenomic strategies to analyze the chicken GIT microbiota composition and its functions related to improving metabolism and health. We summarize methodology of metagenomics in order to obtain bacterial taxonomy and functional inferences of the GIT microbiota and suggest a set of indicator genes for monitoring and manipulating the microbiota to promote host health in future.

  6. Molecular analysis of the bacterial diversity in a specialized consortium for diesel oil degradation

    Energy Technology Data Exchange (ETDEWEB)

    Paixao, Douglas Antonio Alvaredo; Accorsini, Fabio Raphael; Vidotti, Maria Benincasa; Lemos, Eliana Gertrudes de Macedo [Universidade Estadual Paulista (FCAV/UNESP), Jaboticabal, SP (Brazil). Fac. de Ciencias Agrarias e Veterinarias], Emails: douglas_unespfcav@yahoo.com.br, vidotti@netsite.com.bregerle@fcav.unesp.br; Dimitrov, Mauricio Rocha [Universidade de Sao Paulo (USP), SP (Brazil)], Email: mau_dimitrov@yahoo.com.br; Pereira, Rodrigo Matheus [EMBRAPARA Soybean - Empresa Brasileira de Pesquisa Agropecuaria (EMBRAPA - Soja), Londrina, PR (Brazil)], Email: poetbr@gmail.com

    2010-05-15

    Diesel oil is a compound derived from petroleum, consisting primarily of hydrocarbons. Poor conditions in transportation and storage of this product can contribute significantly to accidental spills causing serious ecological problems in soil and water and affecting the diversity of the microbial environment. The cloning and sequencing of the 16S rRNA gene is one of the molecular techniques that allows estimation and comparison of the microbial diversity in different environmental samples. The aim of this work was to estimate the diversity of microorganisms from the Bacteria domain in a consortium specialized in diesel oil degradation through partial sequencing of the 16S rRNA gene. After the extraction of DNA metagenomics, the material was amplified by PCR reaction using specific oligonucleotide primers for the 16S rRNA gene. The PCR products were cloned into a pGEM-T-Easy vector (Promega), and Escherichia coli was used as the host cell for recombinant DNAs. The partial clone sequencing was obtained using universal oligonucleotide primers from the vector. The genetic library obtained generated 431 clones. All the sequenced clones presented similarity to phylum Proteobacteria, with Gammaproteobacteria the most present group (49.8 % of the clones), followed by Alphaproteobacteira (44.8 %) and Betaproteobacteria (5.4 %). The Pseudomonas genus was the most abundant in the metagenomics library, followed by the Parvibaculum and the Sphingobium genus, respectively. After partial sequencing of the 16S rRNA, the diversity of the bacterial consortium was estimated using DOTUR software. When comparing these sequences to the database from the National Center for Biotechnology Information (NCBI), a strong correlation was found between the data generated by the software used and the data deposited in NCBI. (author)

  7. FANTOM: Functional and taxonomic analysis of metagenomes

    Directory of Open Access Journals (Sweden)

    Sanli Kemal

    2013-02-01

    Full Text Available Abstract Background Interpretation of quantitative metagenomics data is important for our understanding of ecosystem functioning and assessing differences between various environmental samples. There is a need for an easy to use tool to explore the often complex metagenomics data in taxonomic and functional context. Results Here we introduce FANTOM, a tool that allows for exploratory and comparative analysis of metagenomics abundance data integrated with metadata information and biological databases. Importantly, FANTOM can make use of any hierarchical database and it comes supplied with NCBI taxonomic hierarchies as well as KEGG Orthology, COG, PFAM and TIGRFAM databases. Conclusions The software is implemented in Python, is platform independent, and is available at http://www.sysbio.se/Fantom.

  8. Pathway-Based Functional Analysis of Metagenomes

    Science.gov (United States)

    Bercovici, Sivan; Sharon, Itai; Pinter, Ron Y.; Shlomi, Tomer

    Metagenomic data enables the study of microbes and viruses through their DNA as retrieved directly from the environment in which they live. Functional analysis of metagenomes explores the abundance of gene families, pathways, and systems, rather than their taxonomy. Through such analysis researchers are able to identify those functional capabilities most important to organisms in the examined environment. Recently, a statistical framework for the functional analysis of metagenomes was described that focuses on gene families. Here we describe two pathway level computational models for functional analysis that take into account important, yet unaddressed issues such as pathway size, gene length and overlap in gene content among pathways. We test our models over carefully designed simulated data and propose novel approaches for performance evaluation. Our models significantly improve over current approach with respect to pathway ranking and the computations of relative abundance of pathways in environments.

  9. 宏基因组学及其在口腔微生物研究领域中的应用%The research methods of metagenomic and the application of metagenomic study in the oral microbiota

    Institute of Scientific and Technical Information of China (English)

    陆剑; 李宇红; 杜民权

    2013-01-01

    Metagenomics is the genomes of the total microbiota found in nature. Metagenome libraries were con-structed by directly extracting DNA from environmental samples, Such as soil, oceans, human gastrointestinal tract and mouth, etc., and transforming to surrogate host. The libraries were screened for novel bioactive compounds or genes. Therefore, Metagenomics can effectively detect oral microbial community structure, and also can enormously amplified the space of microbial resource utilization and enhanced the opportunity of obtain novel bioactive com-pounds. This review included the research progress of the methods of metagenomic and the application of metage-nomic study in the oral microbiota.%  宏基因组学研究特定生物环境中全部微小生物的基因组,直接从土壤和海水以及人体胃肠道和口腔等环境中获取样品 DNA,利用适宜的载体将其克隆到替代宿主细胞中构建宏基因文库,以筛选新的活性物质和新的基因;因此,利用宏基因组学技术不仅能够有效地检测口腔微生物群落结构,同时还极大地扩展了口腔微生物资源的利用空间,增加了获得新的生物活性物质和基因的机会。本文就宏基因组学的研究方法和宏基因组学口腔微生物研究领域中的应用等研究进展作一综述。

  10. [Metagenomics in studying gastrointestinal tract microorganism].

    Science.gov (United States)

    Xu, Bo; Yang, Yunjuan; Li, Junjun; Tang, Xianghua; Mu, Yuelin; Huang, Zunxi

    2013-12-01

    Animal gastrointestinal tract contains a complex community of microbes, whose composition ultimately reflects the co-evolution of microorganisms with their animal host. The gut microbial community of humans and animals has received significant attention from researchers because of its association with health and disease. The application of metagenomics technology enables researchers to study not only the microbial composition but also the function of microbes in the gastrointestinal tract. In this paper, combined with our own findings, we summarized advances in studying gastrointestinal tract microorganism with metagenomics and the bioinformatics technology.

  11. Metagenomic discovery of novel enzymes and biosurfactants in a slaughterhouse biofilm microbial community.

    Science.gov (United States)

    Thies, Stephan; Rausch, Sonja Christina; Kovacic, Filip; Schmidt-Thaler, Alexandra; Wilhelm, Susanne; Rosenau, Frank; Daniel, Rolf; Streit, Wolfgang; Pietruszka, Jörg; Jaeger, Karl-Erich

    2016-06-08

    DNA derived from environmental samples is a rich source of novel bioactive molecules. The choice of the habitat to be sampled predefines the properties of the biomolecules to be discovered due to the physiological adaptation of the microbial community to the prevailing environmental conditions. We have constructed a metagenomic library in Escherichia coli DH10b with environmental DNA (eDNA) isolated from the microbial community of a slaughterhouse drain biofilm consisting mainly of species from the family Flavobacteriaceae. By functional screening of this library we have identified several lipases, proteases and two clones (SA343 and SA354) with biosurfactant and hemolytic activities. Sequence analysis of the respective eDNA fragments and subsequent structure homology modelling identified genes encoding putative N-acyl amino acid synthases with a unique two-domain organisation. The produced biosurfactants were identified by NMR spectroscopy as N-acyltyrosines with N-myristoyltyrosine as the predominant species. Critical micelle concentration and reduction of surface tension were similar to those of chemically synthesised N-myristoyltyrosine. Furthermore, we showed that the newly isolated N-acyltyrosines exhibit antibiotic activity against various bacteria. This is the first report describing the successful application of functional high-throughput screening assays for the identification of biosurfactant producing clones within a metagenomic library.

  12. Unbiased Detection of Respiratory Viruses by Use of RNA Sequencing-Based Metagenomics: a Systematic Comparison to a Commercial PCR Panel.

    Science.gov (United States)

    Graf, Erin H; Simmon, Keith E; Tardif, Keith D; Hymas, Weston; Flygare, Steven; Eilbeck, Karen; Yandell, Mark; Schlaberg, Robert

    2016-04-01

    Current infectious disease molecular tests are largely pathogen specific, requiring test selection based on the patient's symptoms. For many syndromes caused by a large number of viral, bacterial, or fungal pathogens, such as respiratory tract infections, this necessitates large panels of tests and has limited yield. In contrast, next-generation sequencing-based metagenomics can be used for unbiased detection of any expected or unexpected pathogen. However, barriers for its diagnostic implementation include incomplete understanding of analytical performance and complexity of sequence data analysis. We compared detection of known respiratory virus-positive (n= 42) and unselected (n= 67) pediatric nasopharyngeal swabs using an RNA sequencing (RNA-seq)-based metagenomics approach and Taxonomer, an ultrarapid, interactive, web-based metagenomics data analysis tool, with an FDA-cleared respiratory virus panel (RVP; GenMark eSensor). Untargeted metagenomics detected 86% of known respiratory virus infections, and additional PCR testing confirmed RVP results for only 2 (33%) of the discordant samples. In unselected samples, untargeted metagenomics had excellent agreement with the RVP (93%). In addition, untargeted metagenomics detected an additional 12 viruses that were either not targeted by the RVP or missed due to highly divergent genome sequences. Normalized viral read counts for untargeted metagenomics correlated with viral burden determined by quantitative PCR and showed high intrarun and interrun reproducibility. Partial or full-length viral genome sequences were generated in 86% of RNA-seq-positive samples, allowing assessment of antiviral resistance, strain-level typing, and phylogenetic relatedness. Overall, untargeted metagenomics had high agreement with a sensitive RVP, detected viruses not targeted by the RVP, and yielded epidemiologically and clinically valuable sequence information.

  13. METAREP: JCVI metagenomics reports—an open source tool for high-performance comparative metagenomics

    OpenAIRE

    Goll, Johannes; Rusch, Douglas B; Tanenbaum, David M.; Thiagarajan, Mathangi; Li, Kelvin; Methé, Barbara A.; Yooseph, Shibu

    2010-01-01

    Summary: JCVI Metagenomics Reports (METAREP) is a Web 2.0 application designed to help scientists analyze and compare annotated metagenomics datasets. It utilizes Solr/Lucene, a high-performance scalable search engine, to quickly query large data collections. Furthermore, users can use its SQL-like query syntax to filter and refine datasets. METAREP provides graphical summaries for top taxonomic and functional classifications as well as a GO, NCBI Taxonomy and KEGG Pathway Browser. Users can ...

  14. Cultivation-independent comprehensive survey of bacterial diversity in Tulsi Shyam Hot Springs, India

    Directory of Open Access Journals (Sweden)

    Anjana Ghelani

    2015-06-01

    Full Text Available A taxonomic description of bacteria was deduced from 5.78 Mb metagenomic sequence retrieved from Tulsi Shyam hot spring, India using bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP. Metagenome contained 10,893 16S rDNA sequences that were analyzed by MG-RAST server to generate the comprehensive profile of bacteria. Metagenomic data are available at EBI under EBI Metagenomics database with accession no. ERP009559. Metagenome sequences represented the 98.2% bacteria origin, 1.5% of eukaryotic and 0.3% were unidentified. A total of 16 bacterial phyla demonstrating 97 families and 287 species were revealed in the hot spring metagenome. Most abundant phyla were Firmicutes (65.38%, Proteobacteria (21.21% and unclassified bacteria (10.69%. Whereas, Peptostreptococcaceae (37.33%, Clostridiaceae (23.36%, and Enterobacteriaceae (16.37% were highest reported families in metagenome. Ubiquitous species were Clostridium bifermentans (17.47%, Clostridium lituseburense (13.93% and uncultured bacterium (10.15%. Our data provide new information on hot spring bacteria and shed light on their abundance, diversity, distribution and coexisting organisms.

  15. Cultivation-independent comprehensive survey of bacterial diversity in Tulsi Shyam Hot Springs, India.

    Science.gov (United States)

    Ghelani, Anjana; Patel, Rajesh; Mangrola, Amitsinh; Dudhagara, Pravin

    2015-06-01

    A taxonomic description of bacteria was deduced from 5.78 Mb metagenomic sequence retrieved from Tulsi Shyam hot spring, India using bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). Metagenome contained 10,893 16S rDNA sequences that were analyzed by MG-RAST server to generate the comprehensive profile of bacteria. Metagenomic data are available at EBI under EBI Metagenomics database with accession no. ERP009559. Metagenome sequences represented the 98.2% bacteria origin, 1.5% of eukaryotic and 0.3% were unidentified. A total of 16 bacterial phyla demonstrating 97 families and 287 species were revealed in the hot spring metagenome. Most abundant phyla were Firmicutes (65.38%), Proteobacteria (21.21%) and unclassified bacteria (10.69%). Whereas, Peptostreptococcaceae (37.33%), Clostridiaceae (23.36%), and Enterobacteriaceae (16.37%) were highest reported families in metagenome. Ubiquitous species were Clostridium bifermentans (17.47%), Clostridium lituseburense (13.93%) and uncultured bacterium (10.15%). Our data provide new information on hot spring bacteria and shed light on their abundance, diversity, distribution and coexisting organisms.

  16. A metagenomic-based survey of microbial (de)halogenation potential in a German forest soil.

    Science.gov (United States)

    Weigold, Pascal; El-Hadidi, Mohamed; Ruecker, Alexander; Huson, Daniel H; Scholten, Thomas; Jochmann, Maik; Kappler, Andreas; Behrens, Sebastian

    2016-06-29

    In soils halogens (fluorine, chlorine, bromine, iodine) are cycled through the transformation of inorganic halides into organohalogen compounds and vice versa. There is evidence that these reactions are microbially driven but the key enzymes and groups of microorganisms involved are largely unknown. Our aim was to uncover the diversity, abundance and distribution of genes encoding for halogenating and dehalogenating enzymes in a German forest soil by shotgun metagenomic sequencing. Metagenomic libraries of three soil horizons revealed the presence of genera known to be involved in halogenation and dehalogenation processes such as Bradyrhizobium or Pseudomonas. We detected a so far unknown diversity of genes encoding for (de)halogenating enzymes in the soil metagenome including specific and unspecific halogenases as well as metabolic and cometabolic dehalogenases. Genes for non-heme, no-metal chloroperoxidases and haloalkane dehalogenases were the most abundant halogenase and dehalogenase genes, respectively. The high diversity and abundance of (de)halogenating enzymes suggests a strong microbial contribution to natural halogen cycling. This was also confirmed in microcosm experiments in which we quantified the biotic formation of chloroform and bromoform. Knowledge on microorganisms and genes that catalyze (de)halogenation reactions is critical because they are highly relevant to industrial biotechnologies and bioremediation applications.

  17. The metagenomic approach and causality in virology

    Directory of Open Access Journals (Sweden)

    Silvana Beres Castrignano

    2015-01-01

    Full Text Available Nowadays, the metagenomic approach has been a very important tool in the discovery of new viruses in environmental and biological samples. Here we discuss how these discoveries may help to elucidate the etiology of diseases and the criteria necessary to establish a causal association between a virus and a disease.

  18. Snowball: Strain aware gene assembly of Metagenomes

    NARCIS (Netherlands)

    Gregor, I.; Schönhuth, A.; McHardy, A.C.

    2015-01-01

    Gene assembly is an important step in functional analysis of shotgun metagenomic data. Nonetheless, strain aware assembly remains a challenging task, as current assembly tools often fail to distinguish among strain variants or require closely related reference genomes of the studied species to be av

  19. Biomolecular and metagenomic analyses of biofouling communities

    Science.gov (United States)

    Despite the decades of research that have focused on understanding the formation of biofouling communities, relatively little is known about the soft fouling consortia that are responsible for their formation and function. In this study, we used PhyloChip microbial profiling, metagenomic DNA sequenc...

  20. Assembly of viral genomes from metagenomes

    NARCIS (Netherlands)

    S.L. Smits (Saskia); R. Bodewes (Rogier); A. Ruiz-Gonzalez (Aritz); V. Baumgärtner (Volkmar); M.P.G. Koopmans D.V.M. (Marion); A.D.M.E. Osterhaus (Albert); A. Schürch (Anita)

    2014-01-01

    textabstractViral infections remain a serious global health issue. Metagenomic approaches are increasingly used in the detection of novel viral pathogens but also to generate complete genomes of uncultivated viruses. In silico identification of complete viral genomes from sequence data would allow r

  1. Assembly of viral genomes from metagenomes

    Directory of Open Access Journals (Sweden)

    Saskia L Smits

    2014-12-01

    Full Text Available Viral infections remain a serious global health issue. Metagenomic approaches are increasingly used in the detection of novel viral pathogens but also to generate complete genomes of uncultivated viruses. In silico identification of complete viral genomes from sequence data would allow rapid phylogenetic characterization of these new viruses. Often, however, complete viral genomes are not recovered, but rather several distinct contigs derived from a single entity, some of which have no sequence homology to any known proteins. De novo assembly of single viruses from a metagenome is challenging, not only because of the lack of a reference genome, but also because of intrapopulation variation and uneven or insufficient coverage. Here we explored different assembly algorithms, remote homology searches, genome-specific sequence motifs, k-mer frequency ranking, and coverage profile binning to detect and obtain viral target genomes from metagenomes. All methods were tested on 454-generated sequencing datasets containing three recently described RNA viruses with a relatively large genome which were divergent to previously known viruses from the viral families Rhabdoviridae and Coronaviridae. Depending on specific characteristics of the target virus and the metagenomic community, different assembly and in silico gap closure strategies were successful in obtaining near complete viral genomes.

  2. A catalog of the mouse gut metagenome

    DEFF Research Database (Denmark)

    Xiao, Liang; Feng, Qiang; Liang, Suisha;

    2015-01-01

    We established a catalog of the mouse gut metagenome comprising ∼2.6 million nonredundant genes by sequencing DNA from fecal samples of 184 mice. To secure high microbiome diversity, we used mouse strains of diverse genetic backgrounds, from different providers, kept in different housing laborato...

  3. Functional metagenomics: recent advances and future challenges.

    Science.gov (United States)

    Chistoserdovai, Ludmila

    2010-01-01

    Metagenomics is a relatively new but fast growing field within environmental biology directed at obtaining knowledge on genomes of environmental microbes as well as of entire microbial communities. With the sequencing technologies improving steadily, generating large amounts of sequence is becoming routine. However, it remains difficult to connect specific microbial phyla to specific functions in the environment. A number of 'functional metagenomics' approaches have been implemented in the recent years that allow high-resolution genomic analysis of uncultivated microbes, connecting them to specific functions in the environment. These include analysis of niche-specialized low complexity communities, reactor enrichments, and the use labeling technologies. Metatranscriptomics and metaproteomics are the newest sub-disciplines within the metagenomics field that provide further levels of resolution for functional analysis of uncultivated microbes and communities. The recent emergence of new (next generation) sequencing technologies, resulting in higher sequence output and dramatic drop in the price of sequencing, will be defining a new era in metagenomics. At this time the sequencing effort will be taken to a new level to allow addressing new, previously unattainable biological questions as well as accelerating genome-based discovery for medical and biotechnological applications.

  4. The metagenomic approach and causality in virology

    Science.gov (United States)

    Castrignano, Silvana Beres; Nagasse-Sugahara, Teresa Keico

    2015-01-01

    Nowadays, the metagenomic approach has been a very important tool in the discovery of new viruses in environmental and biological samples. Here we discuss how these discoveries may help to elucidate the etiology of diseases and the criteria necessary to establish a causal association between a virus and a disease. PMID:25902566

  5. 深海沉积物宏基因组文库中产蛋白酶克隆CAPRO2的筛选及酶学性质分析%Screening and characterization of a protease-producing clone CAPRO2 from metagenomic library of deep sea sediments

    Institute of Scientific and Technical Information of China (English)

    徐辉; 赵晶; 曾润颖

    2011-01-01

    A protease-producing clone (CAPRO2) was screened from metagenomic library of deep sea sediments u-sing selective medium, and the characteristics of protease was studied. The results showed that the optimal temper-ature and pH for CAPR02 protease activity were 65℃, and 9.0, respectively. The enzyme had excellent thermal stability. It was stable at temperature below 40t, and retained about 60% of residual activity after 6 hours incubation at 50t and 30 minutes incubation at 601. The presence of Ca2+ , Mg2+ , Sr2+ and Co2+ increased the activity obviously, and Ca2 + enhanced the thermal stability distinctly. Three ions, Ca2 + , Sr2 + and Co2 + , increased the activity of the enzyme significantly at concentration of 3.0 mmol/dm3, and the activity-increasing effect was weak when the concentrations of these 3 ions were higher than 3.0 mmol/dm3. The ions Hg2+, Fe3 + and Cu2+ inhibited the enzyme activity notably. The CAPRO2 protease showed higher activity at pH 7. 5 ~ 9. 5 , retaining 80% and60% of residual activity at pH 7.5 and 9. 5 , respectively. The residual activity decreased rapidly at pH higher than 9.5 and reduced to about 15% at pH 10.0. It indicates that the enzyme belongs to alkaline protease. The serine protease inhibitors, PMSF, E-64 and AEBSF, showed no inhibition to the CAPRO2 enzyme activity, which indicates that the enzyme was not serine protease. On the other hand, EDTA inhibited the enzyme activity strongly, indicating that the enzyme belongs to metalloproteinase.%利用选择性筛选培养基从所构建的深海沉积物宏基因组文库中筛选得到一株产蛋白酶的克隆( CAPR0002),对其进行了酶学性质分析.结果表明该酶的最适作用温度为65℃,最适作用pH值为9.0.该蛋白酶具有较好的热稳定性,在40℃以下的温度中可长期保持稳定,在50℃中处理6h后仍能保持60%的活力,在60℃下保温30 min后仍能保持约60%的活力.Ca2+、Mg2+、Sr2+、Co2+对该蛋白酶有

  6. Library Automation

    OpenAIRE

    Dhakne, B. N.; Giri, V. V.; Waghmode, S. S.

    2010-01-01

    New technologies library provides several new materials, media and mode of storing and communicating the information. Library Automation reduces the drudgery of repeated manual efforts in library routine. By use of library automation collection, Storage, Administration, Processing, Preservation and communication etc.

  7. Gut bacterial profile in patients newly diagnosed with treatment-naïve Crohn's disease

    Directory of Open Access Journals (Sweden)

    Ricanek P

    2012-09-01

    Full Text Available Petr Ricanek,1,2 Sheba M Lothe,1 Stephan A Frye,1 Andreas Rydning,2 Morten H Vatn,3,4 Tone Tønjum1,51Centre for Molecular Biology and Neuroscience and Department of Microbiology, Oslo University Hospital, Rikshospitalet, Oslo, 2Department of Gastroenterology, Akershus University Hospital, Lørenskog and Faculty Division Akershus University Hospital, University of Oslo, Lørenskog, 3EpiGen Institute, Faculty Division Akershus University Hospital, University of Oslo, Lørenskog, 4Department of Medicine, Oslo University Hospital, Rikshospitalet, Oslo, 5Centre for Molecular Biology and Neuroscience and Department of Microbiology, University of Oslo, Oslo, NorwayObjectives: The aim of this study was to define the composition of the gut bacterial flora in Norwegian patients with early stage Crohn's disease (CD. Methods: By using a nonselective metagenomics approach, the general bacterial composition in mucosal biopsies from the ileum and the colon of five subjects, four patients with different phenotypes of CD, and one noninflammatory bowel disease control, was characterized. After partial 16S ribosomal RNA (rRNA gene sequencing, BLAST homology searches for species identification and phylogenetic analysis were performed.Results: An overall biodiversity of 106 different bacterial operational taxonomic units (OTUs was detected in the cloned libraries. Nearly all OTUs belonged to the phylae Bacteroidetes (42% in CD, 71% in the control or Firmicutes (42% in CD, 28% in the control, except for some OTUs that belonged to the phylum Proteobacteria (15% in CD, 0% in the control and a few OTUs that could not be assigned to a phylum (2% in CD, 1% in the control.Conclusion: Based on the high incidence of inflammatory bowel disease (IBD in Norway, this pilot study represents a relevant determination of the gut microbiota in Norwegian patients compared to previous findings in other countries. The bacterial profile of Norwegian CD patients was found to be similar

  8. [Comparative Metagenomics of BIOLAK and A2O Activated Sludge Based on Next-generation Sequencing Technology].

    Science.gov (United States)

    Tian, Mei; Liu, Han-hu; Shen, Xin

    2016-02-15

    This is the first report of comparative metagenomic analyses of BIOLAK sludge and anaerobic/anoxic/oxic (A2O) sludge. In the BIOLAK and A2O sludge metagenomes, 47 and 51 phyla were identified respectively, more than the numbers of phyla identified in Australia EBPR (enhanced biological phosphorus removal), USA EBPR and Bibby sludge. All phyla found in the BIOLAK sludge were detected in the A2O sludge, but four phyla were exclusively found in the A20 sludge. The proportion of the phylum Ignavibacteriae in the A2O sludge was 2.0440%, which was 3.2 times as much as that in the BIOLAK sludge (0.6376%). Meanwhile, the proportion of the bacterial phylum Gemmatimonadetes in the BIOLAK sludge was 2.4673%, which was >17 times as much as that in the A2O sludge (0.1404%). The proportion of the bacterial phylum Chlamydiae in the BIOLAK metagenome (0.2192%) was >6 times higher than that in the A2O (0.0360%). Furthermore, 167 genera found in the A20 sludge were not detected in the BIOLAK sludge. And 50 genera found in the BIOLAK sludge were not detected in the A20 sludge. From the analyses of both the phylum and genus levels, there were huge differences between the two biological communities of A2O and BIOLAK sludge. However, the proportions of each group of functional genes associated with metabolism of nitrogen, phosphor, sulfur and aromatic compounds in BIOLAK were very similar to those in A2O sludge. Moreover, the rankings of all six KEGG (Kyoto Encyclopedia for Genes and Genomes) categories were identical in the two sludges. In addition, the analyses of functional classification and pathway related nitrogen metabolism showed that the abundant enzymes had identical ranking in the BIOLAK and A2O metagenomes. Therefore, comparative metagenomics of BIOLAK and A2O activated sludge indicated similar function assignments from the two different biological communities.

  9. What Can We Learn from a Metagenomic Analysis of a Georgian Bacteriophage Cocktail?

    DEFF Research Database (Denmark)

    Zschach, Henrike; Joensen, Katrine Grimstrup; Lindhard, Barbara

    2015-01-01

    Phage therapy, a practice widespread in Eastern Europe, has untapped potential in the combat against antibiotic-resistant bacterial infections. However, technology transfer to Western medicine is proving challenging. Bioinformatics analysis could help to facilitate this endeavor. In the present...... study, the Intesti phage cocktail, a key commercial product of the Eliava Institute, Georgia, has been tested on a selection of bacterial strains, sequenced as a metagenomic sample, de novo assembled and analyzed by bioinformatics methods. Furthermore, eight bacterial host strains were infected...... with the cocktail and the resulting lysates sequenced and compared to the unamplified cocktail. The analysis identified 23 major phage clusters in different abundances in the cocktail, among those clusters related to the ICTV genera T4likevirus, T5likevirus, T7likevirus, Chilikevirus and Twortlikevirus, as well...

  10. eSNaPD: a versatile, web-based bioinformatics platform for surveying and mining natural product biosynthetic diversity from metagenomes.

    Science.gov (United States)

    Reddy, Boojala Vijay B; Milshteyn, Aleksandr; Charlop-Powers, Zachary; Brady, Sean F

    2014-08-14

    Environmental Surveyor of Natural Product Diversity (eSNaPD) is a web-based bioinformatics and data aggregation platform that aids in the discovery of gene clusters encoding both novel natural products and new congeners of medicinally relevant natural products using (meta)genomic sequence data. Using PCR-generated sequence tags, the eSNaPD data-analysis pipeline profiles biosynthetic diversity hidden within (meta)genomes by comparing sequence tags to a reference data set of characterized gene clusters. Sample mapping, molecule discovery, library mapping, and new clade visualization modules facilitate the interrogation of large (meta)genomic sequence data sets for diverse downstream analyses, including, but not limited to, the identification of environments rich in untapped biosynthetic diversity, targeted molecule discovery efforts, and chemical ecology studies. eSNaPD is designed to generate a global atlas of biosynthetic diversity that can facilitate a systematic, sequence-based interrogation of nature's biosynthetic potential.

  11. Evolutionary dynamics of clustered irregularly interspaced short palindromic repeat systems in the ocean metagenome.

    Science.gov (United States)

    Sorokin, Valery A; Gelfand, Mikhail S; Artamonova, Irena I

    2010-04-01

    Clustered regularly interspaced short palindromic repeats (CRISPRs) form a recently characterized type of prokaryotic antiphage defense system. The phage-host interactions involving CRISPRs have been studied in experiments with selected bacterial or archaeal species and, computationally, in completely sequenced genomes. However, these studies do not allow one to take prokaryotic population diversity and phage-host interaction dynamics into account. This gap can be filled by using metagenomic data: in particular, the largest existing data set, generated from the Sorcerer II Global Ocean Sampling expedition. The application of three publicly available CRISPR recognition programs to the Global Ocean metagenome produced a large proportion of false-positive results. To address this problem, a filtering procedure was designed. It resulted in about 200 reliable CRISPR cassettes, which were then studied in detail. The repeat consensuses were clustered into several stable classes that differed from the existing classification. Short fragments of DNA similar to the cassette spacers were more frequently present in the same geographical location than in other locations (P, CRISPR-forming events and reconstructed the likely evolutionary history of cassettes that had common spacers. Metagenomic collections allow for relatively unbiased analysis of phage-host interactions and CRISPR evolution. The results of this study demonstrate that CRISPR cassettes retain the memory of the local virus population at a particular ocean location. CRISPR evolution may be described using a limited vocabulary of elementary events that have a natural biological interpretation.

  12. Metagenomics study of endophytic bacteria in Aloe vera using next-generation technology

    Directory of Open Access Journals (Sweden)

    Mushafau Adewale Akinsanya

    2015-12-01

    Full Text Available Next generation sequencing (NGS enables rapid analysis of the composition and diversity of microbial communities in several habitats. We applied the high throughput techniques of NGS to the metagenomics study of endophytic bacteria in Aloe vera plant, by assessing its PCR amplicon of 16S rDNA sequences (V3–V4 regions with the Illumina metagenomics technique used to generate a total of 5,199,102 reads from the samples. The analyses revealed Proteobacteria, Firmicutes, Actinobacteria and Bacteriodetes as the predominant genera. The roots have the largest composition with 23% not present in other tissues. The stems have more of the genus—Pseudomonas and the unclassified Pseudomonadaceae. The α-diversity analysis indicated the richness and inverse Simpson diversity index of the bacterial endophyte communities for the leaf, root and stem tissues to be 2.221, 6.603 and 1.491 respectively. In a similar study on culturable endophytic bacteria in the same A. vera plants (unpublished work, the dominance of Pseudomonas and Bacillus genera was similar, with equal proportion of four species each in root, stem and leaf tissues. It is evident that NGS technology captured effectively the metagenomics of microbiota in plant tissues and this can improve our understanding of the microbial–plant host interactions.

  13. A potential source for cellulolytic enzyme discovery and environmental aspects revealed through metagenomics of Brazilian mangroves.

    Science.gov (United States)

    Thompson, Claudia Elizabeth; Beys-da-Silva, Walter Orlando; Santi, Lucélia; Berger, Markus; Vainstein, Marilene Henning; Guima Rães, Jorge Almeida; Vasconcelos, Ana Tereza Ribeiro

    2013-01-01

    The mangroves are among the most productive and biologically important environments. The possible presence of cellulolytic enzymes and microorganisms useful for biomass degradation as well as taxonomic and functional aspects of two Brazilian mangroves were evaluated using cultivation and metagenomic approaches. From a total of 296 microorganisms with visual differences in colony morphology and growth (including bacteria, yeast and filamentous fungus), 179 (60.5%) and 117 (39.5%) were isolated from the Rio de Janeiro (RJ) and Bahia (BA) samples, respectively. RJ metagenome showed the higher number of microbial isolates, which is consistent with its most conserved state and higher diversity. The metagenomic sequencing data showed similar predominant bacterial phyla in the BA and RJ mangroves with an abundance of Proteobacteria (57.8% and 44.6%), Firmicutes (11% and 12.3%) and Actinobacteria (8.4% and 7.5%). A higher number of enzymes involved in the degradation of polycyclic aromatic compounds were found in the BA mangrove. Specific sequences involved in the cellulolytic degradation, belonging to cellulases, hemicellulases, carbohydrate binding domains, dockerins and cohesins were identified, and it was possible to isolate cultivable fungi and bacteria related to biomass decomposition and with potential applications for the production of biofuels. These results showed that the mangroves possess all fundamental molecular tools required for building the cellulosome, which is required for the efficient degradation of cellulose material and sugar release.

  14. Determining virus-host interactions and glycerol metabolism profiles in geographically diverse solar salterns with metagenomics

    Science.gov (United States)

    Moller, Abraham G.

    2017-01-01

    Solar salterns are excellent model ecosystems for studying virus-microbial interactions because of their low microbial diversity, environmental stability, and high viral density. By using the power of CRISPR spacers to link viruses to their prokaryotic hosts, we explored virus-host interactions in geographically diverse salterns. Using taxonomic profiling, we identified hosts such as archaeal Haloquadratum, Halorubrum, and Haloarcula and bacterial Salinibacter, and we found that community composition related to not only salinity but also local environmental dynamics. Characterizing glycerol metabolism genes in these metagenomes suggested Halorubrum and Haloquadratum possess most dihydroxyacetone kinase genes while Salinibacter possesses most glycerol-3-phosphate dehydrogenase genes. Using two different methods, we detected fewer CRISPR spacers in Haloquadratum-dominated compared with Halobacteriaceae-dominated saltern metagenomes. After CRISPR detection, spacers were aligned against haloviral genomes to map virus to host. While most alignments for each saltern metagenome linked viruses to Haloquadratum walsbyi, there were also alignments indicating interactions with the low abundance taxa Haloarcula and Haloferax. Further examination of the dinucleotide and trinucleotide usage differences between paired viruses and their hosts confirmed viruses and hosts had similar nucleotide usage signatures. Detection of cas genes in the salterns supported the possibility of CRISPR activity. Taken together, our studies suggest similar virus-host interactions exist in different solar salterns and that the glycerol metabolism gene dihydroxyacetone kinase is associated with Haloquadratum and Halorubrum. PMID:28097058

  15. Functional Metagenomics as a Tool for Identification of New Antibiotic Resistance Genes from Natural Environments.

    Science.gov (United States)

    Dos Santos, Débora Farage Knupp; Istvan, Paula; Quirino, Betania Ferraz; Kruger, Ricardo Henrique

    2017-02-01

    Antibiotic resistance has become a major concern for human and animal health, as therapeutic alternatives to treat multidrug-resistant microorganisms are rapidly dwindling. The problem is compounded by low investment in antibiotic research and lack of new effective antimicrobial drugs on the market. Exploring environmental antibiotic resistance genes (ARGs) will help us to better understand bacterial resistance mechanisms, which may be the key to identifying new drug targets. Because most environment-associated microorganisms are not yet cultivable, culture-independent techniques are essential to determine which organisms are present in a given environmental sample and allow the assessment and utilization of the genetic wealth they represent. Metagenomics represents a powerful tool to achieve these goals using sequence-based and functional-based approaches. Functional metagenomic approaches are particularly well suited to the identification new ARGs from natural environments because, unlike sequence-based approaches, they do not require previous knowledge of these genes. This review discusses functional metagenomics-based ARG research and describes new possibilities for surveying the resistome in environmental samples.

  16. Determining virus-host interactions and glycerol metabolism profiles in geographically diverse solar salterns with metagenomics

    Directory of Open Access Journals (Sweden)

    Abraham G. Moller

    2017-01-01

    Full Text Available Solar salterns are excellent model ecosystems for studying virus-microbial interactions because of their low microbial diversity, environmental stability, and high viral density. By using the power of CRISPR spacers to link viruses to their prokaryotic hosts, we explored virus-host interactions in geographically diverse salterns. Using taxonomic profiling, we identified hosts such as archaeal Haloquadratum, Halorubrum, and Haloarcula and bacterial Salinibacter, and we found that community composition related to not only salinity but also local environmental dynamics. Characterizing glycerol metabolism genes in these metagenomes suggested Halorubrum and Haloquadratum possess most dihydroxyacetone kinase genes while Salinibacter possesses most glycerol-3-phosphate dehydrogenase genes. Using two different methods, we detected fewer CRISPR spacers in Haloquadratum-dominated compared with Halobacteriaceae-dominated saltern metagenomes. After CRISPR detection, spacers were aligned against haloviral genomes to map virus to host. While most alignments for each saltern metagenome linked viruses to Haloquadratum walsbyi, there were also alignments indicating interactions with the low abundance taxa Haloarcula and Haloferax. Further examination of the dinucleotide and trinucleotide usage differences between paired viruses and their hosts confirmed viruses and hosts had similar nucleotide usage signatures. Detection of cas genes in the salterns supported the possibility of CRISPR activity. Taken together, our studies suggest similar virus-host interactions exist in different solar salterns and that the glycerol metabolism gene dihydroxyacetone kinase is associated with Haloquadratum and Halorubrum.

  17. IMG/M: integrated genome and metagenome comparative data analysis system.

    Science.gov (United States)

    Chen, I-Min A; Markowitz, Victor M; Chu, Ken; Palaniappan, Krishna; Szeto, Ernest; Pillay, Manoj; Ratner, Anna; Huang, Jinghua; Andersen, Evan; Huntemann, Marcel; Varghese, Neha; Hadjithomas, Michalis; Tennessen, Kristin; Nielsen, Torben; Ivanova, Natalia N; Kyrpides, Nikos C

    2017-01-04

    The Integrated Microbial Genomes with Microbiome Samples (IMG/M: https://img.jgi.doe.gov/m/) system contains annotated DNA and RNA sequence data of (i) archaeal, bacterial, eukaryotic and viral genomes from cultured organisms, (ii) single cell genomes (SCG) and genomes from metagenomes (GFM) from uncultured archaea, bacteria and viruses and (iii) metagenomes from environmental, host associated and engineered microbiome samples. Sequence data are generated by DOE's Joint Genome Institute (JGI), submitted by individual scientists, or collected from public sequence data archives. Structural and functional annotation is carried out by JGI's genome and metagenome annotation pipelines. A variety of analytical and visualization tools provide support for examining and comparing IMG/M's datasets. IMG/M allows open access interactive analysis of publicly available datasets, while manual curation, submission and access to private datasets and computationally intensive workspace-based analysis require login/password access to its expert review (ER) companion system (IMG/M ER: https://img.jgi.doe.gov/mer/). Since the last report published in the 2014 NAR Database Issue, IMG/M's dataset content has tripled in terms of number of datasets and overall protein coding genes, while its analysis tools have been extended to cope with the rapid growth in the number and size of datasets handled by the system.

  18. Comparative Metagenomics of Toxic Freshwater Cyanobacteria Bloom Communities on Two Continents

    Energy Technology Data Exchange (ETDEWEB)

    Steffen, Morgan M [ORNL; Li, Zhou [ORNL; Effler, Chad [Department of Microbiology, University of Tennessee; Hauser, Loren John [ORNL; Boyer, Gergory [College of Environmental Science and Forestry, State University of New York, Syracuse; Wilhelm, Steven W [ORNL

    2012-01-01

    Toxic cyanobacterial blooms have persisted in freshwater systems around the world for centuries and appear to be globally increasing in frequency and severity. Toxins produced by bloom-associated cyanobacteria can have drastic impacts on the ecosystem and surrounding communities, and bloom biomass can disrupt aquatic food webs and act as a driver for hypoxia. Little is currently known regarding the genomic content of the Microcystis strains that form blooms or the companion heterotrophic community associated with bloom events. To address these issues, we examined the bloomassociated microbial communities in single samples from Lake Erie (North America), Lake Tai (Taihu, China), and Grand Lakes St. Marys (OH, USA) using comparative metagenomics. Together the Cyanobacteria and Proteobacteria comprised .90% of each bloom bacterial community sample, although the dominant phylum varied between systems. Relative to the existing Microcystis aeruginosa NIES 843 genome, sequences from Lake Erie and Taihu revealed a number of metagenomic islands that were absent in the environmental samples. Moreover, despite variation in the phylogenetic assignments of bloomassociated organisms, the functional potential of bloom members remained relatively constant between systems. This pattern was particularly noticeable in the genomic contribution of nitrogen assimilation genes. In Taihu, the genetic elements associated with the assimilation and metabolism of nitrogen were predominantly associated with Proteobacteria, while these functions in the North American lakes were primarily contributed to by the Cyanobacteria. Our observations build on an emerging body of metagenomic surveys describing the functional potential of microbial communities as more highly conserved than that of their phylogenetic makeup within natural systems.

  19. Quantifying the biases in metagenome mining for realistic assessment of microbial ecology of naturally fermented foods

    Science.gov (United States)

    Keisam, Santosh; Romi, Wahengbam; Ahmed, Giasuddin; Jeyaram, Kumaraswamy

    2016-01-01

    Cultivation-independent investigation of microbial ecology is biased by the DNA extraction methods used. We aimed to quantify those biases by comparative analysis of the metagenome mined from four diverse naturally fermented foods (bamboo shoot, milk, fish, soybean) using eight different DNA extraction methods with different cell lysis principles. Our findings revealed that the enzymatic lysis yielded higher eubacterial and yeast metagenomic DNA from the food matrices compared to the widely used chemical and mechanical lysis principles. Further analysis of the bacterial community structure by Illumina MiSeq amplicon sequencing revealed a high recovery of lactic acid bacteria by the enzymatic lysis in all food types. However, Bacillaceae, Acetobacteraceae, Clostridiaceae and Proteobacteria were more abundantly recovered when mechanical and chemical lysis principles were applied. The biases generated due to the differential recovery of operational taxonomic units (OTUs) by different DNA extraction methods including DNA and PCR amplicons mix from different methods have been quantitatively demonstrated here. The different methods shared only 29.9–52.0% of the total OTUs recovered. Although similar comparative research has been performed on other ecological niches, this is the first in-depth investigation of quantifying the biases in metagenome mining from naturally fermented foods. PMID:27669673

  20. Metagenomic Assembly of the Dominant Zetaproteobacteria in an Iron-oxidizing Hydrothermal Microbial Mat

    Science.gov (United States)

    Moyer, C. L.; Fullerton, H.

    2013-12-01

    Iron is the fourth most abundant element in the Earth's crust and is potentially one of the most abundant energy sources on the earth as an electron donor for chemolithoautotrophic growth coupled to Fe(II) oxidation. Despite the rapid abiotic oxidation rate of iron, many microbes have adapted to feeding off this fleeting energy source. One such bacterial class is the Zetaproteobacteria. Iron-dominated microbial mat material was collected with a small-scale syringe sampler from Loihi Seamount, Hawaii. From this sample, gDNA was extracted and prepared for paired-end Illumina sequencing. Reconstruction of SSU rDNA genes using EMERGE allowed for comparison to previous SSU rDNA surveys. Clone libraries and qPCR show these microbial mats to be dominated by Zetaproteobacteria. Results from our in silico reconstruction confirm these initial findings. RDP classification of the EMERGE reconstructed sequences resulted in 44% of the community being identified as Zetaproteobacteria. The most abundant SSU rDNA has 99% similarity to Zeta OTU-2, and only a 94% similarity to M. ferrooxidans PV-1. Zeta OTU-2 has been shown to be the most cosmopolitan population in iron-dominated hydrothermal systems from across Pacific Ocean. Metagenomic assembly has resulted in many contigs with high identity to M. ferrooxidans as identified, by BLAST. However, with large differences in SSU rRNA similarity, M. ferrooxidans PV-1 is not an adequate reference. Current work is focusing on reconstruction of the dominant microbial mat member, without the use of a reference genome through an iterative assembly approach. The resulting 'pan-genome' will be compared to other Zetaproteobacteria (at the class level) and the functional ecology of this cosmopolitan microbial mat community member will be extrapolated. Thus far, we have detected multiple housekeeping genes involved in DNA replication, transcription and translation. The most abundant metabolic gene we have found is Aconitase, a key enzyme in the

  1. Metagenomics as a preliminary screen for antimicrobial bioprospecting

    KAUST Repository

    Al Amoudi, Soha

    2016-09-16

    Since the composition of soil directs the diversity of the contained microbiome and its potential to produce bioactive compounds, many studies has been focused on sediment types with unique features characteristic of extreme environments. However, not much is known about the potential of microbiomes that inhabit the highly saline and hot Red Sea lagoons. This case study explores mangrove mud and the microbial mat of sediments collected from the Rabigh harbor lagoon and Al Kharrar lagoon for antimicrobial bioprospecting. Rabigh harbor lagoon appears the better location, and the best sediment type for this purpose is mangrove mud. On the other hand, Al Kharrar lagoon displayed increased anaerobic hydrocarbon degradation and an abundance of bacterial DNA associated with antibiotic resistance. Moreover, our findings show an identical shift in phyla associated with historic hydrocarbon contamination exposure reported in previous studies (that is, enrichment of Gamma-and Delta-proteobacteria), but we also report that bacterial DNA sequences associated with antibiotic synthesis enzymes are derived from Gamma-, Delta-and Alpha-proteobacteria. This suggests that selection pressure associated with hydrocarbon contamination tend to enrich the bacterial classes DNA associated with antibiotic synthesis enzymes. Although Actinobacteria tends to be the common target for research when it comes to antimicrobial bioprospecting, our study suggests that Firmicutes (Bacilli and Clostridia), Bacteroidetes, Cyanobacteria, and Proteobacteria should be antimicrobial bioprospecting targets as well. To the best of our knowledge, this is the first metagenomic study that analyzed the microbiomes in Red Sea lagoons for antimicrobial bioprospecting. (C) 2016 The Authors. Published by Elsevier B.V.

  2. Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms

    Directory of Open Access Journals (Sweden)

    Tang Xian-Lai

    2010-11-01

    Full Text Available Abstract Background Fumarase catalyzes the reversible hydration of fumarate to L-malate and is a key enzyme in the tricarboxylic acid (TCA cycle and in amino acid metabolism. Fumarase is also used for the industrial production of L-malate from the substrate fumarate. Thermostable and high-activity fumarases from organisms that inhabit extreme environments may have great potential in industry, biotechnology, and basic research. The marine environment is highly complex and considered one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms are inaccessible in nature and are not easily cultivated in the laboratory. Metagenomic approaches provide a powerful tool to isolate and identify enzymes with novel biocatalytic activities for various biotechnological applications. Results A plasmid metagenomic library was constructed from uncultivated marine microorganisms within marine water samples. Through sequence-based screening of the DNA library, a gene encoding a novel fumarase (named FumF was isolated. Amino acid sequence analysis revealed that the FumF protein shared the greatest homology with Class II fumarate hydratases from Bacteroides sp. 2_1_33B and Parabacteroides distasonis ATCC 8503 (26% identical and 43% similar. The putative fumarase gene was subcloned into pETBlue-2 vector and expressed in E. coli BL21(DE3pLysS. The recombinant protein was purified to homogeneity. Functional characterization by high performance liquid chromatography confirmed that the recombinant FumF protein catalyzed the hydration of fumarate to form L-malate. The maximum activity for FumF protein occurred at pH 8.5 and 55°C in 5 mM Mg2+. The enzyme showed higher affinity and catalytic efficiency under optimal reaction conditions: Km= 0.48 mM, Vmax = 827 μM/min/mg, and kcat/Km = 1900 mM/s. Conclusions We isolated a novel fumarase gene, fumF, from a sequence-based screen of a plasmid metagenomic library from uncultivated

  3. Shotgun pyrosequencing metagenomic analyses of dusts from swine confinement and grain facilities.

    Directory of Open Access Journals (Sweden)

    Robert J Boissy

    Full Text Available Inhalation of agricultural dusts causes inflammatory reactions and symptoms such as headache, fever, and malaise, which can progress to chronic airway inflammation and associated diseases, e.g. asthma, chronic bronchitis, chronic obstructive pulmonary disease, and hypersensitivity pneumonitis. Although in many agricultural environments feed particles are the major constituent of these dusts, the inflammatory responses that they provoke are likely attributable to particle-associated bacteria, archaebacteria, fungi, and viruses. In this study, we performed shotgun pyrosequencing metagenomic analyses of DNA from dusts from swine confinement facilities or grain elevators, with comparisons to dusts from pet-free households. DNA sequence alignment showed that 19% or 62% of shotgun pyrosequencing metagenomic DNA sequence reads from swine facility or household dusts, respectively, were of swine or human origin, respectively. In contrast only 2% of such reads from grain elevator dust were of mammalian origin. These metagenomic shotgun reads of mammalian origin were excluded from our analyses of agricultural dust microbiota. The ten most prevalent bacterial taxa identified in swine facility compared to grain elevator or household dust were comprised of 75%, 16%, and 42% gram-positive organisms, respectively. Four of the top five swine facility dust genera were assignable (Clostridium, Lactobacillus, Ruminococcus, and Eubacterium, ranging from 4% to 19% relative abundance. The relative abundances of these four genera were lower in dust from grain elevators or pet-free households. These analyses also highlighted the predominance in swine facility dust of Firmicutes (70% at the phylum level, Clostridia (44% at the Class level, and Clostridiales at the Order level (41%. In summary, shotgun pyrosequencing metagenomic analyses of agricultural dusts show that they differ qualitatively and quantitatively at the level of microbial taxa present, and that the

  4. Metagenomic Surveys of Gut Microbiota

    Directory of Open Access Journals (Sweden)

    Rahul Shubhra Mandal

    2015-06-01

    Full Text Available Gut microbiota of higher vertebrates is host-specific. The number and diversity of the organisms residing within the gut ecosystem are defined by physiological and environmental factors, such as host genotype, habitat, and diet. Recently, culture-independent sequencing techniques have added a new dimension to the study of gut microbiota and the challenge to analyze the large volume of sequencing data is increasingly addressed by the development of novel computational tools and methods. Interestingly, gut microbiota maintains a constant relative abundance at operational taxonomic unit (OTU levels and altered bacterial abundance has been associated with complex diseases such as symptomatic atherosclerosis, type 2 diabetes, obesity, and colorectal cancer. Therefore, the study of gut microbial population has emerged as an important field of research in order to ultimately achieve better health. In addition, there is a spontaneous, non-linear, and dynamic interaction among different bacterial species residing in the gut. Thus, predicting the influence of perturbed microbe–microbe interaction network on health can aid in developing novel therapeutics. Here, we summarize the population abundance of gut microbiota and its variation in different clinical states, computational tools available to analyze the pyrosequencing data, and gut microbe–microbe interaction networks.

  5. Metagenomic Surveys of Gut Microbiota

    Institute of Scientific and Technical Information of China (English)

    Rahul Shubhra Mandal; Sudipto Saha; Santasabuj Das

    2015-01-01

    Gut microbiota of higher vertebrates is host-specific. The number and diversity of the organisms residing within the gut ecosystem are defined by physiological and environmental factors, such as host genotype, habitat, and diet. Recently, culture-independent sequencing techniques have added a new dimension to the study of gut microbiota and the challenge to analyze the large volume of sequencing data is increasingly addressed by the development of novel computational tools and methods. Interestingly, gut microbiota maintains a constant relative abundance at operational tax-onomic unit (OTU) levels and altered bacterial abundance has been associated with complex diseases such as symptomatic atherosclerosis, type 2 diabetes, obesity, and colorectal cancer. Therefore, the study of gut microbial population has emerged as an important field of research in order to ulti-mately achieve better health. In addition, there is a spontaneous, non-linear, and dynamic interac-tion among different bacterial species residing in the gut. Thus, predicting the influence of perturbed microbe–microbe interaction network on health can aid in developing novel therapeutics. Here, we summarize the population abundance of gut microbiota and its variation in different clinical states, computational tools available to analyze the pyrosequencing data, and gut microbe–microbe inter-action networks.

  6. 宏基因组技术在筛选未培养微生物中新型酶的研究进展%Research Progress on Metagenomic Technology in Screening New Enzymes from Uncultured Microorganisms

    Institute of Scientific and Technical Information of China (English)

    姜海琴; 范彩云; 李吕木; 程建波

    2011-01-01

    介绍了宏基因组文库的构建及酶基因的筛选方法,对瘤胃、土壤及其他环境中微生物宏基因组文库的构建和酶基因的筛选研究进行了综述,同时就宏基因组技术在未培养微生物研究中的应用进行了展望.%The metagenomic library construction and gene screening methods were described, and the microbiol metagenomic library construction and gene screening strudies of rumen,soil and extreme environments were reviewed. Meanwhile,the applications of metagenomic in uncultured microbial research were discussed.

  7. An Experimental Metagenome Data Management and AnalysisSystem

    Energy Technology Data Exchange (ETDEWEB)

    Markowitz, Victor M.; Korzeniewski, Frank; Palaniappan, Krishna; Szeto, Ernest; Ivanova, Natalia N.; Kyrpides, Nikos C.; Hugenholtz, Philip

    2006-03-01

    The application of shotgun sequencing to environmental samples has revealed a new universe of microbial community genomes (metagenomes) involving previously uncultured organisms. Metagenome analysis, which is expected to provide a comprehensive picture of the gene functions and metabolic capacity of microbial community, needs to be conducted in the context of a comprehensive data management and analysis system. We present in this paper IMG/M, an experimental metagenome data management and analysis system that is based on the Integrated Microbial Genomes (IMG) system. IMG/M provides tools and viewers for analyzing both metagenomes and isolate genomes individually or in a comparative context.

  8. Algal biogeography: metagenomics shows distribution of a picoplanktonic pelagophyte.

    Science.gov (United States)

    Raven, John A

    2012-09-11

    How can we determine the distribution of uncultured marine microorganisms? Targeted metagenomics has provided the complete chloroplast genome sequence, and the distribution, for a picoplanktonic pelagophyte alga.

  9. A novel D-amino acid oxidase from a contaminated agricultural soil metagenome and its characterization.

    Science.gov (United States)

    Ou, Qian; Liu, Yao; Deng, Jie; Chen, Gao; Yang, Ying; Shen, Peihong; Wu, Bo; Jiang, Chengjian

    2015-06-01

    A novel D-amino acid oxidase (DAAO) gene designated as daoE was cloned by the sequence-based screening of a plasmid metagenomic library of uncultured microorganisms from contaminated agricultural soil. The deduced amino acid sequence comparison and phylogenetic analysis indicated that daoE and other putative DAAOs are closely related. The putative DAAO gene was subcloned into a pETBlue-2 vector and overexpressed in Escherichia coli Tunner(DE3)pLacI. The recombinant protein was purified to homogeneity. The maximum activity of DaoE protein occurred at pH 8.0 and 37 °C. DaoE recombinant protein had an apparent K m of 2.96 mM, V max of 0.018 mM/min, k cat of 10.9/min, and k cat/K m of 1.16 × 10(4)/mol/min. The identification of this novel DAAO gene demonstrated the importance of metagenomic libraries in exploring new D-amino acid oxidases from environmental microorganisms to optimize their applications.

  10. Functional metagenomic analysis reveals rivers are a reservoir for diverse antibiotic resistance genes.

    Science.gov (United States)

    Amos, G C A; Zhang, L; Hawkey, P M; Gaze, W H; Wellington, E M

    2014-07-16

    The environment harbours a significant diversity of uncultured bacteria and a potential source of novel and extant resistance genes which may recombine with clinically important bacteria disseminated into environmental reservoirs. There is evidence that pollution can select for resistance due to the aggregation of adaptive genes on mobile elements. The aim of this study was to establish the impact of waste water treatment plant (WWTP) effluent disposal to a river by using culture independent methods to study diversity of resistance genes downstream of the WWTP in comparison to upstream. Metagenomic libraries were constructed in Escherichia coli and screened for phenotypic resistance to amikacin, gentamicin, neomycin, ampicillin and ciprofloxacin. Resistance genes were identified by using transposon mutagenesis. A significant increase downstream of the WWTP was observed in the number of phenotypic resistant clones recovered in metagenomic libraries. Common β-lactamases such as blaTEM were recovered as well as a diverse range of acetyltransferases and unusual transporter genes, with evidence for newly emerging resistance mechanisms. The similarities of the predicted proteins to known sequences suggested origins of genes from a very diverse range of bacteria. The study suggests that waste water disposal increases the reservoir of resistance mechanisms in the environment either by addition of resistance genes or by input of agents selective for resistant phenotypes.

  11. Genomics and metagenomics in medical microbiology.

    Science.gov (United States)

    Padmanabhan, Roshan; Mishra, Ajay Kumar; Raoult, Didier; Fournier, Pierre-Edouard

    2013-12-01

    Over the last two decades, sequencing tools have evolved from laborious time-consuming methodologies to real-time detection and deciphering of genomic DNA. Genome sequencing, especially using next generation sequencing (NGS) has revolutionized the landscape of microbiology and infectious disease. This deluge of sequencing data has not only enabled advances in fundamental biology but also helped improve diagnosis, typing of pathogen, virulence and antibiotic resistance detection, and development of new vaccines and culture media. In addition, NGS also enabled efficient analysis of complex human micro-floras, both commensal, and pathological, through metagenomic methods, thus helping the comprehension and management of human diseases such as obesity. This review summarizes technological advances in genomics and metagenomics relevant to the field of medical microbiology.

  12. Metagenomics: advances in ecology and biotechnology.

    Science.gov (United States)

    Steele, Helen L; Streit, Wolfgang R

    2005-06-15

    This review highlights the significant advances which have been made in prokaryotic ecology and biotechnology due to the application of metagenomic techniques. It is now possible to link processes to specific microorganisms in the environment, such as the detection of a new phototrophic process in marine bacteria, and to characterise the metabolic cooperation which takes place in mixed species biofilms. The range of prokaryote derived products available for biotechnology applications is increasing rapidly. The knowledge gained from analysis of biosynthetic pathways provides valuable information about enzymology and allows engineering of biocatalysts for specific processes. The expansion of metagenomic techniques to include alternative heterologous hosts for gene expression and the development of sophisticated assays which enable screening of thousands of clones offers the possibility to find out even more valuable information about the prokaryotic world.

  13. Bioprospecting hot spring metagenome: lipase for the production of biodiesel.

    Science.gov (United States)

    Sahoo, Rajesh Kumar; Kumar, Mohit; Sukla, Lala Behari; Subudhi, Enketeswara

    2017-02-01

    Screening of metagenomic library from Taptapani Hot Spring (Odisha) yielded a positive lipase clone (pUC-lip479). Sequence analysis showed an ORF (RK-lip479) of 416 amino acid residues which was overexpressed in Escherichia coli BL21 (DE3). Optimum pH and temperature of purified lipase RK-lip479 were 8.0 and 65 °C, respectively, and found to be stable over a pH range of 7.0-9.0 and temperatures 55-75 °C. RK-lip479 could hydrolyse a wide range of 4-nitrophenyl esters (4-nitrophenyoctanoate, 4-nitrophenyldodecanoate, 4-nitrophenylpalmitate, 4-nitrophenylmyristate and 4-nitrophenylstearate), and maximum activity was observed with 4-nitrophenyldodecanoate. RK-lip479 was resistant to many organic solvents, especially isopropanol, DMSO, methanol, DMF, ethanol, dichloromethane, acetone, glycerol and ethyl acetate. RK-lip479 also showed activity in the presence of monovalent (Na(+) and K(+)), divalent (Mg(2+), Mn(2+), Ca(2+), Hg(2+), Cu(2+), Co(2+), Zn(2+) and Ag(2+) ) and trivalent cations (Fe(3+) and Al(3+)). Yield of biodiesel production was in the range of 40-76% using various waste oils with RK-Lip479 under optimized conditions.

  14. Research on the Preparation of Bacterial Artificial Chromosome Library Genomic DNA%细菌人工染色体文库基因组DNA制备技术

    Institute of Scientific and Technical Information of China (English)

    陈献伟; 王会; 关伟军; 高剑峰

    2010-01-01

    试验旨在对基因组DNA的制备进行研究,为细菌人工染色体(bacterial artificial charomosome,BAc)文库的构建奠定基础.以豁眼鹅全血为试验材料,提取高质量的基因组DNA,分别采用Hind Ⅲ、EcoR Ⅰ和BamHⅠ3种限制性内切酶对所提基因组DNA进行部分酶切,并利用控制酶切时间、设置不同的Hind Ⅲ酶切浓度梯度对基因组DNA进行部分酶切.结果表明,Hind Ⅲ为最佳限制性内切酶,并得到了最佳酶切用量(40U/μL).该方法所制备的基因组DNA质量较好,可用于BAC文库的构建.

  15. Development and validation of a Xanthomonas axonopodis pv. citri DNA microarray platform (XACarray generated from the shotgun libraries previously used in the sequencing of this bacterial genome

    Directory of Open Access Journals (Sweden)

    Zaini Paulo A

    2010-05-01

    Full Text Available Abstract Background From shotgun libraries used for the genomic sequencing of the phytopathogenic bacterium Xanthomonas axonopodis pv. citri (XAC, clones that were representative of the largest possible number of coding sequences (CDSs were selected to create a DNA microarray platform on glass slides (XACarray. The creation of the XACarray allowed for the establishment of a tool that is capable of providing data for the analysis of global genome expression in this organism. Findings The inserts from the selected clones were amplified by PCR with the universal oligonucleotide primers M13R and M13F. The obtained products were purified and fixed in duplicate on glass slides specific for use in DNA microarrays. The number of spots on the microarray totaled 6,144 and included 768 positive controls and 624 negative controls per slide. Validation of the platform was performed through hybridization of total DNA probes from XAC labeled with different fluorophores, Cy3 and Cy5. In this validation assay, 86% of all PCR products fixed on the glass slides were confirmed to present a hybridization signal greater than twice the standard deviation of the deviation of the global median signal-to-noise ration. Conclusions Our validation of the XACArray platform using DNA-DNA hybridization revealed that it can be used to evaluate the expression of 2,365 individual CDSs from all major functional categories, which corresponds to 52.7% of the annotated CDSs of the XAC genome. As a proof of concept, we used this platform in a previously work to verify the absence of genomic regions that could not be detected by sequencing in related strains of Xanthomonas.

  16. Generating viral metagenomes from the coral holobiont

    Directory of Open Access Journals (Sweden)

    Karen Dawn Weynberg

    2014-05-01

    Full Text Available Reef-building corals comprise multipartite symbioses where the cnidarian animal is host to an array of eukaryotic and prokaryotic organisms, and the viruses that infect them. These viruses are critical elements of the coral holobiont, serving not only as agents of mortality, but also as potential vectors for lateral gene flow, and as elements encoding a variety of auxiliary metabolic functions. Consequently, understanding the functioning and health of the coral holobiont requires detailed knowledge of the associated viral assemblage and its function. Currently, the most tractable way of uncovering viral diversity and function is through metagenomic approaches, which is inherently difficult in corals because of the complex holobiont community, an extracellular mucus layer that all corals secrete, and the variety of sizes and structures of nucleic acids found in viruses. Here we present the first protocol for isolating, purifying and amplifying viral nucleic acids from corals based on mechanical disruption of cells. This method produces at least 50% higher yields of viral nucleic acids, has very low levels of cellular sequence contamination and captures wider viral diversity than previously used chemical-based extraction methods. We demonstrate that our mechanical-based method profiles a greater diversity of DNA and RNA genomes, including virus groups such as Retro-transcribing and ssRNA viruses, which are absent from metagenomes generated via chemical-based methods. In addition, we briefly present (and make publically available the first paired DNA and RNA viral metagenomes from the coral Acropora tenuis.

  17. Year-long metagenomic study of river microbiomes across land use and water quality

    Directory of Open Access Journals (Sweden)

    Thea eVan Rossum

    2015-12-01

    Full Text Available Select bacteria, such as Escherichia coli or coliforms, have been widely used as sentinels of low water quality; however, there are concerns regarding their predictive accuracy for the protection of human and environmental health. To develop improved monitoring systems, a greater understanding of bacterial community structure, function and variability across time is required in the context of different pollution types, such as agricultural and urban contamination. Here, we present a year-long survey of free-living bacterial DNA collected from seven sites along rivers in three watersheds with varying land use in Southwestern Canada. This is the first study to examine the bacterial metagenome in flowing freshwater (lotic environments over such a time span, providing an opportunity to describe bacterial community variability as a function of land use and environmental conditions. Characteristics of the metagenomic data, such as sequence composition and average genome size, vary with sampling site, environmental conditions, and water chemistry. For example, average genome size was correlated with hours of daylight in the agricultural watershed and, across the agriculturally and urban-affected sites, k-mer composition clustering corresponded to nutrient concentrations. In addition to indicating a community shift, this change in average genome size has implications in terms of the normalisation strategies required, and considerations surrounding such strategies in general are discussed. When comparing abundances of gene functional groups between high- and low-quality water samples collected from an agricultural area, the latter had a higher abundance of nutrient metabolism and bacteriophage groups, possibly reflecting an increase in agricultural runoff. This work presents a valuable dataset representing a year of monthly sampling across watersheds and an analysis targeted at establishing a foundational understanding of how bacterial lotic communities

  18. Metagenomic analysis of the rumen microbial community following inhibition of methane formation by a halogenated methane analog.

    Science.gov (United States)

    Denman, Stuart E; Martinez Fernandez, Gonzalo; Shinkai, Takumi; Mitsumori, Makoto; McSweeney, Christopher S

    2015-01-01

    Japanese goats fed a diet of 50% Timothy grass and 50% concentrate with increasing levels of the anti-methanogenic compound, bromochloromethane (BCM) were investigated with respect to the microbial population and functional shifts in the rumen. Microbial ecology methods identified species that exhibited positive and negative responses to the increasing levels of BCM. The methane-inhibited rumen appeared to adapt to the higher H2 levels by shifting fermentation to propionate which was mediated by an increase in the population of H2-consuming Prevotella and Selenomonas spp. Metagenomic analysis of propionate production pathways was dominated by genomic content from these species. Reductive acetogenic marker gene libraries and metagenomics analysis indicate that reductive acetogenic species do not play a major role in the BCM treated rumen.

  19. Metagenomic analysis of the rumen microbial community following inhibition of methane formation by a halogenated methane analogue

    Directory of Open Access Journals (Sweden)

    Stuart E Denman

    2015-10-01

    Full Text Available Japanese goats fed a diet of 50% Timothy grass and 50% concentrate with increasing levels of the anti-methanogenic compound, bromochloromethane (BCM were investigated with respect to the microbial shifts in the rumen. Microbial ecology methods identified many species that exhibited positive and negative responses to the increasing levels of BCM. The methane-inhibited rumen appeared to adapt to the higher H2 levels by shifting fermentation to propionate which was mediated by an increase in the population of hydrogen-consuming Prevotella and Selenomonas spp. Metagenomic analysis of propionate production pathways was dominated by genomic content from these species. Reductive acetogenic marker gene libraries and metagenomics analysis indicate that reductive acetogenic species do not play a major role in the BCM treated rumen.

  20. Biomedical Libraries

    Science.gov (United States)

    Pizer, Irwin H.

    1978-01-01

    Biomedical libraries are discussed as a distinct and specialized group of special libraries and their unique services and user interactions are described. The move toward professional standards, as evidenced by the Medical Library Association's new certification program, and the current state of development for a new section of IFLA established…

  1. Digital Libraries

    CERN Document Server

    Papy, Fabrice

    2008-01-01

    Of vital interest to all librarians and information specialists, this book presents all aspects of the effects of digitization of today's and tomorrow's libraries. From social to technical issues, Digital Libraries includes chapters on the growth of the role of librarian, the reader experience, cataloging, search engines, OPAC, law, ergonomic studies, and the future of libraries.

  2. Metagenomic Sequencing of the Chronic Obstructive Pulmonary Disease Upper Bronchial Tract Microbiome Reveals Functional Changes Associated with Disease Severity.

    Science.gov (United States)

    Cameron, Simon J S; Lewis, Keir E; Huws, Sharon A; Lin, Wanchang; Hegarty, Matthew J; Lewis, Paul D; Mur, Luis A J; Pachebat, Justin A

    2016-01-01

    Chronic Obstructive Pulmonary Disease (COPD) is a major source of mortality and morbidity worldwide. The microbiome associated with this disease may be an important component of the disease, though studies to date have been based on sequencing of the 16S rRNA gene, and have revealed unequivocal results. Here, we employed metagenomic sequencing of the upper bronchial tract (UBT) microbiome to allow for greater elucidation of its taxonomic composition, and revealing functional changes associated with the disease. The bacterial metagenomes within sputum samples from eight COPD patients and ten 'healthy' smokers (Controls) were sequenced, and suggested significant changes in the abundance of bacterial species, particularly within the Streptococcus genus. The functional capacity of the COPD UBT microbiome indicated an increased capacity for bacterial growth, which could be an important feature in bacterial-associated acute exacerbations. Regression analyses correlated COPD severity (FEV1% of predicted) with differences in the abundance of Streptococcus pneumoniae and functional classifications related to a reduced capacity for bacterial sialic acid metabolism. This study suggests that the COPD UBT microbiome could be used in patient risk stratification and in identifying novel monitoring and treatment methods, but study of a longitudinal cohort will be required to unequivocally relate these features of the microbiome with COPD severity.

  3. Megraft: A software package to graft ribosomal small subunit (16S/18S) fragments onto full-length sequences for accurate species richness and sequencing depth analysis in pyrosequencing-length metagenomes

    Science.gov (United States)

    Metagenomic libraries represent subsamples of the total DNA found at a study site and offer unprecedented opportunities to study ecological and functional aspects of microbial communities. To examine the depth of the sequencing effort, rarefaction analysis of the ribosomal small sub-unit (SSU/16S/18...

  4. The functional potential of high Arctic permafrost revealed by metagenomic sequencing, qPCR and microarray analyses.

    Science.gov (United States)

    Yergeau, Etienne; Hogues, Hervé; Whyte, Lyle G; Greer, Charles W

    2010-09-01

    The fate of the carbon stocked in permafrost following global warming and permafrost thaw is of major concern in view of the potential for increased CH(4) and CO(2) emissions from these soils. Complex carbon compound degradation and greenhouse gas emissions are due to soil microbial communities, but no comprehensive study has yet addressed their composition and functional potential in permafrost. Here, a 2-m deep permafrost sample and its overlying active layer soil were subjected to metagenomic sequencing, quantitative PCR (qPCR) and microarray analyses. The active layer soil and the 2-m permafrost microbial community structures were very similar, with Actinobacteria being the dominant phylum. The two samples also possessed a highly similar spectrum of functional genes, especially when compared with other already published metagenomes. Key genes related to methane generation, methane oxidation and organic matter degradation were highly diverse for both samples in the metagenomic libraries and some (for example, pmoA) showed relatively high abundance in qPCR assays. Genes related to nitrogen fixation and ammonia oxidation, which could have important roles following climatic change in these nitrogen-limited environments, showed low diversity but high abundance. The 2-m permafrost showed lower abundance and diversity for all the assessed genes and taxa. Experimental biases were also evaluated using qPCR and showed that the whole-community genome amplification technique used caused representational biases in the metagenomic libraries by increasing the abundance of Bacteroidetes and decreasing the abundance of Actinobacteria. This study describes for the first time the detailed functional potential of permafrost-affected soils.

  5. Genometa--a fast and accurate classifier for short metagenomic shotgun reads.

    Directory of Open Access Journals (Sweden)

    Colin F Davenport

    Full Text Available Metagenomic studies use high-throughput sequence data to investigate microbial communities in situ. However, considerable challenges remain in the analysis of these data, particularly with regard to speed and reliable analysis of microbial species as opposed to higher level taxa such as phyla. We here present Genometa, a computationally undemanding graphical user interface program that enables identification of bacterial species and gene content from datasets generated by inexpensive high-throughput short read sequencing technologies. Our approach was first verified on two simulated metagenomic short read datasets, detecting 100% and 94% of the bacterial species included with few false positives or false negatives. Subsequent comparative benchmarking analysis against three popular metagenomic algorithms on an Illumina human gut dataset revealed Genometa to attribute the most reads to bacteria at species level (i.e. including all strains of that species and demonstrate similar or better accuracy than the other programs. Lastly, speed was demonstrated to be many times that of BLAST due to the use of modern short read aligners. Our method is highly accurate if bacteria in the sample are represented by genomes in the reference sequence but cannot find species absent from the reference. This method is one of the most user-friendly and resource efficient approaches and is thus feasible for rapidly analysing millions of short reads on a personal computer.The Genometa program, a step by step tutorial and Java source code are freely available from http://genomics1.mh-hannover.de/genometa/ and on http://code.google.com/p/genometa/. This program has been tested on Ubuntu Linux and Windows XP/7.

  6. Construction of BAC libraries from flow-sorted chromosomes

    OpenAIRE

    Šafář, J.; Šimková, H; Doležel, J

    2016-01-01

    Cloned DNA libraries in bacterial artificial chromosome (BAC) are the most widely used form of large-insert DNA libraries. BAC libraries are typically represented by ordered clones derived from genomic DNA of a particular organism. In the case of large eukaryotic genomes, whole-genome libraries consist of a hundred thousand to a million clones, which make their handling and screening a daunting task. The labor and cost of working with whole-genome libraries can be greatly reduced by construct...

  7. Comparative Metagenomics of Eight Geographically Remote Terrestrial Hot Springs.

    Science.gov (United States)

    Menzel, Peter; Gudbergsdóttir, Sóley Ruth; Rike, Anne Gunn; Lin, Lianbing; Zhang, Qi; Contursi, Patrizia; Moracci, Marco; Kristjansson, Jakob K; Bolduc, Benjamin; Gavrilov, Sergey; Ravin, Nikolai; Mardanov, Andrey; Bonch-Osmolovskaya, Elizaveta; Young, Mark; Krogh, Anders; Peng, Xu

    2015-08-01

    Hot springs are natural habitats for thermophilic Archaea and Bacteria. In this paper, we present the metagenomic analysis of eight globally distributed terrestrial hot springs from China, Iceland, Italy, Russia, and the USA with a temperature range between 61 and 92 (∘)C and pH between 1.8 and 7. A comparison of the biodiversity and community composition generally showed a decrease in biodiversity with increasing temperature and decreasing pH. Another important factor shaping microbial diversity of the studied sites was the abundance of organic substrates. Several species of the Crenarchaeal order Thermoprotei were detected, whereas no single bacterial species was found in all samples, suggesting a better adaptation of certain archaeal species to different thermophilic environments. Two hot springs show high abundance of Acidithiobacillus, supporting the idea of a true thermophilic Acidithiobacillus species that can thrive in hyperthermophilic environments. Depending on the sample, up to 58 % of sequencing reads could not be assigned to a known phylum, reinforcing the fact that a large number of microorganisms in nature, including those thriving in hot environments remain to be isolated and characterized.

  8. Metagenomics of the Methane Ice Worm, Hesiocaeca methanicola

    Science.gov (United States)

    Goodwin, K. D.; Edsall, L.; Xin, W.; Head, S. R.; Gelbart, T.; Wood, A. M.; Gaasterland, T.

    2012-12-01

    The methane ice worm (Hesiocaeca methanicola) is a polychaete found on methane hydrate deposits for which there appears to be no publically available genomic or metagenomic data. Methane ice worms were collected in 2009 by the Johnson-Sea-Link submersible (543m depth; N 27:44.7526 W 91:13.3168). Next-generation sequencing (HiSeq2000) was applied to samples of tissue and gut contents. A subset of the assembled data (40M reads, randomly selected) was run through MG-RAST. Preliminary results for the gut content (1,269,153 sequences, average length 202 bp) indicated that 0.1% of the sequences contained ribosomal RNA genes with the majority (67%) classified as Bacteria, a relatively small per cent (1.4%) as Archae, and 31% as Eukaryota. Campylobacterales was the predominant order (14%), with unclassified (7.5%) and Desulfobacterales (4%) being the next dominant. Preliminary results for the worm tissue (2,716,461 sequences, average length 241 bp) indicated that the majority of sequences were Eukaryota (73%), with 256 sequences classified as phylum Annelida and 58% of those belonging to class Polychaeta. For the bacterial sequences obtained from the tissue samples, the predominant order was Actinomycetales (2.7%). For both the tissue and gut content samples, the majority of proteins were classified as clustering-based subsystems. This preliminary analysis will be compared to an assembly consisting of 40M of the highest quality reads.; methane ice worms on methane hydrate

  9. Investigation of bacterial diversity of hot springs of Odisha, India.

    Science.gov (United States)

    Sahoo, Rajesh Kumar; Subudhi, Enketeswara; Kumar, Mohit

    2015-12-01

    16S rRNA deep sequencing analysis, targeting V3 region was performed using Illumina bar coded sequencing. Sediment samples from two hot springs (Atri and Taptapani) were collected. Atri and Taptapani metagenomes were classified into 50 and 51 bacterial phyla. Proteobacteria (45.17%) dominated the Taptapani sample metagenome followed by Bacteriodetes (23.43%) and Cyanobacteria (10.48%) while in the Atri sample, Chloroflexi (52.39%), Nitrospirae (10.93%) and Proteobacteria (9.98%) dominated. A large number of sequences remained taxonomically unresolved in both hot springs, indicating the presence of potentially novel microbes in these two unique habitats thus unraveling the importance of the current study. Metagenome sequence information is now available at NCBI, SRA database accession no. SRP057428.

  10. Investigation of bacterial diversity of hot springs of Odisha, India

    Directory of Open Access Journals (Sweden)

    Rajesh Kumar Sahoo

    2015-12-01

    Full Text Available 16S rRNA deep sequencing analysis, targeting V3 region was performed using Illumina bar coded sequencing. Sediment samples from two hot springs (Atri and Taptapani were collected. Atri and Taptapani metagenomes were classified into 50 and 51 bacterial phyla. Proteobacteria (45.17% dominated the Taptapani sample metagenome followed by Bacteriodetes (23.43% and Cyanobacteria (10.48% while in the Atri sample, Chloroflexi (52.39%, Nitrospirae (10.93% and Proteobacteria (9.98% dominated. A large number of sequences remained taxonomically unresolved in both hot springs, indicating the presence of potentially novel microbes in these two unique habitats thus unraveling the importance of the current study. Metagenome sequence information is now available at NCBI, SRA database accession no. SRP057428.

  11. Glucose-tolerant β-glucosidase retrieved from the metagenome

    Directory of Open Access Journals (Sweden)

    Taku eUchiyama

    2015-06-01

    Full Text Available β-glucosidases (BGLs hydrolyze cellooligosaccharides to glucose and play a crucial role in the enzymatic saccharification of cellulosic biomass. Despite their significance for the production of glucose, most identified BGLs are commonly inhibited by low (~mM concentrations of glucose. Therefore, BGLs that are insensitive to glucose inhibition have great biotechnological merit. We applied a metagenomic approach to screen for such rare glucose-tolerant BGLs. A metagenomic library was created in Escherichia coli (approximately 10,000 colonies and grown on LB agar plates containing 5-bromo-4-chloro-3-indolyl-β-D-glucoside, yielding 828 positive (blue colonies. These were then arrayed in 96-well plates, grown in LB, and secondarily screened for activity in the presence of 10% (w/v glucose. Seven glucose-tolerant clones were identified, each of which contained a single bgl gene. The genes were classified into two groups, differing by two nucleotides. The deduced amino acid sequences of these genes were identical (452 aa and found to belong to the glycosyl hydrolase family 1. The recombinant protein (Ks5A7 was overproduced in E. coli as a C-terminal 6 × His-tagged protein and purified to apparent homogeneity. The molecular mass of the purified Ks5A7 was determined to be 54 kDa by SDS-PAGE, and 160 kDa by gel filtration analysis. The enzyme was optimally active at 45°C and pH 5.0–6.5 and retained full or 1.5–2-fold enhanced activity in the presence of 0.1–0.5 M glucose. It had a low KM (78 µM with p-nitrophenyl β-D-glucoside; 0.36 mM with cellobiose and high Vmax (91 µmol min-1 mg-1 with p-nitrophenyl β-D-glucoside; 155 µmol min-1 mg-1 with cellobiose among known glucose-tolerant BGLs and was free from substrate (0.1 M cellobiose inhibition. The efficient use of Ks5A7 in conjunction with Trichoderma reesei cellulases in enzymatic saccharification of alkaline-treated rice straw was demonstrated by increased production of glucose.

  12. SmashCommunity: A metagenomic annotation and analysis tool

    DEFF Research Database (Denmark)

    Arumugam, Manimozhiyan; Harrington, Eoghan D; Foerstner, Konrad U;

    2010-01-01

    the quantitative phylogenetic and functional compositions of metagenomes, to compare compositions of multiple metagenomes and to produce intuitive visual representations of such analyses. AVAILABILITY: SmashCommunity is freely available at http://www.bork.embl.de/software/smash CONTACT: bork@embl.de....

  13. Unlocking the potential of metagenomics through replicated experimental design

    NARCIS (Netherlands)

    Knight, R.; Jansson, J.; Field, D.; Fierer, N.; Desai, N.; Fuhrman, J.A.; Hugenholtz, P.; Van der Lelie, D.; Meyer, F.; Stevens, R.; Bailey, M.J.; Gordon, J.I.; Kowalchuk, G.A.; Gilbert, J.A.

    2012-01-01

    Metagenomics holds enormous promise for discovering novel enzymes and organisms that are biomarkers or drivers of processes relevant to disease, industry and the environment. In the past two years, we have seen a paradigm shift in metagenomics to the application of cross-sectional and longitudinal s

  14. Genomic standards consortium workshop: metagenomics, metadata and metaanalysis (M3).

    Science.gov (United States)

    Sterk, Peter; Hirschman, Lynette; Field, Dawn; Wooley, John

    2010-01-01

    The M3 workshop has, as its primary focus, the rapidly growing area of metagenomics, including the metadata standards and the meta-analysis approaches needed to organize, process and interpret metagenomics data. The PSB Workshop builds on the first M3 meeting, a Special Interest Group (SIG) meeting at ISMB 2009, organized by the Genomics Standards Consortium.

  15. Functional assignment of metagenomic data: challenges and applications.

    Science.gov (United States)

    Prakash, Tulika; Taylor, Todd D

    2012-11-01

    Metagenomic sequencing provides a unique opportunity to explore earth's limitless environments harboring scores of yet unknown and mostly unculturable microbes and other organisms. Functional analysis of the metagenomic data plays a central role in projects aiming to explore the most essential questions in microbiology, namely 'In a given environment, among the microbes present, what are they doing, and how are they doing it?' Toward this goal, several large-scale metagenomic projects have recently been conducted or are currently underway. Functional analysis of metagenomic data mainly suffers from the vast amount of data generated in these projects. The shear amount of data requires much computational time and storage space. These problems are compounded by other factors potentially affecting the functional analysis, including, sample preparation, sequencing method and average genome size of the metagenomic samples. In addition, the read-lengths generated during sequencing influence sequence assembly, gene prediction and subsequently the functional analysis. The level of confidence for functional predictions increases with increasing read-length. Usually, the most reliable functional annotations for metagenomic sequences are achieved using homology-based approaches against publicly available reference sequence databases. Here, we present an overview of the current state of functional analysis of metagenomic sequence data, bottlenecks frequently encountered and possible solutions in light of currently available resources and tools. Finally, we provide some examples of applications from recent metagenomic studies which have been successfully conducted in spite of the known difficulties.

  16. Antibiotic Resistome: Improving Detection and Quantification Accuracy for Comparative Metagenomics.

    Science.gov (United States)

    Elbehery, Ali H A; Aziz, Ramy K; Siam, Rania

    2016-04-01

    The unprecedented rise of life-threatening antibiotic resistance (AR), combined with the unparalleled advances in DNA sequencing of genomes and metagenomes, has pushed the need for in silico detection of the resistance potential of clinical and environmental metagenomic samples through the quantification of AR genes (i.e., genes conferring antibiotic resistance). Therefore, determining an optimal methodology to quantitatively and accurately assess AR genes in a given environment is pivotal. Here, we optimized and improved existing AR detection methodologies from metagenomic datasets to properly consider AR-generating mutations in antibiotic target genes. Through comparative metagenomic analysis of previously published AR gene abundance in three publicly available metagenomes, we illustrate how mutation-generated resistance genes are either falsely assigned or neglected, which alters the detection and quantitation of the antibiotic resistome. In addition, we inspected factors influencing the outcome of AR gene quantification using metagenome simulation experiments, and identified that genome size, AR gene length, total number of metagenomics reads and selected sequencing platforms had pronounced effects on the level of detected AR. In conclusion, our proposed improvements in the current methodologies for accurate AR detection and resistome assessment show reliable results when tested on real and simulated metagenomic datasets.

  17. Online semi-supervised learning: algorithm and application in metagenomics

    NARCIS (Netherlands)

    S. Imangaliyev; B. Keijser; W. Crielaard; E. Tsivtsivadze

    2013-01-01

    As the amount of metagenomic data grows rapidly, online statistical learning algorithms are poised to play key role in metagenome analysis tasks. Frequently, data are only partially labeled, namely dataset contains partial information about the problem of interest. This work presents an algorithm an

  18. Online Semi-Supervised Learning: Algorithm and Application in Metagenomics

    NARCIS (Netherlands)

    Imangaliyev, S.; Keijser, B.J.F.; Crielaard, W.; Tsivtsivadze, E.

    2013-01-01

    As the amount of metagenomic data grows rapidly, online statistical learning algorithms are poised to play key rolein metagenome analysis tasks. Frequently, data are only partially labeled, namely dataset contains partial information about the problem of interest. This work presents an algorithm and

  19. Recovering full-length viral genomes from metagenomes

    NARCIS (Netherlands)

    S.L. Smits (Saskia); R. Bodewes (Rogier); A. Ruiz-Gonzalez (Aritz); V. Baumgärtner (Volkmar); M.P.G. Koopmans D.V.M. (Marion); A.D.M.E. Osterhaus (Albert); A. Schürch (Anita)

    2015-01-01

    textabstractInfectious disease metagenomics is driven by the question: "what is causing the disease?" in contrast to classical metagenome studies which are guided by "what is out there?" In case of a novel virus, a first step to eventually establishing etiology can be to recover a full-length viral

  20. Cross-cutting activities: Soil quality and soil metagenomics