High-resolution metagenomics targets specific functional types in complex microbial communities.

Department of Microbiology, University of Washington, Benjamin Hall IRB, 616 NE Northlake Place, Seattle, Washington 98105, USA.
Nature Biotechnology (Impact Factor: 39.08). 09/2008; 26(9):1029-34. DOI: 10.1038/nbt.1488
Source: PubMed

ABSTRACT Most microbes in the biosphere remain unculturable. Whole genome shotgun (WGS) sequencing of environmental DNA (metagenomics) can be used to study the genetic and metabolic properties of natural microbial communities. However, in communities of high complexity, metagenomics fails to link specific microbes to specific ecological functions. To overcome this limitation, we developed a method to target microbial subpopulations by labeling DNA through stable isotope probing (SIP), followed by WGS sequencing. Metagenome analysis of microbes from Lake Washington in Seattle that oxidize single-carbon (C1) compounds shows specific sequence enrichments in response to different C1 substrates, revealing the ecological roles of individual phylotypes. We also demonstrate the utility of our approach by extracting a nearly complete genome of a novel methylotroph, Methylotenera mobilis, reconstructing its metabolism and conducting genome-wide analyses. This high-resolution, targeted metagenomics approach may be applicable to a wide variety of ecosystems.

Download full-text


Available from: Susannah G Tringe, Jul 07, 2015
1 Follower
  • Source
    • "Analysis based on 16S rRNA gene sequences is informative regarding the composition and diversity of a microbial community; however, its ability to elucidate the functional gene content and metabolic pathways is limited (Kalyuzhnaya et al., 2008). Metagenomics can provide detailed information on global metabolic functions; however, it typically achieves limited depth of coverage for any one pathway or gene family of interest, and as a result has low ability to detect small polymorphisms or rare genes (Kalyuzhnaya et al., 2008). Neither of these approaches has thus yet provided detailed information on the GH profile of the human gut. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Glycoside hydrolases (GHs), the enzymes that breakdown complex carbohydrates, are a highly diversified class of key enzymes associated with the gut microbiota and its metabolic functions. To learn more about the diversity of GHs and their potential role in a variety of gut microbiomes, we used a combination of 16S, metagenomic and targeted amplicon sequencing data to study one of these enzyme families in detail. Specifically, we employed a functional gene-targeted metagenomic approach to the 1-4-α-glucan-branching enzyme (gBE) gene in the gut microbiomes of four host species (human, chicken, cow and pig). The characteristics of operational taxonomic units (OTUs) and operational glucan-branching units (OGBUs) were distinctive in each of hosts. Human and pig were most similar in OTUs profiles while maintaining distinct OGBU profiles. Interestingly, the phylogenetic profiles identified from 16S and gBE gene sequences differed, suggesting the presence of different gBE genes in the same OTU across different vertebrate hosts. Our data suggest that gene-targeted metagenomic analysis is useful for an in-depth understanding of the diversity of a particular gene of interest. Specific carbohydrate metabolic genes appear to be carried by distinct OTUs in different individual hosts and among different vertebrate species' microbiomes, the characteristics of which differ according to host genetic background and/or diet.The ISME Journal advance online publication, 10 October 2013; doi:10.1038/ismej.2013.167.
    The ISME Journal 10/2013; DOI:10.1038/ismej.2013.167 · 9.27 Impact Factor
  • Source
    • "The rapid development of DNA sequencing technologies has also facilitated SIP study combined with metagenome sequencing for the full characterization of microorganisms in their metagenomic contexts. In particular , the almost complete genome of the methylotroph Methylotenera mobilis was obtained from DNA labelled with 13 C1 compounds using an SIP method combined with metagenome sequencing (Kalyuzhnaya et al., 2008). The MT Hebei Spirit oil spill (ca. 10 900 tons of crude oil) occurred near the coast of Taean in the Yellow Sea in December 2007, and most of the western side of the Korean Peninsula with tidal flats was heavily contaminated by the oil spill. "
    [Show abstract] [Hide abstract]
    ABSTRACT: DNA stable isotope probing and metagenomic sequencing were used to assess the metabolic potential of iron-reducing bacteria involved in anaerobic aromatic hydrocarbon degradation in oil spill-affected tidal flats. In a microcosm experiment, (13) C-toluene was degraded with the simultaneous reduction of Fe(III)-NTA, which was also verified by quasi-stoichiometric (13) C-CO2 release. The metabolic potential of the dominant member affiliated with the genus Desulfuromonas in the heavy DNA fraction was inferred using assembled scaffolds (designated TF genome, 4.40 Mbp with 58.8 GC mol%), which were obtained by Illumina sequencing. The gene clusters with peripheral pathways for toluene and benzoate conversion possessed the features of strict and facultative anaerobes. In addition to the class II-type benzoyl-CoA reductase (Bam) of strict anaerobes, the class I-type (Bcr) of facultative anaerobes was encoded. Genes related to the utilization of various anaerobic electron acceptors, including iron, nitrate (to ammonia), sulfur and fumarate, were identified. Furthermore, genes encoding terminal oxidases (caa3 , cbb3 and bd) and a diverse array of genes for oxidative stress responses were detected in the TF genome. This metabolic versatility may be an adaptation to the fluctuating availability of electron acceptors and donors in tidal flats.
    Environmental Microbiology 09/2013; 16(1). DOI:10.1111/1462-2920.12277 · 6.24 Impact Factor
  • Source
    • "As expected from prior analyses (Kalyuzhnaya et al., 2008), the community in the unamended sample revealed high complexity, being represented by a total of 1,486 sequence clusters (97% sequence identity; Kunin & Hugenholtz, 2010). The community was dominated by Proteobacteria (33.3%), of which phylotypes of the Methylococcaceae family that represents one class (called type I) of methane oxidizing bacteria were most prominently present (10% of all sequences). "
    [Show abstract] [Hide abstract]
    ABSTRACT: We investigated microbial communities active in methane oxidation in lake sediment at different oxygen tensions and their response to the addition of nitrate, via stable isotope probing combined with deep metagenomic sequencing. Communities from a total of four manipulated microcosms were analyzed, supplied with (13)C-methane in, respectively, ambient air, ambient air with the addition of nitrate, nitrogen atmosphere and nitrogen atmosphere with the addition of nitrate, and these were compared to the community from an unamended sediment sample. We found that the major group involved in methane oxidation in both aerobic and microaerobic conditions were members of the family Methylococcaceae, dominated by species of the genus Methylobacter, and these were stimulated by nitrate in aerobic but not microaerobic conditions. In aerobic conditions, we also noted a pronounced response to both methane and nitrate by members of the family Methylophilaceae that are non-methane-oxidizing methylotrophs, and predominantly by the members of the genus Methylotenera. The relevant abundances of the Methylococcaceae and the Methylophilaceae and their coordinated response to methane and nitrate suggest that these species may be engaged in cooperative behavior, the nature of which remains unknown.
    02/2013; 1:e23. DOI:10.7717/peerj.23