High resolution metagenomics targets major functional types in complex microbial communities. Nat Biotechnol

Department of Microbiology, University of Washington, Benjamin Hall IRB, 616 NE Northlake Place, Seattle, Washington 98105, USA.
Nature Biotechnology (Impact Factor: 41.51). 09/2008; 26(9):1029-34. DOI: 10.1038/nbt.1488
Source: PubMed


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.


Available from: Susannah G Tringe
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    • "Interestingly, almost all sequences of methanotrophs residing in the mat were affiliated to type I methanotrophs. These have been found to dominate in high-methane environments such as lake sediments [32], wetland and landfill soils [33] where they carried methane oxidation. In our analysis the methane concentrations were mostly higher in deep samples. "
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    ABSTRACT: A metatranscriptomic approach was used to study community gene expression in a naturally occurring iron-rich microbial mat. Total microbial community RNA was reversely transcribed and sequenced by pyrosequencing. Characterization of expressed gene sequences provided accurate and detailed information of the composition of the transcriptionally active community and revealed phylogenetic and functional stratifications within the mat. Comparison of 16S rRNA reads and delineation of OTUs showed significantly lower values of metatranscriptomic-based richness and diversity in the upper parts of the mat than in the deeper regions. Taxonomic affiliation of rRNA sequences and mRNA genome recruitments indicated that iron-oxidizing bacteria affiliated to the genus Leptothrix, dominated the community in the upper layers of the mat. Surprisingly, type I methanotrophs contributed to the majority of the sequences in the deep layers of the mat. Analysis of mRNA expression patterns showed that genes encoding the three subunits of the particulate methane monooxygenase (pmoCAB) were the most highly expressed in our dataset. These results provide strong hints that iron-oxidation and methane-oxidation occur simultaneously in microbial mats and that both groups of microorganisms are major players in the functioning of this ecosystem.
    PLoS ONE 07/2014; 9(7):e102561. DOI:10.1371/journal.pone.0102561 · 3.23 Impact Factor
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    • "The relative number of OTUs attributed to Actinobacteria and Verrucomicrobia was higher among the shotgun sequences than the PCR-based sequences and corresponded well with the lineages identified from comparison to the freshwater 16S database (above), indicating that the shotgun library is sometimes able to capture more taxonomic diversity than 16S amplicons. Our observations corroborate those seen by others that the 16S rRNA gene sequences derived from metagenomic datasets vs. PCR amplification are roughly similar at broad taxonomic classifications (e.g., [49]), but that the number of OTUs identified by 16S rRNA gene sequencing is larger simply by virtue of the number of sequences obtained and that shotgun approaches capture greater diversity due to the lack of PCR primer specificity [50]. "
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    PLoS ONE 04/2014; 9(4):e93827. DOI:10.1371/journal.pone.0093827 · 3.23 Impact Factor
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    • "This approach was elegantly demonstrated in a study that identified genes associated with carbon uptake in sediments taken from Lake Washington [39]. Cultures were grown in the presence of single carbon C13-labeled substrates to enrich the organisms responsible for carbon uptake and their genomic DNA isolated by density gradient centrifugation [39]. Target gene enrichment has also been achieved using degenerate PCR primers to amplify the gene families or pathways of interest using phylogenetically conserved priming sites [40]. "
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    ABSTRACT: Rewiring and optimization of metabolic networks to enable the production of commercially valuable chemicals is a central goal of metabolic engineering. This prospect is challenged by the complexity of metabolic networks, lack of complete knowledge of gene function(s), and the vast combinatorial genotype space that is available for exploration and optimization. Various approaches have thus been developed to aid in the efficient identification of genes that contribute to a variety of different phenotypes, allowing more rapid design and engineering of traits desired for industrial applications. This review will highlight recent technologies that have enhanced capabilities to map genotype-phenotype relationships on a genome wide scale and emphasize how such approaches enable more efficient design and engineering of complex phenotypes.
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