Most microbes in the biosphere remain unculturable1. Whole genome shotgun (WGS) sequencing of environmental DNA (metagenomics) can be used to study the genetic and metabolic properties of natural microbial communities2,3,4. 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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.



  1. 1.

    The New Science of Metagenomics Revealing the Secrets of Our Microbial Planet (Committee on Metagenomics, Board of Life Sciences, Division of Earth and Life Studies, The National Academies Press, 2007).

  2. 2.

    et al. Community structure and metabolism through reconstruction of microbial genomes from the environment. Nature 428, 37–43 (2004).

  3. 3.

    et al. Comparative metagenomics of microbial communities. Science 308, 554–557 (2005).

  4. 4.

    et al. The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific. PLoS Biol. 5, e77 (2007).

  5. 5.

    & Methanotrophic bacteria. Microbiol. Rev. 60, 439–471 (1996).

  6. 6.

    The contribution of reactive carbon emissions from vegetation to the carbon balance of terrestrial ecosystems. Chemosphere 49, 837–844 (2002).

  7. 7.

    Aerobic methylotrophic procaryotes. in The Prokaryotes (eds. Balows, A., Truper, H.G., Dworkin, M., Harder, W. & Schleifer, K.-H.) 613–634 (Springer, New York, 2006).

  8. 8.

    , , & Molecular ecology techniques for the study of aerobic methanotrophs. Appl. Environ. Microbiol. 74, 1305–1315 (2008).

  9. 9.

    et al. Genomic insights into methanotrophy: the complete genome sequence of Methylococcus capsulatus (Bath). PLoS Biol. 2, e303 (2004).

  10. 10.

    et al. Whole-genome analysis of Methyl tert-Butyl Ether (MTBE)-degrading beta-proteobacterium Methylibium petroleiphilum PM1. J. Bacteriol. 189, 1931–1945 (2007).

  11. 11.

    et al. The genome of Methylobacillus flagellatus, the molecular basis for obligate methylotrophy, and the polyphyletic origin of methylotrophy. J. Bacteriol. 189, 4020–4027 (2007).

  12. 12.

    , , & Stable-isotope probing as a tool in microbial ecology. Nature 403, 646–649 (2000).

  13. 13.

    , , & Molecular characterization of methanotrophic isolates from freshwater lake sediment. Appl. Environ. Microbiol. 66, 5259–5266 (2000).

  14. 14.

    , , , & Methylotenera mobilis gen. nov., sp. nov, an obligately methylamine-utilizing bacterium within the family Methylophilaceae. Int. J. Syst. Evol. Microbiol. 56, 2819–2823 (2006).

  15. 15.

    , & Real-time detection of actively metabolizing microbes via redox sensing as applied to methylotroph populations in Lake Washington. ISME J. 2, 696–706 (2008).

  16. 16.

    , , , & Accurate phylogenetic classification of variable-length DNA fragments. Nat. Methods 4, 63–72 (2007).

  17. 17.

    , , & Novel formaldehyde-activating enzyme in Methylobacterium extorquens AM1 required for growth on methanol. J. Bacteriol. 182, 6645–6650 (2000).

  18. 18.

    et al. DMS formation by dimethylsulfoniopropionate route in freshwater. Environ. Sci. Technol. 32, 2130–2136 (1998).

  19. 19.

    et al. Genomic and genetic analysis of Bordetella bacteriophages encoding reverse transcriptase-mediated tropism-switching cassettes. J. Bacteriol. 186, 1503–1517 (2004).

  20. 20.

    et al. Genome characterization of lipid-containing marine bacteriophage PM2 by transposon insertion mutagenesis. J. Virol. 80, 9270–9278 (2006).

  21. 21.

    , , & Methylobacter tundripaludum sp. nov., a methane-oxidizing bacterium from Arctic wetland soil on the Svalbard islands, Norway (78° N). Int. J. Syst. Evol. Microbiol. 56, 109–113 (2006).

  22. 22.

    et al. Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutopha H16. Nat. Biotechnol. 24, 1257–1262 (2006).

  23. 23.

    , , , & Methane oxidation at 55°C and pH 2 by a thermoacidophilic bacterium belonging to the Verrucomicrobiaphylum. Proc. Natl. Acad. Sci. USA 105, 300–304 (2008).

  24. 24.

    et al. Methane oxidation by an extremely acidophilic bacterium of the phylum Verrucomicrobia. Nature 450, 879–882 (2007).

  25. 25.

    et al. Methanotrophy below pH 1 by a new Verrucomicrobia species. Nature 450, 874–878 (2007).

  26. 26.

    & Methanol: coenzyme M methyltransferase from Methanosarcina barkeri. Zinc dependence and thermodynamics of the methanol:cob(I)alamin methyltransferase reaction. Eur. J. Biochem. 249, 280–285 (1997).

  27. 27.

    et al. Characterization of a corrinoid protein involved in the C1 metabolism of strict anaerobic bacterium Moorella thermoacetica. Proteins: Struct. Funct. Bioinform. 67, 167–176 (2007).

  28. 28.

    et al. Diversity and abundance of aerobic and anaerobic methane oxidizers at the Haakon Mosby mud volcano, Barents Sea. Appl. Environ. Microbiol. 73, 3348–3362 (2007).

  29. 29.

    , , , & Bacterial populations active in metabolism of C1 compounds in the sediment of Lake Washington, a freshwater lake. Appl. Environ. Microbiol. 71, 6885–6899 (2005).

  30. 30.

    & DNA sequence quality trimming and vector removal. Bioinformatics 17, 1093–1104 (2001).

Download references


This research was supported by the National Science Foundation as part of the Microbial Observatories program (MCB-0604269). This work was performed, in part, under the auspices of the US Department of Energy's Office of Science Biological and Environmental Research Program, and by the University of California, Lawrence Livermore National Laboratory under contract no. W-7405-Eng-48, Lawrence Berkeley National Laboratory under contract no. DE-AC02-05CH11231 and Los Alamos National Laboratory under contract no. DE-AC02-06NA25396. The sequencing for the project was provided through the US Department of Energy Community Sequencing Program (

Author information

Author notes

    • Alice C McHardy

    Present address: Computational Genomics and Epidemiology Group, Max Planck Institute for Computer Science, Campus E1 4, 66123 Saarbruecken, Germany.


  1. Department of Microbiology, University of Washington, Benjamin Hall IRB, 616 NE Northlake Place, Seattle, Washington 98105, USA.

    • Marina G Kalyuzhnaya
    •  & Mary E Lidstrom
  2. Department of Chemical Engineering, University of Washington, Benjamin Hall IRB, 616 NE Northlake Place, Seattle, Washington 98105, USA.

    • Samuel R Levine
    • , Mary E Lidstrom
    •  & Ludmila Chistoserdova
  3. Production Genomics Facility, DOE Joint Genome Institute, 2800 Mitchell Drive, Bldg. 400, Walnut Creek, California 94596, USA.

    • Alla Lapidus
    • , Natalia Ivanova
    • , Alex C Copeland
    • , Asaf Salamov
    • , Igor V Grigoriev
    • , Susannah G Tringe
    •  & Paul M Richardson
  4. Bioinformatics and Pattern Discovery Group, IBM Thomas J. Watson Research Center, 1101 Kitchawan Road, Yorktown Heights, New York 10598, USA.

    • Alice C McHardy
    •  & Isidore Rigoutsos
  5. Biological Data Management and Technology Center, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Mail Stop 50A-1148, Berkeley, California 94720, USA.

    • Ernest Szeto
    •  & Victor M Markowitz
  6. Combimatrix Corporation, 6500 Harbour Heights Pkwy., Mukilteo, Washington 98275, USA.

    • Dominic Suciu
  7. DOE Joint Genome Institute, Los Alamos National Laboratory, PO Box 1663, Los Alamos, New Mexico 87545, USA.

    • David C Bruce


  1. Search for Marina G Kalyuzhnaya in:

  2. Search for Alla Lapidus in:

  3. Search for Natalia Ivanova in:

  4. Search for Alex C Copeland in:

  5. Search for Alice C McHardy in:

  6. Search for Ernest Szeto in:

  7. Search for Asaf Salamov in:

  8. Search for Igor V Grigoriev in:

  9. Search for Dominic Suciu in:

  10. Search for Samuel R Levine in:

  11. Search for Victor M Markowitz in:

  12. Search for Isidore Rigoutsos in:

  13. Search for Susannah G Tringe in:

  14. Search for David C Bruce in:

  15. Search for Paul M Richardson in:

  16. Search for Mary E Lidstrom in:

  17. Search for Ludmila Chistoserdova in:


M.G.K., M.E.L. and L.C. conceived the project. M.E.L. and L.C. coordinated project execution. M.G.K. collected samples, performed SIP, purified DNA for sequencing and performed microarray hybridizations. D.C.B. and P.M.R. oversaw library construction and sequencing. S.G.T. oversaw sequence assembly and analysis. A.C.C. and A.L. carried out assemblies. A.S. and I.V.G. conducted gene prediction and annotation. A.C.M. and I.R. carried out binning. E.S. and V.M.M. carried out data processing and loading into IMG/M. L.C. and N.I. carried out metabolic reconstruction. S.R.L. and M.G.K. performed species richness estimates. D.S. carried our microarray design. M.G.K., M.E.L. and L.C. wrote the initial draft of the paper; all other authors contributed.

Corresponding author

Correspondence to Ludmila Chistoserdova.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Figures 1–7, Tables 1–11, Methods

About this article

Publication history