Analysis of Active Methylotrophic Communities: When DNA-SIP Meets High-Throughput Technologies

  • Martin Taubert
  • Carolina Grob
  • Alexandra M. Howat
  • Oliver J. Burns
  • Yin Chen
  • Josh D. Neufeld
  • J. Colin MurrellEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1399)


Methylotrophs are microorganisms ubiquitous in the environment that can metabolize one-carbon (C1) compounds as carbon and/or energy sources. The activity of these prokaryotes impacts biogeochemical cycles within their respective habitats and can determine whether these habitats act as sources or sinks of C1 compounds. Due to the high importance of C1 compounds, not only in biogeochemical cycles, but also for climatic processes, it is vital to understand the contributions of these microorganisms to carbon cycling in different environments. One of the most challenging questions when investigating methylotrophs, but also in environmental microbiology in general, is which species contribute to the environmental processes of interest, or “who does what, where and when?” Metabolic labeling with C1 compounds substituted with 13C, a technique called stable isotope probing, is a key method to trace carbon fluxes within methylotrophic communities. The incorporation of 13C into the biomass of active methylotrophs leads to an increase in the molecular mass of their biomolecules. For DNA-based stable isotope probing (DNA-SIP), labeled and unlabeled DNA is separated by isopycnic ultracentrifugation. The ability to specifically analyze DNA of active methylotrophs from a complex background community by high-throughput sequencing techniques, i.e. targeted metagenomics, is the hallmark strength of DNA-SIP for elucidating ecosystem functioning, and a protocol is detailed in this chapter.

Key words

Carbon-13 DNA stable isotope probing DNA-SIP High-throughput sequencing Isotopic labeling Methylotrophy Metagenomics One-carbon compounds 



This work was possible thanks to financial support from the Gordon and Betty Moore Foundation Marine Microbiology Initiative Grant GBMF3303 to J. Colin Murrell and Yin Chen and through the Earth and Life Systems Alliance, Norwich Research Park, Norwich, UK.


  1. 1.
    Carpenter LJ, Archer SD, Beale R (2012) Ocean–atmosphere trace gas exchange. Chem Soc Rev 41:6473–6506PubMedCrossRefGoogle Scholar
  2. 2.
    Heikes BG, Chang WN, Pilson MEQ, Swift E, Singh HB, Guenther A, Jacob DJ, Field BD, Fall R, Riemer D, Brand L (2002) Atmospheric methanol budget and ocean implication. Global Biogeochem Cycles 16:8001–8013CrossRefGoogle Scholar
  3. 3.
    Carini P, White AE, Campbell EO, Giovannoni SJ (2014) Methane production by phosphate-starved SAR11 chemoheterotrophic marine bacteria. Nat Commun 5:4346PubMedCrossRefGoogle Scholar
  4. 4.
    Chen Y, McAleer KL, Murrell JC (2010) Monomethylamine as a nitrogen source for a nonmethylotrophic bacterium, Agrobacterium tumefaciens. Appl Environ Microbiol 76:4102–4104PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Kiene RP, Linn LJ, Bruton JA (2000) New and important roles for DMSP in marine microbial communities. J Sea Res 43:209–224CrossRefGoogle Scholar
  6. 6.
    Anthony C (1982) The biochemistry of methylotrophs. Academic, New YorkGoogle Scholar
  7. 7.
    Neufeld JD, Wagner M, Murrell JC (2007) Who eats what, where and when? Isotope-labelling experiments are coming of age. ISME J 1:103–110PubMedCrossRefGoogle Scholar
  8. 8.
    Chistoserdova L (2011) Modularity of methylotrophy, revisited. Environ Microbiol 13:2603–2622PubMedCrossRefGoogle Scholar
  9. 9.
    Holmes AJ, Costello A, Lidstrom ME, Murrell JC (1995) Evidence that participate methane monooxygenase and ammonia monooxygenase may be evolutionarily related. FEMS Microbiol Lett 132:203–208PubMedCrossRefGoogle Scholar
  10. 10.
    McDonald IR, Murrell JC (1997) The methanol dehydrogenase structural gene mxaF and its use as a functional gene probe for methanotrophs and methylotrophs. Appl Environ Microbiol 63:3218–3224PubMedPubMedCentralGoogle Scholar
  11. 11.
    Costello AM, Lidstrom ME (1999) Molecular characterization of functional and phylogenetic genes from natural populations of methanotrophs in lake sediments. Appl Environ Microbiol 65:5066–5074PubMedPubMedCentralGoogle Scholar
  12. 12.
    Auman AJ, Stolyar S, Costello AM, Lidstrom ME (2000) Molecular characterization of methanotrophic isolates from freshwater lake sediment. Appl Environ Microbiol 66:5259–5266PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Hutchens E, Radajewski S, Dumont MG, McDonald IR, Murrell JC (2004) Analysis of methanotrophic bacteria in Movile Cave by stable isotope probing. Environ Microbiol 6:111–120PubMedCrossRefGoogle Scholar
  14. 14.
    Neufeld JD, Schafer H, Cox MJ, Boden R, McDonald IR, Murrell JC (2007) Stable-isotope probing implicates Methylophaga spp. and novel Gammaproteobacteria in marine methanol and methylamine metabolism. ISME J 1:480–491PubMedCrossRefGoogle Scholar
  15. 15.
    Wischer D, Kumaresan D, Johnston A, El Khawand M, Stephenson J, Hillebrand-Voiculescu AM, Chen Y, Colin Murrell J (2014) Bacterial metabolism of methylated amines and identification of novel methylotrophs in Movile Cave. ISME J 9:195–206PubMedPubMedCentralCrossRefGoogle Scholar
  16. 16.
    Shokralla S, Spall JL, Gibson JF, Hajibabaei M (2012) Next-generation sequencing technologies for environmental DNA research. Mol Ecol 21:1794–1805PubMedCrossRefGoogle Scholar
  17. 17.
    Lüke C, Frenzel P (2011) Potential of pmoA amplicon pyrosequencing for methanotroph diversity studies. Appl Environ Microbiol 77:6305–6309PubMedPubMedCentralCrossRefGoogle Scholar
  18. 18.
    Kolb S, Stacheter A (2013) Prerequisites for amplicon pyrosequencing of microbial methanol utilizers in the environment. Front Microbiol 4:1–12CrossRefGoogle Scholar
  19. 19.
    Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D, Eisen JA, Wu D, Paulsen I, Nelson KE, Nelson W, Fouts DE, Levy S, Knap AH, Lomas MW, Nealson K, White O, Peterson J, Hoffman J, Parsons R, Baden-Tillson H, Pfannkoch C, Rogers YH, Smith HO (2004) Environmental genome shotgun sequencing of the Sargasso Sea. Science 304:66–74PubMedCrossRefGoogle Scholar
  20. 20.
    Chistoserdova L (2014) Is metagenomics resolving identification of functions in microbial communities? Microb Biotechnol 7:1–4PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    Boschker H, Nold S, Wellsbury P, Bos D, De Graaf W, Pel R, Parkes R, Cappenberg T (1998) Direct linking of microbial populations to specific biogeochemical processes by 13C-labelling of biomarkers. Nature 392:801–805CrossRefGoogle Scholar
  22. 22.
    Radajewski S, Ineson P, Parekh NR, Murrell JC (2000) Stable-isotope probing as a tool in microbial ecology. Nature 403:646–649PubMedCrossRefGoogle Scholar
  23. 23.
    Neufeld JD, Vohra J, Dumont MG, Lueders T, Manefield M, Friedrich MW, Murrell JC (2007) DNA stable-isotope probing. Nat Protoc 2:860–866PubMedCrossRefGoogle Scholar
  24. 24.
    Murrell JC, Whiteley AS (2011) Stable isotope probing and related technologies. ASM, Washington, DCCrossRefGoogle Scholar
  25. 25.
    Neufeld JD, Dumont MG, Vohra J, Murrell JC (2007) Methodological considerations for the use of stable isotope probing in microbial ecology. Microb Ecol 53:435–442:2027Google Scholar
  26. 26.
    Dunford EA, Neufeld JD (2010) DNA stable-isotope probing (DNA-SIP). J Vis Exp 42:2027Google Scholar
  27. 27.
    Neufeld JD, Chen Y, Dumont MG, Murrell JC (2008) Marine methylotrophs revealed by stable-isotope probing, multiple displacement amplification and metagenomics. Environ Microbiol 10:1526–1535PubMedCrossRefGoogle Scholar
  28. 28.
    Kalyuzhnaya MG, Lapidus A, Ivanova N, Copeland AC, McHardy AC, Szeto E, Salamov A, Grigoriev IV, Suciu D, Levine SR, Markowitz VM, Rigoutsos I, Tringe SG, Bruce DC, Richardson PM, Lidstrom ME, Chistoserdova L (2008) High-resolution metagenomics targets specific functional types in complex microbial communities. Nat Biotechnol 26:1029–1034PubMedCrossRefGoogle Scholar
  29. 29.
    Green SJ, Leigh MB, Neufeld JD (2010) Denaturing Gradient Gel Electrophoresis (DGGE) for microbial community analysis. In: Timmis K (ed) Handbook of hydrocarbon and lipid microbiology. Springer, Berlin Heidelberg, pp 4137–4158CrossRefGoogle Scholar
  30. 30.
    Binga EK, Lasken RS, Neufeld JD (2008) Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology. ISME J 2:233–241PubMedCrossRefGoogle Scholar
  31. 31.
    Muyzer G, De Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59:695–700Google Scholar
  32. 32.
    Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541PubMedPubMedCentralCrossRefGoogle Scholar
  33. 33.
    Edgar RC (2013) UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods 10:996–998PubMedCrossRefGoogle Scholar
  34. 34.
    Cebron A, Bodrossy L, Stralis-Pavese N, Singer AC, Thompson IP, Prosser JI, Murrell JC (2007) Nutrient amendments in soil DNA stable isotope probing experiments reduce the observed methanotroph diversity. Appl Environ Microbiol 73:798–807PubMedPubMedCentralCrossRefGoogle Scholar
  35. 35.
    Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedPubMedCentralCrossRefGoogle Scholar
  36. 36.
    Meyer F, Paarmann D, D'Souza M, Olson R, Glass EM, Kubal M, Paczian T, Rodriguez A, Stevens R, Wilke A, Wilkening J, Edwards RA (2008) The metagenomics RAST server - a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinformatics 9:386PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    Bartram A, Poon C, Neufeld J (2009) Nucleic acid contamination of glycogen used in nucleic acid precipitation and assessment of linear polyacrylamide as an alternative co-precipitant. Biotechniques 47:1019–1022PubMedCrossRefGoogle Scholar
  38. 38.
    Huson DH, Mitra S, Ruscheweyh HJ, Weber N, Schuster SC (2011) Integrative analysis of environmental sequences using MEGAN4. Genome Res 21:1552–1560PubMedPubMedCentralCrossRefGoogle Scholar
  39. 39.
    Dumont MG, Lüke C, Deng YC, Frenzel P (2014) Classification of pmoA amplicon pyrosequences using BLAST and the lowest common ancestor method in MEGAN. Front Microbiol 5:1–11CrossRefGoogle Scholar
  40. 40.
    Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Pena AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Yilmaz S, Allgaier M, Hugenholtz P (2010) Multiple displacement amplification compromises quantitative analysis of metagenomes. Nat Methods 7:943–944PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Martin Taubert
    • 1
    • 5
  • Carolina Grob
    • 1
  • Alexandra M. Howat
    • 1
  • Oliver J. Burns
    • 2
  • Yin Chen
    • 3
  • Josh D. Neufeld
    • 4
  • J. Colin Murrell
    • 1
    Email author
  1. 1.School of Environmental SciencesUniversity of East AngliaNorwichUK
  2. 2.School of Biological SciencesUniversity of East AngliaNorwichUK
  3. 3.School of Life SciencesUniversity of WarwickCoventryUK
  4. 4.Department of BiologyUniversity of WaterlooWaterlooCanada
  5. 5.Institute for EcologyFriedrich Schiller University JenaJenaGermany

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