Advertisement

Generating Enriched Metagenomes from Active Microorganisms with DNA Stable Isotope Probing

  • Carolina Grob
  • Martin Taubert
  • Alexandra M. Howat
  • Oliver J. Burns
  • Yin Chen
  • Josh D. Neufeld
  • J. Colin MurrellEmail author
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Cultivation-dependent methods have long been used to characterise the physiology of microorganisms after they have been isolated from the environment. In contrast, cultivation-independent methods help better identify the microorganisms that regulate specific biogeochemical processes in situ, regardless of their ability to grow in the laboratory. In this chapter, we describe how the cultivation-independent technique of DNA stable isotope probing (DNA-SIP) can be combined with targeted functional gene probing and metagenomic analyses to characterise the metabolic potential of active microbes from environmental samples, including those that assimilate labelled hydrocarbons. DNA-SIP enables the separation of DNA from microbes that have assimilated a given 13C-labelled substrate into their biomass (i.e., heavy DNA) from those that have not (i.e., light DNA). The heavy DNA can be used subsequently for PCR amplicon sequencing and metagenome sequencing, which can potentially lead to retrieving genomes of uncultivated and active microbial representatives.

Keywords:

DNA stable isotope probing High-throughput sequencing Metabolic labelling Metagenomics 

Notes

Acknowledgements

This work was possible thanks to the financial support from the Gordon and Betty Moore Foundation Marine Microbiology Initiative Grant GBMF3303 to J. Colin Murrell and Yin Chen, and the Earth and Life Systems Alliance, Norwich Research Park, Norwich, UK. Josh Neufeld acknowledges a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC).

References

  1. 1.
    Glöckner FO, Gasol JM, McDonough N, Calewaert J-B (2012) Marine microbial diversity and its role in ecosystem functioning and environmental change. In: Calewaert JB, McDonough N (eds) Marine Board-ESF Position Paper 17. Marine Board-ESF, OstendGoogle Scholar
  2. 2.
    Yarza P, Yilmaz P, Pruesse E, Glöckner FO, Ludwig W, Schleifer K-H, Whitman WB, Euzéby J, Amann R, Rosselló-Móra R (2014) Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nat Rev Microbiol 12(9):635–645CrossRefPubMedGoogle Scholar
  3. 3.
    Murrell JC, Whiteley AS (2011) Stable isotope probing and related technologies. ASM Press, Washington, DCCrossRefGoogle Scholar
  4. 4.
    Neufeld JD, Vohra J, Dumont MG, Lueders T, Manefield M, Friedrich MW, Murrell JC (2007) DNA stable isotope probing. Nat Protoc 2(4):860–866CrossRefPubMedGoogle Scholar
  5. 5.
    Dunford EA, Neufeld JD (2010) DNA stable-isotope probing (DNA-SIP). J Vis Exp 42:e2027Google Scholar
  6. 6.
    Lueders T (2010) Stable isotope probing of hydrocarbon-degraders. In: Handbook of hydrocarbon and lipid microbiology. Springer, Berlin, pp 4011–4026Google Scholar
  7. 7.
    Chen Y, Murrell JC (2011) DNA stable isotope probing. In: Murrell JC, Whiteley AS (eds) Stable isotope probing and related technologies. ASM press, Washington, DC, pp 3–24CrossRefGoogle Scholar
  8. 8.
    Madsen E (2011) Stable isotope probing techniques and bioremediation. In: Murrell JC, Whiteley AS (eds) Stable isotope probing and related technologies. ASM Press, Washington, DC, pp 165–202CrossRefGoogle Scholar
  9. 9.
    Gutierrez T, Singleton DR, Berry D, Yang T, Aitken MD, Teske A (2013) Hydrocarbon-degrading bacteria enriched by the Deepwater Horizon oil spill identified by cultivation and DNA-SIP. ISME J 7(11):2091–2104CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Singleton DR, Jones MD, Richardson SD, Aitken MD (2013) Pyrosequence analyses of bacterial communities during simulated in situ bioremediation of polycyclic aromatic hydrocarbon-contaminated soil. Appl Microbiol Biotechnol 97(18):8381–8391CrossRefPubMedGoogle Scholar
  11. 11.
    Schäfer H, Muyzer G (2001) Denaturing gradient gel electrophoresis in marine microbial ecology. Method Microbiol 30:425–468CrossRefGoogle Scholar
  12. 12.
    Radajewski S, Ineson P, Parekh NR, Murrell JC (2000) Stable isotope probing as a tool in microbial ecology. Nature 403(6770):646–649CrossRefPubMedGoogle Scholar
  13. 13.
    Neufeld JD, Boden R, Moussard H, Schäfer H, Murrell JC (2008) Substrate-specific clades of active marine methylotrophs associated with a phytoplankton bloom in a temperate coastal environment. Appl Environ Microbiol 74(23):7321–7328CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Kleindienst S, Herbst F-A, Stagars M, von Netzer F, von Bergen M, Seifert J, Peplies J, Amann R, Musat F, Lueders T, Knittel K (2014) Diverse sulfate-reducing bacteria of the Desulfosarcina/Desulfococcus clade are the key alkane degraders at marine seeps. ISME J 8(10):2029–2044. doi: 10.1038/ismej.2014.51 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Neufeld JD, Chen Y, Dumont MG, Murrell JC (2008) Marine methylotrophs revealed by stable isotope probing, multiple displacement amplification and metagenomics. Environ Microbiol 10(6):1526–1535CrossRefPubMedGoogle Scholar
  16. 16.
    Chen Y, Murrell JC (2010) When metagenomics meets stable-isotope probing: progress and perspectives. Trends Microbiol 18(4):157–163CrossRefPubMedGoogle Scholar
  17. 17.
    Kalyuzhnaya MG, Lapidus A, Ivanova N, Copeland AC, McHardy AC, Szeto E, Salamov A, Grigoriev IV, Suciu D, Levine SR (2008) High-resolution metagenomics targets specific functional types in complex microbial communities. Nat Biotechnol 26(9):1029–1034CrossRefPubMedGoogle Scholar
  18. 18.
    Binga EK, Lasken RS, Neufeld JD (2008) Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology. ISME J 2(3):233–241CrossRefPubMedGoogle Scholar
  19. 19.
    Chen Y, Kumaresan D, Murrell JC (2011) Stable-isotope probing and its application in soil microbiology. In: Huang P, Li Y, Sumner M (eds) Handbook of soil science, 2nd edn, Vols 3 and 28. Taylor & Francis, London, pp 30–38Google Scholar
  20. 20.
    Verastegui Y, Cheng J, Engel K, Kolczynski D, Mortimer S, Lavigne J, Montalibet J, Romantsov T, Hall M, McConkey B (2014) Multisubstrate isotope labeling and metagenomic analysis of active soil bacterial communities. Mbio 5(4):e01157-01114CrossRefGoogle Scholar
  21. 21.
    Neufeld JD, Schäfer 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(6):480–491CrossRefPubMedGoogle Scholar
  22. 22.
    Griffiths RI, Whiteley AS, O'Donnell AG, Bailey MJ (2000) Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA-and rRNA-based microbial community composition. Appl Environ Microbiol 66(12):5488–5491CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Gaillard C, Fo S (1990) Ethanol precipitation of DNA with linear polyacrylamide as carrier. Nucleic Acids Res 18(2):378CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    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(3):695–700PubMedPubMedCentralGoogle Scholar
  25. 25.
    Klindworth A, Pruesse E, Schweer T, Peplies J, Quast C, Horn M, Glöckner FO (2012) Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res 41(1):e1. doi: 10.1093/nar/gks808 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75(23):7537–7541CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Pena AG, Goodrich JK, Gordon JI (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7(5):335–336CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Lynch MD, Masella AP, Hall MW, Bartram AK, Neufeld JD (2013) AXIOME: automated exploration of microbial diversity. GigaScience 2(1):3CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Meyer F, Paarmann D, D'Souza M, Olson R, Glass EM, Kubal M, Paczian T, Rodriguez A, Stevens R, Wilke A (2008) The metagenomics RAST server–a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinformat 9(1):386CrossRefGoogle Scholar
  30. 30.
    Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73(16):5261–5267CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19(5):455–477CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Namiki T, Hachiya T, Tanaka H, Sakakibara Y (2012) MetaVelvet: an extension of Velvet assembler to de novo metagenome assembly from short sequence reads. Nucleic Acids Res 40(20):e155CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M (2008) The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9(1):75CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Neufeld JD, Dumont MG, Vohra J, Murrell JC (2007) Methodological considerations for the use of stable isotope probing in microbial ecology. Microb Ecol 53(3):435–442CrossRefPubMedGoogle Scholar
  35. 35.
    Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, Chichester, pp 115–175Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Carolina Grob
    • 1
  • Martin Taubert
    • 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 Sciences, University of East Anglia, Norwich Research ParkNorwichUK
  2. 2.School of Biological Sciences, University of East Anglia, Norwich Research ParkNorwichUK
  3. 3.School of Life Sciences, University of WarwickCoventryUK
  4. 4.Department of BiologyUniversity of WaterlooWaterlooCanada

Personalised recommendations