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Stable isotope probing — linking microbial identity to function

Nature Reviews Microbiology volume 3, pages 499–504 (2005)Cite this article

Abstract

Stable isotope probing (SIP) is a technique that is used to identify the microorganisms in environmental samples that use a particular growth substrate. The method relies on the incorporation of a substrate that is highly enriched in a stable isotope, such as 13C, and the identification of active microorganisms by the selective recovery and analysis of isotope-enriched cellular components. DNA and rRNA are the most informative taxonomic biomarkers and 13C-labelled molecules can be purified from unlabelled nucleic acid by density-gradient centrifugation. The future holds great promise for SIP, particularly when combined with other emerging technologies such as microarrays and metagenomics.

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Figure 1: Cultivation-independent identification of microorganisms using radioisotopes.
Figure 2: DNA-based stable isotope probing (SIP) and 13C-phospholipid fatty acids (PLFA) analyses.

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References

  1. Rappé, M. S. & Giovannoni, S. J. The uncultured microbial majority. Annu. Rev. Microbiol. 57, 369–394 (2003).

    Article  Google Scholar 

  2. Zengler, K. et al. Cultivating the uncultured. Proc. Natl Acad. Sci. USA 99, 15681–15686 (2002).

    Article  CAS  Google Scholar 

  3. Cole, J. R. et al. The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis. Nucleic Acids Res. 33, 294–296 (2005).

    Article  Google Scholar 

  4. Schloss, P. D. & Handelsman, J. Status of the microbial census. Microbiol. Mol. Biol. Rev. 68, 686–691 (2004).

    Article  Google Scholar 

  5. Ouverney, C. C. & Fuhrman, J. A. Combined microautoradiography–16S rRNA probe technique for determination of radioisotope uptake by specific microbial cell types in situ. Appl. Environ. Microbiol. 65, 1746–1752 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Lee, N. et al. Combination of fluorescent in situ hybridization and microautoradiography — a new tool for structure–function analyses in microbial ecology. Appl. Environ. Microbiol. 65, 1289–1297 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Daims, H., Nielsen, J. L., Nielsen, P. H., Schleifer, K. H. & Wagner, M. In situ characterization of Nitrospira-like nitrite-oxidizing bacteria active in wastewater treatment plants. Appl. Environ. Microbiol. 67, 5273–5284 (2001).

    Article  CAS  Google Scholar 

  8. Adamczyk, J. et al. The isotope array, a new tool that employs substrate-mediated labeling of rRNA for determination of microbial community structure and function. Appl. Environ. Microbiol. 69, 6875–6887 (2003).

    Article  CAS  Google Scholar 

  9. Orphan, V. J., House, C. H., Hinrichs, K. U., McKeegan, K. D. & DeLong, E. F. Methane-consuming Archaea revealed by directly coupled isotopic and phylogenetic analysis. Science 293, 484–487 (2001).

    Article  CAS  Google Scholar 

  10. Boetius, A. et al. A marine microbial consortium apparently mediating anaerobic oxidation of methane. Nature 407, 623–626 (2000).

    Article  CAS  Google Scholar 

  11. Boschker, H. T. S. et al. Direct linking of microbial populations to specific biogeochemical processes by 13C-labelling of biomarkers. Nature 392, 801–805 (1998).

    Article  CAS  Google Scholar 

  12. Radajewski, S., Ineson, P., Parekh, N. R. & Murrell, J. C. Stable-isotope probing as a tool in microbial ecology. Nature 403, 646–649 (2000).

    Article  CAS  Google Scholar 

  13. Meselson, M. & Stahl, F. W. The replication of DNA in Escherichia coli. Proc. Natl Acad. Sci. USA 44, 671–682 (1958).

    Article  CAS  Google Scholar 

  14. Schildkraut, C. L. in Methods in Enzymology (eds Grossman, L. & Moldave, K.) 695–699 (Academic Press, New York, 1967).

    Google Scholar 

  15. Radajewski, S. & Murrell, J. C. Stable isotope probing for detection of methanotrophs after enrichment with 13CH4 . Methods Mol. Biol. 179, 149–157 (2002).

    CAS  PubMed  Google Scholar 

  16. Bodrossy, L. et al. Development and validation of a diagnostic microbial microarray for methanotrophs. Environ. Microbiol. 5, 566–582 (2003).

    Article  CAS  Google Scholar 

  17. Dedysh, S. N. et al. Isolation of acidophilic methane-oxidizing bacteria from northern peat wetlands. Science 282, 281–284 (1998).

    Article  CAS  Google Scholar 

  18. Morris, S. A., Radajewski, S., Willison, T. W. & Murrell, J. C. Identification of the functionally active methanotroph population in a peat soil microcosm by stable-isotope probing. Appl. Environ. Microbiol. 68, 1446–1453 (2002).

    Article  CAS  Google Scholar 

  19. Radajewski, S. et al. Identification of active methylotroph populations in an acidic forest soil by stable-isotope probing. Microbiology 148, 2331–2342 (2002).

    Article  CAS  Google Scholar 

  20. Hutchens, E., Radajewski, S., Dumont, M. G., McDonald, I. R. & Murrell, J. C. Analysis of methanotrophic bacteria in Movile Cave by stable isotope probing. Environ. Microbiol. 6, 111–120 (2004).

    Article  CAS  Google Scholar 

  21. Lin, J. -L. et al. Molecular diversity of methanotrophs in Transbaikal soda lake sediments and identification of potentially active populations by stable isotope probing. Environ. Microbiol. 6, 1049–1060 (2004).

    Article  CAS  Google Scholar 

  22. Whitby, C. B. et al. 13C incorporation into DNA as a means of identifying the active components of ammonia-oxidizer populations. Lett. Appl. Microbiol. 32, 398–401 (2001).

    Article  CAS  Google Scholar 

  23. Miller, L. G. et al. Degradation of methyl bromide and methyl chloride in soil microcosms: use of stable C isotope fractionation and stable isotope probing to identify reactions and the responsible microorganisms. Geochim. Cosmochim. Acta 68, 3271–3283 (2004).

    Article  CAS  Google Scholar 

  24. McDonald, I. R. et al. A review of bacterial methyl halide degradation: biochemistry, genetics and molecular ecology. Environ. Microbiol. 4, 193–203 (2002).

    Article  CAS  Google Scholar 

  25. Ginige, M. P. et al. Use of stable-isotope probing, full-cycle rRNA analysis, and fluorescence in situ hybridization–microautoradiography to study a methanol-fed denitrifying microbial community. Appl. Environ. Microbiol. 70, 588–596 (2004).

    Article  CAS  Google Scholar 

  26. Padmanabhan, P. et al. Respiration of 13C-labeled substrates added to soil in the field and subsequent 16S rRNA gene analysis of 13C-labeled soil DNA. Appl. Environ. Microbiol. 69, 1614–1622 (2003).

    Article  CAS  Google Scholar 

  27. Jeon, C. O. et al. Discovery of a bacterium, with distinctive dioxygenase, that is responsible for in situ biodegradation in contaminated sediment. Proc. Natl Acad. Sci. USA 100, 13591–13596 (2003).

    Article  CAS  Google Scholar 

  28. Wackett, L. P. Stable isotope probing in biodegradation research. Trends Biotechnol. 22, 153–154 (2004).

    Article  CAS  Google Scholar 

  29. Manefield, M., Whiteley, A. S. & Bailey, M. J. What can stable isotope probing do for bioremediation? Inter. Biodeter. Biodegradation 54, 163–166 (2004).

    Article  CAS  Google Scholar 

  30. Lueders, T., Pommerenke, B. & Friedrich, M. W. Stable-isotope probing of microorganisms thriving at thermodynamic limits: syntrophic propionate oxidation in flooded soil. Appl. Environ. Microbiol. 70, 5778–5786 (2004).

    Article  CAS  Google Scholar 

  31. Manefield, M., Whiteley, A. S., Griffiths, R. I. & Bailey, M. J. RNA stable isotope probing, a novel means of linking microbial community function to phylogeny. Appl. Environ. Microbiol. 68, 5367–5373 (2002).

    Article  CAS  Google Scholar 

  32. Manefield, M., Whiteley, A. S., Ostle, N., Ineson, P. & Bailey, M. J. Technical considerations for RNA-based stable isotope probing: an approach to associating microbial diversity with microbial community function. Rapid Commun. Mass Spectrom. 16, 2179–2183 (2002).

    Article  CAS  Google Scholar 

  33. Mahmood, S., Paton, G. I. & Prosser, J. I. Cultivation-independent in situ molecular analysis of bacteria involved in degradation of pentachlorophenol in soil. Environ. Microbiol. (in the press).

  34. Lueders, T., Manefield, M. & Friedrich, M. W. Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients. Environ. Microbiol. 6, 73–78 (2004).

    Article  CAS  Google Scholar 

  35. Lueders, T., Wagner, B., Claus, P. & Friedrich, M. W. Stable isotope probing of rRNA and DNA reveals a dynamic methylotroph community and trophic interactions with fungi and protozoa in oxic rice field soil. Environ. Microbiol. 6, 60–72 (2004).

    Article  CAS  Google Scholar 

  36. Singh, B. K., Millard, P., Whiteley, A. S. & Murrell, J. C. Unravelling rhizosphere–microbial interactions: opportunities and limitations. Trends Microbiol. 12, 386–393 (2004).

    Article  CAS  Google Scholar 

  37. Griffiths, R. I. et al. 13CO2 pulse labelling of plants in tandem with stable isotope probing: methodological considerations for examining microbial function in the rhizosphere. J. Microbiol. Methods 58, 119–129 (2004).

    Article  CAS  Google Scholar 

  38. Rangel-Castro, J. I. et al. Stable isotope probing analysis of the influence of liming on root exudate utilization by soil microorganisms. Environ. Microbiol. (in the press).

  39. Schloss, P. D. & Handelsman, J. Biotechnological prospects from metagenomics. Curr. Opin. Biotechnol. 14, 303–310 (2003).

    Article  CAS  Google Scholar 

  40. Handelsman, J. Metagenomics: application of genomics to uncultured microorganisms. Microbiol. Mol. Biol. Rev. 68, 669–685 (2004).

    Article  CAS  Google Scholar 

  41. Rondon, M. R. et al. Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms. Appl. Environ. Microbiol. 66, 2541–2547 (2000).

    Article  CAS  Google Scholar 

  42. Venter, J. C. et al. Environmental genome shotgun sequencing of the Sargasso sea. Science 304, 66–74 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  44. Radajewski, S., McDonald, I. R. & Murrell, J. C. Stable-isotope probing of nucleic acids: a window to the function of uncultured microorganisms. Curr. Opin. Biotechnol. 14, 296–302 (2003).

    Article  CAS  Google Scholar 

  45. Bull, I. D., Parekh, N. R., Hall, G. H., Ineson, P. & Evershed, R. P. Detection and classification of atmospheric methane oxidizing bacteria in soil. Nature 405, 175–178 (2000).

    Article  CAS  Google Scholar 

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Acknowledgements

M.G.D. received financial support during his Ph.D. from the Fonds de Recherche sur la Nature et les Technologies (Quebec, Canada). J.C.M. gratefully acknowledges support from the Natural Environment Research Council, the Biotechnology and Biological Sciences Research Council and the European Union for funding work in his laboratory.

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  1. Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK

    Marc G. Dumont & J. Colin Murrell

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  1. Marc G. Dumont
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Correspondence to J. Colin Murrell.

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Glossary

CONSORTIUM

Physical association between cells of two or more types of microorganisms. Such an association might be advantageous to at least one of the microorganisms.

SYNTROPHIC ASSOCIATION

An association in which the growth of one organism depends on, or is improved by, growth factors or substrates provided (or in some cases removed) by another organism growing nearby.

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Dumont, M., Murrell, J. Stable isotope probing — linking microbial identity to function. Nat Rev Microbiol 3, 499–504 (2005). https://doi.org/10.1038/nrmicro1162

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