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Stable-isotope probing as a tool in microbial ecology

Abstract

Microorganisms are responsible for driving the biogeochemical cycling of elements on Earth. Despite their importance and vast diversity1, the taxonomic identity of the microorganisms involved in any specific process has usually been confined to that small fraction of the microbiota that has been isolated and cultivated. The recent coupling of molecular biological methods with stable-isotope abundance in biomarkers has provided a cultivation-independent means of linking the identity of bacteria with their function in the environment2,3. Here we show that 13C-DNA, produced during the growth of metabolically distinct microbial groups on a 13C-enriched carbon source, can be resolved from 12C-DNA by density-gradient centrifugation. DNA isolated from the target group of microorganisms can be characterized taxonomically and functionally by gene probing and sequence analysis. Application of this technique to investigate methanol-utilizing microorganisms in soil demonstrated the involvement of members of two phylogenetically distinct groups of eubacteria; the α-proteobacterial and Acidobacterium lineages. Stable-isotope probing thus offers a powerful new technique for identifying microorganisms that are actively involved in specific metabolic processes under conditions which approach those occurring in situ .

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Figure 1: Equilibrium centrifugation of isotopically labelled DNA in CsCl/ethidium bromide density gradients.
Figure 2: Phylogenetic analysis of bacterial 16S rRNA gene sequences amplified from 13C-DNA.
Figure 3: Phylogenetic analysis of deduced amino-acid sequences corresponding to the mxaF genes amplified from 13C-DNA.

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Acknowledgements

We thank D. P. Kelly for discussion, C. B. Miguez for Methylobacterium extorquens strain AM155 and Forest Research for access to the study site. Funding was from the Natural Environment Research Council (NERC) EDGE Programme (S.R., P.I. and J.C.M.), NERC (N.R.P.) with further support from the European Union 4th Framework Programme (J.C.M.).

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Correspondence to J. Colin Murrell.

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Radajewski, S., Ineson, P., Parekh, N. et al. Stable-isotope probing as a tool in microbial ecology. Nature 403, 646–649 (2000). https://doi.org/10.1038/35001054

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