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MOLECULAR-BASED APPROACHES TO SOIL MICROBIOLOGY

Stable Isotope Probing: Linking Functional Activity to Specific Members of Microbial Communities

Pacific Northwest National Lab., 999 Battelle Blvd., P8-13, Richland, WA 99352

^* Corresponding author (Helen.Kreuzer{at}pnl.gov).

Linking organisms or groups of organisms to specific functions within natural environments is a fundamental challenge in microbial ecology. Advances in technology for manipulating and analyzing nucleic acids have made it possible to characterize the members of microbial communities without the intervention of laboratory culturing. Results from such studies have shown that the vast majority of soil organisms have never been cultured, highlighting the risks of culture-based approaches in community analysis. The development of culture-independent techniques for following the flow of substrates through microbial communities therefore represents an important advance. These techniques, collectively known as stable isotope probing (SIP), involve introducing a stable isotope-labeled substrate into a microbial community and following the fate of the substrate by extracting diagnostic molecular species such as fatty acids and nucleic acids from the community and determining which specific molecules have incorporated the isotope. The molecules in which the isotope label appears provide identifying information about the organism that incorporated the substrate. Stable isotope probing allows direct observations of substrate assimilation in minimally disturbed communities, and thus represents an exciting new tool for linking microbial identity and function. The use of lipids or nucleic acids as the diagnostic molecule brings different strengths and weaknesses to the experimental approach, and necessitates the use of significantly different instrumentation and analytical techniques. This short review provides an overview of the lipid and nucleic acid approaches, discusses their strengths and weaknesses, gives examples of applications in various settings, and looks at prospects for the future of SIP technology.

Abbreviations: DGGE, denaturing gradient gel electrophoresis • FISH, fluorescent in situ hybridization • GC, gas chromatography • GC-C-IRMS, gas chromatography–combustion–isotope ratio mass spectrometry • GC–MS, gas chromatography–mass spectrometry • GC-Py-IRMS, gas chromatography–pyrolysis–isotope ratio mass spectrometry • IRMS, isotope ratio mass spectrometry or isotope ratio mass spectrometer • NA-SIP, stable isotope probing of nucleic acids • PCR, polymerase chain reaction • PLFA, polar lipid-derived fatty acids • rDNA, DNA encoding ribosomal RNA • rRNA, ribosomal RNA • RT-PCR, reverse transcription polymerase chain reaction • SIMS, secondary ion mass spectrometry • SIP, stable isotope probing • SSU, small subunit