Stable-isotope probing as a tool in microbial ecology

Microorganisms are responsible for driving the biogeochemical cycling of el ements on Earth. Despite their importance and vast diversity(1), the taxono mic identity of the microorganisms involved in any specific process has usu ally been confined to that small fraction of the microbiota that has been i solated and cultivated. The recent coupling of molecular biological methods with stable-isotope abundance in biomarkers has provided a cultivation-ind ependent means of linking the identity of bacteria with their function in t he environment(2,3). Here we show that C-13-DNA, produced during the growth of metabolically distinct microbial groups on a C-13-enriched carbon sourc e, can be resolved from C-12- DNA by density-gradient centrifugation. DNA i solated from the target group of microorganisms can be characterized taxono mically 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 g roups of eubacteria; the alpha-proteobacterial and Acidobacterium lineages. Stable-isotope probing thus offers a powerful new technique for identifyin g microorganisms that are actively involved in specific metabolic processes under conditions which approach those occurring in situ.