Identification of in situ 2,4-dichlorophenoxyacetic acid-degrading soil microorganisms using DNA-stable isotope probing

Microorganisms are responsible for driving the biogeochemical cycling of elements on Earth. Despite their importance and vast diversity, 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 environment. 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 alpha-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.

Identifying microorganisms responsible for recognized environmental processes remains a great challenge in contemporary microbial ecology. Only in the last few years have methodological innovations provided access to the relationship between the function of a microbial community and the phylogeny of the organisms accountable for it. In this study stable-isotope-labeled [13C]phenol was fed into a phenol-degrading community from an aerobic industrial bioreactor, and the 13C-labeled RNA produced was used to identify the bacteria responsible for the process. Stable-isotope-labeled RNA was analyzed by equilibrium density centrifugation in concert with reverse transcription-PCR and denaturing gradient gel electrophoresis. In contradiction with findings from conventional methodologies, this unique approach revealed that phenol degradation in the microbial community under investigation is dominated by a member of the Thauera genus. Our results suggest that this organism is important for the function of this bioreactor.