The greatest challenge to understanding microbial processes in nature, including microbial biodegradation of pollutants, is to determine which populations and genes are responsible for the catalysis While advances in molecular methods applied to microbial ecology have certainly enriched our understanding of microbial diversity and community composition, there are barriers to extending our knowledge to identifying the active populations and their catalytic genes. The limitations are caused by the current lack of methods to separate or distinguish the small portion of active members of a community from the large background of inactive members. Methods are needed that will detect both the culturable and uncultured active members, are more sensitive than current methods and provide information on both the active organisms and their active catalytic genes. We propose to use stable isotope probing (SIP) as the front end and couple this to a suite of nucleic acid methods. Briefly, the active populations in biodegradation are detected by their assimilation of 13C substrates which is followed by separation of their heavier RNA and DNA from that of the non-active populations (with 12C-RNA/DNA) by density gradient centrifugation. The heavy nucleic acids are then interrogated by (a) sequencing of the ribosomal RNA to determine phylogeny, (b) quantifying the growth of those populations by real-time PCR using primers designed from the rRNA gene sequences in the active fraction, (c) using the heavy DNA as PCR targets for functional gene sequences developing catch probes based on these sequences to more sensitively detect expression of the active genes and (d) finally using the heavy DNA on DNA microarrays to detect in a more parallel manner which of the subclasses of functional genes are present. Our research will address the following specific aims: (1) advancing 13C-SIP-based technology to attain comprehensive and multidimensional information on active biodegrading communities in soil. (2) improvement of the sensitivity and quantification of gene expressions in soil by using catch probe technology, and (3) to determine the validity of DNA microarrays for detecting functional genes and their expressions in soil. The result will be an integrated and versatile suite of methods to analyze the DNA and RNA produced only by the active populations. The research team includes microbial ecologists experienced in biodegradation, a stable isotope chemist with excellent facilities to advance this work and an engineer experienced in microarrays and complexity analysis of molecular data.
