Detection, enumeration, and selection of aromatic oxygenase genes

Our ability to detect and enumerate pollutant biodegrading microorganisms in the environment is rapidly advancing with the development of molecular genetic techniques. Improved in-situ microbial characterization will facilitate assessment of the impact of remediation technologies on indigenous microbial populations, provide more accurate assessment of intrinsic pollution biodegradation, and enhance studies of contaminated site ecology. The research project focused on development of multiplex and real-time polymerase chain reaction (PCR) procedures to quantify aromatic catabolic genes that were then used to investigate the selection of aromatic catabolic pathways in laboratory microcosms and environmental samples from petroleum-contaminated sites. Aromatic oxygenases were chosen as the indicator genes because they mediate the first and rate-limiting step in aromatic hydrocarbon biodegradation and their DNA sequences are conserved within families of oxygenase genes. PCR primer sets were chosen from conserved regions unique to each family of oxygenases observed during alignments of known gene sequences. Thus each primer set is specific for a family of oxygenase genes (e.g. toluene dioxygenase) without excluding closely related but uncharacterized oxygenase genes. In all, primer sets were identified which allowed amplification of an initial oxygenase gene from pathways for the catabolism of naphthalene, biphenyl, benzene, toluene, xylenes, and phenol. With positive control strains, the length of the observed amplification product matched that predicted from published sequences and specificity was confirmed by hybridization for all primer sets. Optimization of polymerization temperatures for real-time PCR greatly reduced background fluorescence signals allowing detection limits of 103 gene copies per reaction. Following development of PCR assays, laboratory microcosms with single aromatic substrates (enrichment substrates) were prepared to test the PCR assay with uncharacterized bacteria and evaluate the selective pressure exerted on the soil microbial community by aromatic hydrocarbon contamination. For each microcosm, at least one family of oxygenase genes responsible for the biodegradation of the enrichment substrate was amplified using the primers developed. Results from the microcosm study gave insight into the selection of aromatic catabolic pathways in the environment and indicated that primers were specific for their targets in a complex pool of unknown DNA. Finally, groundwater samples from two gasoline-contaminated sites were studied. In field samples, aromatic oxygenase genes were detected in groundwater monitoring wells with current or recent petroleum contamination but not in wells with no history of contamination, confirming that this technology is appropriate for monitoring pollutant biodegradation