Functional Biomarker Genes for the Detection of PAH-degrading Bacteria in Coastal Marine Environments of Patagonia

DIONISI H.; LOZADA M.; MARCOS M.; LOVISO, C.

Dalian

Congreso; BIT's 1st Annual World Congress of Petromicrobiology; 2010

Molecular tools suitable for providing quantitative and timely information on polycyclic aromatic hydrocarbon (PAH) biodegradation during the bioremediation of hydrocarbon polluted sites are greatly needed. In order to gain insight into functional genes that could be used as biomarkers for PAH biodegradation in cold marine environments, we targeted gene fragments encoding the large subunit of the catalytic component of ring-hydroxylating oxygenases (RHOs). The area chosen for this study, Ushuaia Bay, is located on the Big Island of Tierra del Fuego, Argentina, in the southernmost tip of South America, and is chronically polluted with hydrocarbons. We used different primer sets to amplify RHO gene fragments from metagenomic DNA extracted from Ushuaia Bay sediments, cloned and sequenced these amplicons. We identified 22 different groups of gene fragments, most of them presenting low to intermediate sequence similarities with previously described dioxygenase sequences (33 to 68% identity at the amino acid level). These groups were also divergent among them, and were classified as belonging to three out of the five different RHO enzyme types known up to date, indicating a great diversity of genes related to aromatic hydrocarbon biodegradation present in these sediments. In order to evaluate the relative abundance of some of these novel genes in sediment samples of Ushuaia Bay, we designed qPCR assays targeting these genes. In addition, we designed a qPCR assay to quantify the phnA1-like genes from the genus Cycloclasticus, obligate marine hydrocarbonoclastic bacteria with a well established role in aromatic hydrocarbon biodegradation, which had also been detected in sediments of Ushuaia Bay. As a reference value and to rule out qPCR inhibition, we quantified bacterial 16S rRNA genes using broad-specificity primers. Novel genes were detected at levels comparable to the ones found for the phnA1 gene in polluted sediments, suggesting that microbial populations carrying these genes could be playing an important role in aromatic hydrocarbon biodegradation in this cold marine environment. Further studies are being performed in this direction.