Genetic Diversity of PAH-Degrading Bacteria in Marine Sediments of Ushuaia Bay, Argentina

GUERRERO, L.D.; DI MARZIO, W.; DIONISI, H.M.

Atlanta, GA, EEUU

Congreso; 105th General Meeting American Society for Microbiology; 2005

American Society for Microbiology

Polycyclic aromatic hydrocarbons (PAHs), which consist of two or more fused aromatic rings, are widespread pollutants in the marine environment. Although PAH biodegradation has been extensively studied in soil and groundwater, much less is known about the biodegradation process of these compounds in marine ecosystems. The objective of this study was to explore the diversity of PAH-degrading microorganisms in marine sediments using culture-independent techniques. Surficial intertidal sediment samples were collected from Ushuaia city, Tierra del Fuego, Argentina. Chemical analysis using gas chromatography-mass spectrometry indicated the presence of low concentrations of naphthalene, phenanthrene, fluorene, pyrene, fluoranthene, acenaphthene, and benzoanthracene, with a total PAH concentration of 0.03 mg/kg dry weight sediment. Total DNA was purified using the FastDNA® SPIN kit for soil (Q-BIOgene). The majority of the DNA was in the size range of 5 to 20 kb, and was suitable for PCR amplification using bacterial 16S rRNA gene primers. Alpha subunits of initial PAH dioxygenases were amplified from the purified DNA using different primer sets, selected from the literature or designed in this work. After the amplification, the PCR products were cloned and sequenced. A primer set originally designed to amplify nahAc homologs rendered in the library three different insert sizes of 442, 479 and 480 bp, respectively. However, only those clones containing a 479-bp insert (44% of the clones in the library) showed high homology with initial PAH-dioxygenases. These clones were closely related to phnAc genes cloned from Burkholderia sp. Ch1-1, Ch3-5, Cs1-4 and Eh1-1 (AY367784-7; 97.3 to 99.8% similarity at the nucleotide level) and Alcaligenes faecalis (AB024945; 95.7-95.9% similarity). In addition, a primer set designed to target the phnA1 gene from Cycloclasticus sp. A5 (AB102786) and close relatives amplified from the same sample a 483 bp gene fragment closely related to the phnA1 gene (99.5% similarity). These results start to unravel the diversity of the PAH-degrading bacteria coexisting in this cold, coastal marine environment.