Dioxygenase Genes from Uncultured Bacteria Similar to the phnAc gene from Alcaligenes faecalis Detected in Marine and Freshwater Sediments.

HEBE M. DIONISI; WALTER DI MARZIO; FU MIN MENN; JAMES P. EASTER; GARY S. SAYLER

San Francisco, CA, EEUU

Congreso; IUMS 2005. Joint Meeting of the 3 divisions of the International Union of Microbiological Societies 2005; 2005

International Union of Microbiological Societies

Background: Many polycyclic aromatic hydrocarbons (PAHs), organic compounds consisting of two or more fused aromatic rings, are known to be toxic, mutagenic or carcinogenic. Recent studies involving the isolation of PAH-degrading bacteria and the amplification of catabolic genes from environmental samples indicate a large diversity in these microorganisms, and may still be many unidentified PAH-degrading bacteria in the environment. The objective of this study was to explore the diversity of PAH-degrading microorganisms in different environments using culture-independent techniques. Methods: Chemical analyses of PAHs in marine and freshwater sediment samples were performed using an accelerated solvent extractor and gas chromatography-mass spectrometry (GC-MS). Total DNA was purified from 0.5 g sediment in duplicate or triplicate using the FastDNA® SPIN kit for soil (Q-BIOgene). Dioxygenase genes were amplified using the degenerate primer set Ac114f and Ac596r [Wilson et al. (1999) Appl. Environ. Microbiol. 65, 80-87], cloned and sequenced. Results: GC-MS indicated a phenanthrene concentration of 0.01 mg/kg dry weight sediment in an intertidal marine sediment sample from Ushuaia, Argentina. Low concentrations of other PAHs were also detected in this sample. Surficial freshwater sediment samples from Chattanooga Creek contained 180 and 11,450 mg/kg dry weight phenanthrene, with high concentrations of other PAHs. A primer set originally, designed to amplify nahAc homologs, amplified from marine sediment DNA dioxygenase sequences 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).  Dioxygenase sequences found in freshwater sediment libraries had higher sequence similarities with the phnAc gene from A. faecalis (99.1 to 99.3% similarity) than with phnAc genes cloned from Burkholderia sp. Ch1-1, Ch3-5, Cs1-4 and Eh1-1 (95.9 to 95.3% similarity). Conclusions: These results indicate the presence of microorganisms carrying A. faecalis-type PAH biodegradation pathways in a coastal marine environment, as well as in freshwater sediments.