Q-185. Isolation of a Phenanthrene-Degrading Acidovorax Strain and Identification of Associated Genetic Elements

D. R. Singleton, L. Guzman Ramirez, M. D. Aitken;
Univ. of North Carolina, Chapel Hill, NC.

A bacterium was isolated from phenanthrene-enriched, polycyclic aromatic hydrocarbon (PAH)-contaminated soil from the site of a former manufactured gas plant. The organism was identified as a member of the Acidovorax genus whose 16S rRNA gene sequence was highly similar to sequences previously recovered during stable-isotope probing with 13C-labeled phenanthrene. The pure culture was capable of at least partial mineralization of a number of PAH including phenanthrene, chrysene, benz[a]anthracene, and benzo[a]pyrene and additionally could convert indole to indigo. PCR screening of extracted pure-culture DNA using primers for nah, nid, and other PAH-related ring-hydroxylating dioxygenases (RHD) did not produce a product. A genomic fosmid library was constructed and colony hybridizations using the nahAc gene from Pseudomonas putida G7 as a probe successfully identified a clone containing a homologous gene. Subsequent sequencing indicated that this Acidovorax isolate contained phn-type RHD genes related to those in various Burkholderia isolates. Additionally, the sequences and arrangement of other PAH-degradation genes in the cluster were similar to Alcaligenes faecalis AFK2. Contrary to the Alcaligenes isolate, which contains a plasmid capable of conferring the phenanthrene-degradation phenotype, pulsed-field gel electrophoresis (PFGE) and southern blot hybridization suggest that the phn genes of Acidovorax sp. strain NA3 are located on the chromosome.