Molecular analysis of phenanthrene degradation by Comamonas testosteroni GZ38 and Acidovorax sp. GZ39

Polycyclic aromatic hydrocarbons (PAHs) are toxic pollutants that are present in a variety of environments. Some bacterial species have the ability to metabolize these compounds and use them as energy sources for growth. Comamonas testosteroni GZ38 and Acidovorax sp. GZ39 were isolated from the Passaic River, NJ, based on their ability to grow on phenanthrene. These isolates are unusual in that they can grow on phenanthrene but not on naphthalene. The genes that code for phenanthrene degradation in GZ38 and GZ39 were identified. The DNA sequence of the genes is unique with respect to other sequences in the GenBank database with a single match to a putative phenanthrene degradation gene cluster from the phenanthrene degrader Alcaligenes faecalis AFK2 (GenBank accession number AB024945). BLAST matches to the putative genes in both operons suggest that phenanthrene degradation proceeds through phthalate. The putative phthalate degradation gene clusters from GZ38 and GZ39 have also been identified and sequenced. RT-PCR analysis of the phenanthrene degradation genes of GZ39 showed that the genes are expressed as one continuous transcript beginning with a transposase that is upstream from the start of the phenanthrene operon. The phenanthrene degradation genes of GZ38 are also expressed as one continuous transcript but it does not include a transposase located upstream from the operon. For both GZ38 and GZ39, expression of the genes was observed with both phenanthrene and succinate as growth substrates. RT-qPCR results for GZ38 showed that the phenanthrene degradation genes were equally expressed on phenanthrene and succinate. A lacZ assay demonstrated that the promoter region upstream of the phnAb gene in GZ39 is constitutively active. A 5' RACE assay was performed in order to map the transcription start site of the phenanthrene operon in GZ39. Inactivation of the phthalate dioxygenase in GZ39 resulted in the accumulation of phthalate when grown on phenanthrene and provides strong evidence that phenanthrene degradation proceeds through phthalate. Although the phenanthrene degradation genes of GZ38, GZ39, and AFK2 are nearly identical, this is the first report of functional information relating to these genes.