Characterization of tdt genes for the degradation of tricyclic diterpenes by Pseudomonas diterpeniphila A19-6a

Resin acids are tricyclic diterpenes that are toxic to aquatic life when released in high concentrations in pulp mill effluents. These naturally formed organic acids are readily degraded by bacteria and fungi; nevertheless, many of the mechanisms involved are still unknown. We report the localization, cloning, and sequencing of genes for abietane degradation (9.18 kb; designated tdt (tricyclic diterpene) LRSABCD) from the gamma-Proteobacterium Pseudomonas diterpeniphila A19-6a. Using gene knockout mutants, we demonstrate that tdtL, encoding a putative CoA ligase, is required for growth on abietic and dehydroabietic acids. A second gene knockout in tdtD, encoding a putative cytochrome P450 monooxygenase, reduced the growth of strain A19-6a on abietic and dehydroabietic acids as sole sources of carbon and energy, but did not eliminate growth. The degree of homology between P450(TdtD) and P450(TerpC), the closest known P450 homologue to TdtD, identifies TdtD as a new member of the P450 superfamily. Hybridization of six of the tdt genes to genomic DNA of a related resin acid degrading bacterium Pseudomonas abietaniphila BKME-9 identified tdt homologues in this strain that utilizes aromatic ring dioxygenase genes (dit) to open the ring structure of abietic and dehydroabietic acids. These results suggest the tdt and dit genes may function in concert to allow these Pseudomonas strains to degrade resin acids. Homologues of several of the tdt genes were detected in resin acid degrading Ralstonia and Comamonas species within the beta- and gamma-Proteobacteria.