Characterization oftdtgenes for the degradation of tricyclic diterpenes byPseudomonas diterpeniphilaA19-6a

Abstract

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 P450TdtD and P450TerpC, 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.

A Large Gene Cluster in Burkholderia xenovorans Encoding Abietane Diterpenoid Catabolism

“…Interestingly, one of these strains did not contain a megaplasmid, implying that the dit cluster was encoded on chromosome 1 or 2 of that strain. As mentioned above, at least part of the LB400 dit cluster is conserved in P. abietaniphila BKME-9 (33) and P. diterpeniphila A19-6a (25). Further, a cluster of genes with high sequence identity and similar gene arrangement to that of the LB400 dit cluster was identified in the recently sequenced genome of P. aeruginosa 2192 (Pseudomonas aeruginosa 2192 Sequencing Project, Broad Institute of Harvard and MIT [http://www.broad.mit .edu]).…”

“…The LB400 dit cluster contains homologs of all but one gene previously associated with abietane catabolism. These comprise 22 genes of the Pseudomonas abietaniphila BKME-9 dit cluster (33), seven of which are also in the Pseudomonas diterpeniphila A19-6a tdt cluster (25). The sole outstanding gene not found in LB400 is ditE from BKME-9, encoding a putative permease of the major facilitator superfamily.…”

“…Through previous gene knockout analysis, very similar orthologs of six additional genes of the LB400 dit cluster were shown to be required for abietane diterpenoid metabolism. These orthologs include ditA3, ditC, ditF, ditH, and ditI in BKME-9 (20), as well as tdtL (orthologous to ditJ) in A19-6a (25).…”

A Cytochrome P450 Involved in the Metabolism of Abietane Diterpenoids by Pseudomonas abietaniphila BKME-9

“…2; Tables 2 and 3). A region of the 10.4-kbp extension corresponds very closely to the tdt cluster (17), having the same ORF arrangement and greater than 72% identity of deduced amino acid sequences. ditP is a short coding region that was identified by COG analysis as an ancient conserved region common to two or more phylogenetic branches (Table 3).…”

“…P450s are heme-thiolate proteins that function as the catalytic component of an electron transport chain generally including a ferredoxin and a ferredoxin reductase in bacterial systems (19). A putative P450, encoded by the tdtD gene, that may function in abietane diterpenoid degradation was recently identified in Pseudomonas diterpeniphila A19-6a, another resin acid-degrading bacterium closely related to BKME-9 (17,18). Morgan and Wyndham (17) reported that a tdtD knockout mutant of A19-6a was retarded in its removal of DhA or AbA from its growth medium when compared to that of the wild type.…”