A Large Gene Cluster in Burkholderia xenovorans Encoding Abietane Diterpenoid Catabolism

Abstract

Abietane diterpenoids are defense compounds synthesized by trees that are abundant in natural environments and occur as significant pollutants from pulp and paper production. Burkholderia xenovorans LB400 has diverse catabolic capabilities and represents an important group of heterotrophic bacteria in soil environments. The genome sequence of LB400 revealed homologs of the dit genes of Pseudomonas abietaniphila BKME-9, which encode abietane diterpenoid degradation. LB400 grew on abietic acid (AbA), dehydroabietic acid (DhA), palustric acid (PaA), and 7-oxo-DhA. A Xeotron microarray set, with probes for 8450 of the estimated 9000 LB400 genes, was used to compare the transcriptomes of LB400 growing on DhA versus on succinate. On DhA, 97 genes were upregulated, 43 of which were within an 80-kb cluster located on the 1.47-Mbp megaplasmid of LB400. Upregulated genes in this cluster encode a permease, a ring-hydroxylating dioxygenase system (DitA), a ring-cleavage dioxygenase (DitC), a P450 monooxygenase (DitQ), and enzymes catalyzing beta-oxidation-type reactions. Disruption of the ditA1 gene, encoding the alpha-subunit of DitA, abolished growth on these abietanes. Analyses of the metabolism of abietanes by cell suspensions of wild-type LB400 and the ditA1 mutant indicate a convergent pathway, with 7-oxo-DhA as a common intermediate for ring hydroxylation by DitA. Also, 7-oxo-PaA was identified as a metabolite of both AbA and PaA. Sequence analysis indicates that genes encoding this pathway have been horizontally transferred among Betaproteobacteria and Gammaproteobacteria.

Dynamics of Pseudomonas aeruginosa genome evolution

“…The ortholog of PA2G 02002 in P. abietaniphila is DitE (absent in B. xenovorans). PA2G 02028 shares significant similarity (57%) with B. xenovorans Bxe C0645 whose expression was shown to be induced by growth of the bacteria on dehydroabeitic acid (25).…”

“…12). These compounds are synthesized by trees as defense molecules and several bacteria, including Pseudomonas abietaniphila and Burkholderia xenovorans, can use abietane diterpenoids as the sole carbon source (24,25). Twenty-three of the P. aeruginosa dit genes (PA2G 01983 to PA2G 02008) are highly homologous to genes in P. abietaniphila BKME-9 (62-91% sequence identity of their protein products) with conserved synteny (26).…”

Distinct Roles for Two CYP226 Family Cytochromes P450 in Abietane Diterpenoid Catabolism by Burkholderia xenovorans LB400

“…Gene replacement. The gene encoding DitQ and the gene encoding DitU were disrupted by replacement as previously described (38). The primers and plasmids used are listed in Tables 1 and 2.…”

“…The possible presence of functional P450 homologs with complementary activities may explain how the P450 knockout strains described above retained a capacity for reduced growth on abietanes. However, transcriptomic analysis of LB400 indicated that ditQ, but not ditU, was up-regulated during growth of LB400 on DhA (38). Thus, the patterns of expression and substrate specificities of various P450s likely dictate distinct roles for the enzymes in abietane metabolism, and these roles remain to be elucidated.…”

“…Recent investigation of LB400 identified a large cluster of abietane catabolic genes and suggested that bacteria may employ multiple P450s to degrade the diterpenoids (38). The LB400 gene cluster encodes two homologs of DitQ BKME-9 : DitQ LB400 (CYP226A1) and DitU (CYP226A2).…”