Genetic Investigation of the Catabolic Pathway for Degradation of Abietane Diterpenoids by Pseudomonas abietaniphila BKME-9

Abstract

We have cloned and sequenced the dit gene cluster encoding enzymes of the catabolic pathway for abietane diterpenoid degradation by Pseudomonas abietaniphila BKME-9. The dit gene cluster is located on a 16.7-kb DNA fragment containing 13 complete open reading frames (ORFs) and 1 partial ORF. The genes ditA1A2A3 encode the ␣ and ␤ subunits and the ferredoxin of the dioxygenase which hydroxylates 7-oxodehydroabietic acid to 7-oxo-11,12-dihydroxy-8,13-abietadien acid. The dioxygenase mutant strain BKME-941 (ditA1::Tn5) did not grow on nonaromatic abietanes, and transformed palustric and abietic acids to 7-oxodehydroabietic acid in cell suspension assays. Thus, nonaromatic abietanes are aromatized prior to further degradation. Catechol 2,3-dioxygenase activity of xylE transcriptional fusion strains showed induction of ditA1 and ditA3 by abietic, dehydroabietic, and 7-oxodehydroabietic acids, which support the growth of strain BKME-9, as well as by isopimaric and 12,14-dichlorodehydroabietic acids, which are diterpenoids that do not support the growth of strain BKME-9. In addition to the aromatic-ring-hydroxylating dioxygenase genes, the dit cluster includes ditC, encoding an extradiol ring cleavage dioxygenase, and ditR, encoding an IclR-type transcriptional regulator. Although ditR is not strictly required for the growth of strain BKME-9 on abietanes, a ditR::Km r mutation in a ditA3::xylE reporter strain demonstrated that it encodes an inducer-dependent transcriptional activator of ditA3. An ORF with sequence similarity to genes encoding permeases (ditE) is linked with genes involved in abietane degradation.The pulping of wood to extract fibers used to make paper produces toxic wastewaters (23). The majority of the acute toxicity can be attributed to resin acids found in these wastewaters (18). Resin acids, a class of diterpenoids found in wood extractives, can be grouped into abietanes and pimeranes. Abietane-type acids have an isopropyl chain at the C-13 carbon atom, while pimerane-type acids have methyl and vinyl substituents at this position. Although these compounds are abundant in nature, they are problematic in the pulp and paper industry because of their unnaturally high concentrations in wastewaters. Therefore, resin acids must be removed from wastewater prior to its discharge to the environment. Biological treatment using aerated lagoons is the most common method used for detoxifying the wastewater (19). Several bacteria isolated from such biotreatment systems have been reported to use resin acids as growth substrates, and this catabolic activity appears to be widespread (22,25).Pseudomonas abietaniphila BKME-9, a bacterium isolated from a pulp and paper wastewater treatment system, is able to use dehydroabietic acid, an abietane-type resin acid, as a sole source of carbon and reductant (4). A novel three-component aromatic-ring-hydroxylating dioxygenase from this strain has been cloned and expressed (21). In strain BKME-9, dehydroabietic acid is first oxidized by an unidentified enzyme at the C-7 positi...

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

“…In fact, a pimerane diterpenoid, isopimaric acid, and a chlorinated diterpenoid, 12,14-dichlorodehydroabietic acid, also induced ditQ, despite these two compounds not being growth substrates for BKME-9. However, this is not surprising considering that the same inducers were identified for ditA1 and ditA3, the genes encoding the ␣ subunit and ferredoxin of the ring-hydroxylating dioxygenase, respectively (14). As previ- ously seen with the dioxygenase components, nonditerpenoid compounds did not induce expression of ditQ above the level of the pyruvate control.…”

“…Previously, it was reported that wild-type BKME-9 shows no endogenous catechol-2,3-dioxygenase (C23O) activity and that activity served as an adequate reporter for gene induction studies (14). For C23O assays, strain P450KO was grown on mineral medium supplemented with 1 g of sodium pyruvate/liter to an OD 600 between 0.15 and 0.3 and then spiked with a potential inducer, 150 mg of DhA/liter, 150 mg of AbA/liter, 158 mg of 7-oxo-DhA/liter, 150 mg of isopimaric acid/liter, 37 mg of 12,14-dichlorodehydroabietic acid/liter, 15.4 mg of biphenyl/liter, 12.0 mg of naphthalene/liter, or 17.8 mg of phenanthrene/liter.…”

“…DitA catalyzes the hydroxylation of the aromatic ring to form a cisdihydrodiol intermediate (15). Evidence for a convergent pathway came from studies of a ditA1 knockout mutant (14). 7-Oxo-DhA accumulated in cell suspensions of the ditA1 mutant strain incubated with AbA or DhA, while DhA and 7-oxo-DhA accumulated in cell suspensions incubated with PaA.…”

“…The dit gene cluster of BKME-9 encodes enzymes required for the catabolism of these compounds (14,15). Several genes of the dit cluster were previously sequenced and characterized, and a convergent pathway for abietane diterpenoid metabolism was proposed.…”

Dynamics of Pseudomonas aeruginosa genome evolution

“…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). Several orthologs are found in the B. xenovorans genome, although synteny with the PA2192 is less conserved.…”