Identification and characterization of genes encoding polycyclic aromatic hydrocarbon dioxygenase and polycyclic aromatic hydrocarbon dihydrodiol dehydrogenase in Pseudomonas putida OUS82

Abstract

Naphthalene and phenanthrene are transformed by enzymes encoded by the pah gene cluster ofPseudomonas putida OUS82. The pahA and pahB genes, which encode the first and second enzymes, dioxygenase and cis-dihydrodiol dehydrogenase, respectively, were identified and sequenced. The DNA sequences showed that pahA and pahB were clustered and that pah/A consisted of four cistrons, pah/Aa paMAb, pahAc, and pahAd, which encode ferredoxin reductase, ferredoxin, and two subunits of the iron-sulfur protein, respectively.Pseudomonas putida OUS82 can assimilate naphthalene and phenanthrene as its sole carbon sources. The strain converts naphthalene and phenanthrene to salicylate and 1-hydroxy-2-naphthoate, respectively, by a shared catabolic pathway (the upper pathway; Fig. 1). Salicylate and 1-hydroxy-2-naphthoate are further degraded by other catabolic enzymes. The enzymes in the upper pathway have broad substrate specificities, and various polycyclic aromatic hydrocarbons other than naphthalene and phenanthrene are oxidized by a high-density suspension of OUS82 cells (9).Previously, we cloned the gene cluster encoding the enzymes of the upper pathway and named it pah (polycyclic aromatic hydrocarbon; 9). The pah region strongly hybridized to a corresponding region of plasmid NAH7 of P. putida G7, which degrades naphthalene (4). All recombinant plasmids carrying pahA have 6.5-and 3.0-kb Sall fragments. The two fragments were seen to be necessary for the dioxygenase phenotype (PahA). A restriction endonuclease map of a region in the fragments resembles that of the nahA region of NAH7 and pDTG1 in P. putida G7 and NCIB 9816-4, which degrade naphthalene (2, 4, 23). The pahA gene was expected to be in that region.Here, we describe the identification and characterization of the pahA and pahB genes, which encode dioxygenase PahA, which is the first enzyme of the pathway and converts polycyclic aromatic hydrocarbon (PAH) to the corresponding cis-dihydrodiol, and dehydrogenase PahB, the second enzyme of the pathway, which converts the product of PahA to the corresponding diol. P. putida OUS8211 (trp-82 Apah-821), a derivative of strain OUS82 that is defective in naphthalene and phenanthrene utilization, and plasmid pDIl, which carries the pahAB gene cluster, were described previously (9). Plasmid NAH7 was described elsewhere (4, 5). Escherichia coli JM109 and plasmid pUC119 were described by Yanisch-Perron et al. (21)

Polychlorinated Biphenyl/Biphenyl Degrading Gene Clusters inRhodococcussp. K37, HA99, and TA431 Are Different from Well-KnownbphGene Clusters of Rhodococci

“…[21][22][23] Its phd gene cluster is localized on the chromosome, and the order of the phd genes is quite different from that of other reported analogous gene sets for polycyclic aromatic hydrocarbon degradation. [24][25][26] The phenanthrene dioxygenase (phdABCD) genes of Nocardioides sp. KP7 induced in E. coli do not transform monocyclic aromatic hydrocarbons including biphenyl, but they do convert tricyclic fused aromatic hydrocarbons such as phenanthrene, anthracene, and fluorene.…”

Differential detection of key enzymes of polyaromatic-hydrocarbon-degrading bacteria using PCR and gene probes

“…The following reference strains were used in this study: P. putida OUS82 (Kiyohara et al, 1994) and P. putida NCIB 9816 (Simon et al, 1993) ; both are reported to possess an initial dioxygenase. P. putida F1 is reported to possess a toluene dioxygenase but not PAH dioxygenase (Zylstra & Gibson, 1989).…”

“…When comparing the DNA sequences of Pseudomonas dioxygenase large iron-sulfur subunits, the similarity of calculated sequence pair distances of P. putida NCIB 9816 (GenBank accession no. M23914), P. putida G7 (M83949), P. putida OUS82 (D16629) and P. aeruginosa PaKl (D84146) PAH dioxygenases with toluene dioxygenase of P. putida F1 (J04996), benzene dioxygenase of P. putida ML2 (L04642) and biphenyl dioxygenase of P. putida LB400 (M84348) is between 45% and 49% (Simon el al., 1993;Kiyohara et al, 1994).…”

Recent Advances in Petroleum Microbiology

“…High levels (Ϸ90%) of homology and a conserved gene arrangement are observed in the nah, ndo, pah, and dox sequences (63,64,147,333,355,601). In fact, it has been proposed that the dox plasmid, which encodes a dibenzothiophene (DBT) metabolic pathway analogous to the naphthalene catabolic pathway, may in fact be a naphthalene catabolic plasmid (163).…”