A 25-kb DNA SalI fragment cloned from the chromosomal DNA of Pseudomonas putida OUS82, which utilizes phenanthrene (Phn+) and naphthalene (Nah+), carried all of the genes necessary for upper naphthalene catabolism. Cosmid recombinant pIP7 complemented both the Nah- and Phn- defects of OUS8211 (Trp-Nah-Phn-Sal+[salicylate utilizing]Hna+[1-hydroxy-2-naphthoate utilizing]) and only the Phn- defect of OUS8212 (Trp-Nah-Phn-Sal-Hna+). The results indicate that strain OUS82 uses different pathways after o-hydroxycarboxylic aromatics in the catabolism of naphthalene and phenanthrene.

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)