A rapid procedure was devised for detecting on solid media bacteria able to degrade water-insoluble, solid hydrocarbons such as the polycyclic aromatic hydrocarbons phenanthrene, anthracene, and biphenyl. After Alcaligenesfaecalis AFK2 was inoculated on a plate containing mineral salts agar, an ethereal solution of phenanthrene (about 10%, wt/vol) was sprayed on the surface of the plate, and the plate was incubated at 30°C for 2 to 3 days. Colonies showing degradation were surrounded with clear zones on the opaque plate. A similar clear zone also was formed around colonies which had been grown on a succinate-mineral salts agar or nutrient agar, followed by spraying of the ethereal solution of phenanthrene and further incubating for 1 day. Other phenanthrene-assimilating bacteria, including Beijerinckia Bwt and Pseudomonas SPM64, also formed clear zones on phenanthrene-covered agar plates. This method was applicable to detection of bacteria able to assimilate anthracene, naphthalene, and biphenyl.
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)
Thirteen strains of bacteria able to grow on phenanthrene were isolated from soil; they included fluorescent and non-fluorescent pseudomonads, vibrios and unidentified bacteria. Two of the pseudomonads, like Aeromonas sp. ~4 5~1 , also grew on naphthalene. In all strains, growth on phenanthrene induced the enzyme responsible for the conversion of 1-hydroxy-2-naphthoate to 2-carboxybenzaldehyde, NAD-dependent 2-carboxybenzaldehyde dehydrogenase and protocatechuate oxygenase, but not salicylate hydroxylase, catechol oxygenase or NAD(P)H-dependent 1-hydroxy-2-naphthoate hydroxylase. Growth on naphthalene induced salicylate hydroxylase and catechol oxygenase. It is suggested that the catabolism of phenanthrene occurs via protocatechuate in all these bacteria, and that the pathways for degradation of phenanthrene and naphthalene are separate. I N T R O D U C T I O NWith the development of the petroleum industry, there has been an increase in the amount of polyaromatic hydrocarbons released into the environment. As part of a study of the mechanism of degradation of hydrocarbons by micro-organisms, we have examined the catabolism of phenanthrene by bacteria. The enzymic steps involved in the conversion of phenanthrene to 1 -hydroxy-2-naphthoate by a soil pseudomonad were elucidated by Evans, Fernley & Griffiths (1965). The naphthoate was then presumed to be oxidatively decarboxylated to 1,2-dihydroxynaphthalene which would be further degraded through the naphthalene-catabolizing pathway described by Davies & Evans (1964). However, Aeromonas sp. ~4 5~1 , which was isolated from soil by Kiyohara, Nagao & Nomi (1976), did not convert 1-hydroxy-2-naphthoate to 1,2-dihydroxynaphthalene but instead metabolized it to 2-carboxybenzaldehyde which was then oxidized to o-phthalate, by an NAD-dependent dehydrogenase, and finally to protocatechuate (Fig. 1). The cleavage of the naphthoate (Kiyohara & Nagao, 1977) involves the fission of the bond between the carbon atoms bearing the hydroxyl and carboxyl groups by an intradiol type of dioxygenase which acts in a similar way to gentisate oxidase (EC 1 ,13.1.4) (Sugiyama et al., 1958).The work described in the present paper shows that the protocatechuate pathway is the only pathway for degradation of phenanthrene in 13 independent isolates of soil bacteria. The relationship between the pathways for degradation of phenanthrene and naphthalene is also described. METHODSBacteriaZ strains and media. Bacteria able to grow on phenanthrene were isolated from soil by enrichment culture in phenanthrene/salts medium, followed by plating on Pseudomonas F agar (Difco). The salts medium contained (yo, w/v, in tap water): (NH&HP04, 0-5; KH2P04, 0.15; Na,HPO,. 12H20, 0.15; MgS04. 7H20, 0.02; NaCI, 0.05; the pH was 7-3 without adjustment. Phenanthrene (0.3 %, w/v) was added to the medium before autoclaving at 120 "C for 15 min. The phenanthrene was melted by the heat but was finely dispersed by vigorously shaking the flasks after cooling. Naphthalene, which was crystallized from
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