The reactions involved in the bacterial metabolism of naphthalene to salicylate have been reinvestigated by using recombinant bacteria carrying genes cloned from plasmid NAH7. When intact cells of Pseudomonas aeruginosa PAO1 carrying DNA fragments encoding the first three enzymes of the pathway were incubated with naphthalene, they formed products of the dioxygenase-catalyzed ring cleavage of 1,2-dihydroxynaphthalene. These products were separated by chromatography on Sephadex G-25 and were identified by 'H and 13C nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry as 2-hydroxychromene-2-carboxylate (HCCA) and trans-o-hydroxybenzylidenepyruvate (tHBPA Naphthalene is the simplest fused polycyclic aromatic hydrocarbon. Information obtained from studies of bacterial degradation of naphthalene has been valuable for understanding and predicting the pathways used in the metabolism of the structurally more complex polycyclic aromatic hydrocarbons and related heterocyclic aromatic compounds. However, much about the bacterial metabolism of naphthalene has remained unclear, in particular the steps in the pathway by which 1,2-dihydroxynaphthalene (Fig. 1, compound III) is metabolized to salicylate.In the currently accepted naphthalene metabolic pathway ( Fig. 1) (3, 42, 53), 1,2-dihydroxynaphthalene is cleaved by a dioxygenase (Fig. 1, enzyme C) to an unstable ring cleavage product (Fig. 1, compound V), which spontaneously recyclizes to 2-hydroxychromene-2-carboxylate (HCCA) (compound VII). This compound is subsequently converted by means of an isomerase (enzyme D) to cis-o-hydroxybenzylidenepyruvate (cHBPA) (compound VII), which is cleaved by an aldolase (enzyme E), yielding salicylaldehyde and pyruvate. An NAD+-requiring aldehyde dehydrogenase then transforms salicylaldehyde to salicylate. This pathway for the metabolism of 1,2-dihydroxynaphthalene is based primarily on the work of Barnsley (3) and differs from the pathway proposed earlier by Davies and Evans (7), in which the unstable ring cleavage product (compound V) is rearomatized to give cHBPA (compound VIII), which is then metabolized in two sequential steps (hydration followed by aldol cleavage to salicylaldehyde Davies and Evans (7) and Barnsley (3) reached these different conclusions despite taking similar experimental approaches. These workers incubated cell extracts with low concentrations of 1,2-dihydroxynaphthalene for a short time and then rapidly took steps to purify and stabilize the product. Davies and Evans made the perchlorate derivative, which they crystallized, while Barnsley separated the product from cell extracts by chromatography on Sephadex G-25 followed by freeze-drying. Both Davies and Evans (7) and Barnsley (3) obtained HCCA. Barmsley identified this compound as the initial product of ring cleavage, but Davies and Evans recognized it as the hemiacetal (actually the hemiketal) of cHBPA and considered it to be an artifact of their isolation procedure. Both Davies and Evans (7) and Barnsley (3) demonstrated that HC...

Several 2-substituted benzoates (including 2-trifluoromethyl-, 2-chloro-, 2-bromo-, 2-iodo-, 2-nitro-, 2-methoxy-, and 2-acetyl-benzoates) were converted by phthalate-grown Arthrobacter keyseri (formerly Micrococcus sp.) 12B to the corresponding 2-substituted 3,4-dihydroxybenzoates (protocatechuates). Because these products lack a carboxyl group at the 2 position, they were not substrates for the next enzyme of the phthalate catabolic pathway, 3,4-dihydroxyphthalate 2-decarboxylase, and accumulated. When these incubations were carried out in iron-containing minimal medium, the products formed colored chelates. This chromogenic response was subsequently used to identify recombinant Escherichia coli strains carrying genes encoding the responsible enzymes, phthalate 3,4-dioxygenase and 3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase, from the 130-kbp plasmid pRE1 of strain 12B. Beginning with the initially cloned 8.14-kbp PstI fragment of pRE824 as a probe to identify recombinant plasmids carrying overlapping fragments, a DNA segment of 33.5 kbp was cloned from pRE1 on several plasmids and mapped using restriction endonucleases. From these plasmids, the sequence of 26,274 contiguous bp was determined. Sequenced DNA included several genetic units: tnpR, pcm operon, ptr genes, pehA, norA fragment, and pht operon, encoding a transposon resolvase, catabolism of protocatechuate (3,4-dihydroxybenzoate), a putative ATP-binding cassette transporter, a possible phthalate ester hydrolase, a fragment of a norfloxacin resistance-like transporter, and the conversion of phthalate to protocatechuate, respectively. Activities of the eight enzymes involved in the catabolism of phthalate through protocatechuate to pyruvate and oxaloacetate were demonstrated in cells or cell extracts of recombinant E. coli strains.

Pseudomonas putida F1 utilizes p-cymene (p-isopropyltoluene) by an 11-step pathway through p-cumate (p-isopropylbenzoate) to isobutyrate, pyruvate, and acetyl coenzyme A. The cym operon, encoding the conversion of p-cymene to p-cumate, is located just upstream of the cmt operon, which encodes the further catabolism of p-cumate and is located, in turn, upstream of the tod (toluene catabolism) operon in P. putida F1. The sequences of an 11,236-bp DNA segment carrying the cym operon and a 915-bp DNA segment completing the sequence of the 2,673-bp DNA segment separating the cmt and tod operons have been determined and are discussed here. The cym operon contains six genes in the order cymBCAaAbDE. The gene products have been identified both by functional assays and by comparing deduced amino acid sequences to published sequences. Thus, cymAa and cymAb encode the two components of p-cymene monooxygenase, a hydroxylase and a reductase, respectively; cymB encodes p-cumic alcohol dehydrogenase; cymC encodes p-cumic aldehyde dehydrogenase; cymD encodes a putative outer membrane protein related to gene products of other aromatic hydrocarbon catabolic operons, but having an unknown function in p-cymene catabolism; and cymE encodes an acetyl coenzyme A synthetase whose role in this pathway is also unknown. Upstream of the cym operon is a regulatory gene, cymR. By using recombinant bacteria carrying either the operator-promoter region of the cym operon or the cmt operon upstream of genes encoding readily assayed enzymes, in the presence or absence of cymR, it was demonstrated that cymR encodes a repressor which controls expression of both the cym and cmt operons and is inducible by p-cumate but not p-cymene. Short (less than 350 bp) homologous DNA segments that are located upstream of cymR and between the cmt and tod operons may have been involved in recombination events that led to the current arrangement of cym, cmt, and tod genes in P. putida F1.

Pseudomonas putida F1 utilizes p-cumate (p-isopropylbenzoate) as a growth substrate by means of an eightstep catabolic pathway. A 35.75-kb DNA segment, within which the cmt operon encoding the catabolism of p-cumate is located, was cloned as four separate overlapping restriction fragments and mapped with restriction endonucleases. By examining enzyme activities in recombinant bacteria carrying these fragments and subcloned fragments, genes encoding most of the enzymes of the p-cumate pathway were located. Subsequent sequence analysis of 11,260 bp gave precise locations of the 12 genes of the cmt operon. The first three genes, cmtAaAbAc, and the sixth gene, cmtAd, encode the components of p-cumate 2,3-dioxygenase (ferredoxin reductase, large subunit of the terminal dioxygenase, small subunit of the terminal dioxygenase, and ferredoxin, respectively); these genes are separated by cmtC, which encodes 2,3-dihydroxy-p-cumate 3,4-dioxygenase, and cmtB, coding for 2,3-dihydroxy-2,3-dihydro-p-cumate dehydrogenase. The ring cleavage product, 2-hydroxy-3-carboxy-6-oxo-7-methylocta-2,4-dienoate, is acted on by a decarboxylase encoded by the seventh gene, cmtD, which is followed by a large open reading frame, cmtI, of unknown function. The next four genes, cmtEFHG, encode 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate hydrolase, 2-hydroxypenta-2,4-dienoate hydratase, 4-hydroxy-2-oxovalerate aldolase, and acetaldehyde dehydrogenase, respectively, which transform the decarboxylation product to amphibolic intermediates. The deduced amino acid sequences of all the cmt gene products except CmtD and CmtI have a recognizable but low level of identity with amino acid sequences of enzymes catalyzing analogous reactions in other catabolic pathways. This identity is highest for the last two enzymes of the pathway (4-hydroxy-2-oxovalerate aldolase and acetaldehyde dehydrogenase [acylating]), which have identities of 66 to 77% with the corresponding enzymes from other aromatic meta-cleavage pathways. Recombinant bacteria carrying certain restriction fragments bordering the cmt operon were found to transform indole to indigo. This reaction, known to be catalyzed by toluene 2,3-dioxygenase, led to the discovery that the tod operon, encoding the catabolism of toluene, is located 2.8 kb downstream from and in the same orientation as the cmt operon in P. putida F1.

A Pseudomonas putida strain designated RE204, able to utilize isopropylbenzene as the sole carbon and energy source, was isolated. TnS transposon mutagenesis by means of the suicide transposon donor plasmid pLG221 yielded mutant derivatives defective in isopropylbenzene metabolism. These were characterized by the identification of the products which they accumulated when grown in the presence of isopropylbenzene and by the assay of enzyme activities in cell extracts. Based on the results obtained, the following metabolic pathway is proposed: isopropylbenzene --2,3-dihydro-2,3-dihydroxyisopropylbenzene 3-isopropylcatechol -) 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate -> isobutyrate + 2-oxopent-4-enoate amphibolic intermediates.Plasmid DNA was isolated from strain RE204 and mutant derivatives and characterized by restriction enzyme cleavage analysis. Isopropylbenzene-negative isolates carried a TnS insert within a 15-kilobase region of a 105-kilobase plasmid designated pRE4. DNA fragments of pRE4 carrying genes encoding isopropylbenzene catabolic enzymes were cloned in Escherichia coli with various plasmid vectors; clones were identified by (i) selection for TnS-encoded kanamycin resistance in the case of TnS mutant plasmids, (ii) screening for isopropylbenzene dioxygenase-catalyzed oxidation of indole to indigo, and (iii) use of a TnS-carrying restriction fragment, derived from a pRE4::TnS mutant plasmid, as a probe for clones carrying wild-type restriction fragments. These clones were subsequently used to generate a transposon insertion and restriction enzyme cleavage map of the isopropylbenzene metabolic region of pRE4.The monoalkylbenzenes isopropylbenzene (cumene), ethylbenzene, and toluene are important industrially as synthetic intermediates and solvents (32). As a consequence of their widespread use and subsequent introduction into the environment, the latter two compounds were included on the U.S. Environmental Protection Agency's list of priority pollutants (39).The bacterial metabolism of toluene has been shown to occur by two different pathways. One, exemplified by that encoded by the well-studied TOL plasmid (54), is initiated by a multistep oxidation of the methyl group to give benzoate (40); the other, which is apparently chromosome encoded (26), is initiated by a dioxygenase-catalyzed oxidation of the aromatic ring to give 2,3-dihydro-2,3-dihydroxytoluene which is subsequently dehydrogenated to give 3-methylcatechol (28,29). Similarly, ethylbenzene has been shown to be primarily metabolized through 2,3-dihydro-2,3-dihydroxyethylbenzene to 3-ethylcatechol (27). A general pathway for the complete metabolism of these and other monoalkylbenzenes in which the side chain is not oxidized before ring cleavage has been described (Fig. 1), and evidence for this is summarized in two recent reviews (31, 47). Some evidence has been presented for the metabolism of isopropylbenzene through the ring-cleavage product IV depicted in Fig. 1 (29) and for the metabolism of this compound to isobutyrate, pyruvate, and ace...