Utilization of 3-chloro-2-methylbenzoic acid by Pseudomonas cepacia MB2 through the meta fission pathway.

Pseudomonas cepacia MB2 grew on 3-chloro-2-methylbenzoate as a sole carbon source by metabolism through the meta fission pathway with the subsequent liberation of chloride. meta pyrocatechase activity in cell extracts was induced strongly by 3-chloro-2-methylbenzoate, but not by nongrowth analogs 4- or 5-chloro-2-methylbenzoate. Although rapid turnover of metabolites precluded direct identification, a mutant strain MB2-G5 lacking meta pyrocatechase activity produced 4-chloro-3-methylcatechol when incubated with 3-chloro-2-methylbenzoate. The catecholic product, confirmed by nuclear magnetic resonance and mass spectral analyses, produced a transient meta fission product (lambda max = 391 nm) from cell extracts of the wild-type MB2 strain. Further confirmation of meta pyrocatechase activity was noted by conversion of 4-chlorocatechol to 2-hydroxy-5-chloromuconic semialdehyde, which was not further metabolized. In contrast to 3-chlorocatechol, which was not metabolized and is known to generate suicidal products, 4-chlorocatechols do not generate acyl halides. Thus, further metabolism of the ring fission products is governed in strain MB2 by their suitability as substrates for the hydrolase.     

Degradation of 2-methylbenzoic acid by Pseudomonas cepacia MB2.

We report the isolation of Pseudomonas cepacia MB2, believed to be the first microorganism to utilize 2-methylbenzoic acid as the sole carbon source. Its growth range included all mono- and dimethylbenzoates (with the exception of 2,5- and 2,6-dimethylbenzoates) and 3-chloro-2-methylbenzoate (but not 4- or 5-chloro-2-methylbenzoate) but not chlorobenzoates lacking a methyl group. 2-Chlorobenzoate, 3-chlorobenzoate, and 2,3-, 2,4-, and 3,4-dichlorobenzoates inhibited growth of MB2 on 2-methylbenzoate as a result of cometabolism to the corresponding chlorinated catechols which blocked the key enzyme catechol 2,3-dioxygenase. A metapyrocatechase-negative mutant, MB2-G5, showed accumulation of dimethylcatechols from 2,3- and 3,4-dimethylbenzoates, and phenols were detected in resting-cell transformation extracts bearing the same substitution pattern as the original substrate, presumably following thermal degradation of the intermediate dihydrodiol. 2-Methylphenol was also found in extracts of the mutant cells with 2-methylbenzoate. These observations suggested a major route of methylbenzoate metabolism to be dioxygenation to a carboxy-hydrodiol which then forms a catechol derivative. In addition, the methyl group of 2-methylbenzoate was oxidized to isobenzofuranone (by cells of MB2-G5) and to phthalate (by cells of a separate mutant that could not utilize phthalate, MB2-D2). This pathway also generated a chlorinated isobenzofuranone from 3-chloro-2-methylbenzoate.     

Degradation of 2-methylbenzoic acid by Pseudomonas cepacia MB2.

We report the isolation of Pseudomonas cepacia MB2, believed to be the first microorganism to utilize 2-methylbenzoic acid as the sole carbon source. Its growth range included all mono- and dimethylbenzoates (with the exception of 2,5- and 2,6-dimethylbenzoates) and 3-chloro-2-methylbenzoate (but not 4- or 5-chloro-2-methylbenzoate) but not chlorobenzoates lacking a methyl group. 2-Chlorobenzoate, 3-chlorobenzoate, and 2,3-, 2,4-, and 3,4-dichlorobenzoates inhibited growth of MB2 on 2-methylbenzoate as a result of cometabolism to the corresponding chlorinated catechols which blocked the key enzyme catechol 2,3-dioxygenase. A metapyrocatechase-negative mutant, MB2-G5, showed accumulation of dimethylcatechols from 2,3- and 3,4-dimethylbenzoates, and phenols were detected in resting-cell transformation extracts bearing the same substitution pattern as the original substrate, presumably following thermal degradation of the intermediate dihydrodiol. 2-Methylphenol was also found in extracts of the mutant cells with 2-methylbenzoate. These observations suggested a major route of methylbenzoate metabolism to be dioxygenation to a carboxy-hydrodiol which then forms a catechol derivative. In addition, the methyl group of 2-methylbenzoate was oxidized to isobenzofuranone (by cells of MB2-G5) and to phthalate (by cells of a separate mutant that could not utilize phthalate, MB2-D2). This pathway also generated a chlorinated isobenzofuranone from 3-chloro-2-methylbenzoate.     

Formation of chlorocatechol meta cleavage products by a pseudomonad during metabolism of monochlorobiphenyls.

Pseudomonas cepacia P166 was able to metabolize all monochlorobiphenyls to the respective chlorobenzoates. Although they transiently accumulated, the chlorobenzoate degradation intermediates were further metabolized to chlorocatechols, which in turn were meta cleaved. 2- and 3-Chlorobiphenyl both produced 3-chlorocatechol, which was transformed to an acyl halide upon meta cleavage. 3-Chlorocatechol metabolism was toxic to the cells and impeded monochlorobiphenyl metabolism. In the case of 2-chlorobiphenyl, toxicity was manifested as a diminished growth rate, which nevertheless effected rapid substrate utilization. In the case of 3-chlorobiphenyl, which generates 3-chlorocatechol more rapidly than does 2-chlorobiphenyl, toxicity was manifested as a decrease in viable cells during substrate utilization. 4-Chlorobenzoate was transformed to 4-chlorocatechol, which was metabolized by a meta cleavage pathway leading to dehalogenation. Chloride release from 4-chlorocatechol metabolism, however, was slow and did not coincide with rapid 4-chlorocatechol turnover. Growth experiments with strain P166 on monochlorobiphenyls illustrated the difficulties of working with hydrophobic substrates that generate toxic intermediates. Turbidity could not be used to measure the growth of bacteria utilizing monochlorobiphenyls because high turbidities were routinely measured from cultures with very low viable-cell counts.     

Degradation of 2-chlorobenzoate by in vivo constructed hybrid pseudomonads

Abstract 5-Chlorosalicylate degrading bacteria were obtained from the mating between Pseudomonas sp. strain WR401 and Pseudomonas sp. strain B13. Further selection of the hybrid organisms for growth on 2-chlorobenzoate allowed the isolation of strains such as JH230. During growth on 2-chlorobenzoate stoichiometric amounts of chloride were released. Steps in the pathway for 2-chlorobenzoate degradation were determined by simultaneous adaptation studies, assays of enzymes in cell extracts and cooxidation of the analogous substrate 2-methylbenzoate. Results indicate that 2-chlorobenzoate was degraded to 3-chlorocatechol. Ring cleavage of 3-chlorocatechol was by a catechol 1,2-dioxygenase to from 2-chloro- cis, cis - muconate. Further degradation runs via 4-carboxymethylenebut-2-en-4-olide.     

Metabolism of 2,4-dichlorophenoxyacetic acid, 4-chloro-2-methylphenoxyacetic acid and 2-methylphenoxyacetic acid by Alcaligenes eutrophus JMP 134

Of eleven substituted phenoxyacetic acids tested, only three (2,4-dichloro-, 4-chloro-2-methyl- and 2-methylphenoxyacetic acid) served as growth substrates for Alcaligenes eutrophus JMP 134. Whereas only one enzyme seems to be responsible for the initial cleavage of the ether bond, there was evidence for the presence of three different phenol hydroxylases in this strain. 3,5-Dichlorocatechol and 5-chloro-3-methylcatechol, metabolites of the degradation of 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid, respectively, were exclusively metabolized via the ortho-cleavage pathway. 2-Methylphenoxyacetic acid-grown cells showed simultaneous induction of meta- and ortho-cleavage enzymes. Two catechol 1,2-dioxygenases responsible for ortho-cleavage of the intermediate catechols were partially purified and characterized. One of these enzymes converted 3,5-dichlorocatechol considerably faster than catechol or 3-chlorocatechol. A new enzyme for the cycloisomerisation of muconates was found, which exhibited high activity against the ring-cleavage products of 3,5-dichlorocatechol and 4-chlorocatechol, but low activities against 2-chloromuconate and muconate.Non-standard abbreviations MCPA 4-chloro-2-methylphenoxyacetic acid - 2MPA 2-methylphenoxyacetic acid - PA phenoxyacetic acid     

Construction of a 3-chlorobiphenyl-utilizing recombinant from an intergeneric mating.

Recombinant Pseudomonas sp. strain CB15, which grows on 3-chlorobiphenyl (3CB), was constructed from Pseudomonas sp. strain HF1, which grows on 3-chlorobenzoate, and from Acinetobacter sp. strain P6, which grows on biphenyl, by using a continuous amalgamated culture apparatus. DNA from strains CB15 and HF1 hybridized very strongly to each other, while hybridization between both parental strains, HF1 and P6, was negligible. However, DNA from the recombinant CB15 hybridized moderately to strongly with three specific fragments of parental strain P6. Strains HF1 and P6 did not grow on 3CB, but recombinant strain CB15 mineralized this compound and released inorganic chloride. When growing on 3CB, strain CB15 accumulated brown products, one of which was identified as 3-chloro-5-(2'-hydroxy-3'-chlorophenyl)-1,2-benzoquinone by mass spectrometry. Emulsification and mechanical fragmentation greatly increased the rate of 3CB mineralization by strain CB15. At least three methods of inhibition from catecholic intermediates may account for slow growth on 3CB. The meta fission of 2,3-dihydroxybiphenyl (the nonchlorinated analog of the metabolic intermediate 3-chloro-2',3'-dihydroxybiphenyl) was affected by substrate inhibition (Vmax = 359 nmol.min-1.mg-1, Km = 114 microM, Kss [the inhibition constant] = 951 microM) and was also inhibited by 3-chlorocatechol. The ortho fission of 3-chlorocatechol, a degradation product, followed Michaelis-Menten kinetics (Vmax = 365 nmol.min-1.mg-1, Km = 1 microM), but the addition of 2,3-dihydroxybiphenyl inhibited the reaction (Ki = 0.87 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of 4-chlorophenol by Azotobacter sp. GP1: Structure of the meta cleavage product of 4-chlorocatechol

Abstract A mutant strain of Azobacter sp. GP1 converted 4-chlorphenol to 4-chlorocatechol under cometabolic conditions. Under the same conditions the wild-type strain accumulated yellow compound, which by chemical and spectroscopic methods was identified as 5-chloro-2-hydroxy-6-oxohexadienoic acid (5-chloro-2-hydroxy-muconic semialdehyde). The structure of this compound indicates a meta -proximal cleavage of 4-chlorocatechol.     

Construction of a 3-chlorobiphenyl-utilizing recombinant from an intergeneric mating.

Recombinant Pseudomonas sp. strain CB15, which grows on 3-chlorobiphenyl (3CB), was constructed from Pseudomonas sp. strain HF1, which grows on 3-chlorobenzoate, and from Acinetobacter sp. strain P6, which grows on biphenyl, by using a continuous amalgamated culture apparatus. DNA from strains CB15 and HF1 hybridized very strongly to each other, while hybridization between both parental strains, HF1 and P6, was negligible. However, DNA from the recombinant CB15 hybridized moderately to strongly with three specific fragments of parental strain P6. Strains HF1 and P6 did not grow on 3CB, but recombinant strain CB15 mineralized this compound and released inorganic chloride. When growing on 3CB, strain CB15 accumulated brown products, one of which was identified as 3-chloro-5-(2'-hydroxy-3'-chlorophenyl)-1,2-benzoquinone by mass spectrometry. Emulsification and mechanical fragmentation greatly increased the rate of 3CB mineralization by strain CB15. At least three methods of inhibition from catecholic intermediates may account for slow growth on 3CB. The meta fission of 2,3-dihydroxybiphenyl (the nonchlorinated analog of the metabolic intermediate 3-chloro-2',3'-dihydroxybiphenyl) was affected by substrate inhibition (Vmax = 359 nmol.min-1.mg-1, Km = 114 microM, Kss [the inhibition constant] = 951 microM) and was also inhibited by 3-chlorocatechol. The ortho fission of 3-chlorocatechol, a degradation product, followed Michaelis-Menten kinetics (Vmax = 365 nmol.min-1.mg-1, Km = 1 microM), but the addition of 2,3-dihydroxybiphenyl inhibited the reaction (Ki = 0.87 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

A meta cleavage pathway for 4-chlorobenzoate, an intermediate in the metabolism of 4-chlorobiphenyl by Pseudomonas cepacia P166.

Bacterial degradation of biphenyl and polychlorinated biphenyls proceeds by a well-studied pathway which produces benzoate and 2-hydroxypent-2,4-dienoate (or, in the case of polychlorinated biphenyls, the chlorinated derivatives of these compounds). Pseudomonas cepacia P166 utilizes 4-chlorobiphenyl for growth and produces 4-chlorobenzoate as a central intermediate. In this study we found that strain P166 further transforms 4-chlorobenzoate to 4-chlorocatechol, which is mineralized by a meta cleavage pathway. Key metabolites which we identified include the meta cleavage product (5-chloro-2-hydroxymuconic semialdehyde), 5-chloro-2-hydroxymuconate, 5-chloro-2-oxopent-4-enoate, 5-chloro-4-hydroxy-2-oxopentanoate, and chloroacetate. Chloroacetate accumulated transiently, and slow but stoichiometric dehalogenation was observed.     

Pathways for 3-chloro- and 4-chlorobenzoate degradationin Pseudomonas aeruginosa 3mT

A bacterial isolate, Pseudomonas aeruginosa 3mT, exhibited the ability to degrade high concentrations of 3-chlorobenzoate (3-CBA, 8 g l^-1) and 4-chlorobenzoate (4-CBA 12 g l^-1) (Ajithkumar 1998). In this study, by delineating the initial biochemical steps involved in the degradation of these compounds, we investigated how this strain can do so well. Resting cells, permeabilised cells as well as cell-free extracts failed to dechlorinate both 3-CBA and 4-CBA under anaerobic conditions, whereas the former two readily degraded both compounds under aerobic conditions. Accumulation of any intermediary metabolite was not observed during growth as well as reaction with resting cells under highly aerated conditions. However, on modification of reaction conditions, 3-chlorocatechol (3-CC) and 4-chlorocatechol (4-CC) accumulated in 3-CBA and 4-CBA flasks, respectively. Fairly high titres of pyrocatechase II (chlorocatechol 1,2-dioxygenase) activity were obtained in extracts of cells grown on 3-CBA and 4-CBA. Meta-pyrocatechase (catechol 2,3-dioxygenase) activity against4-CC and catechol, but not against 3-CC, was also detected in low titres. Accumulation of small amounts of 2-chloro-5-hydroxy muconic semialdehyde, the meta-cleavage product of 4-CC, was detected in the medium, when 4-CBA concentration was 4 mM or greater, indicating the presence of a minor meta-pathway in strain 3mT. However, 3-CBA exclusively, and more than 99% of 4-CBA were degraded through the formation of the respective chlorocatechol, via a modified ortho-pathway. This defies the traditional view that the microbes that follow chlorocatechol pathways are not very good degraders of chlorobenzoates. 4-Hydroxybenzoatewas readily (and 3-hydroxybenzoate to a lesser extent) degraded by the strain, through the formation of protocatechuate and gentisate, respectively, as intermediary dihydroxy metabolites.     

Chemical structure and biodegradability of halogenated aromatic compounds. Two catechol 1,2-dioxygenases from a 3-chlorobenzoate-grown pseudomonad.

1. Two catechol 1,2-dioxygenases, pyrocatechase I and pyrocatechase II, were found in 3-chlorobenzoate-grown cells of Pseudomonas sp. B 13. The latter enzyme showed high relative activities with 3- and 4-chlorocatechol compared with catechol. 2. In benzoate-grown cells, only pyrocatechase I was induced. It was purified 29-fold with a final specific activity of 20 mumol of catechol oxygenated/min per mg of protein and an overall yield of 22%. Because of the instability of pyrocatechase II on chromatography and dialysis, no increase of specific activity was obtained during the purification experiments. 3. Molecular weights of pyrocatechase I and pyrocatechase II were 82000 and 67000 respectively. 4. For both pyrocatechases the pH optimum was found to be at 8.0.5. Inhibitions of the two pyrocatechases by Cu2+ and Hg2+ ions and p-chloromercuribenzoate were different. The effect on pyrocatechase I after incubation for 20 h with the heavy metals was decreased by addition of 1 mM-2-mercaptoethanol to the reaction mixture. The inhibition of pyrocatechase II was even enhanced under these conditions. 6. Extradiol cleavage of 3-methylcatechol in addition to intradiol fission at a ratio of 1:14 was observed only with pyrocatechase I.     

Transformation and mineralization of halophenols by Penicillium simplicissimum SK9117

The metabolism of monohalophenols by Penicillium simplicissimum SK9117, isolated from a sewage plant was investigated. In submerged cultures, 3-, 4-chlorophenol, and 4-bromophenol were metabolized in the presence of phenol. 3-Chlorophenol was transformed to chlorohydroquinone, 4-chlorocatechol, 4-chloro-1,2,3-trihydroxybenzene, and 5-chloro-1,2,3-trihydroxybenzene. With 4-chlorophenol only 4-chlorocatechol was observed as transient product. A release of chloride ions was not observed. Whereas monobromo-, and monochlorophenols could not support growth as sole carbon and energy source, growth and release of fluoride ions were observed with monofluorophenols as substrates. In presence of phenol, the degradation of all monofluorophenols was enhanced. Substrate and cosubstrate disappeared simultaneously. 3-Fluorophenol and 4-fluorophenol were completely mineralized as shown by the equimolar release of fluoride ions.     

Transformation and mineralization of halophenols by Penicillium simplicissimum SK9117

The metabolism of monohalophenols by Penicillium simplicissimum SK9117, isolated from a sewage plant was investigated. In submerged cultures, 3-, 4-chlorophenol, and 4-bromophenol were metabolized in the presence of phenol. 3-Chlorophenol was transformed to chlorohydroquinone, 4-chlorocatechol, 4-chloro-1,2,3-trihydroxybenzene, and 5-chloro-1,2,3-trihydroxybenzene. With 4-chlorophenol only 4-chlorocatechol was observed as transient product. A release of chloride ions was not observed. Whereas monobromo-, and monochlorophenols could not support growth as sole carbon and energy source, growth and release of fluoride ions were observed with monofluorophenols as substrates. In presence of phenol, the degradation of all monofluorophenols was enhanced. Substrate and cosubstrate disappeared simultaneously. 3-Fluorophenol and 4-fluorophenol were completely mineralized as shown by the equimolar release of fluoride ions.Parts of the results have been presented at the annual meeting of the VAAM in Stuttgart, Germany, March 1995.     

Degradation of 4-Chlorophenol via the meta Cleavage Pathway by Comamonas testosteroni JH5

Comamonas testosteroni JH5 used 4-chlorophenol (4-CP) as its sole source of energy and carbon up to a concentration of 1.8 mM, accompanied by the stoichiometric release of chloride. The degradation of 4-CP mixed with the isomeric 2-CP by resting cells led to the accumulation of 3-chlorocatechol (3-CC), which inactivated the catechol 2,3-dioxygenase. As a result, further 4-CP breakdown was inhibited and 4-CC accumulated as a metabolite. In the crude extract of 4-CP-grown cells, catechol 1,2-dioxygenase and muconate cycloisomerase activities were not detected, whereas the activities of catechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, 2-hydroxymuconic semialdehyde hydrolase, and 2-oxopent-4-enoate hydratase were detected. These enzymes of the meta cleavage pathway showed activity with 4-CC and with 5-chloro-2-hydroxymuconic semialdehyde. The activities of the dioxygenase and semialdehyde dehydrogenase were constitutive. Two key metabolites of the meta cleavage pathway, the meta cleavage product (5-chloro-2-hydroxymuconic semialdehyde) and 5-chloro-2-hydroxymuconic acid, were detected. Thus, our previous postulation that C. testosteroni JH5 uses the meta cleavage pathway for the complete mineralization of 4-CP was confirmed.     

Metabolism of Phenol and Cresols by Mutants of Pseudomonas putida

Mutant strains of Pseudomonas putida strain U have been obtained which are deficient in enzymes of the degradative pathways of phenol and cresols. Mutant strains deficient in catechol 2, 3-oxygenase accumulated the appropriate catechol derivative from cresols. A mutant strain which would not grow on either phenol or a cresol was shown to be deficient in both 2-hydroxymuconic semialdehyde hydrolase and a nicotinamide adenine dinucleotide, oxidized form, (NAD(+))-dependent aldehyde dehydrogenase. When this strain was grown in the presence of phenol or a cresol, the appropriate product of meta fission of these compounds accumulated in the growth medium. A partial revertant of this mutant strain, which was able to grow on ortho- and meta-cresol but not para-cresol, was shown to have regained only the hydrolase activity. This strain was used to show that the products of meta ring fission of the cresols and phenol are metabolized as follows: (i) ortho- and meta-cresol exclusively by a hydrolase; (ii) para-cresol exclusively by a NAD(+)-dependent aldehyde dehydrogenase; (iii) phenol by both a NAD(+)-dependent dehydrogenase and a hydrolase in the approximate ratio of 5 to 1. This conclusion is supported by the substrate specificity and enzymatic activity of the hydrolase and NAD(+)-dependent aldehyde dehydrogenase enzymes of the wild-type strain. The results are discussed in terms of the physiological significance of the pathway. Properties of some of the mutant strains isolated are discussed.     

Bacterial metabolism of hydroxylated biphenyls

Isolates able to grow on 3- or 4-hydroxybiphenyl (HB) as the sole carbon source were obtained by enrichment culture. The 3-HB degrader Pseudomonas sp. strain FH12 used an NADPH-dependent monooxygenase restricted to 3- and 3,3'-HBs to introduce an ortho-hydroxyl. The 4-HB degrader Pseudomonas sp. strain FH23 used either a mono- or dioxygenase to generate a 2,3-diphenolic substitution pattern which allowed meta-fission of the aromatic ring. By using 3-chlorocatechol to inhibit catechol dioxygenase activity, it was found that 2- and 3-HBs were converted by FH23 to 2,3-HB, whereas biphenyl and 4-HB were attacked by dioxygenation. 4-HB was metabolized to 2,3,4'-trihydroxybiphenyl. Neither organism attacked chlorinated HBs. The degradation of 3- and 4-HBs by these strains is therefore analogous to the metabolism of biphenyl, 2-HB, and naphthalene in the requirement for 2,3-catechol formation.     

微生物降解对氯苯胺的一条新代谢途径

迄今为止的研究报道表明,对氯苯胺的生物降解只能以邻位途径或修饰邻位途径进行。采用HPLC、液相色谱质谱联用技术(LC/MS)对Diaphorobacter PCA039菌株降解对氯苯胺的中间代谢产物进行了分析和鉴定,结果表明,对氯苯胺经PCA039菌株的降解形成了氯代邻苯二酚,5-氯-4草酰巴豆酸,5-氯-2-氧戊烯酸,5-氯-2-氧-4-羟戊酸,氯代乙酸等中间代谢产物,这些都是典型的间位代谢途径(meta-pathway)的中间物质,说明Diaphorobacter PCA039菌株以间位裂解途径对对氯苯胺进行降解。这对于对氯代胺的生物降解代谢研究、代谢机理及其遗传表达调控研究具有意义。     

Bacterial metabolism of hydroxylated biphenyls.

Isolates able to grow on 3- or 4-hydroxybiphenyl (HB) as the sole carbon source were obtained by enrichment culture. The 3-HB degrader Pseudomonas sp. strain FH12 used an NADPH-dependent monooxygenase restricted to 3- and 3,3'-HBs to introduce an ortho-hydroxyl. The 4-HB degrader Pseudomonas sp. strain FH23 used either a mono- or dioxygenase to generate a 2,3-diphenolic substitution pattern which allowed meta-fission of the aromatic ring. By using 3-chlorocatechol to inhibit catechol dioxygenase activity, it was found that 2- and 3-HBs were converted by FH23 to 2,3-HB, whereas biphenyl and 4-HB were attacked by dioxygenation. 4-HB was metabolized to 2,3,4'-trihydroxybiphenyl. Neither organism attacked chlorinated HBs. The degradation of 3- and 4-HBs by these strains is therefore analogous to the metabolism of biphenyl, 2-HB, and naphthalene in the requirement for 2,3-catechol formation.     

Three different 2,3-dihydroxybiphenyl-1,2-dioxygenase genes in the gram-positive polychlorobiphenyl-degrading bacterium Rhodococcus globerulus P6.

Rhodococcus globerulus P6 (previously designated Acinetobacter sp. strain P6, Arthrobacter sp. strain M5, and Corynebacterium sp. strain MB1) is able to degrade a wide range of polychlorinated biphenyl (PCB) congeners. The genetic and biochemical analyses of the PCB catabolic pathway reported here have revealed the existence of a PCB gene cluster--bphBC1D--and two further bphC genes--bphC2 and bphC3--that encode three narrow-substrate-specificity enzymes (2,3-dihydroxybiphenyl dioxygenases) that meta cleave the first aromatic ring. None of the bphC genes show by hybridization homology to each other or to bphC genes in other bacteria, and the three bphC gene products have different kinetic parameters and sensitivities to inactivation by 3-chlorocatechol. This suggests that there exists a wide diversity in PCB meta cleavage enzymes.