Identification and Characterization of a Pseudomonas Strain Capable of Metabolizing Phenoxybenzoates

3-Phenoxybenzoate is a transient metabolite from the breakdown of a number of pyrethroid insecticides in soil. In this study, we identified and characterized a bacterium which could grow on 3-phenoxybenzoate, converting it to phenol. On the basis of morphological and biochemical features, the 3-phenoxybenzoatedegrading isolate was determined to be a Pseudomonas species, probably a strain of Pseudomonas delafieldii, now designated Pseudomonas strain ET1. Pseudomonas strain ET1 grew on 3-phenoxybenzoate with a generation time of 3 h and a specific rate of metabolism of (2.6 ± 0.9) × 10^-13 g of 3-phenoxybenzoate consumed cell^-1 h^-1. The K_m for 3-phenoxybenzoate metabolism was 1.4 ± 0.8 μM. The metabolism of 3-phenoxybenzoate was constitutive and not subject to catabolite repression. The metabolism of a variety of substituted diaryl ether compounds was examined. 3- and 4-Phenoxybenzoates were metabolized, but 2-phenoxybenzoate was not. Phenoxy-substituted benzyl aldehyde was metabolized, but phenoxy-substituted benzyl alcohol, benzene, phenol, and aniline were not. Derivatives of 3-phenoxybenzoate substituted in the 4′ position with hydroxyl, methyl, or chlorine were metabolized, yielding the corresponding 4-substituted phenol. 3-(2-Hydroxyphenoxy)benzoate was not metabolized, but 3-phenoxy-4-fluorobenzoate was. These results indicate that the metabolism of the tested diaryl ether compounds was restricted to 4-phenoxybenzoate, 3-phenoxybenzyl aldehyde, and 3-phenoxybenzoate derivatives without a substitution in the 2′ position.     

Degradation of cocaine by a mixed culture of Pseudomonas fluorescens MBER and Comamonas acidovorans MBLF.

A mixed culture that could utilize cocaine as the sole source of carbon and energy for growth was isolated by selective enrichment. The individual microorganisms within this mixed culture were identified as Pseudomonas fluorescens (termed MBER) and Comamonas acidovorans (termed MBLF). Each microorganism was shown to be unable to grow to any appreciable extent on 10 mM cocaine in the absence of the other. C. acidovorans MBLF was found to possess an inducible cocaine esterase which catalyzed the hydrolysis of cocaine to ecgonine methyl ester and benzoate. C. acidovorans was capable of growth on benzoate at concentrations below 5 mM but was unable to metabolize ecgonine methyl ester. P. fluorescens MBER was capable of growth on either benzoate as the sole source of carbon or ecgonine methyl ester as the sole source of carbon and nitrogen. P. fluorescens MBER was found to initiate the degradation of ecgonine methyl ester via ecgonine, pseudoecgonine, and pseudoecgonyl-coenzyme A. Subcellular studies resulted in the identification of an ecgonine methyl esterase, an ecgonine epimerase, and a pseudoecgonyl-coenzyme A synthetase which were induced by growth on ecgonine methyl ester or ecgonine. Further metabolism of the ecgonine moiety is postulated to involve nitrogen debridging, with the production of carbonyl-containing intermediates.     

benK encodes a hydrophobic permease-like protein involved in benzoate degradation by Acinetobacter sp. strain ADP1.

The chromosomal benK gene was identified within a supraoperonic gene cluster involved in benzoate degradation by Acinetobacter sp. strain ADP1, and benK was expressed in response to a benzoate metabolite, cis,cis-muconate. The disruption of benK reduced benzoate uptake and impaired the use of benzoate or benzaldehyde as the carbon source. BenK was homologous to several aromatic compound transporters.     

A Mycobacterium strain with extended capacities for degradation of gasoline hydrocarbons

A bacterial strain (strain IFP 2173) was selected from a gasoline-polluted aquifer on the basis of its capacity to use 2,2, 4-trimethylpentane (isooctane) as a sole carbon and energy source. This isolate, the first isolate with this capacity to be characterized, was identified by 16S ribosomal DNA analysis, and 100% sequence identity with a reference strain of Mycobacterium austroafricanum was found. Mycobacterium sp. strain IFP 2173 used an unusually wide spectrum of hydrocarbons as growth substrates, including n-alkanes and multimethyl-substituted isoalkanes with chains ranging from 5 to 16 carbon atoms long, as well as substituted monoaromatic hydrocarbons. It also attacked ethers, such as methyl t-butyl ether. During growth on gasoline, it degraded 86% of the substrate. Our results indicated that strain IFP 2173 was capable of degrading 3-methyl groups, possibly by a carboxylation and deacetylation mechanism. Evidence that it attacked the quaternary carbon atom structure by an as-yet-undefined mechanism during growth on 2,2,4-trimethylpentane and 2,2-dimethylpentane was also obtained.     

Biotransformation of 2,7-dichloro- and 1,2,3,4-tetrachlorodibenzo-p-dioxin by Sphingomonas wittichii RW1

Aerobic biotransformation of the diaryl ethers 2,7-dichlorodibenzo-p-dioxin and 1,2,3,4-tetrachlorodibenzo-p-dioxin by the dibenzo-p-dioxin-utilizing strain Sphingomonas wittichii RW1, producing corresponding metabolites, was demonstrated for the first time. Our strain transformed 2,7-dichlorodibenzo-p-dioxin, yielding 4-chlorocatechol, and 1,2,3,4-tetrachlorodibenzo-p-dioxin, producing 3,4,5,6-tetrachlorocatechol and 2-methoxy-3,4,5,6-tetrachlorophenol; all of these compounds were unequivocally identified by mass spectrometry both before and after N,O-bis(trimethylsilyl)-trifluoroacetamide derivatization by comparison with authentic standards. Additional experiments showed that strain RW1 formed a second metabolite, 2-methoxy-3,4,5,6-tetrachlorophenol, from the original degradation product, 3,4,5,6-tetrachlorocatechol, by methylation of one of the two hydroxy substituents.     

A cold active (2R,3R)-(−)-di-O-benzoyl-tartrate hydrolyzing esterase from Rhodotorula mucilaginosa

In a screening procedure a pink-colored yeast was isolated from enrichment cultures with (2R,3R)-(−)-di-O-benzoyl-tartrate (benzoyl-tartrate) as the sole carbon source. The organism saar1 was identified by morphological, physiological, and 18S ribosomal DNA/internal transcribed spacer analysis as Rhodotorula mucilaginosa, a basidiomycetous yeast. During growth the yeast hydrolyzed the dibenzoyl ester stoichiometrically to the monoester using the separated benzoate as the growth substrate, before the monoester was further cleaved into benzoate and tartrate, which were both metabolized. The corresponding benzoyl esterase was purified from the culture supernatant and characterized as a monomeric glycosylated 86-kDa protein with an optimum pH of 7.5 and an optimum temperature of 45 °C. At 0 °C the esterase still exhibited 20% of the corresponding activity at 30 °C, which correlates it to psychrophilic enzymes. The esterase could hydrolyze short chain p-nitrophenyl-alkyl esters and several benzoyl esters like benzoyl-methyl ester, ethylene-glycol-dibenzoyl ester, phenyl-benzoyl ester, cocaine, and 1,5-anhydro-d-fructose-tribenzoyl ester. However feruloyl-ethyl ester was not hydrolyzed. The activity characteristics let the enzyme appear as a promising tool for synthesis of benzoylated compounds for pharmaceutical, cosmetic, or fine chemical applications, even at low temperatures.     

Expression of the 2,4-D degradative pathway of pJP4 in an alkaliphilic, moderately halophilic soda lake isolate, Halomonas sp. EF43

The broad host range plasmid pJP4, which carries genes for the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid, and 3-chlorobenzoic acid, was used in conjugation experiments with mixed cultures enriched from water and sediment samples from an alkaline pond in the area of Szegedi Fehértó, a soda lake in south Hungary. pJP4-encoded mercury resistance was used as a selection marker. One of the transconjugants, the alkaliphilic, moderately halophilic strain EF43, stably maintained the plasmid and was able to degrade 2,4-D and 3-chlorobenzoate under alkaline conditions in the presence of an additional carbon source such as pyruvate, benzoate, or alpha-ketoglutarate, indicating that the degradative genes of pJP4 were expressed in this strain. However, it was unable to grow on these chloroaromatic substrates when the substrate was the sole source of carbon and energy. Chemostat cultivation experiments revealed that the 2,4-D degradation rate during growth on benzoate or pyruvate was limited by the low activity of chlorocatechol-degrading enzymes, particularly chloromuconate cycloisomerase. Strain EF43 was identified as Halomonas sp. on the basis of 16S rRNA sequencing and additional taxonomic studies. 16S rRNA sequence analysis revealed that strain EF43 is closely related to typical soda lake isolates belonging to the genus Halomonas.     

Interspecies Acetate Transfer Influences the Extent of Anaerobic Benzoate Degradation by Syntrophic Consortia

Benzoate degradation by an anaerobic, syntrophic bacterium, strain SB, in coculture with Desulfovibrio sp. strain G-11 reached a threshold value which depended on the amount of acetate added and ranged from about 2.5 to 29.9 (mu)M. Increasing acetate concentrations also uncompetitively inhibited benzoate degradation. The apparent V(infmax) and apparent K(infm) for benzoate degradation decreased with increasing acetate concentration, but the benzoate degradation capacities (V(infmax)/K(infm)) of cell suspensions remained comparable. The addition of an acetate-using bacterium to cocultures after the threshold was reached resulted in the degradation of benzoate to below the detection limit. Mathematical simulations showed that the benzoate threshold was not predicted by the inhibitory effect of acetate on benzoate degradation kinetics. With nitrate instead of sulfate as the terminal electron acceptor, no benzoate threshold was observed in the presence of 20 mM acetate even though the kinetics of benzoate degradation were slower with nitrate rather than sulfate as the electron acceptor. When strain SB was grown with Desulfovibrio sp. strain DG2 that had a fourfold-lower V(infmax) for hydrogen use than strain G-11, the V(infmax) for benzoate degradation was 37-fold lower than that of strain SB-G-11 cocultures. The Gibb's free energy for benzoate degradation was less negative in cell suspensions with a threshold than in suspensions without a threshold. These studies showed that the threshold was not a function of the inhibition of benzoate degradation by acetate or the toxicity of the undissociated form of acetate. Rather, a critical or minimal Gibb's free energy may exist where thermodynamic constraints preclude further benzoate degradation.     

Nitrobenzoates and aminobenzoates are chemoattractants for Pseudomonas strains

Three Pseudomonas strains were tested for the ability to sense and respond to nitrobenzoate and aminobenzoate isomers in chemotaxis assays. Pseudomonas putida PRS2000, a strain that grows on benzoate and 4-hydroxybenzoate by using the beta-ketoadipate pathway, has a well-characterized beta-ketoadipate-inducible chemotactic response to aromatic acids. PRS2000 was chemotactic to 3- and 4-nitrobenzoate and all three isomers of aminobenzoate when grown under conditions that induce the benzoate chemotactic response. P. putida TW3 and Pseudomonas sp. strain 4NT grow on 4-nitrotoluene and 4-nitrobenzoate by using the ortho (beta-ketoadipate) and meta pathways, respectively, to complete the degradation of protocatechuate derived from 4-nitrotoluene and 4-nitrobenzoate. However, based on results of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase assays, both strains were found to use the beta-ketoadipate pathway for the degradation of benzoate. Both strains were chemotactic to benzoate, 3- and 4-nitrobenzoate, and all three aminobenzoate isomers after growth with benzoate but not succinate. Strain TW3 was chemotactic to the same set of aromatic compounds after growth with 4-nitrotoluene or 4-nitrobenzoate. In contrast, strain 4NT did not respond to any aromatic acids when grown with 4-nitrotoluene or 4-nitrobenzoate, apparently because these substrates are not metabolized to the inducer (beta-ketoadipate) of the chemotaxis system. The results suggest that strains TW3 and 4NT have a beta-ketoadipate-inducible chemotaxis system that responds to a wide range of aromatic acids and is quite similar to that present in PRS2000. The broad specificity of this chemotaxis system works as an advantage in strains TW3 and 4NT because it functions to detect diverse carbon sources, including 4-nitrobenzoate.     

Anaerobic degradation of halogenated benzoic acids by photoheterotrophic bacteria

Abstract From light-exposed enrichment cultures containing benzoate and a mixture of chlorobenzoates, a pure culture was obtained able to grow with 3-chlorobenzoate (3-CBA) or 3-bromobenzoate (3-BrBA) as the sole growth substrate anaerobically in the light. The thus isolated organism is a photoheterotroph, designated isolate DCP3. It is preliminarily identified as a Rhodopseudomonas palustris strain. It differs from Rhodopseudomonas palustris WS17, the only other known photoheterotroph capable of using 3-CBA for growth, in its independence of benzoate for growth with 3-CBA and in its wider substrate range: if grown on 3-CBA, it can also use 2-CBA, 4-CBA or 3,5-CBA.     

Regulation of terephthalate catabolism in Rhodococcus rubropertinctus

The regulation of terephthalate catabolism was studied in Rhodococcus rubropertinctus which decomposed this synthetic monomer. The pathway (a) of terephthalate (TP) catabolism is as follows: TP----benzoate----4-hydroxybenzoate----protocatechuate----pyrocatechol-- --cycle ortho-cleavage. The following results were obtained when studying why two other catabolic pathways were realized if benzoate and 4-hydroxybenzoate were taken as a sole carbon source, namely, (b) benzoate----pyrocatechol----cycle cleavage and (c) 4-hydroxybenzoate----protocatechuate----cycle cleavage. TP seemed to cause the divergence of pathways (a) and (b) by repressing the system of benzoate oxidation to pyrocatechol. In pathway (c), benzoate repressed the synthesis of enzymes which catalysed protocatechuate oxidation. Pathway (b) was switched over to (a) when the strain was grown in a medium containing TP and benzoate at a benzoate concentration above 5 mM. Here, the concentration of benzoate (first exogenous and later formed from TP) played a key role. R. rubropertinctus growth in a medium with TP and glucose had diauxic characteristics.     

Identification of a cocaine esterase in a strain of Pseudomonas maltophilia.

A strain of Pseudomonas maltophilia (termed MB11L) which was capable of using cocaine as its sole carbon and energy source was isolated by selective enrichment. An inducible esterase catalyzing the hydrolysis of cocaine to ecgonine methyl ester and benzoic acid was identified and purified 22-fold. In the presence of the solubilizing agent cholate, cocaine esterase had a native Mr of 110,000 and was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be a monomer. In the absence of cholate, cocaine esterase had a native Mr of 410,000 and probably existed as a tetramer. The pH optimum of the enzyme was 8.0, and the Km values for cocaine, ethyl benzoate, and ethyl 2-hydroxybenzoate were 0.36, 1.89, and 1.75 mM, respectively. Inhibition studies indicated that the enzyme was a serine esterase, possibly possessing a cation-binding site similar to those of mammalian acetylcholinesterase and the atropine esterase of Pseudomonas putida PMBL-1. The cocaine esterase of P. maltophilia MB11L showed no activity with atropine, despite the structural similarity of cocaine and atropine.     

Identification of enzymes involved in anaerobic benzene degradation by a strictly anaerobic iron‐reducing enrichment culture

Anaerobic benzene degradation was studied with a highly enriched iron‐reducing culture (BF) composed of mainly Peptococcaceae‐related Gram‐positive microorganisms. The proteomes of benzene‐, phenol‐ and benzoate‐grown cells of culture BF were compared by SDS‐PAGE. A specific benzene‐expressed protein band of 60 kDa, which could not be observed during growth on phenol or benzoate, was subjected to N‐terminal sequence analysis. The first 31 amino acids revealed that the protein was encoded by ORF 138 in the shotgun sequenced metagenome of culture BF. ORF 138 showed 43% sequence identity to phenylphosphate carboxylase subunit PpcA of Aromatoleum aromaticum strain EbN1. A LC/ESI‐MS/MS‐based shotgun proteomic analysis revealed other specifically benzene‐expressed proteins with encoding genes located adjacent to ORF 138 on the metagenome. The protein products of ORF 137, ORF 139 and ORF 140 showed sequence identities of 37% to phenylphosphate carboxylase PpcD of A. aromaticum strain EbN1, 56% to benzoate‐CoA ligase (BamY) of Geobacter metallireducens and 67% to 3‐octaprenyl‐4‐hydroxybenzoate carboxy‐lyase (UbiD/UbiX) of A. aromaticum strain EbN1 respectively. These genes are proposed as constituents of a putative benzene degradation gene cluster (～17 kb) composed of carboxylase‐related genes. The identified gene sequences suggest that the initial activation reaction in anaerobic benzene degradation is probably a direct carboxylation of benzene to benzoate catalysed by putative anaerobic benzene carboxylase (Abc). The putative Abc probably consists of several subunits, two of which are encoded by ORFs 137 and 138, and belongs to a family of carboxylases including phenylphosphate carboxylase (Ppc) and 3‐octaprenyl‐4‐hydroxybenzoate carboxy‐lyase (UbiD/UbiX).     

A Mycobacterium Strain with Extended Capacities for Degradation of Gasoline Hydrocarbons

A bacterial strain (strain IFP 2173) was selected from a gasoline-polluted aquifer on the basis of its capacity to use 2,2,4-trimethylpentane (isooctane) as a sole carbon and energy source. This isolate, the first isolate with this capacity to be characterized, was identified by 16S ribosomal DNA analysis, and 100% sequence identity with a reference strain of Mycobacterium austroafricanum was found. Mycobacterium sp. strain IFP 2173 used an unusually wide spectrum of hydrocarbons as growth substrates, including n-alkanes and multimethyl-substituted isoalkanes with chains ranging from 5 to 16 carbon atoms long, as well as substituted monoaromatic hydrocarbons. It also attacked ethers, such as methyl t-butyl ether. During growth on gasoline, it degraded 86% of the substrate. Our results indicated that strain IFP 2173 was capable of degrading 3-methyl groups, possibly by a carboxylation and deacetylation mechanism. Evidence that it attacked the quaternary carbon atom structure by an as-yet-undefined mechanism during growth on 2,2,4-trimethylpentane and 2,2-dimethylpentane was also obtained.     

Evidence for anaerobic syntrophic benzoate degradation threshold and isolation of the syntrophic benzoate degrader.

An anaerobic, motile, gram-negative, rod-shaped, syntrophic, benzoate-degrading bacterium, strain SB, was isolated in pure culture with crotonate as the energy source. Benzoate was degraded only in association with an H2-using bacterium. The kinetics of benzoate degradation by cell suspensions of strain SB in coculture with Desulfovibrio strain G-11 was studied by using progress curve analysis. The coculture degraded benzoate to a threshold concentration of 214 nM to 6.5 microM, with no further benzoate degradation observed even after extended incubation times. The value of the threshold depended on the amount of benzoate added and, consequently, the amount of acetate produced. The addition of sodium acetate, but not that of sodium chloride, affected the threshold value; higher acetate concentrations resulted in higher threshold values for benzoate. When a cell suspension that had reached a threshold benzoate concentration was reamended with benzoate, benzoate was used without a lag. The hydrogen partial pressure was very low and formate was not detected in cell suspensions that had degraded benzoate to a threshold value. The Gibbs free energy change calculations showed that the degradation of benzoate was favorable when the threshold was reached. These studies showed that the threshold for benzoate degradation was not caused by nutritional limitations, the loss of metabolic activity, or inhibition by hydrogen or formate. The data are consistent with a thermodynamic explanation for the existence of a threshold, but a kinetic explanation based on acetate inhibition may also account for the existence of a threshold.     

Biosynthesis of isoprenoid wax ester in Marinobacter hydrocarbonoclasticus DSM 8798: identification and characterization of isoprenoid coenzyme A synthetase and wax ester synthases

Marinobacter hydrocarbonoclasticus DSM 8798 has been reported to synthesize isoprenoid wax ester storage compounds when grown on phytol as the sole carbon source under limiting nitrogen and/or phosphorous conditions. We hypothesized that isoprenoid wax ester synthesis involves (i) activation of an isoprenoid fatty acid by a coenzyme A (CoA) synthetase and (ii) ester bond formation between an isoprenoid alcohol and isoprenoyl-CoA catalyzed, most likely, by an isoprenoid wax ester synthase similar to an acyl wax ester synthase, wax ester synthase/diacylglycerol acyltransferase (WS/DGAT), recently described from Acinetobacter sp. strain ADP1. We used the recently released rough draft genome sequence of a closely related strain, M. aquaeolei VT8, to search for WS/DGAT and acyl-CoA synthetase candidate genes. The sequence information from putative WS/DGAT and acyl-CoA synthetase genes identified in this strain was used to clone homologues from the isoprenoid wax ester synthesizing Marinobacter strain. The activities of the recombinant enzymes were characterized, and two new isoprenoid wax ester synthases capable of synthesizing isoprenoid ester and acyl/isoprenoid hybrid ester in vitro were identified along with an isoprenoid-specific CoA synthetase. One of the Marinobacter wax ester synthases displays several orders of magnitude higher activity toward acyl substrates than any previously characterized acyl-WS and may reflect adaptations to available carbon sources in their environments.     

Microbial production of cis,cis-muconic acid from benzoic acid

Summary The authors isolated numerous microorganisms with the capacity to assimilate large amounts of benzoate from many soil samples. Several of them were selected and subjected to mutation mainly by ultraviolet irradiation. One mutant lacking active muconate-lactonizing enzyme, the parent strain of which was identified as belonging to the genus Arthrobacter, was isolated and found to be capable of producing cis, cis-muconic acid with a quantitative yield of 44.1 g/l over 48 h in a 30 1 jar fermentor by successive feeding of small amounts of benzoate. This mutant, however, was more sensitive to high concentrations of the substrate than the parent strain. As few intermediates and isomers other than cis, cis-muconic acid were accumulated in the large fermentor, a large amount of pure cis, cis-muconic acid was easily obtained from the broth by salting out and recrystallization at a high recovery rate.     

Degradation of mono-, di-, and trihalogenated benzoic acids by Pseudomonas aeruginosa JB2.

Pseudomonas aeruginosa JB2 was isolated from a polychlorinated biphenyl-contaminated soil by enrichment culture containing 2-chlorobenzoate as the sole carbon source. Strain JB2 was subsequently found also to grow on 3-chlorobenzoate, 2,3- and 2,5-dichlorobenzoates, 2,3,5-trichlorobenzoate, and a wide range of other mono- and dihalogenated benzoic acids. Cometabolism of 2,4-dichlorobenzoate was also observed. Chlorocatechols were the central intermediates of all chlorobenzoate catabolic pathways. Degradation of 2-chlorobenzoate was routed through 3-chlorocatechol, whereas 4-chlorocatechol was identified from the metabolism of both 2,3- and 2,5-dichlorobenzoate. The initial attack on chlorobenzoates was oxygen dependent and most likely mediated by dioxygenases. Although plasmids were not detected in strain JB2, spontaneous mutants were detected in 70% of glycerol-grown colonies. The mutants were all of the following phenotype: benzoate+, 3-chlorobenzoate+, 2-chlorobenzoate-, 2,3-dichlorobenzoate-, 2,5-dichlorobenzoate-. While chlorocatechols were oxidized by the mutants at wild-type levels, oxidation of 2-chloro- and 2,3- and 2,5-dichlorobenzoates was substantially diminished. These findings suggested that strain JB2 possessed, in addition to the benzoate dioxygenase, a halobenzoate dioxygenase that was necessary for the degradation of chlorobenzoates substituted in the ortho position.     

Degradation of mono-, di-, and trihalogenated benzoic acids by Pseudomonas aeruginosa JB2

Pseudomonas aeruginosa JB2 was isolated from a polychlorinated biphenyl-contaminated soil by enrichment culture containing 2-chlorobenzoate as the sole carbon source. Strain JB2 was subsequently found also to grow on 3-chlorobenzoate, 2,3- and 2,5-dichlorobenzoates, 2,3,5-trichlorobenzoate, and a wide range of other mono- and dihalogenated benzoic acids. Cometabolism of 2,4-dichlorobenzoate was also observed. Chlorocatechols were the central intermediates of all chlorobenzoate catabolic pathways. Degradation of 2-chlorobenzoate was routed through 3-chlorocatechol, whereas 4-chlorocatechol was identified from the metabolism of both 2,3- and 2,5-dichlorobenzoate. The initial attack on chlorobenzoates was oxygen dependent and most likely mediated by dioxygenases. Although plasmids were not detected in strain JB2, spontaneous mutants were detected in 70% of glycerol-grown colonies. The mutants were all of the following phenotype: benzoate+, 3-chlorobenzoate+, 2-chlorobenzoate-, 2,3-dichlorobenzoate-, 2,5-dichlorobenzoate-. While chlorocatechols were oxidized by the mutants at wild-type levels, oxidation of 2-chloro- and 2,3- and 2,5-dichlorobenzoates was substantially diminished. These findings suggested that strain JB2 possessed, in addition to the benzoate dioxygenase, a halobenzoate dioxygenase that was necessary for the degradation of chlorobenzoates substituted in the ortho position.