Conversion of 3-Chlorocatechol by Various Catechol 2,3-Dioxygenases and Sequence Analysis of the Chlorocatechol Dioxygenase Region of Pseudomonas putida GJ31

Pseudomonas putida GJ31 contains an unusual catechol 2,3-dioxygenase that converts 3-chlorocatechol and 3-methylcatechol, which enables the organism to use both chloroaromatics and methylaromatics for growth. A 3.1-kb region of genomic DNA of strain GJ31 containing the gene for this chlorocatechol 2,3-dioxygenase (cbzE) was cloned and sequenced. The cbzE gene appeared to be plasmid localized and was found in a region that also harbors genes encoding a transposase, a ferredoxin that was homologous to XylT, an open reading frame with similarity to a protein of a meta-cleavage pathway with unknown function, and a 2-hydroxymuconic semialdehyde dehydrogenase. CbzE was most similar to catechol 2,3-dioxygenases of the 2.C subfamily of type 1 extradiol dioxygenases (L. D. Eltis and J. T. Bolin, J. Bacteriol. 178:5930–5937, 1996). The substrate range and turnover capacity with 3-chlorocatechol were determined for CbzE and four related catechol 2,3-dioxygenases. The results showed that CbzE was the only enzyme that could productively convert 3-chlorocatechol. Besides, CbzE was less susceptible to inactivation by methylated catechols. Hybrid enzymes that were made of CzbE and the catechol 2,3-dioxygenase of P. putida UCC2 (TdnC) showed that the resistance of CbzE to suicide inactivation and its substrate specificity were mainly determined by the C-terminal region of the protein.     

Degradation of Chloroaromatics: Purification and Characterization of a Novel Type of Chlorocatechol 2,3-Dioxygenase of Pseudomonas putida GJ31

A purification procedure for a new kind of extradiol dioxygenase, termed chlorocatechol 2,3-dioxygenase, that converts 3-chlorocatechol productively was developed. Structural and kinetic properties of the enzyme, which is part of the degradative pathway used for growth of Pseudomonas putida GJ31 with chlorobenzene, were investigated. The enzyme has a subunit molecular mass of 33.4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the native M_r value under nondenaturating conditions by gel filtration gave a molecular mass of 135 ± 10 kDa, indicating a homotetrameric enzyme structure (4 × 33.4 kDa). The pI of the enzyme was estimated to be 7.1 ± 0.1. The N-terminal amino acid sequence (43 residues) of the enzyme was determined and exhibits 70 to 42% identity with other extradiol dioxygenases. Fe(II) seems to be a cofactor of the enzyme, as it is for other catechol 2,3-dioxygenases. In contrast to other extradiol dioxygenases, the enzyme exhibited great sensitivity to temperatures above 40°C. The reactivity of this enzyme toward various substituted catechols, especially 3-chlorocatechol, was different from that observed for other catechol 2,3-dioxygenases. Stoichiometric displacement of chloride occurred from 3-chlorocatechol, leading to the production of 2-hydroxymuconate.     

Ferredoxin-mediated reactivation of the chlorocatechol 2,3-dioxygenase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> GJ31

In the chlorobenzene degrader Pseudomonas putida GJ31, chlorocatechol is formed as an intermediate and cleaved by a meta-cleavage extradiol chlorocatechol dioxygenase, which has previously been shown to be exceptionally resistant to inactivation by substituted catechols. The gene encoding this dioxygenase ( cbzE) is preceded by a gene ( cbzT) potentially encoding a ferredoxin, the function of which was studied. The cbzT gene product was overproduced in Escherichia coli and purified in recombinant form. Two homologous proteins, CdoT and AtdS, encoded by genes identified in strains degrading nitrobenzene and aniline, respectively, were also purified and characterized. All three proteins showed spectroscopic properties typical for [2Fe-2S] ferredoxins. The chlorocatechol dioxygenase from strain GJ31 (CbzE) was fully inactivated when 4-methylcatechol was used as substrate. Inactivated CbzE could be rapidly reactivated in vitro in the presence of purified CbzT and a source of reductant. It is inferred that the ability of strain GJ31 to metabolize both chlorobenzene and toluene might depend on the regeneration of the chlorocatechol dioxygenase activity mediated by CbzT. Three CbzT-like ferredoxins, including AtdS, were found to be competent in the reactivation of CbzE, whereas XylT, a protein known to mediate reactivation of the catechol dioxygenase from P. putida mt2 (XylE), was ineffective. Accordingly, CbzT formed a covalent complex with CbzE when cross-linked with a carbodiimide, whereas XylT did not. In the reverse situation, CbzT was found to reactivate XylE as efficiently as XylT and formed an heterologous covalent complex with this enzyme upon cross-linking. We conclude that CbzT, CdoT and AtdS are isofunctional ferredoxins that appear to be involved in the reactivation of their cognate catechol dioxygenases. Based on primary structure comparisons, residues of the ferredoxins possibly involved in the molecular interaction with catechol dioxygenases were identified and their significance is discussed.     

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.     

Microbial degradation of chloroaromatics: use of the meta-cleavage pathway for mineralization of chlorobenzene.

Pseudomonas putida GJ31 is able to simultaneously grow on toluene and chlorobenzene. When cultures of this strain were inhibited with 3-fluorocatechol while growing on toluene or chlorobenzene, 3-methylcatechol or 3-chlorocatechol, respectively, accumulated in the medium. To establish the catabolic routes for these catechols, activities of enzymes of the (modified) ortho- and meta-cleavage pathways were measured in crude extracts of cells of P. putida GJ31 grown on various aromatic substrates, including chlorobenzene. The enzymes of the modified ortho-cleavage pathway were never present, while the enzymes of the meta-cleavage pathway were detected in all cultures. This indicated that chloroaromatics and methylaromatics are both converted via the meta-cleavage pathway. Meta cleavage of 3-chlorocatechol usually leads to the formation of a reactive acylchloride, which inactivates the catechol 2,3-dioxygenase and blocks further degradation of catechols. However, partially purified catechol 2,3-dioxygenase of P. putida GJ31 converted 3-chlorocatechol to 2-hydroxy-cis,cis-muconic acid. Apparently, P. putida GJ31 has a meta-cleavage enzyme which is resistant to inactivation by the acylchloride, providing this strain with the exceptional ability to degrade both toluene and chlorobenzene via the meta-cleavage pathway.     

Genetic and biochemical analyses of chlorobenzene degradation gene clusters in <Emphasis Type="Italic">Pandoraea</Emphasis> sp. strain MCB032

Pandoraea sp. strain MCB032 was isolated as an emerging chlorobenzene degrader from a functionally stable bioreactor where species succession had occurred. In this study, two gene clusters encoding chlorobenzene metabolic functions have been cloned. Within the cbs gene cluster, CbsA and CbsB are similar to the chlorobenzene dioxygenase and the cis-chlorobenzene dihydrodiol dehydrogenase in Ralstonia sp. JS705 and shown to transform chlorobenzene to 3-chlorocatechol. The clc gene cluster shows strong similarity to the clc genes of Ralstonia sp. JS705 and encodes chlorocatechol 1,2-dioxygenase (ClcA) and other enzymes, which catalyze the conversion of chlorocatechol to 3-oxoadipate. The Michaelis constants (K _m) values of ClcA for catechol, 3-methylcatechol and 3-chlorocatechol were determined as 10.0, 8.9 and 3.4 μM, respectively. CbsX, a putative transport protein present in the cbs cluster of strain MCB032 but not in those of other chlorobenzene degraders, shows 76 and 53% identities to two previously identified transport proteins involved in toluene degradation, TbuX from Ralstonia pickettii PKO1 and TodX from Pseudomonas putida F1. The presence of the transport protein in strain MCB032 likely provides a mechanistic explanation for its higher chlorobenzene affinity and may well be the basis for the competitive advantage of this strain in the bioreactor.     

Detection of chlorocatechol 1,2-dioxygenase genes in proteobacteria by PCR and gene probes

In various bacterial strains belonging to the β-subdivision of proteobacteria which are capable of degrading chlorinated monoaromatic compounds, chlorocatechol 1,2-dioxygenase genes were detected by PCR and Southern hybridization. Using PCR primers derived from the conserved sequence motifs of chlorocatechol 1,2-dioxygenase genes tfdC, clcA and tcbC, PCR products of the expected size were obtained with the test strains, but not with negative control strains. The specificity of the PCR products was verified by hybridization using an oligonucleotide probe for an internal sequence motif which is evolutionarily conserved among chlorocatechol 1,2-dioxygenases and some other dioxygenases that catalyze the intradiol aromatic-ring-cleavage. Hybridization with the tfdC PCR product from the 2,4-D degradative plasmid pJP4 under stringent conditions revealed different extents of homology of the chlorocatechol 1,2-dioxygenase genes to the canonical tfdC sequence in the various strains. These findings were confirmed by the nucleotide sequence analysis of the tfdC-specific PCR products. From our results, we conclude that the PCR primer set is more suitable than the hybridization with pJP4-derived gene probes for the detection of diverse chlorocatechol 1,2-dioxygenase genes in proteobacteria.     

Characterisation of a chromosomally encoded catechol 1,2-dioxygenase (E.C. 1.13.11.1) from Alcaligenes eutrophus CH34

Alcaligenes eutrophus CH34 used benzoate as a sole source of carbon and energy, degrading it through the 3-oxoadipate pathway. All the enzymes required for this degradation were shown to be encoded by chromosomal genes. Catechol 1,2-dioxygenase activity was induced by benzoate, catechol, 4-chlorocatechol, and muconate. The enzyme is most likely a homodimer, with an apparent molecular weight of 76,000 ± 500. According to several criteria, its properties are intermediate between those of catechol 1,2-dioxygenases (CatA) and chlorocatechol 1,2-dioxygenases (ClcA). The determined K _m for catechol is the lowest among known catechol and chlorocatechol dioxygenases. Similar K _m values were found for para-substituted catechols, although the catalytic constants were much lower. The catechol 1,2-dioxygenase from strain CH34 is unique in its property to transform tetrachlorocatechol; however, excess substrate led to a marked reversible inhibition. Some meta- and multi-substituted catechols behaved similarly. The determined K _m (or K _i) values for para- or meta-substituted catechols suggest that the presence of an electron-withdrawing substituent at one of these positions results in a higher affinity of the enzyme for the ligand. Results of studies of recognition by the enzyme of various nonmetabolised aromatic compounds are also discussed. Received: 20 November 1996 / Accepted: 11 April 1996     

Tetrameric structure and cellular location of catechol 2,3-dioxygenase

Catechol 2,3-dioxygenase from the meta-cleavage pathway encoded on the TOL plasmid of Pseudomonas putida (pWWO) was investigated by electron microscopy. Negatively stained samples of the purified catechol 2,3-dioxygenase revealed that the enzyme consists of four subunits arranged in a tetrahedral conformation. Monoclonal antibodies raised against catechol 2,3-dioxygenase showed highly specific reactions and were used to localize the enzyme in Escherichia coli (pAW31) and P. putida (pWWO), using the protein A-gold technique carried out as a post-embedding immunoelectron microscopy procedure. Our in situ labeling studies revealed a cytoplasmic location of the catechol 2,3-dioxygenase in both cell types.Abbreviations C23O Catechol 2,3-dioxygenase - 3MB 3 Methylbenzoate - AK1 Anti-C23O-IgG-antibody - G Gold particle     

The meta cleavage operon of TOL degradative plasmid pWWO comprises 13 genes

Summary The meta-cleavage operon of TOL plasmid pWWO of Pseudomonas putida encodes a set of enzymes which transform benzoate/toluates to Krebs cycle intermediates via extradiol (meta-) cleavage of (methyl)catechol. The genetic organization of the operon was characterized by cloning of the meta-cleavage genes into an expression vector and identification of their products in Escherichia coli maxicells. This analysis showed that the meta-cleavage operon contains 13 genes whose order and products (in kilodaltons) are The xyIXYZ genes encode three subunits of toluate 1,2-dioxygenase. The xylL, xyIE, xyIG, xylF, xylJ, xylK, xylI and xylH genes encode 1,2-dihydroxy-3,5-cyclohexadiene-1-carboxylate dehydrogenase, catechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, 2-hydroxymuconic semialdehyde hydrolase, 2-oxopent-4-enoate hydratase, 4-hydroxy-2-oxovalerate aldolase, 4-oxalocrotonate decarboxylase and 4-oxalocrotonate tautomerase, respectively. The functions of xyIT and xylQ are not known at present. The comparison of the coding capacity and the sizes of the products of the meta-cleavage operon genes indicated that most of the DNA between xyIX and xyIH consists of coding sequences.     

Isolation and characterization of phenanthrene-degrading <Emphasis Type="Italic">Sphingomonas paucimobilis</Emphasis> strain ZX4

Phenanthrene-degrading bacterium strain ZX4 was isolated from an oil-contaminated soil, and identified as Sphingomonas paucimobilis based on 16S rDNA sequence, cellular fatty acid composition, mol% G + C and Biolog-GN tests. Besides phenanthrene, strain ZX4 could also utilize naphthalene, fluorene and other aromatic compounds. The growth on salicylic acid and catechol showed that the strain degraded phenanthrene via salicylate pathway, while the assay of catechol 2, 3-dioxygenase revealed catechol could be metabolized through meta-cleavage pathway. Three genes, including two of meta-cleavage operon genes and one of GST encoding gene were obtained. The order of genes arrangement was similar to S-type meta-pathway operons. The phylogenetic trees based on 16S rDNA sequence and meta-pathway gene both revealed that strain ZX4 is clustered with strains from genus Sphingomonas.     

Cloning and characterization of a chromosome-encoded catechol 2,3-dioxygenase gene from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> ZD 4-3

Catechol 2,3-dioxygenase (C23O), an extradiol-type dioxygenase cleaving the aromatic C—C bond at the meta-position of dihydroxylated aromatic substrates, catalyzes the conversion of catechol to 2-hydroxy-muconic semialdehyde. Based on a curing experiment, PCR identification, and Southern hybridization, the gene responsible for C23O was localized on a 3.5-kb EcoRI/BamHI fragment and cloned from Pseudomonas aeruginosa ZD 4-3, which was able to degrade both single and bicyclic compounds via a meta-cleavage path-way. A complete nucleotide sequence analysis of the C23O revealed that it has one ORF, which showed a strong overall amino acid similarity to the known gram-negative bacterial mesophilic C23Os. The alignment analysis indicated a distinct difference between the C23O in this study and the 2,3-dihydroxybiphenyl dioxygenases that cleave bicyclic aromatic compounds. The heterogeneous expression of the pheB gene in E. Coli BL21(DE3) demonstrated that this C23O possesses a meta-cleavage activity.From Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 802–809.Original English Text Copyright © 2004 by Chen, Liu, Zhu, Jin.This article was submitted by the authors in English.     

Biochemical and Genetic Evidence for meta-Ring Cleavage of 2,4,5-Trihydroxytoluene in Burkholderia sp. Strain DNT

2,4,5-Trihydroxytoluene (THT) oxygenase from Burkholderia sp. strain DNT catalyzes the conversion of THT to an unstable ring fission product. Biochemical and genetic studies of THT oxygenase were undertaken to elucidate the mechanism of the ring fission reaction. The THT oxygenase gene (dntD) was previously localized to the 1.2-kb DNA insert subcloned in the recombinant plasmid designated pJS76 (W. C. Suen and J. C. Spain, J. Bacteriol. 175:1831–1837, 1993). Analysis of the deduced amino acid sequence of DntD revealed the presence of the highly conserved residues characteristic of the catechol 2,3-dioxygenase gene family I. The deduced amino acid sequence of DntD corresponded to a molecular mass of 35 kDa. The native molecular masses for the THT oxygenase estimated by using gel filtration chromatography and nondenaturing gel electrophoresis were 67.4 and 77.8 kDa, respectively. The results suggested that the native protein consists of two identical subunits. The colorless protein contained 2 mol of iron per mol of protein. Stimulation of activity in the presence of ferrous iron and ascorbate suggested a requirement for ferrous iron in the active site. The properties of the enzyme are similar to those of the catechol 2,3-dioxygenases (meta-cleavage dioxygenases). In addition to THT, the enzyme exhibited activity towards 1,2,4-benzenetriol, catechol, 3- and 4-methylcatechol, and 3- and 4-chlorocatechol. The chemical analysis of the THT ring cleavage product showed that the product was 2,4-dihydroxy-5-methyl-6-oxo-2,4-hexadienoic acid, consistent with extradiol ring fission of THT.     

Induction of ortho- and meta-cleavage pathways in Pseudomonas in biodegradation of high benzoate concentration: MS identification of catabolic enzymes

The degradation pathways of benzoate at high concentration in Pseudomonas putida P8 were directly elucidated through mass spectrometric identification of some key catabolic enzymes. Proteins from P. putida P8 grown on benzoate or succinate were separated using two-dimensional gel electrophoresis. For cells grown on benzoate, eight distinct proteins, which were absent in the reference gel patterns from succinate-grown cells, were found. All the eight proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as catabolic enzymes involved in benzoate degradation. Among them, CatB (EC5.5.1.1), PcaI (EC2.8.3.6), and PcaF (EC2.3.1.174) were the enzymes involved in the ortho-cleavage pathway; DmpC (EC1.2.1.32), DmpD (EC3.1.1.-), DmpE (EC4.2.1.80), DmpF (EC1.2.1.10), and DmpG (EC4.1.3.-) were the meta-cleavage pathway enzymes. In addition, enzyme activity assays showed that the activities of both catechol 1,2-dioxygenase (C12D; EC1.13.11.1) and catechol 2,3-dioxygenase (C23D; EC1.13.11.2) were detected in benzoate-grown P. putida cells, undoubtedly suggesting the simultaneous expression of both the ortho- and the meta-cleavage pathways in P. putida P8 during the biodegradation of benzoate at high concentration.     

Initial reactions in the mineralization of 2-sulfobenzoate by Pseudomonas sp. RW611

Abstract Pseudomonas sp. strain RW611 utilized the ammonium salt of 2-sulfobenzoate as sole source of carbon, sulfur, nitrogen, and energy. The xenobiotic sulfo substituent was dioxygenolytically eliminated as sulfite, which was then slowly oxidized to sulfate. 2,3-Dihydroxybenzoate, which resulted from desulfonation underwent meta -cleavage, mediated by 2,3-dihydroxybenzoate 3,4-dioxygenase activity. This enzyme was inhibited by 3-chlorocatechol and 2,3,4-trihydroxybenzoate.     

Cooxidation of chloro- and methylphenols by Alcaligenes xylosoxidans JH1

Alcaligenes xylosoxidans subspecies denitrificans JH1 was enriched with 2-chlorophenol from a mixed culture degrading different chloro- and methylphenols. The strain used all monochloro- and monomethylphenols apart from 2-methylphenol as sole source of energy and carbon with stoichiometric release of chloride. 4-Chlorophenol was mineralized up to a concentration of 1.3 mM. Degradation of mixtures of monochloro- and monomethylphenols occurred at least partially except for the mixture of 2-chlorophenol and 3-methylphenol. Depending upon the growth substrates used, enzymes of the ortho and/or meta cleavage pathway catalysed the degradation of the phenols. The transformation of chlorophenols was concluded to occur exclusively via the ortho cleavage pathway because no chlorocatechol 2,3-dioxygenase activity was found in chlorophenol-grown cells. Degradation of 4-methylphenol in strain JH1 occurred both by the ortho and meta cleavage pathway as indicated by the finding that the ortho- and meta-cleaving dioxygenases were expressed in 4-methylphenol-grown cells. Transformation of methylphenols by the ortho cleavage pathway led to the accumulation of methyllactones as dead-end products. Mixtures of methyl- and chlorophenols were metabolized mainly by the ortho cleavage pathway because chlorocatechols formed inactivated the constitutive catechol 2,3-dioxygenase which caused channelling of methylphenols into the ortho cleavage pathway.     

恶臭假单胞菌ND6菌株catA基因的克隆和表达及其儿茶酚裂解途径探讨

恶臭假单胞菌ND6菌株的萘降解质粒pND6-1中编码儿茶酚1,2-双加氧酶的catA基因在大肠杆菌中进行了克隆和表达,并研究表达产物的酶学性质。结果表明:酶的K_m为0.019μmol/L,V_max为1.434μmol/(min.mg);具有很好的耐热性,在50℃保温45min后仍能够保留酶活力的93.7%;Fe²⁺对酶活性有显著的促进作用,其比活力是对照反应的292%;酶对4-氯儿茶酚的催化活性非常低,属于Ⅰ型儿茶酚1,2-双加氧酶。以萘为底物生长时,ND6菌株的细胞提取液中既存在催化邻位裂解途径的儿茶酚1,2-双加氧酶活性,也存在催化间位裂解途径的儿茶酚2,3-双加氧酶活性。以苯甲酸、对羟基苯甲酸和苯乙酸为唯一碳源生长时,ND6菌株细胞提取液的儿茶酚1,2-双加氧酶活性远远大于儿茶酚2,3-双加氧酶活性。表明ND6菌株既能通过儿茶酚间位裂解途径降解萘,也能通过儿茶酚邻位裂解途径降解萘,而以苯甲酸、对羟基苯甲酸和苯乙酸为诱导物时只利用儿茶酚邻位裂解途径。     

Genetic and Biochemical Analyses of the tec Operon Suggest a Route for Evolution of Chlorobenzene Degradation Genes

The TecA broad-spectrum chlorobenzene dioxygenase of Burkholderia sp. strain PS12 catalyzes the first step in the mineralization of 1,2,4,5-tetrachlorobenzene. The catabolic genes were localized on a small plasmid that belongs to the IncPβ incompatibility group. PCR analysis of the genetic environment of the tec genes indicated high similarity to the transposon-organized catabolic tcb chlorobenzene degradation genes of Pseudomonas sp. strain P51. Sequence analysis of the regions flanking the tecA genes revealed an upstream open reading frame (ORF) with high similarity to the todF 2-hydroxy-6-oxo-2,4-heptadienoate hydrolase gene of Pseudomonas putida F1 and a discontinuous downstream ORF showing high similarity to the todE catechol 2,3-dioxygenase gene of strain F1. Both homologues in strain P51 exist only as deletion remnants. We suggest that different genetic events thus led to inactivation of the perturbing meta-cleavage enzymes in strains P51 and PS12 during the evolution of efficient chlorobenzene degradation pathways. Biochemical characterization of TodF-like protein TlpF and a genetically refunctionalized TodE-like protein, TlpE, produced in Escherichia coli provided data consistent with the proposed relationships.     

Catechol ring-cleavage in Pseudomonas cepacia: the simultaneous induction of ortho and meta pathways

This work demonstrates the ring-cleavage pathways of catechol on Pseudomonas cepacia ATCC 29351, formed upon its growth on salicylate and benzoate, each as a sole carbon source. When grown on salicylate, P. cepacia induces only the catechol ortho pathway by its induction of catechol 1,2-dioxygenase. However, interestingly, benzoate-grown cells induce the ortho and meta pathways for the biodegradation of catechol, by inducing simultaneously catechol 1,2-dioxygenase and 2,3-dioxygenase, respectively, in the ratio of 7:1. The results indicate that P. cepacia ATCC 29351 possesses the genetic capacity for enzymes of both the ortho- and meta-cleavage pathways of benzoate degradation, although the phenotypic expression for the ortho pathway is higher. The simultaneous induction of catechol 1,2- and 2,3-dioxygenase is not detected in salicylate degradation. Although catechol is the metabolic intermediate for both salicylate and benzoate, catechol did not induce either pathway when used as a sole carbon source.