Degradation of 2-methylaniline in Rhodococcus rhodochrous: cloning and expression of two clustered catechol 2,3-dioxygenase genes from strain CTM

Rhodococcus rhodochrous strain CTM degrades 2-methylaniline mainly via the meta-cleavage pathway. Conversion of the metabolite 3-methylcatechol was catalysed by an Mr 156,000 catechol 2,3-dioxygenase (C23OI) comprising four identical subunits of Mr 39,000. The corresponding gene was detected by using an oligonucleotide as a gene probe. This oligonucleotide was synthesized on the basis of a partial amino acid sequence obtained from the purified enzyme from R. rhodochrous. The structural gene of C23OI was located on a 3.5 kb BglII restriction fragment of plasmid pTC1. On the same restriction fragment the gene for a second catechol 2,3-dioxygenase, designated C23OII, was found. This gene coded for the synthesis of the Mr 40,000 polypeptide of the Mr 158,000 tetrameric C23OII. More precise mapping of the structural genes showed that the C23OI gene was located on a 1.2 kb BglII-SmaI fragment and the C23OII gene on the adjacent 1.15 kb SmaI fragment. Comprehensive substrate range analysis showed that C23OII accepted all the substrates that C23OI did, but additionally cleaved 2,3-dihydroxybiphenyl and catechols derived from phenylcarboxylic acids. C23OI exhibited highest activity towards methylcatechols, whereas C23OII cleaved unsubstituted catechol preferentially.     

Degradation of 2-chlorobenzoate by Pseudomonas cepacia 2CBS

A bacterium was isolated from water by enrichment on 2-chlorobenzoate as sole source of carbon and energy. Based on morphological and physiological properties, this microorganism was assigned to the species Pseudomonas cepacia. The organism was designated Pseudomonas cepacia 2CBS. During growth on 2-chlorobenzoate, the chlorine substituent was released quantitatively, and a small amount of 2,3-dihydroxybenzoate accumulated in the culture medium. Mutants of Pseudomonas cepacia 2CBS were induced by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. Some of these mutants produced catechol from 2-chlorobenzoate. Other mutants accumulated the meta-cleavage product of catechol, 2-hydroxy-cis,cis-muconic acid semialdehyde. In crude cell-free extracts of Pseudomonas cepacia 2CBS, an enzyme was detected which catalysed the conversion of 2-chlorobenzoate to catechol. Molecular oxygen, NADH and exogenous Fe2+ were required for activity. Stoichiometric amounts of chloride were released. Experiments with 18O2 revealed that both oxygen atoms in the hydroxyl groups of the product were derived from molecular oxygen. Thus, the enzyme catalysing the conversion of 2-chlorobenzoate was identified as 2-chlorobenzoate 1,2-dioxygenase (1,2-hydroxylating, dehalogenating, decarboxylating). 2-Chlorobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS was shown to be a multicomponent enzyme system. The activities of catechol 2,3-dioxygenase and catechol 1,2-dioxygenase were detected in crude cell-free extracts. The activity of catechol 2,3-dioxygenase was 60 times higher than the activity of catechol 1,2-dioxygenase, indicating that catechol is mainly degraded via meta-cleavage in Pseudomonas cepacia 2CBS. No enzyme was found which converted 2,3-dihydroxybenzoate, suggesting that this compound is a dead-end metabolite of 2-chlorobenzoate catabolism. A pathway for the degradation of 2-chlorobenzoate by Pseudomonas cepacia 2CBS is proposed.     

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.     

In vivo reactivation of catechol 2,3-dioxygenase mediated by a chloroplast-type ferredoxin: a bacterial strategy to expand the substrate specificity of aromatic degradative pathways.

The meta-cleavage operon of the TOL plasmid pWW0 of Pseudomonas putida contains 13 genes responsible for the oxidation of benzoate and toluates to Krebs cycle intermediates via estradiol (meta) cleavage of (methyl)catechol. The functions of all the genes are known with the exception of xylT. We constructed pWW0 mutants defective in the xylT gene, and found that these mutants were not able to grow on p-toluate while they were still capable of growing on benzoate and m-toluate. In the xylT mutants, all the meta-cleavage enzymes were induced by p-toluate with the exception of catechol 2,3-dioxygenase whose activity was 1% of the p-toluate-induced activity in wild-type cells. Addition of 4-methylcatechol to m-toluate-grown wild-type and xylT cells resulted in the inactivation of catechol 2,3-dioxygenase in these cells. In the wild-type strain but not in the xylT mutant, the catechol 2,3-dioxygenase activity was regenerated in a short time. The regeneration of the catechol 2,3-dioxygenase activity was also observed in H2O2-treated wild-type cells, but not in H2O2-treated xylT cells. We concluded that the xylT product is required for the regeneration of catechol 2,3-dioxygenase.     

Degradation of phenanthrene via meta-cleavage of 2-hydroxy-1-naphthoic acid by Ochrobactrum sp. strain PWTJD

The present study describes the assimilation of phenanthrene by an aerobic bacterium, Ochrobactrum sp. strain PWTJD, isolated from municipal waste-contaminated soil sample utilizing phenanthrene as a sole source of carbon and energy. The isolate was identified as Ochrobactrum sp. based on the morphological, nutritional and biochemical characteristics as well as 16S rRNA gene sequence analysis. A combination of chromatographic analyses, oxygen uptake assay and enzymatic studies confirmed the degradation of phenanthrene by the strain PWTJD via 2-hydroxy-1-naphthoic acid, salicylic acid and catechol. The strain PWTJD could also utilize 2-hydroxy-1-naphthoic acid and salicylic acid, while the former was metabolized by a ferric-dependent meta-cleavage dioxygenase. In the lower pathway, salicylic acid was metabolized to catechol and was further degraded by catechol 2,3-dioxygenase to 2-hydroxymuconoaldehyde acid, ultimately leading to tricarboxylic acid cycle intermediates. This is the first report of the complete degradation of a polycyclic aromatic hydrocarbon molecule by Gram-negative Ochrobactrum sp. describing the involvement of the meta-cleavage pathway of 2-hydroxy-1-naphthoic acid in phenanthrene assimilation.     

Microbial mineralization of ring-substituted anilines through an ortho-cleavage pathway.

Moraxella sp. strain G is able to utilize as sole source of carbon and nitrogen aniline, 4-fluoroaniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline (PCA), and 4-bromoaniline but not 4-iodoaniline, 4-methylaniline, 4-methoxyaniline, or 3,4-dichloroaniline. The generation time on PCA was 6 h. The pathway for the degradation of PCA was investigated by analysis of catabolic intermediates and enzyme activities. Mutants of strain G were isolated to enhance the accumulation of specific pathway intermediates. PCA was converted by an aniline oxygenase to 4-chlorocatechol, which in turn was degraded via a modified ortho-cleavage pathway. Synthesis of the aniline oxygenase was inducible by various anilines. This enzyme exhibited a broad substrate specificity. Its specific activity towards substituted anilines seemed to be correlated more with the size than with the electron-withdrawing effect of the substituent and was very low towards anilines having substituents larger than iodine or a methyl group. The initial enzyme of the modified ortho-cleavage pathway, catechol 1,2-dioxygenase, had similar characteristics to those of corresponding enzymes of pathways for the degradation of chlorobenzoic acid and chlorophenol, that is, a broad substrate specificity and high activity towards chlorinated and methylated catechols.     

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。     

Microbial mineralization of ring-substituted anilines through an ortho-cleavage pathway

Moraxella sp. strain G is able to utilize as sole source of carbon and nitrogen aniline, 4-fluoroaniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline (PCA), and 4-bromoaniline but not 4-iodoaniline, 4-methylaniline, 4-methoxyaniline, or 3,4-dichloroaniline. The generation time on PCA was 6 h. The pathway for the degradation of PCA was investigated by analysis of catabolic intermediates and enzyme activities. Mutants of strain G were isolated to enhance the accumulation of specific pathway intermediates. PCA was converted by an aniline oxygenase to 4-chlorocatechol, which in turn was degraded via a modified ortho-cleavage pathway. Synthesis of the aniline oxygenase was inducible by various anilines. This enzyme exhibited a broad substrate specificity. Its specific activity towards substituted anilines seemed to be correlated more with the size than with the electron-withdrawing effect of the substituent and was very low towards anilines having substituents larger than iodine or a methyl group. The initial enzyme of the modified ortho-cleavage pathway, catechol 1,2-dioxygenase, had similar characteristics to those of corresponding enzymes of pathways for the degradation of chlorobenzoic acid and chlorophenol, that is, a broad substrate specificity and high activity towards chlorinated and methylated catechols.     

A comparison of biodegradation of phenol and homologous compounds by Pseudomonas vesicularis and Staphylococcus sciuri strains

Pseudomonas vesicularis and Staphylococcus sciuri were isolated as dominant strains from phenol-acclimated activated sludge. P. vesicularis was an efficient degrader of phenol, catechol, p-cresol, sodium benzoate and sodium salicylate in a single substrate system. Under similar conditions S. sciuri degraded only phenol and catechol from among aromatic compounds that were tested. Cell-free extracts of P. vesicularis grown on phenol (376 mg l(-1)), sodium benzoate (576 mg l(-1)) and sodium salicylate (640 mg l(-1)) showed catechol 2,3-dioxygenase activity initiating an extradiol (meta) splitting pathway. The degradative intradiol (ortho) pathway as a result of catechol 1,2-dioxygenase synthesis was induced in P. vesicularis cells grown on catechol (440 mg l(-1)) orp-cresol (432 mg l(-1)). Catechol 1,2-dioxygenase and the ortho-cleavage has been also reported in S. sciuri cells capable of degrading phenol (376 mg l(-1)) or catechol (440 mg l(-1)). In cell-free extracts of S. sciuri no meta-cleavage enzyme activity was detected. These results demonstrated that gram-positive S. sciuri strain was able to effectively metabolize some phenols as do many bacteria of the genus Pseudomonas but have a different capacity for degrading of these compounds.     

Molecular characterisation of a Rhodococcus ohp operon

The ohp operon of Rhodococcus strain V49 consists of five genes, ohpR, ohpA, ohpB, ohpC and ohpD which encode putative regulator and transport proteins and confirmed monooxygenase, hydroxymuconic semialdehyde hydrolase and catechol 2,3-dioxygenase enzymes, respectively. These enzymes catalyse the conversion of 3-(2- hydroxyphenyl)propionic acid to the corresponding linear product via a meta-cleavage pathway. Confirmation that the ohp gene cluster formed an operon was provided by gene disruption during which expression of Bacillus levansucrase was confirmed in Rhodococcus. Following biochemical assays of cell-free extracts from recombinant Escherichia coli expressing ohpB (monooxygenase), ohpC (hydroxymuconic-semialdehyde hydrolase) and ohpD (catechol 2,3-dioxygenase), the ortho-hydroxyphenylpropionic acid catabolic pathway in Rhodococcus strain V49 (ATCC 19070) has been predicted.     

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。     

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     

Isolation and characterization of a rhodococcus species strain able to grow on ortho- and para-xylene

Rhodococcus sp. strain YU6 was isolated from soil for the ability to grow on o-xylene as the sole carbon and energy source. Unlike most other o-xylene-degrading bacteria, YU6 is able to grow on p-xylene. Numerous growth substrate range experiments, in addition to the ring-cleavage enzyme assay data, suggest that YU6 initially metabolizes o- and p-xylene by direct aromatic ring oxidation. This leads to the formation of dimethylcatechols, which was further degraded largely through meta-cleavage pathway. The gene encoding meta-cleavage dioxygenase enzyme was PCR cloned from genomic YU6 DNA using previously known gene sequence data from the o-xylene-degrading Rhodococcus sp. strain DK17. Subsequent sequencing of the 918-bp PCR product revealed a 98% identity to the gene, encoding methylcatechol 2,3-dioxygenase from DK17. PFGE analysis followed by Southern hybridization with the catechol 2,3-dioxygenase gene demonstrated that the gene is located on an approximately 560-kb megaplasmid, designated pJYJ1.     

Influence of para-substituents on the oxidative metabolism of o-nitrophenols by Pseudomonas putida B2.

Pseudomonas putida B2 is able to grow on o-nitrophenol (ONP) as the sole source of carbon and nitrogen. ONP was converted by a nitrophenol oxygenase to nitrite and catechol. Catechol was then attacked by a catechol 1,2-dioxygenase and further degraded through an ortho-cleavage pathway. ONP derivatives which were para-substituted with a methyl-, chloro-, carboxy-, formyl- or nitro-group failed to support growth of strain B2. Relevant catabolic enzymes were characterized to analyze why these derivatives were not mineralized. Nitrophenol oxygenase of strain B2 is a soluble, NADPH-dependent enzyme that is stimulated by magnesium, manganese, and calcium ions. It is active toward ONP, 4-methyl-, 4-chloro-, and to a lesser extent, 4-formyl-ONP but not toward 4-carboxy- or 4-nitro-ONP. In addition, 4-formyl-, 4-carboxy-, and 4-nitro-ONP failed to induce the formation of nitrophenol oxygenase. Catechol 1,2-dioxygenase of strain B2 is active toward catechol and 4-methyl-catechol but only poorly active toward chlorinated catechols. 4-Methyl-catechol is likely to be degraded to methyl-lactones, which are often dead-end metabolites in bacteria. Thus, of the compounds tested, only unsubstituted ONP acts as an inducer and substrate for all of the enzymes of a productive catabolic pathway.     

Influence of para-substituents on the oxidative metabolism of o-nitrophenols by Pseudomonas putida B2

Pseudomonas putida B2 is able to grow on o-nitrophenol (ONP) as the sole source of carbon and nitrogen. ONP was converted by a nitrophenol oxygenase to nitrite and catechol. Catechol was then attacked by a catechol 1,2-dioxygenase and further degraded through an ortho-cleavage pathway. ONP derivatives which were para-substituted with a methyl-, chloro-, carboxy-, formyl- or nitro-group failed to support growth of strain B2. Relevant catabolic enzymes were characterized to analyze why these derivatives were not mineralized. Nitrophenol oxygenase of strain B2 is a soluble, NADPH-dependent enzyme that is stimulated by magnesium, manganese, and calcium ions. It is active toward ONP, 4-methyl-, 4-chloro-, and to a lesser extent, 4-formyl-ONP but not toward 4-carboxy- or 4-nitro-ONP. In addition, 4-formyl-, 4-carboxy-, and 4-nitro-ONP failed to induce the formation of nitrophenol oxygenase. Catechol 1,2-dioxygenase of strain B2 is active toward catechol and 4-methyl-catechol but only poorly active toward chlorinated catechols. 4-Methyl-catechol is likely to be degraded to methyl-lactones, which are often dead-end metabolites in bacteria. Thus, of the compounds tested, only unsubstituted ONP acts as an inducer and substrate for all of the enzymes of a productive catabolic pathway.     

Hybrid pathway for chlorobenzoate metabolism in Pseudomonas sp. B13 derivatives.

Derivatives of Pseudomonas sp. B13 which had acquired the capability to utilize 4-chloro- and 3,5-dichlorobenzoate as a consequence of the introduction of genes of the TOL plasmid of Pseudomonas putida mt-2 were studied. The utilization of these substrates, a property not shared by the parent strains, was shown to depend upon the combined activities of enzymes from the donor and from the recipient. During growth on 3-chloro-, 4-chloro-, and 3,5-dichlorobenzoate, predominantly the toluate 1,2-deoxygenase and both dihydrodihydroxybenzoate dehydrogenases of the parent strains were induced. On the other hand, no catechol 2,3-dioxygenase from P. putida mt-2 was detectable, so that degradation of chlorocatechols by the nonproductive meta-cleavage pathway was avoided. Instead of that, chlorocatechols were subject to ortho cleavage and totally degraded by the preexisting enzymes of Pseudomonas sp. B13.     

Degradation of 4-chloro-2-methylphenol by an activated sludge isolate and its taxonomic description

The Gram-negative strain S1, isolated from activated sludge, metabolized 4-chloro-2-methylphenol by an inducible pathway via a modifiedortho-cleavage route as indicated by a transiently secreted intermediate, identified as 2-methyl-4-carboxymethylenebut-2-en-4-olide by gas chromatography/mass spectrometry. Beside 4-chloro-2-methylphenol only 2,4-dichlorophenol and 4-chlorophenol were totally degraded, without an accumulation of intermediates. The chlorinated phenols tested induced activities of 2,4-dichlorophenol hydroxylase and catechol 1,2-dioxygenase type II. Phenol itself appeared to be degraded more efficiently via a separate, inducibleortho-cleavage pathway. The strain was characterized with respect to its physiological and chemotaxonomic properties. The fatty acid profile, the presence of spermidine as main polyamine, and of ubiquinone Q-10 allowed the allocation of the strain into the -2 subclass of theProteobacteria. Ochrobactrum anthropi was indicated by fatty acid analysis as the most similar organism, however, differences in a number of physiological features (e.g. absence of nitrate reduction) and pattern of soluble proteins distinguished strain S1 from this species.     

Chloromethylmuconolactones as critical metabolites in the degradation of chloromethylcatechols: recalcitrance of 2-chlorotoluene

To elucidate possible reasons for the recalcitrance of 2-chlorotoluene, the metabolism of chloromethylcatechols, formed after dioxygenation and dehydrogenation by Ralstonia sp. strain PS12 tetrachlorobenzene dioxygenase and chlorobenzene dihydrodiol dehydrogenase, was monitored using chlorocatechol dioxygenases and chloromuconate cycloisomerases partly purified from Ralstonia sp. strain PS12 and Wautersia eutropha JMP134. Two chloromethylcatechols, 3-chloro-4-methylcatechol and 4-chloro-3-methylcatechol, were formed from 2-chlorotoluene. 3-Chloro-4-methylcatechol was transformed into 5-chloro-4-methylmuconolactone and 2-chloro-3-methylmuconolactone. For mechanistic reasons neither of these cycloisomerization products can be dehalogenated by chloromuconate cycloisomerases, with the result that 3-chloro-4-methylcatechol cannot be mineralized by reaction sequences related to catechol ortho-cleavage pathways known thus far. 4-Chloro-3-methylcatechol is only poorly dehalogenated during enzymatic processing due to the kinetic properties of the chloromuconate cycloisomerases. Thus, degradation of 2-chlorotoluene via a dioxygenolytic pathway is evidently problematic. In contrast, 5-chloro-3-methylcatechol, the major dioxygenation product formed from 3-chlorotoluene, is subject to quantitative dehalogenation after successive transformation by chlorocatechol 1,2-dioxygenase and chloromuconate cycloisomerase, resulting in the formation of 2-methyldienelactone. 3-Chloro-5-methylcatechol is transformed to 2-chloro-4-methylmuconolactone.     

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.