Critical Reactions in Fluorobenzoic Acid Degradation by Pseudomonas sp. B13

3-Chlorobenzoate-grown cells of Pseudomonas sp. B13 readily cometabolized monofluorobenzoates. A catabolic pathway for the isomeric fluorobenzoates is proposed on the basis of key metabolites isolated. Only 4-fluorobenzoate was utilized and totally degraded after a short period of adaptation. The isoenzymes for total degradation of chlorocatechols, being found during growth with 3-chlorobenzoate or 4-chlorophenol, were not induced in the presence of fluorobenzoates. Correspondingly, only the ordinary enzymes of the benzoate pathway were detected in 4-fluorobenzoate-grown cells. Ring cleavage of 3-fluorocatechol was recognized as a critical step in 3-fluorobenzoate degradation. 2-Fluoro-cis,cis-muconic acid was identified as a dead-end metabolite from 2- and 3-fluorobenzoate catabolism. During 2-fluorobenzoate cometabolism, fluoride is eliminated by the initial dioxygenation.     

Degradation of 2-fluorobenzoate by a pseudomonad

Pseudomonas (spp), isolated from a complex petrochemical sludge, was able to utilize 2-fluorobenzoate as its sole source of carbon and energy. At the end of the growth phase, about 42% of the organically bound fluoride was released. Catechol, 3-fluorocatechol, and 6-fluorodihydrodihydroxybenzoate were confirmed as intermediates by chromatographic and spectral analyses. During 2-fluorobenzoate metabolism, fluoride is eliminated before the aromaticity of the ring is lost. Twofold higher levels of catechol 1,2-oxygenase were detected in 2-fluorobenzoate-grown cells compared with cells grown on benzoate. When used as assay substrates, 3-chlorocatechol showed less catechol 1,2-oxygenase activity than catechol or 4-chlorocatechol. The ability to degrade 4-fluorobenzoate could be transferred toPseudomonas (spp) by the conjugal transfer of plasmid pWR1 fromPseudomonas sp. B13.     

Evolution of chlorocatechol catabolic pathways

The aerobic bacterial degradation of chloroaromatic compounds often involves chlorosubstituted catechols as central intermediates. They are converted to 3-oxoadipate in a series of reactions similar to that for catechol catabolism and therefore designated as modifiedortho-cleavage pathway. Among the enzymes of this catabolic route, the chlorocatechol 1,2-dioxygenases are known to have a relaxed substrate specificity. In contrast, several chloromuconate cycloisomerases are more specific, and the dienelactone hydrolases of chlorocatechol catabolic pathways do not even convert the corresponding intermediate of catechol degradation, 3-oxoadipate enol-lactone. While the sequences of chlorocatechol 1,2-dioxygenases and chloromuconate cycloisomerases are very similar to those of catechol 1,2-dioxygenases and muconate cycloisomerases, respectively, the relationship between dienelactone hydrolases and 3-oxoadipate enol-lactone hydrolases is more distant. They seem to share an / hydrolase fold, but the sequences comprising the fold are quite dissimilar. Therefore, for chlorocatechol catabolism, dienelactone hydrolases might have been recruited from some other, preexisting pathway. Their relationship to dienelactone (hydrolases identified in 4-fluorobenzoate utilizing strains ofAlcaligenes andBurkholderia (Pseudomonas) cepacia is investigated). Sequence evidence suggests that the chlorocatechol catabolic operons of the plasmids pJP4, pAC27, and pP51 have been derived from a common precursor. The latter seems to have evolved for the purpose of halocatechol catabolism, and may be considerably older than the chemical industry.     

Metabolism of monofluorobenzoates by Acinetobacter calcoaceticus N.C.I.B. 8250 formation of monofluorocatechols

None of the monofluorobenzoates serves as sole source of carbon and energy for growth of Acinetobacter calcoaceticus but all can contribute to growth on other substrates. The monofluorobenzoates are oxidised by bacteria pre-induced for benzoate oxidation and can themselves induce the appropriate enzymes. The initial products of oxidation have been separated and identified by gas-liquid chromatography. 2-Fluorobenzoate is oxidised to catechol, fluoride and 3-fluorocatechol; 3-fluorobenzoate gives 3- and 4-fluorocatechol; 4-fluorobenzoate gives 4-fluorocatechol. The fluorocatechols appear to be partially oxidised beyond the stage of 3-oxoadipate by suitably pre-induced bacteria.     

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     

Evidence for an isomeric muconolactone isomerase involved in the metabolism of 4-methylmuconolactone by Alcaligenes eutrophus JMP134

An enzyme specifically induced during 4-methylmuconolactone metabolism by Alcaligenes eutrophus JMP 134 and that exhibited muconolactone isomerizing activity was purified to homogeneity. The enzyme, involved in the isomerization of 3-methylmuconolactone had a high degree of sequence similarity with muconolactone isomerase of Alcaligenes eutrophus JMP 134 and other previously described muconolactone isomerases of the 3-oxoadipate pathway. Kinetic analysis showed that the enzyme has a substrate spectrum and a reaction mechanism similar to those of the muconolactone isomerase, but that it has distinct kinetic properties. Received: 5 November 1996 / Accepted: 13 January 1997     

A gene cluster involved in degradation of substituted salicylates via ortho cleavage in Pseudomonas sp. strain MT1 encodes enzymes specifically adapted for transformation of 4-methylcatechol and 3-methylmuconate

Pseudomonas sp. strain MT1 has recently been reported to degrade 4- and 5-chlorosalicylate by a pathway assumed to consist of a patchwork of reactions comprising enzymes of the 3-oxoadipate pathway. Genes encoding the initial steps in the degradation of salicylate and substituted derivatives were now localized and sequenced. One of the gene clusters characterized (sal) showed a novel gene arrangement, with salA, encoding a salicylate 1-hydroxylase, being clustered with salCD genes, encoding muconate cycloisomerase and catechol 1,2-dioxygenase, respectively, and was expressed during growth on salicylate and chlorosalicylate. A second gene cluster (cat), exhibiting the typical catRBCA arrangement of genes of the catechol branch of the 3-oxoadipate pathway in Pseudomonas strains, was expressed during growth on salicylate. Despite their high sequence similarities with isoenzymes encoded by the cat gene cluster, the catechol 1,2-dioxygenase and muconate cycloisomerase encoded by the sal cluster showed unusual kinetic properties. Enzymes were adapted for turnover of 4-chlorocatechol and 3-chloromuconate; however, 4-methylcatechol and 3-methylmuconate were identified as the preferred substrates. Investigation of the substrate spectrum identified 4- and 5-methylsalicylate as growth substrates, which were effectively converted by enzymes of the sal cluster into 4-methylmuconolactone, followed by isomerization to 3-methylmuconolactone. The function of the sal gene cluster is therefore to channel both chlorosubstituted and methylsubstituted salicylates into a catechol ortho cleavage pathway, followed by dismantling of the formed substituted muconolactones through specific pathways.     

Degradation of 1,2,4-Trichloro- and 1,2,4,5-Tetrachlorobenzene by Pseudomonas Strains

Two Pseudomonas sp. strains, capable of growth on chlorinated benzenes as the sole source of carbon and energy, were isolated by selective enrichment from soil samples of an industrial waste deposit. Strain PS12 grew on monochlorobenzene, all three isomeric dichlorobenzenes, and 1,2,4-trichlorobenzene (1,2,4-TCB). Strain PS14 additionally used 1,2,4,5-tetrachlorobenzene (1,2,4,5-TeCB). During growth on these compounds both strains released stoichiometric amounts of chloride ions. The first steps of the catabolism of 1,2,4-TCB and 1,2,4,5-TeCB proceeded via dioxygenation of the aromatic nuclei and furnished 3,4,6-trichlorocatechol. The intermediary cis-3,4,6-trichloro-1,2-dihydroxycyclohexa-3,5-diene (TCB dihydrodiol) formed from 1,2,4-TCB was rearomatized by an NAD⁺-dependent dihydrodiol dehydrogenase activity, while in the case of 1,2,4,5-TeCB oxidation the catechol was obviously produced by spontaneous elimination of hydrogen chloride from the initially formed 1,3,4,6-tetrachloro-1,2-dihydroxycyclohexa-3,5-diene. Subsequent ortho cleavage was catalyzed by a type II catechol 1,2-dioxygenase producing the corresponding 2,3,5-trichloromuconate which was channeled into the tricarboxylic acid pathway via an ordinary degradation sequence, which in the present case included 2-chloro-3-oxoadipate. From the structure-related compound 2,4,5-trichloronitrobenzene the nitro group was released as nitrite, leaving the above metabolite as 3,4,6-trichlorocatechol. Enzyme activities for the oxidation of chlorobenzenes and halogenated metabolites were induced by both strains during growth on these haloaromatics and, to a considerable extent, during growth of strain PS12 on acetate.     

Metabolism of monofluorobenzoates by Acinetobacter calcoaceticus N.C.I.B. 8250. Formation of monofluorocatechols.

None of the monofluorobenzoates serves as sole source of carbon and energy for growth of Acinetobacter calcoaceticus but all can contribute to growth on other substrates. The monofluorobenzoates are oxidised by bacteria pre-induced for benzoate oxidation and can themselves induce the appropriate enzymes. The initial products of oxidation have been separated and identified by gas-liquid chromatography. 2-Flurobenzoate is oxidised to catechol, fluoride and 3-fluorocatechol; 3-fluorobenzoate gives 3- and 4-fluorocatechol; 4-fluorobenzoate gives 4-fluorocatechol. The fluorocatechols appear to be partially oxidised beyond the stage of 3-oxoadipate by suitably pre-induced bacteria.     

Different types of dienelactone hydrolase in 4-fluorobenzoate-utilizing bacteria

Of various benzoate-utilizing bacteria tested, Alcaligenes eutrophus 335, A. eutrophus H16, A. eutrophus JMP222, A. eutrophus JMP134, Alcaligenes strain A7, and Pseudomonas cepacia were able to grow with 4-fluorobenzoate as the sole source of carbon and energy. P. cepacia also utilizes 3-fluorobenzoate. Except for A. eutrophus JMP134, which is known to grow with 2,4-dichlorophenoxyacetate and 3-chlorobenzoate (R. H. Don and J. M. Pemberton, J. Bacteriol. 145:681-686, 1981), the strains were unable to grow at the expense of these compounds or 4-chlorobenzoate. Assays of cell extracts revealed that all strains express dienelactone hydrolase and maleylacetate reductase activities in addition to enzymes of the catechol branch of the 3-oxoadipate pathway when growing with 4-fluorobenzoate. Induction of dienelactone hydrolase and maleylacetate reductase apparently is not necessarily connected to synthesis of catechol 1,2-dioxygenase type II and chloromuconate cycloisomerase activities, which are indispensable for the degradation of chlorocatechols. Substrate specificities of the dienelactone hydrolases provisionally differentiate among three types of this activity. (i) Extracts of A. eutrophus 335, A. eutrophus H16, A. eutrophus JMP222, and Alcaligenes strain A7 convert trans-4-carboxymethylenebut-2-en-4-olide (trans-dienelactone) much faster than the cis-isomer (type I). (ii) The enzyme present in P. cepacia shows the opposite preference for the isomeric substrates (type II). (iii) Cell extracts of A. eutrophus JMP134, as well as purified dienelactone hydrolase from Pseudomonas strain B13 (E. Schmidt and H.-J. Knackmuss, Biochem. J. 192:339-347, 1980), hydrolyze both dienelactones at rates that are of the same order of magnitude (type III). This classification implies that A. eutrophus JMP134 possesses at least two different dienelactone hydrolases, one of type III encoded by the plasmid pJP4 and one of type I, which is also present in the cured strain JMP222.     

Microbial degradation of alkylbenzenesulphonates. Metabolism of homologues of short alkyl-chain length by an Alcaligenes sp

1. An organism isolated from sewage and identified as an Alcaligenes sp. utilized benzenesulphonate, toluene-p-sulphonate or phenylethane-p-sulphonate as sole source of carbon and energy for growth. Higher alkylbenzenesulphonate homologues and the hydrocarbons, benzene, toluene, phenylethane and 1-phenyldodecane were not utilized. 2. 2-Phenylpropanesulphonate was metabolized to 4-isopropylcatechol. 3. 1-Phenylpropanesulphonate was metabolized to an ortho-diol, which was tentatively identified, in the absence of an authentic specimen, as 4-n-propylcatechol. 4. In the presence of 4-isopropylcatechol, which inhibited catechol 2,3-dioxygenase, 4-ethylcatechol accumulated in cultures growing on phenylethane-p-sulphonate. 5. Authentic samples of catechol, 3-methylcatechol, 4-methylcatechol, 4-ethylcatechol and 3-isopropylcatechol were oxidized by heat-treated extracts to the corresponding 2-hydroxyalkylmuconic semialdehydes. Ring cleavage occurred between C-2 and C-3. 6. The catechol derived from 1-phenylpropanesulphonate was oxygenated by catechol 2,3-dioxygenase to a compound with all the properties of a 2-hydroxyalkylmuconic semialdehyde, but it was not rigorously identified. 7. The catechol 2,3-dioxygenase induced by growth on benzenesulphonate, toluene-p-sulphonate or phenylethane-p-sulphonate showed a constant ratio of specific activities with catechol, 3-methylcatechol, 4-methylcatechol and 4-ethylcatechol that was independent of the growth substrate. At 60°C, activity towards these substrates declined at an identical first-order rate. 8. Enzymes of the `ortho' pathway of catechol metabolism were present in small amounts in cells grown on benzenesulphonate, toluene-p-sulphonate or phenylethane-p-sulphonate. 9. The catechol 1,2-dioxygenase oxidized the alkylcatechols, but the rates and the total extents of oxidation were less than for catechol itself. The oxidation products of these alkylcatechols were not further metabolized.     

Analysis of bacterial degradation pathways for long-chain alkylphenols involving phenol hydroxylase, alkylphenol monooxygenase and catechol dioxygenase genes

Eighteen 4-t-octylphenol-degrading bacteria were isolated and screened for the presence of degradative genes by polymerase chain reaction method using four designed primer sets. The primer sets were designed to amplify specific fragments from multicomponent phenol hydroxylase, single component monooxygenase, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase genes. Seventeen of the 18 isolates exhibited the presence of a 232 bp amplicon that shared 61-92% identity to known multicomponent phenol hydroxylase gene sequences from short and/or medium-chain alkylphenol-degrading strains. Twelve of the 18 isolates were positive for a 324 bp region that exhibited 78-95% identity to the closest published catechol 1,2-dioxygenase gene sequences. The two strains, Pseudomonas putida TX2 and Pseudomonas sp. TX1, contained catechol 1,2-dioxygenase genes also have catechol 2,3-dioxygenase genes. Our result revealed that most of the isolated bacteria are able to degrade long-chain alkylphenols via multicomponent phenol hydroxylase and the ortho-cleavage pathway.     

Degradation of guaiacol glyceryl ether (GGE) by Bacillus subtilis

Summary Bacillus subtilis utilized guaiacol glyceryl ether (GGE) as sole carbon source and catabolized it via guaiacol and catechol. Cell free extracts of GGE grown cells contained high levels of catechol 1,2-dioxygenase and cleaved catechol via the ortho pathway. Nutrients such as sugars, organic acids, methanol, nitrogen and phosphate influenced the catabolism of GGE by B. subtilis.     

Chemical structure and biodegradability of halogenated aromatic compounds substituent effects on dehydrogenation of 3,5-cyclohexadiene-1,2,-diol-1-carboxylic acid

The dehydrogenation of substituted 3,5-cyclohexadiene-1,2-diol-1-carboxylic acids by dihydrodihydroxybenzoic acid dehydrogenases from benzoate grown cells of Alcaligenes eutrophus and Pseudomonas sp. B 13 and 3 -chlorobenzoate grown cells of the latter organism was examined. No significant differences (K_m and V_rel values) were detected for the enzymes from both organisms. The same dihydrodihydroxybenzoic acid dehydrogenase is formed in Pseudomonas sp. B13 during growth on benzoate as well as on 3-chlorobenzoate. The lower turnover rates of 3- and 5-chlorodihydrodihydroxybenzoic acid compared to dihydrodihydroxybenzoic acid are counterbalanced by an increase in specific activity. With the exception of 4-substituted dihydrodihydroxybenzoic acids exhibiting relative high K_m values, only slight sterical and electronic substituent effects are evident. Reaction rates were never reduced to a critical level.     

Biodegradation of 3-nitrotoluene by Rhodococcus sp. strain ZWL3NT

A pure bacterial culture was isolated by its ability to utilize 3-nitrotoluene (3NT) as the sole source of carbon, nitrogen, and energy for growth. Analysis of its 16S rRNA gene showed that the organism (strain ZWL3NT) belongs to the genus Rhodococcus. A rapid disappearance of 3NT with concomitant release of nitrite was observed when strain ZWL3NT was grown on 3NT. The isolate also grew on 2-nitrotoluene, 3-methylcatechol and catechol. Two metabolites, 3-methylcatechol and 2-methyl-cis,cis-muconate, in the reaction mixture were detected after incubation of cells of strain ZWL3NT with 3NT. Enzyme assays showed the presence of both catechol 1,2-dioxygenase and catechol 2,3-dioxygenase in strain ZWL3NT. In addition, a catechol degradation gene cluster (catRABC cluster) for catechol ortho-cleavage pathway was cloned from this strain and cell extracts of Escherichia coli expressing CatA and CatB exhibited catechol 1,2-dioxygenase activity and cis,cis-muconate cycloisomerase activity, respectively. These experimental evidences suggest a novel pathway for 3NT degradation with 3-methylcatechol as a key metabolite by Rhodococcus sp. strain ZWL3NT.     

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.     

Production of cis,cis-muconate from benzoate and 2-fluoro-cis,cis-muconate from 3-fluorobenzoate by 3-chlorobenzoate degrading bacteria

Summary 3-Chlorobenzoate grown cells of Pseudomonas sp. strain B13 or Alcaligenes sp. strain A7-2 converted 3-fluorobenzoate to 2-fluoro-cis,cis-muconate with 87% yield. The latter strain produced 1.6 g/l. The type II muconate cycloisomerases of neither strain exhibit acitivity for 2-fluoro-cis,cis-muconate. Succinate grown cells of Pseudomonas sp. strain B13 converted benzoate to cis,cis-muconate (91% yield; 7.4 g/l). Enzyme tests confirmed that no muconate cycloisomerising enzyme was induced within 24 h.     

Conversion of 2-chloromaleylacetate in Alcaligenes eutrophus JMP134

2,4-Dichlorophenoxyacetate (2,4-D) in Alcaligenes eutrophus JMP134 (pJP4) is degraded via 2-chloromaleylacetate as an intermediate. The latter compound was found to be reduced by NADH in a maleylacetate reductase catalyzed reaction. Maleylacetate and chloride were formed as products of 2-chloromaleylacetate reduction, the former being funnelled into the 3-oxoadipate pathway by a second reductive step. There was no indication for an involvement of a pJP4-encoded enzyme in either the reduction or the dechlorination reaction.Abbreviations 2,4-D 2,4-dichlorophenoxyacetate     

Maleylacetate reductase of Pseudomonas sp. strain B13: dechlorination of chloromaleylacetates,metabolites in the degradation of chloroaromatic compounds

The maleylacetate reductase of 3-chlorobenzoate-grown cells of Pseudomonas sp. strain B13 has been purified 50-fold. The enzyme converted 2-chloromaleylacetate to 3-oxoadipate with temporary occurrence of maleylacetate; 1 mol of chloride was eliminated during the conversion of 1 mol of 2-chloro- and 2,3-dichloromaleylacetate; 2 mol of NADH were consumed per mol of 2-chloro- and 2,3-dichloromaleylacetate while only 1 mol was necessary to catalyze the conversion of maleylacetate or 2-methylmaleylacetate. The maleylacetate reductase failed to use fumarylacetate as a substrate. The role of the enzyme in the chloroaromatics degradation is discussed.     

Characterization of a Protocatechuate Catabolic Gene Cluster from Rhodococcus opacus 1CP: Evidence for a Merged Enzyme with 4-Carboxymuconolactone-Decarboxylating and 3-Oxoadipate Enol-Lactone-Hydrolyzing Activity

The catechol and protocatechuate branches of the 3-oxoadipate pathway, which are important for the bacterial degradation of aromatic compounds, converge at the common intermediate 3-oxoadipate enol-lactone. A 3-oxoadipate enol-lactone-hydrolyzing enzyme, purified from benzoate-grown cells of Rhodococcus opacus (erythropolis) 1CP, was found to have a larger molecular mass under denaturing conditions than the corresponding enzymes previously purified from γ-proteobacteria. Sequencing of the N terminus and of tryptic peptides allowed cloning of the gene coding for the 3-oxoadipate enol-lactone hydrolase by using PCR with degenerate primers. Sequencing showed that the gene belongs to a protocatechuate catabolic gene cluster. Most interestingly, the hydrolase gene, usually termed pcaD, was fused to a second gene, usually termed pcaC, which encodes the enzyme catalyzing the preceding reaction, i.e., 4-carboxymuconolactone decarboxylase. The two enzymatic activities could not be separated chromatographically. At least six genes of protocatechuate catabolism appear to be transcribed in the same direction and in the following order: pcaH and pcaG, coding for the subunits of protocatechuate 3,4-dioxygenase, as shown by N-terminal sequencing of the subunits of the purified protein; a gene termed pcaB due to the homology of its gene product to 3-carboxy-cis,cis-muconate cycloisomerases; pcaL, the fused gene coding for PcaD and PcaC activities; pcaR, presumably coding for a regulator of the IclR-family; and a gene designated pcaF because its product resembles 3-oxoadipyl coenzyme A (3-oxoadipyl-CoA) thiolases. The presumed pcaI, coding for a subunit of succinyl-CoA:3-oxoadipate CoA-transferase, was found to be transcribed divergently from pcaH.