Specificity of aFlavobacterium in the metabolism of substituted chlorobenzoates

Summary A strain ofFlavobacterium breve capable of utilizing 3,5-dichlorosalicylate as a sole source of carbon and energy was identified. Degradation of 3,5-dichlorosolicylate, was specific as this strain did not metabolize dicamba (3,6-dichloro-2-methoxybenzoic acid), 3,5-dicamba (3,5-dichloros2-methoxybenzoic acid), or 3,6-dichlorosalicylate. The organism was able to remove completely 3,5-dichlorosalicylate in the presence of three times as much 3,6-dichlorosalicylate being degraded. The organism was able to utilize 3,5-dichlorosalicylate at concentrations up to 1000g/ml. A mixture of 3,5 and 3,6-dichlorosalicylate isomers purified by biological destruction of the unwanted isomer (3,5-dichlorosalicylate) would be useful for producing isomerically pure dicamba, an important herbicide.     

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     

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.     

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.     

Production of halogenated compounds byBjerkandera adusta

The white-rot fungusBjerkandera adusta produces volatile chlorinated phenyl compounds. The main compounds identified were 3-chloro-4-methoxybenzaldehyde (3-chloro-p-anisaldehyde), 3-chloro-4-methoxybenzyl alcohol (3-chloro-p-anisyl alcohol), 3,5-dichloro-4-methoxybenzaldehyde (3,5-dichloro-p-anisaldehyde), and 3,5-dichloro, 4-methoxybenzyl alcohol (3,5-dichloro-p-anisyl alcohol).p-Anisaldehyde, veratraldehyde and the corresponding alcohols,p-anisyl alcohol and veratryl alcohol were produced simultaneously. Even with a very low concentration of chloride in the medium (< 10^–5 m), chlorinated aromatic compounds were still observed. Addition of bromide to the culture medium led to the production of brominated compounds: 3-bromo-4-methoxybenzaldehyde, 3-bromo-4-methoxybenzyl alcohol, 3,5-dibromo-4-methoxybenzaldehyde and 3-bromo-5-chloro-4-methoxybenzaldehyde. These brominated compounds have not previously been reported as natural products. Although iodo-aromatic compounds were not produced by supplementation of the medium with iodide, isovanillin was found in the culture broth under these conditions. This compound may be formed by substitution of the iodine intermediate by a hydroxyl group on the third carbon of the ring. Diiodomethane or chloroiodomethane were also found. It is the first time that the production of halomethane has been related to the production of halogenated aromatic compounds. All the strains tested have these capabilities.     

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

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

Halogenated tyrosines from the cuticle of Limulus polyphemus (L.)

Several halogenated tyrosines have been identified in acid hydrolysates of Limulus polyphemus (L.) cuticle: 3-chlorotyrosine, 3-bromotyrosine, 3,5-dichlorotyrosine, 3-chloro-5-bromotyrosine and 3,5-dibromotyrosine. Tryptophan could be isolated from the cuticle when it was hydrolyzed under basic conditions.     

Enzymatic dehalogenation of chlorinated nitroaromatic compounds.

4-Chlorobenzoate dehalogenase from Pseudomonas sp. strain CBS3 converted 4-chloro-3,5-dinitrobenzoate to 3,5-dinitro-4-hydroxybenzoate and 1-chloro-2,4-dinitrobenzene to 2,4-dinitrophenol. The activities were 0.13 mU/mg of protein for 4-chloro-3,5-dinitrobenzoate and 0.16 mU/mg of protein for 1-chloro-2,4-dinitrobenzene compared with 0.5 mU/mg of protein for 4-chlorobenzoate.     

Degradation of 2-methylaniline and chlorinated isomers of 2-methylaniline by Rhodococcus rhodochrous strain CTM

Rhodococcus rhodochrous strain CTM co-metabolized 2-methylaniline and some of its chlorinated isomers in the presence of ethanol as additional carbon source. Degradation of 2-methylaniline proceeded via 3-methylcatechol, which was metabolized mainly by meta-cleavage. In the case of 3-chloro-2-methylaniline, however, only a small proportion (about 10%) was subjected to meta-cleavage; the chlorinated meta-cleavage product was accumulated in the culture fluid as a dead-end metabolite. In contrast, 4-chloro-2-methylaniline was degraded via ortho-cleavage exclusively. Enzyme assays showed the presence of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase as inducible enzymes in strain CTM. Extended cultivation of strain CTM with 2-methylaniline and 3-chloro-2-methylaniline yielded mutants, including R. rhodochrous strain CTM2, that had lost catechol 2,3-dioxygenase activity; these mutants degraded the aromatic amines exclusively via the ortho-cleavage pathway. DNA hybridization experiments using a gene probe revealed the loss of the catechol 2,3-dioxygenase gene from strain CTM2.     

Enzymatic dehalogenation of chlorinated nitroaromatic compounds

4-Chlorobenzoate dehalogenase from Pseudomonas sp. strain CBS3 converted 4-chloro-3,5-dinitrobenzoate to 3,5-dinitro-4-hydroxybenzoate and 1-chloro-2,4-dinitrobenzene to 2,4-dinitrophenol. The activities were 0.13 mU/mg of protein for 4-chloro-3,5-dinitrobenzoate and 0.16 mU/mg of protein for 1-chloro-2,4-dinitrobenzene compared with 0.5 mU/mg of protein for 4-chlorobenzoate.     

Metabolism of and inhibition by chlorobenzoates in Pseudomonas putida P111.

Pseudomonas putida P111 was isolated by enrichment culture on 2,5-dichlorobenzoate and was also able to grow on 2-chloro-, 3-chloro-, 4-chloro-, 2,3-dichloro-, 2,4-dichloro-, and 2,3,5-trichlorobenzoates. However, 3,5-dichlorobenzoate completely inhibited growth of P111 on all ortho-substituted benzoates that were tested. When 3,5-dichlorobenzoate was added as a cosubstrate with either 3- or 4-chlorobenzoate, cell yields and chloride release were greater than those observed from growth on either monochlorobenzoate alone. Moreover, resting cells of P111 grown on 4-chlorobenzoate released chloride from 3,5-dichlorobenzoate and produced no identifiable intermediate. In contrast, resting cells grown on 2,5-dichlorobenzoate metabolized 3,5-dichlorobenzoate without release of chloride and accumulated a degradation product, which was identified as 1-carboxy-1,2-dihydroxy-3,5-dichlorocyclohexadiene on the basis of gas chromatography-mass spectrometry confirmation of its two acid-hydrolyzed products, 3,5- and 2,4-dichlorophenol. Since 3,5-dichlorocatechol was rapidly metabolized by cells grown on 2,5-dichlorobenzoate, it is apparent that 1-carboxy-1,2-dihydroxy-3,5-dichlorocyclohexadiene is not further metabolized by these cells. Moreover, induction of a functional dihyrodiol dehydrogenase would not be required for growth of P111 on other ortho-chlorobenzoates since the corresponding chlorodihydrodiols produced from a 1,2-dioxygenase attack would spontaneously decompose to the corresponding catechols. In contrast, growth on 3-chloro-, 4-chloro-, or 3,5-dichlorobenzoate requires a functional dihydrodiol dehydrogenase, yet only the two monochlorobenzoates appear to induce for it.     

Metabolism of and inhibition by chlorobenzoates in Pseudomonas putida P111.

Pseudomonas putida P111 was isolated by enrichment culture on 2,5-dichlorobenzoate and was also able to grow on 2-chloro-, 3-chloro-, 4-chloro-, 2,3-dichloro-, 2,4-dichloro-, and 2,3,5-trichlorobenzoates. However, 3,5-dichlorobenzoate completely inhibited growth of P111 on all ortho-substituted benzoates that were tested. When 3,5-dichlorobenzoate was added as a cosubstrate with either 3- or 4-chlorobenzoate, cell yields and chloride release were greater than those observed from growth on either monochlorobenzoate alone. Moreover, resting cells of P111 grown on 4-chlorobenzoate released chloride from 3,5-dichlorobenzoate and produced no identifiable intermediate. In contrast, resting cells grown on 2,5-dichlorobenzoate metabolized 3,5-dichlorobenzoate without release of chloride and accumulated a degradation product, which was identified as 1-carboxy-1,2-dihydroxy-3,5-dichlorocyclohexadiene on the basis of gas chromatography-mass spectrometry confirmation of its two acid-hydrolyzed products, 3,5- and 2,4-dichlorophenol. Since 3,5-dichlorocatechol was rapidly metabolized by cells grown on 2,5-dichlorobenzoate, it is apparent that 1-carboxy-1,2-dihydroxy-3,5-dichlorocyclohexadiene is not further metabolized by these cells. Moreover, induction of a functional dihyrodiol dehydrogenase would not be required for growth of P111 on other ortho-chlorobenzoates since the corresponding chlorodihydrodiols produced from a 1,2-dioxygenase attack would spontaneously decompose to the corresponding catechols. In contrast, growth on 3-chloro-, 4-chloro-, or 3,5-dichlorobenzoate requires a functional dihydrodiol dehydrogenase, yet only the two monochlorobenzoates appear to induce for it.     

Metabolism of 3-chloro-, 4-chloro-, and 3,5-dichlorobenzoate by a pseudomonad.

Pseudomonas sp. WR912 was isolated by continuous enrichment in three steps with 3-chloro-, 4-chloro-, and finally 3,5-dichlorobenzoate as sole source of carbon and energy. The doubling times of the pure culture with these growth substrates were 2.6, 3.3, and 5.2 h, respectively. Stoichiometric amounts of chloride were eliminated during growth. Oxygen uptake rates with chlorinated benzoates revealed low stereospecificity of the initial benzoate 1,2-dioxygenation. Dihydrodi-hydroxybenzoate dehydrogenase, catechol 1,2-dixoygenase, and muconate cycloisomerase activities were found in cell-free extracts. The ortho cleavage activity for catechols appeared to involve induction of isoenzymes with different stereospecificity towards chlorocatechols. A catabolic pathway for chlorocatechols was proposed on the basis of similarity to chlorophenoxyacetate catabolism, and cometabolism of 3,5-dimethylbenzoate by chlorobenzoate-induced cells yielded 2,5-dihydro-2,4-dimethyl-5-oxo-furan-2-acetic acid.     

Metabolism of 3-chloro-, 4-chloro-, and 3,5-dichlorobenzoate by a pseudomonad.

Pseudomonas sp. WR912 was isolated by continuous enrichment in three steps with 3-chloro-, 4-chloro-, and finally 3,5-dichlorobenzoate as sole source of carbon and energy. The doubling times of the pure culture with these growth substrates were 2.6, 3.3, and 5.2 h, respectively. Stoichiometric amounts of chloride were eliminated during growth. Oxygen uptake rates with chlorinated benzoates revealed low stereospecificity of the initial benzoate 1,2-dioxygenation. Dihydrodi-hydroxybenzoate dehydrogenase, catechol 1,2-dixoygenase, and muconate cycloisomerase activities were found in cell-free extracts. The ortho cleavage activity for catechols appeared to involve induction of isoenzymes with different stereospecificity towards chlorocatechols. A catabolic pathway for chlorocatechols was proposed on the basis of similarity to chlorophenoxyacetate catabolism, and cometabolism of 3,5-dimethylbenzoate by chlorobenzoate-induced cells yielded 2,5-dihydro-2,4-dimethyl-5-oxo-furan-2-acetic acid.     

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.     

Pseudomonas aeruginosa strain RW41 mineralizes 4-chlorobenzenesulfonate, the major polar by-product from DDT manufacturing

Pseudomonas aeruginosa RW41 is the first bacterial strain, which could be isolated by virtue of its capability to mineralize 4-chlorobenzenesulfonic acid (4CBSA), the major polar by-product of the chemical synthesis of 1,1,1-trichloro-2,2-bis-(4-chlorophenyl)ethane (DDT). This capability makes the isolate a promising candidate for the development of bioremediation technologies. The bacterial mineralization of 4CBSA proceeds under oxygenolytic desulfonation and transient accumulation of sulfite which then is oxidized to sulfate. High enzyme activities for the turnover of 4-chlorocatechol were measured. The further catabolism proceeded through 3-chloromuconate and, probably, the instable 4-chloromuconolactone, which is directly hydrolyzed to maleylacetate. Detectable levels of maleylacetate reductase were only present when cells were grown with 4CBSA. When the ordinary catechol pathway was induced during growth on benzenesulfonate, catechol was ortho-cleaved to cis,cis-muconate and a partially purified muconate cycloisomerase transformed it to muconolactone in vitro. The same enzyme transformed 3-chloro-cis,cis-muconate into cis-dienelactone (76%) and the antibiotically active protoanemonin (24%). These observations are indicative for a not yet highly evolved catabolism for halogenated substrates by bacterial isolates from environmental samples which, on the other hand, are able to productively recycle sulfur and chloride ions from synthetic haloorganosulfonates.     

Degradation of 2-bromobenzoic acid by a strain of Pseudomonas aeruginosa

A strain of Pseudomonas aeruginosa producing 2-bromobenzoic acid, designated 2-BBZA, was isolated by enrichment culture from municipal sewage. It degraded all four 2-halobenzoates as well as certain 3-halo- and dihalobenzoates, though none of the 4-halobenzoates supported growth of this organism. 3-Hydroxybenzoate and 3-chlorocatechol were respective inhibitors of salicylate and catechol oxidation: when each was added separately to resting cells incubated with 2-bromobenzoate, salicylate and catechol were found. Oxygen uptake data suggest that the same dehalogenase may be involved in the oxidation of 2-bromo-, 2-chloro-, and 2-iodobenzoates.     

Degradation of 2-bromobenzoic acid by a strain of Pseudomonas aeruginosa.

A strain of Pseudomonas aeruginosa producing 2-bromobenzoic acid, designated 2-BBZA, was isolated by enrichment culture from municipal sewage. It degraded all four 2-halobenzoates as well as certain 3-halo- and dihalobenzoates, though none of the 4-halobenzoates supported growth of this organism. 3-Hydroxybenzoate and 3-chlorocatechol were respective inhibitors of salicylate and catechol oxidation: when each was added separately to resting cells incubated with 2-bromobenzoate, salicylate and catechol were found. Oxygen uptake data suggest that the same dehalogenase may be involved in the oxidation of 2-bromo-, 2-chloro-, and 2-iodobenzoates.     

Transformations of Halogenated Aromatic Aldehydes by Metabolically Stable Anaerobic Enrichment Cultures

Metabolically stable enrichment cultures of anaerobic bacteria obtained by elective enrichment of sediment samples from the Baltic Sea and Gulf of Bothnia have been used to study the oxidation and reduction of the aldehyde group of various halogenated aromatic aldehydes. During the transformation of 5- and 6-chlorovanillin, 6-bromovanillin, 3-chloro-4-hydroxybenzaldehyde, 3,5-dichloro-4-hydroxybenzaldehyde, and 3,5-dibromo-4-hydroxybenzaldehyde, it was shown that synthesis of the corresponding carboxylic acids, which were the principal metabolites, was invariably accompanied by partial reduction of the aldehyde to a hydroxymethyl group in yields of between 3 and 30%. Complete reduction to a methyl group was observed with some of the halogenated vanillins, but to an extremely limited extent with the halogenated 4-hydroxybenzaldehydes. One consortium produced both the hydroxymethyl and methyl compounds from both 5- and 6-chlorovanillin: it was therefore assumed that the methyl compound was the ultimate reduction product. On the basis of the kinetics of formation of the metabolites, it was concluded that the oxidation and reduction reactions were mechanistically related. In addition to these oxidations and reductions, dehalogenation was observed with one of the consortia. In contrast to the transformations of 5- and 6-chlorovanillin, which produced chlorinated methylcatechols, the corresponding compounds were not observed with 5- and 6-bromovanillin: the former was debrominated, forming 4-methylcatechol, whereas the latter produced 6-bromovanillyl alcohol without demethylation. Similarly, although 3-chloro-4-hydroxybenzaldehyde formed the chlorinated carboxylic acid and the benzyl alcohol, the 3-bromo compound was debrominated with formation of 4-hydroxybenzoic acid and, ultimately, phenol. On prolonged incubation, the halogenated carboxylic acids were generally decarboxylated, so that the final products from these substrates were halogenated catechols or phenols. Reductive processes of the type revealed in this study might therefore plausibly occur in the environment during anaerobic transformation of halogenated aromatic aldehydes containing hydroxyl and/or methoxyl groups.     

Co-metabolism of methyl- and chloro-substituted catechols by an Achromobacter sp. possessing a new meta-cleaving oxygenase

Co-metabolism of 3-methylcatechol, 4-chlorocatechol and 3,5-dichlorocatechol by an Achromobacter sp. was shown to result in the accumulation of 2-hydroxy-3-methylmuconic semialdehyde, 4-chloro-2-hydroxymuconic semialdehyde and 3,5-dichloro-2-hydroxymuconic semialdehyde respectively. Formation of these products indicated that cleavage of the aromatic nucleus of the substituted catechols was accomplished by a new meta-cleaving enzyme, catechol 1,6-oxygenase. This enzyme was equally active on both chloro- and methyl-substituted catechols.