Reductive dechlorination of 2,4-dichlorobenzoate to 4-chlorobenzoate and hydrolytic dehalogenation of 4-chloro-, 4-bromo-, and 4-iodobenzoate by Alcaligenes denitrificans NTB-1.

Alcaligenes denitrificans NTB-1, previously isolated on 4-chlorobenzoate, also utilized 4-bromo-, 4-iodo-, and 2,4-dichlorobenzoate but not 4-fluorobenzoate as a sole carbon and energy source. During growth, stoichiometric amounts of halide were released. Experiments with whole cells and cell extracts revealed that 4-bromo- and 4-iodobenzoate were metabolized like 4-chlorobenzoate, involving an initial hydrolytic dehalogenation yielding 4-hydroxybenzoate, which in turn was hydroxylated to 3,4-dihydroxybenzoate. The initial step in the metabolism of 2,4-dichlorobenzoate was catalyzed by a novel type of reaction for aerobic organisms, involving inducible reductive dechlorination to 4-chlorobenzoate. Under conditions of low and controlled oxygen concentrations, A. denitrificans NTB-1 converted all 4-halobenzoates and 2,4-dichlorobenzoate almost quantitatively to 4-hydroxybenzoate.     

NADPH-dependent reductive ortho dehalogenation of 2,4-dichlorobenzoic acid in Corynebacterium sepedonicum KZ-4 and Coryneform bacterium strainNTB-1 via 2,4-dichlorobenzoyl coenzyme A.

Corynebacterium sepedonicum KZ-4, described earlier as a strain capable of growth on 2,4-dichlorobenzoate (G.M. Zaitsev and Y.N. Karasevich, Mikrobiologiya 54:356-369, 1985), is known to metabolize this substrate via 4-hydroxybenzoate and protocatechuate, and evidence consistent with an initial reductive dechlorination step to form 4-chlorobenzoate was found in another coryneform bacterium, strain NTB-1 (W.J.J. van den Tweel, J.B. Kok, and J.A.M. de Bont, Appl. Environ. Microbiol. 53:810-815, 1987). 2-Chloro-4-fluorobenzoate was found to be converted stoichiometrically to 4-fluorobenzoate by resting cells of strain KZ-4, compatible with a reductive process. Experiments with cell extracts demonstrated that Mg - ATP and coenzyme A (CoA) were required to stimulate reductive dehalogenation, consistent with the intermediacy of 2-chloro-4-fluoro-benzoyl-CoA and 2,4-dichlorobenzoyl-CoA thioesters. 2,4-Dichlorobenzoyl-CoA was shown to be converted to 4-chlorobenzoyl-CoA in a novel NADPH-dependent reaction in extracts of both KZ-4 and NTB-1. In addition to the ligase and reductive dehalogenase activities, hydrolytic 4-chlorobenzoyl-CoA dehalogenase and thioesterase activities, 4-hydroxybenzoate 3-monooxygenase, and protocatechuate 3,4-dioxygenase activities were demonstrated to be present in the soluble fraction of KZ-4 extracts following ultracentrifugation. We propose that the pathway for 2,4-dichlorobenzoate catabolism in strains KZ-4 and NTB-1 involves formation of 2,4-dichlorobenzoyl-CoA, NADPH-dependent ortho dehalogenation yielding 4-chlorobenzoyl-CoA, hydrolytic removal of chlorine from the para position to generate 4-hydroxybenzoyl-CoA, hydrolysis to form 4-hydroxybenzoate, oxidation to yield protocatechuate, and oxidative ring cleavage.     

Bioformation of 4-hydroxybenzoate from 4-chlorobenzoate by Alcaligenes denitrificans NTB-1

Summary Strain NTB-1, identified as a Alcaligenes denitrificans sp., was isolated from a mixture of soil and sewage samples using 4-chlorobenzoate as sole carbon and energy source. Simultaneous adaptation experiments and enzyme studies revealed that 4-chlorobenzoate was converted to 4-hydroxybenzoate which was further oxidized yielding 3,4-dihydroxybenzoate. Bioformation of 4-hydroxybenzoate from 4-chlorobenzoate when 4-chlorobenzoate-grown cells were incubated with 4-chlorobenzoate under conditions of low and controlled oxygen concentrations.     

Kinetic Aspects Of The Bioconversion Of 4-Chlorobenzoate To 4-Hydroxybenzoate By Alcaligenes Denitrificans Ntb-1 Immobilized In Carrageenan

Alcaligenes denitrificans NTB-1 converts 4-chlorobenzoate to 4-hydroxybenzoate by means of a hydrolytic dehalogenation. In the presence of oxygen the product is further metabolized by the cells. Although it has been shown that the dehalogenation does not require oxygen, no bioproduction of 4-hydroxybenzoate was achieved by whole cells when oxygen was absent. An energy-dependent active transport may explain this anomaly. Little activity was lost after immobilization of whole cells in carrageenan. Diffusion of oxygen within the carrageenan beads rapidly limited the rate of decolorization. This effect was magnified with increasing bead diameters and cell loadings. External transport of oxygen, on the other hand, did not decrease the reaction rate, except at extremely low oxygen concentrations. Maximal dehalogenation activity was observed at 35°C and at pH 8.0 and this was independent of whether free or immobilized cells were used. 4-Hydroxybenzoate significantly inhibited the rate of decolorization, while the chloride formed and the substrate 4-chlorobenzoate did not show inhibitory effects on decolorization. The yield of this bioconversion was mainly dependent on the oxygen concentration. In the case of free cells, 4-hydroxybenzoate was produced only at very low oxygen concentrations, while immobilized cells still produced 4-hydroybenzoate at air saturation as a result of oxygen diffusion limitation.     

Kinetic Aspects Of The Bioconversion Of 4-Chlorobenzoate To 4-Hydroxybenzoate By Alcaligenes Denitrificans Ntb-1 Immobilized In Carrageenan

Alcaligenes denitrificans NTB-1 converts 4-chlorobenzoate to 4-hydroxybenzoate by means of a hydrolytic dehalogenation. In the presence of oxygen the product is further metabolized by the cells. Although it has been shown that the dehalogenation does not require oxygen, no bioproduction of 4-hydroxybenzoate was achieved by whole cells when oxygen was absent. An energy-dependent active transport may explain this anomaly. Little activity was lost after immobilization of whole cells in carrageenan. Diffusion of oxygen within the carrageenan beads rapidly limited the rate of decolorization. This effect was magnified with increasing bead diameters and cell loadings. External transport of oxygen, on the other hand, did not decrease the reaction rate, except at extremely low oxygen concentrations. Maximal dehalogenation activity was observed at 35°C and at pH 8.0 and this was independent of whether free or immobilized cells were used. 4-Hydroxybenzoate significantly inhibited the rate of decolorization, while the chloride formed and the substrate 4-chlorobenzoate did not show inhibitory effects on decolorization. The yield of this bioconversion was mainly dependent on the oxygen concentration. In the case of free cells, 4-hydroxybenzoate was produced only at very low oxygen concentrations, while immobilized cells still produced 4-hydroybenzoate at air saturation as a result of oxygen diffusion limitation.     

Preliminary characterization of four 2-chlorobenzoate-degrading anaerobic bacterial consortia

Dechlorination was the initial step of 2CB biodegradation in four 2-chlorobenzoate-degrading methanogenic consortia. Selected characteristics of ortho reductive dehalogenation were examined in consortia developed from the highest actively dechlorinating dilutions of the original 2CB consortia, designated consortia M34-9, P20-9, P21-9 and M50-7. In addition to 2-chlorobenzote, all four dilution consortia dehalogenated 4 of 32 additional halogenated aromatic substrates tested, including 2-bromobenzoate; 2,6-dichlorobenzoate; 2,4-dichlorobenzoate; and 2-chloro-5-hydroxybenzoate. Dehalogenation occurred exclusively at the ortho position. Both ortho chlorines were removed from 2,6-dichlorobenzoate. Benzoate was detected from 2-bromobenzoate and 2,6-dichlorobenzoate. 4-Chlorobenzoate and 3-hydroxybenzoate were formed from 2,4-dichlorobenzoate and 2-chloro-5-hydroxybenzoate, respectively. Only benzoate was further degraded. Slightly altering the structure of the parent 'benzoate molecule' resulted in observing reductive biotransformations other than dehalogenation. 2-Chlorobenzaldehyde was reduced to 2-chlorobenzyl alcohol by all four consortia. 2-chloroanisole was O-demethoxylated by three of the four consortia forming 2-chlorophenol. GC-MS analysis indicated reduction of the double bond in the propenoic side chain of 2-chlorocinnamate forming 2-chlorohydrocinnamate. None of the reduction products was dechlorinated. The following were not dehalogenated: 3- and 4-bromobenzoate; 3- and 4-chlorobenzoate; 2-, 3-, and 4-fluorobenzoate; 2-, 3-, and 4-iodobenzoate; 2-, 3-, and 4-chlorophenol; 2-chloroaniline; 2-chloro-5-methylbenzoate; 2,3-dichlorobenzoate; 2,5-dichlorobenzoate; 2,4,5-trichlorophenoxyacetic acid; and 2,4-dichlorophenoxyacetic acid. Consortia M34-9, P20-9, P21-9, and M50-7 dechlorinated 2-chlorobenzoate at 4 mm. Dechlorination rates were highest for consortia P20-9 followed by those of M50-7with rates declining above 2 and 3mm 2CB, respectively. The major physiological types of microorganisms in consortia M34-9, P20-9, P21-9, and M50-7 were sulfate-reducing and hydrogen-utilizing anaerobes.     

Bacterial dehalogenation of chlorobenzoates and coculture biodegradation of 4,4'-dichlorobiphenyl

Acinetobacter sp. strain 4CB1 was isolated from a polychlorobiphenyl-contaminated soil sample by using 4-chlorobenzoate as a sole source of carbon and energy. Resting cells of Acinetobacter sp. strain 4CB1 hydrolytically dehalogenated 4-chlorobenzoate under aerobic and anaerobic conditions, but 4-hydroxybenzoate accumulated only under anaerobic conditions. Cell extracts of Acinetobacter sp. strain 4CB1 oxidized 4-hydroxybenzoate by an NADH-dependent monooxygenase to form protocatechuate, which was subsequently oxidized by both ortho- and meta-protocatechuate dioxygenase reactions. When grown on biphenyl, Acinetobacter sp. strain P6 cometabolized 4,4'-dichlorobiphenyl primarily to 4-chlorobenzoate; however, when this strain was grown in a coculture with Acinetobacter sp. strain 4CB1, 4-chlorobenzoate did not accumulate but was converted to inorganic chloride. When resting cells of Acinetobacter sp. strain 4CB1 were incubated anaerobically with 3,4-dichlorobenzoate, they accumulated 4-carboxy-1,2-benzoquinone as a final product. Since 3,4-dichlorobenzoate is a product that is formed from the cometabolism of 3,4-dichloro-substituted tetrachlorobiphenyls by Acinetobacter sp. strain P6, the coculture has a potential application for dehalogenation and mineralization of specific polychlorobiphenyl congeners.     

Bacterial dehalogenation of chlorobenzoates and coculture biodegradation of 4,4'-dichlorobiphenyl.

Acinetobacter sp. strain 4CB1 was isolated from a polychlorobiphenyl-contaminated soil sample by using 4-chlorobenzoate as a sole source of carbon and energy. Resting cells of Acinetobacter sp. strain 4CB1 hydrolytically dehalogenated 4-chlorobenzoate under aerobic and anaerobic conditions, but 4-hydroxybenzoate accumulated only under anaerobic conditions. Cell extracts of Acinetobacter sp. strain 4CB1 oxidized 4-hydroxybenzoate by an NADH-dependent monooxygenase to form protocatechuate, which was subsequently oxidized by both ortho- and meta-protocatechuate dioxygenase reactions. When grown on biphenyl, Acinetobacter sp. strain P6 cometabolized 4,4'-dichlorobiphenyl primarily to 4-chlorobenzoate; however, when this strain was grown in a coculture with Acinetobacter sp. strain 4CB1, 4-chlorobenzoate did not accumulate but was converted to inorganic chloride. When resting cells of Acinetobacter sp. strain 4CB1 were incubated anaerobically with 3,4-dichlorobenzoate, they accumulated 4-carboxy-1,2-benzoquinone as a final product. Since 3,4-dichlorobenzoate is a product that is formed from the cometabolism of 3,4-dichloro-substituted tetrachlorobiphenyls by Acinetobacter sp. strain P6, the coculture has a potential application for dehalogenation and mineralization of specific polychlorobiphenyl congeners.     

Anaerobic bioformation of 4-hydroxybenzoate from 4-chlorobenzoate by the coryneform bacterium NTB-1

Summary Resting cells of the coryneform strain NTB-1, previously incorrectly classified as Alcaligenes denitrificans NTB-1, quantitatively converted 4-chlorobenzoate (4-CBA) to 4-hydroxybenzoate (4-HBA) under strict anaerobic conditions in the presence of ferricyanide or nitrate. 4-HBA formation was enhanced by supplying anaerobic cells with glucose as an energy source. Using permeabilized cells it was shown that energy is not needed to drive the energy-dependent uptake of 4-CBA but also to convert 4-CBA into 4-HBA. In extracts it was subsequently demonstrated that a coenzymeA-thioester of 4-CBA is involved in the metabolism of 4-CBA. Offprint requests to: P. E. J. Groenewegen     

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

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

4-Chlorobenzoate uptake in Comamonas sp. strain DJ-12 is mediated by a tripartite ATP-independent periplasmic transporter

The fcb gene cluster involved in the hydrolytic dehalogenation of 4-chlorobenzoate is organized in the order fcbB-fcbA-fcbT1-fcbT2-fcbT3-fcbC in Comamonas sp. strain DJ-12. The genes are operonic and inducible with 4-chloro-, 4-iodo-, and 4-bromobenzoate. The fcbT1, fcbT2, and fcbT3 genes encode a transporter in the secondary TRAP (tripartite ATP-independent periplasmic) family. An fcbT1T2T3 knockout mutant shows a much slower growth rate on 4-chlorobenzoate compared to the wild type. 4-Chlorobenzoate is transported into the wild-type strain five times faster than into the fcbT1T2T3 knockout mutant. Transport of 4-chlorobenzoate shows significant inhibition by 4-bromo-, 4-iodo-, and 4-fluorobenzoate and mild inhibition by 3-chlorobenzoate, 2-chlorobenzoate, 4-hydroxybenzoate, 3-hydroxybenzoate, and benzoate. Uptake of 4-chlorobenzoate is significantly inhibited by ionophores which collapse the proton motive force.     

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

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

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.     

Cometabolism of 3,4-dichlorobenzoate by Acinetobacter sp. strain 4-CB1

When Acinetobacter sp. strain 4-CB1 was grown on 4-chlorobenzoate (4-CB), it cometabolized 3,4-dichlorobenzoate (3,4-DCB) to 3-chloro-4-hydroxybenzoate (3-C-4-OHB), which could be used as a growth substrate. No cometabolism of 3,4-DCB was observed when Acinetobacter sp. strain 4-CB1 was grown on benzoate. 4-Carboxyl-1,2-benzoquinone was formed as an intermediate from 3,4-DCB and 3-C-4-OHB in aerobic and anaerobic resting-cell incubations and was the major transient intermediate found when cells were grown on 3-C-4-OHB. The first dechlorination step of 3,4-DCB was catalyzed by the 4-CB dehalogenase, while a soluble dehalogenase was responsible for dechlorination of 3-C-4-OHB. Both enzymes were inducible by the respective chlorinated substrates, as indicated by oxygen uptake experiments. The dehalogenase activity on 3-C-4-OHB, observed in crude cell extracts, was 109 and 44 nmol of 3-C-4-OHB min-1 mg of protein-1 under anaerobic and aerobic conditions, respectively. 3-Chloro-4-hydroxybenzoate served as a pseudosubstrate for the 4-hydroxybenzoate monooxygenase by effecting oxygen and NADH consumption without being hydroxylated. Contrary to 4-CB metabolism, the results suggest that 3-C-4-OHB was not metabolized via the protocatechuate pathway. Despite the ability of resting cells grown on 4-CB or 3-C-4-OHB to carry out all of the necessary steps for dehalogenation and catabolism of 3,4-DCB, it appeared that 3,4-DCB was unable to induce the necessary 4-CB dehalogenase for the initial p-dehalogenation step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cometabolism of 3,4-dichlorobenzoate by Acinetobacter sp. strain 4-CB1.

When Acinetobacter sp. strain 4-CB1 was grown on 4-chlorobenzoate (4-CB), it cometabolized 3,4-dichlorobenzoate (3,4-DCB) to 3-chloro-4-hydroxybenzoate (3-C-4-OHB), which could be used as a growth substrate. No cometabolism of 3,4-DCB was observed when Acinetobacter sp. strain 4-CB1 was grown on benzoate. 4-Carboxyl-1,2-benzoquinone was formed as an intermediate from 3,4-DCB and 3-C-4-OHB in aerobic and anaerobic resting-cell incubations and was the major transient intermediate found when cells were grown on 3-C-4-OHB. The first dechlorination step of 3,4-DCB was catalyzed by the 4-CB dehalogenase, while a soluble dehalogenase was responsible for dechlorination of 3-C-4-OHB. Both enzymes were inducible by the respective chlorinated substrates, as indicated by oxygen uptake experiments. The dehalogenase activity on 3-C-4-OHB, observed in crude cell extracts, was 109 and 44 nmol of 3-C-4-OHB min-1 mg of protein-1 under anaerobic and aerobic conditions, respectively. 3-Chloro-4-hydroxybenzoate served as a pseudosubstrate for the 4-hydroxybenzoate monooxygenase by effecting oxygen and NADH consumption without being hydroxylated. Contrary to 4-CB metabolism, the results suggest that 3-C-4-OHB was not metabolized via the protocatechuate pathway. Despite the ability of resting cells grown on 4-CB or 3-C-4-OHB to carry out all of the necessary steps for dehalogenation and catabolism of 3,4-DCB, it appeared that 3,4-DCB was unable to induce the necessary 4-CB dehalogenase for the initial p-dehalogenation step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dehalogenation of 4-chlorobenzoate by 4-chlorobenzoate dehalogenase from pseudomonas sp. CBS3: an ATP/coenzyme A dependent reaction

Pseudomonas sp. CBS3 was grown with 4-chlorobenzoate as sole source of carbon and energy. Freshly prepared cell-free extracts converted 4-chlorobenzoate to 4-hydroxybenzoate. After storage for 16 hours at 25 degrees C only about 50% of the initial activity was left. Treatment at 55 degrees C for 10 minutes, dialysis or desalting of the extracts by gel filtration caused a total loss of the activity of the 4-chlorobenzoate dehalogenase. The activity could be restored by the addition of ATP, coenzyme A and Mg2+. If one of these cofactors was missing, no dehalogenating activity was detectable. The amount of 4-hydroxybenzoate formed was proportional to the amount of ATP available in the test system whereas CoA served as a real coenzyme. A novel ATP/coenzyme A dependent reaction mechanism for the dehalogenation of 4-chlorobenzoate by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 is proposed.     

3-Chloro-, 2,3- and 3,5-dichlorobenzoate co-metabolism in a 2-chlorobenzoate-degrading consortium: role of 3,5-dichlorobenzoate as antagonist of 2-chlorobenzoate degradation

A study was made of the metabolic and co-metabolic intermediates of 2- and 3-chlorobenzoate, 2,3- and 3,5-dichlorobenzoate to elucidate the mechanism(s) involved in the negative effects observed on the growth of a chlorobenzoate-degrading microbial consortium in the presence of mixed chlorobenzoates. 2-Chloro-muconate accumulated as the end-product in the cultural broths of the microbial consortium during growth on 2-chlorobenzoate; the same 2-chloromuconate was identified in the reaction mixtures of resting cells pre-grown on 2-chlorobenzoate and exposed to 3-chloro- and 2,3-dichlorobenzoate, while in similar experiments 1,2-dihydroxy-3,5-dichloro-cyclohexa-3,5-dienoate was detected as dead-end product of 3,5-dichlorobenzoate co-metabolism. These results suggest an initial degradative attack by 2-chlorobenzoate induced dioxygenase(s). The role of 3,5-dichlorobenzoate as an antagonist of 2-chlorobenzoate degradation was also studied: in the presence of mixed 2-chloro- and 3,5-dichlorobenzoate, the 3,5-dichlorobenzoate preferential uptake by the resting cells of the chlorobenzoate-degrading consortium was observed. 2-Chlorobenzoate entered the cells only after the complete removal of the co-substrate. In growing cells experiments, the addition of 1,2-dihydroxy-3,5-dichloro-cyclohexa-3,5-dienoate, the 3,5-dichlorobenzoate co-metabolite, to 2-chlorobenzoate exerted the same antagonistic effect of the parent compound, inhibiting both the microbial growth and the degradative process. These data are discussed, allowing us to attribute the inhibitory effects observed to a substrate/co-substrate competition, though other additional causes may not be totally excluded.     

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 both 4-chlorobenzoate and toluene under denitrifying conditions by a constructed bacterial strain.

T1, a dentrifying bacterium originally isolated for its ability to grow on toluene, can also metabolize 4-hydroxybenzoate and other aromatic compounds under denitrifying conditions. A cosmid clone carrying the three genes that code for the 4-chlorobenzoate dehalogenase enzyme complex isolated from the aerobic bacterium Pseudomonas sp. strain CBS3 was successfully conjugated into strain T1. The cloned enzyme complex catalyzes the hydrolytic dechlorination of 4-chlorobenzoate to 4-hydroxybenzoate. Since molecular oxygen is not required for the dehalogenation reaction, the transconjugate strain of T1 (T1-pUK45-10C) was able to grow on 4-chlorobenzoate in the absence of O2 under denitrifying conditions. 4-Chlorobenzoate was dehalogenated to 4-hydroxybenzoate, which was then further metabolized by strain T1. The dehalogenation and metabolism of 4-chlorobenzoate were nitrate dependent and were coupled to the production of nitrite and nitrogen gas. 4-Bromobenzoate was also degraded by this strain, while 4-iodobenzoate was not. Additionally, when T1-pUK45-10C was presented with a mixture of 4-chlorobenzoate and toluene, simultaneous degradation of the compounds was observed. These results illustrate that dechlorination and degradation of aromatic xenobiotics can be mediated by a pure culture in the absence of oxygen. Furthermore, it is possible to expand the range of xenobiotic substrates degradable by an organism, and it is possible that concurrent metabolism of these substrates can occur.