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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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)     

Enzymic Dehalogenation of 4-Chlorobenzoyl Coenzyme A in Acinetobacter sp. Strain 4-CB1

4-Chlorobenzoate degradation in cell extracts of Acinetobacter sp. strain 4-CB1 occurs by initial synthesis of 4-chlorobenzoyl coenzyme A (4-chlorobenzoyl CoA) from 4-chlorobenzoate, CoA, and ATP. 4-Chlorobenzoyl CoA is dehalogenated to 4-hydroxybenzoyl CoA. Following the dehalogenation reaction, 4-hydroxybenzoyl CoA is hydrolyzed to 4-hydroxybenzoate and CoA. Possible roles for the CoA moiety in the dehalogenation reaction are discussed.     

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)     

The coexistence in rat muscle cells of two distinct classes of Ca2+-dependent K+ channels with different pharmacological properties and different physiological functions

An Arthrobacter sp. has been shown to dehalogenate 4-chlorobenzoate yielding 4-hydroxybenzoate. Experiments with 18O indicate that, in the presence of cell-free extracts, the hydroxyl group which is substituted onto the aromatic nucleus during dehalogenation is derived from water and not from molecular oxygen. Dehalogenation therefore is not catalysed by a mixed-function oxidase; instead a novel aromatic hydroxylase is implicated in the reaction.     

Cloning of Pseudomonas sp. strain CBS3 genes specifying dehalogenation of 4-chlorobenzoate.

The degradation of 4-chlorobenzoate (4-CBA) by Pseudomonas sp. strain CBS3 is thought to proceed first by the dehalogenation of 4-CBA to 4-hydroxybenzoate (4-HBA), which is then metabolized following the protocatechuate branch of the beta-ketoadipate pathway. The cloning of the 4-CBA dehalogenation system was carried out by constructing a gene bank of Pseudomonas sp. strain CBS3 in Pseudomonas putida. Hybrid plasmid pPSA843 contains a 9.5-kilobase-pair fragment derived from the chromosome of Pseudomonas sp. strain CBS3. This plasmid confers on P. putida the ability to dehalogenate 4-CBA and grow on 4-CBA as the only source of carbon. However, pPSA843 did not complement mutants of P. putida unable to grow on 4-HBA (POB-), showing that the genes involved in the metabolism of 4-HBA were not cloned. Subcloning of Pseudomonas sp. strain CBS3 genes revealed that most of the insert is required for the dehalogenation of 4-CBA, suggesting that more than one gene product is involved in this dehalogenation.     

Enzymatic dehalogenation of 4-chlorobenzoate by extracts from Arthrobacter sp. SU DSM 20407

In extracts from Arthrobacter sp. SU DSM 20407 an enzyme was detectable, that converted 4-chlorobenzoate into 4-hydroxybenzoate. This conversion was also observed when no oxygen was present in the reaction mixture. Boiling for 5 min destroyed the enzyme activity. 4-Bromo- and 4-iodobenzoate were substrates for the enzyme too, but not 4-fluorobenzoate, 4-chlorophenylacetate and 4-chlorocinnamic acid. The enzyme showed optimum activity at 16 degrees C and at pH 7-7.5. The specific activity in the extracts varied between 0.5 and 5 mU/mg of protein. Zn2+ and Cu2+ inhibited the enzyme, while H2O2 slightly activated. In contrast to all other 4-chlorobenzoate dehalogenases described before the enzyme was not inhibited by EDTA, nor was it activated by Mn2+. Other divalent ions also had no effect. The molecular mass of the enzyme was 45,000 +/- 5,000 Da as judged by gel-filtration.     

Degradation of 4-Chlorobenzoic Acid by Arthrobacter sp

A mixed population, enriched and established in a defined medium, from a sewage sludge inoculum was capable of complete mineralization of 4-chlorobenzoate. An organism, identified as Arthrobacter sp., was isolated from the consortium and shown to be capable of utilizing 4-chlorobenzoate as the sole carbon and energy source in pure culture. This organism (strain TM-1), dehalogenated 4-chlorobenzoate as the initial step in the degradative pathway. The product, 4-hydroxybenzoate, was further metabolized via protocatechuate. The ability of strain TM-1 to degrade 4-chlorobenzoate in liquid medium at 25°C was improved by the use of continuous culture and repeated sequential subculturing. Other chlorinated benzoates and the parent compound benzoate did not support growth of strain TM-1. An active cell extract was prepared and shown to dehalogenate 4-chloro-, 4-fluoro-, and 4-bromobenzoate. Dehalogenase activity had an optimum pH of 6.8 and an optimum temperature of 20°C and was inhibited by dissolved oxygen and stimulated by manganese (Mn²⁺). Strain improvement resulted in an increase in the specific activity of the cell extract from 0.09 to 0.85 nmol of 4-hydroxybenzoate per min per mg of protein and a decrease in the doubling time of the organism from 50 to 1.6 h.     

Genetic control of degradation of chlorinated benzoic acids in Arthrobacter globiformis, Corynebacterium sepedonicum and Pseudomonas cepacia strains

The strains of Arthrobacter globiformis KZT1, Corynebacterium sepedonicum KZ4 and Pseudomonas cepacia KZ2 capable of early dehalogenation and complete oxidation of 4-chloro-, 2,4-dichloro-and 2-chlorobenzoic acids, respectively, have been analyzed for the origin of the genetic control of degradation. The occurrence and molecular sizes of plasmids in all the strains have been established. Plasmid pBS1501 was shown to control 4-chlorobenzoate dehalogenation in the case of KZT1 strain. The same possibility is proposed for plasmid pBS1502 for dehalogenation of 2,4DCBA by KZ4 strain. The chromosome localization of the genes controlling oxidation of 4-hydroxybenzoate in strain KZT1 is shown. Localization of the whole set of genes responsible for 2CBA degradation in the strain KZ2 chromosome is suggested.     

Characterization of Chloroethylene Dehalogenation by Cell Extracts of Desulfomonile tiedjei and Its Relationship to Chlorobenzoate Dehalogenation

We characterized the reductive dehalogenation of tetrachloroethylene in cell extracts of Desulfomonile tiedjei and compared it with this organism's 3-chlorobenzoate dehalogenation activity. Tetrachloroethylene was sequentially dehalogenated to trichloro- and dichloroethylene; there was no evidence for dichloroethylene dehalogenation. Like the previously characterized 3-chlorobenzoate dehalogenation activity, tetrachloroethylene dehalogenation was heat sensitive, not oxygen labile, and increased in proportion to the amount of protein in assay mixtures. In addition, both dehalogenation activities were dependent on hydrogen or formate as an electron donor and had an absolute requirement for either methyl viologen or triquat as an electron carrier in vitro. Both activities appear to be catalyzed by integral membrane proteins with similar solubilization characteristics. Dehalogenation of tetrachloroethylene was inhibited by 3-chlorobenzoate but not by the structural isomers 2- and 4-chlorobenzoate. The last two compounds are not substrates for D. tiedjei. These findings lead us to suggest that the dehalogenation of tetrachloroethylene in D. tiedjei is catalyzed by a dehalogenase previously thought to be specific for meta-halobenzoates.     

Identification of 4-chlorobenzoyl-coenzyme A as intermediate in the dehalogenation catalysed by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3

The intermediate in the reaction catalysed by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 was identified as 4-chlorobenzoyl-CoA. One component of 4-chlorobenzoate debalogenase worked as a a 4-chlorobenzoyl-CoA ligase catalysing the formation of 4-chlorobenzoyl-CoA from 4-chlorobenzoate, coenzyme A and ATP. This intermediate was detected spectrophotometrically and by HPLC. 4-chlorobenzoyl-CoA was the substrate for the dehalogenase component, which catalysed the conversion to 4-hydroxybenzoate with concomitant release or coenzyme A.     

Relationship between hydrogen consumption, dehalogenation, and the reduction of sulfur oxyanions by Desulfomonile tiedjei.

Resting-cell suspensions of Desulfomonile tiedjei consumed H2 with 3-chloro-, 3-bromo-, and 3-iodobenzoate as electron acceptors with rates of 0.50, 0.44, and 0.04 mumol h-1 mg-1, respectively. However, benzoate and 3-fluorobenzoate were not metabolized by this bacterium. In addition, H2 uptake was at least fourfold faster when sulfate, sulfite, or thiosulfate was available as the electron acceptor instead of a haloaromatic substrate. When sulfite and 3-chlorobenzoate were both available for this purpose, the rate of H2 uptake by D. tiedjei was intermediate between that obtained with either electron acceptor alone. Hydrogen concentrations were reduced to comparably low levels when either 3-chlorobenzoate, sulfate, or sulfite was available as an electron acceptor, but significantly less H2 depletion was evident with benzoate or nitrate. Rates of 3-chlorobenzoate dechlorination increased from an endogenous rate of 14.5 to 17.1, 74.0, 81.1, and 82.3 nmol h-1 mg-1 with acetate, pyruvate, H2, and formate, respectively, as the electron donors. Sulfite and thiosulfate inhibited dehalogenation, but sulfate and NaCl had no effect. Dehalogenation and H2 metabolism were also inhibited by acetylene, molybdate, selenate, and metronidazole. Sulfite reduction and dehalogenation were inhibited by the same respiratory inhibitors. These results suggest that the reduction of sulfite and dehalogenation may share part of the same electron transport chain. The kinetics of H2 consumption and the direct inhibition of dehalogenation by sulfite and thiosulfate in D. tiedjei cells clearly indicate that the reduction of sulfur oxyanions is favored over aryl dehalogenation for the removal of reducing equivalents under anaerobic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)