Reductive metabolism of 1,1,1,2-tetrachloroethane and related chloroethanes by rat liver microsomes

The susceptibility of polychlorinated ethanes to reductive metabolism was evaluated by measuring the amount of each compound consumed during anaerobic incubations with rat live microsomes; 1,1,1,2-tetrachloroethane, pentachloroethane and hexachloroethane were metabolized extensively, 1,1,1,2-tetrachloroethane and the trichloroethanes were metabolized very slowly and the dichloroethanes were not metabolized at a detectable rate. The electron affinity of the chloroethanes was determined by measuring electrochemical half-wave reduction potentials. Chloroethanes with an E1/2 of - 1.35 V or less negative were reduced readily in microsomes while those with an E1/2 equal to or more negative than -1.90 V were not good substrates for enzymatic reduction. Metabolites produced from 1,1,1,2-tetrachloroethane in vitro were 1,1-dichloroethylene (DCE) and 1,1,2-trichloroethane (TCEA) and the ratio DCE/TCEA was about 25:1. These conversions were NADPH-dependent and were inhibited by air, CO and metyrapone. In the presence of SKF 525-A, DCE formation was inhibited by 47%. Microsomes from untreated or beta-naphthoflavone-treated rats were 70-90% less active than microsomes from phenobarbital-treated rats. The Km was 0.50 mM and the Vmax was 66 nmol min-1 mg-1 protein for DCE formation. The results are consistent with the proposal that 1,1,1,2-tetrachloroethane is reduced by hepatic cytochrome(s) P-450 to a free radical intermediate which, for the most part, remains closely associated with the enzyme, is reduced further and undergoes beta-elimination of a chloride ion to form DCE. The occurrence of this reductive pathway in vivo was demonstrated by the quantitation of DCE and TCEA in blood from rats treated with 1,1,1,2-tetrachloroethane.     

A mechanistic perspective on bacterial metabolism of chlorinated methanes

Chlorinated methanes are important environmental pollutants, which can be metabolized by bacteria. The biotransformation of chlorinated methanes by bacteria has been shown to be due either to gratuitous metabolism (cometabolism) or their use as a source of carbon and energy. The reactions which result in carbon-halogen bond cleavage include substitutive, reductive, oxygenative, and gem-elimination mechanisms. Certain methylotrophic bacteria can use dichloromethane as a source of carbon and energy. Dichloromethane dehalogenase catalyzes the first substitutive reaction in this metabolism. The enzyme shows a 10¹⁰-fold rate enhancement over the reaction of the bisulfide anion with dichloromethane in water. Pseudomonas putida G786 synthesizes cytochrome P-450_CAM which catalyzes the gratuitous reduction of chlorinated methanes. These studies with purified enzymes are beginning to reveal more detailed mechanistic features of bacterial chlorinated methane metabolism.Abbreviations DNA deoxyribonucleic acid - k_cat catalytic first order rate constant for an enzyme catalyzed reaction - K_M Michaelis constant for an enzyme catalyzed reaction - MNDO modified neglect of diatomic overlap - PIMA pattern induced multialignment - DCMD dichloromethane dehalogenase     

Metabolism of chlorofluorocarbons and polybrominated compounds by Pseudomonas putida G786(pHG-2) via an engineered metabolic pathway.

The recombinant bacterium Pseudomonas putida G786(pHG-2) metabolizes pentachloroethane to glyoxylate and carbon dioxide, using cytochrome P-450CAM and toluene dioxygenase to catalyze consecutive reductive and oxidative dehalogenation reactions (L.P. Wackett, M.J. Sadowsky, L.N. Newman, H.-G. Hur, and S. Li, Nature [London] 368:627-629, 1994). The present study investigated metabolism of brominated and chlorofluorocarbon compounds by the recombinant strain. Under anaerobic conditions, P. putida G786(pHG-2) reduced 1,1,2,2-tetrabromoethane, 1,2-dibromo-1,2-dichloroethane, and 1,1,1,2-tetrachloro-2,2-difluoroethane to products bearing fewer halogen substituents. Under aerobic conditions, P. putida G786(pHG-2) oxidized cis- and trans-1,2-dibromoethenes, 1,1-dichloro-2,2-difluoroethene, and 1,2-dichloro-1-fluoroethene. Several compounds were metabolized by sequential reductive and oxidative reactions via the constructed metabolic pathway. For example, 1,1,2,2-tetrabromoethane was reduced by cytochrome P-450CAM to 1,2-dibromoethenes, which were subsequently oxidized by toluene dioxygenase. The same pathway metabolized 1,1,1,2-tetrachloro-2,2-difluoroethane to oxalic acid as one of the final products. The results obtained in this study indicate that P. putida G786(pHG-2) metabolizes polyfluorinated, chlorinated, and brominated compounds and further demonstrates the value of using a knowledge of catabolic enzymes and recombinant DNA technology to construct useful metabolic pathways.     

Biochemical and molecular characterization of a tetrachloroethene dechlorinating Desulfitobacterium sp. strain Y51: a review

A strict anaerobic bacterium, Desulfitobacterium sp. strain Y51, is capable of very efficiently dechlorinating tetrachloroethene (PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (cis-DCE) at concentrations as high as 960 μM and as low as 0.06 μM. Dechlorination was highly susceptible to air oxidation and to potential alternative electron acceptors, such as nitrite, nitrate or sulfite. The PCE reductive dehalogenase (encoded by the pceA gene and abbreviated as PceA dehalogenase) of strain Y51 was purified and characterized. The purified enzyme catalyzed the reductive dechlorination of PCE to cis-DCE at a specific activity of 113.6 nmol min⁻¹  mg protein⁻¹ . The apparent K _m values for PCE and TCE were 105.7 and 535.3 μM, respectively. In addition to PCE and TCE, the enzyme exhibited dechlorination activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane and 1,1,2,2-tetrachloroethane. An 8.4-kb DNA fragment cloned from the Y51 genome revealed eight open reading frames, including the pceAB genes. Immunoblot analysis revealed that PceA dehalogenase is localized in the periplasm of Y51 cells. Production of PceA dehalogenase was induced upon addition of TCE. Significant growth inhibition of strain Y51 was observed in the presence of cis-DCE, More interestingly, the pce gene cluster was deleted with high frequency when the cells were grown with cis-DCE.

     

Anaerobic reductive dehalogenation of polychlorinated dioxins

Polychlorinated dibenzo-p-dioxins and -furans (PCDD/Fs) are among the most harmful environmental contaminants. Their widespread distribution due to unintentional or unknown release coincides with environmental persistence, acute and chronic toxicity to living organisms, and long-term effects due to the compounds’ tendency for bioaccumulation and biomagnification. While microbial aerobic degradation of PCDD/Fs is mainly reported for the turnover of low chlorinated congeners, this review focuses on anaerobic reductive dehalogenation, which may constitute a potential remediation strategy for polychlorinated compounds in soils and sediments. Microorganisms in sediments and in microcosms or enrichment cultures have been shown to be involved in the reductive dechlorination of dioxins. Bacteria related to the genus Dehalococcoides are capable of the reductive transformation of dioxins leading to lower chlorinated dioxins including di- and monochlorinated congeners. Thus, reductive dehalogenation might be one of the very few mechanisms able to mediate the turnover of polychlorinated dioxins by reducing their toxicity and paving the way for a subsequent breakdown of the carbon skeleton.     

Anaerobic dehalogenation of halothane by reconstituted liver microsomal cytochrome P-450 enzyme system

Cytochrome P-450 from liver microsomes of phenobarbital-treated rabbits catalyzed anaerobic dehalogenation of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) when combined with NADPH and NADPH-cytochrome P-450 reductase. Cytochromes P-450B₁ and P-448 from liver microsomes of untreated rabbits were less active. Triton X-100 accelerated the reaction. Unlike anaerobic dehalogenation of halothane in microsomes, the major product was 2-chloro-1,1,1-trifluoroethane and 2-chloro-1,1-difluoroethylene was negligible. These products were not detected under aerobic conditions, and dehalogenation activity was inhibited by carbon monoxide, phenyl isocyanide and metyrapone.     

Enhanced dechlorination of chloroethenes by granular sludge under microaerophilic conditions

This study investigates an innovative dechlorination process using anaerobic granular sludge that was partially exposed to oxygen. The exposure supported a synchronously anaerobic and aerobic bioconversion process that combined reductive dechlorination with aerobic co-oxidation in a sludge granule. Experimental results showed that the highest dechlorination rates of tetrachloroethene, trichloroethene, cis-dichloroethene and vinyl chloride were 6.44, 2.98, 1.70 and 0.97 nmol/gVS day, at initial O₂ concentrations of 10, 100, 5 and 0%, respectively. Strictly anaerobic conditions favored the dechlorination of vinyl chloride while absolutely aerobic conditions were preferred for trichloroethene dechlorination. Microaerophilic conditions are suggested to ensure the overall biodegradation of the chlorinated ethenes present in groundwater as a mixture.     

Biochemical and molecular characterization of a tetrachloroethene dechlorinating Desulfitobacterium sp. strain Y51: a review

A strict anaerobic bacterium, Desulfitobacterium sp. strain Y51, is capable of very efficiently dechlorinating tetrachloroethene (PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (cis-DCE) at concentrations as high as 960 microM and as low as 0.06 microM. Dechlorination was highly susceptible to air oxidation and to potential alternative electron acceptors, such as nitrite, nitrate or sulfite. The PCE reductive dehalogenase (encoded by the pceA gene and abbreviated as PceA dehalogenase) of strain Y51 was purified and characterized. The purified enzyme catalyzed the reductive dechlorination of PCE to cis-DCE at a specific activity of 113.6 nmol min(-1) mg protein(-1). The apparent K(m) values for PCE and TCE were 105.7 and 535.3 microM, respectively. In addition to PCE and TCE, the enzyme exhibited dechlorination activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane and 1,1,2,2-tetrachloroethane. An 8.4-kb DNA fragment cloned from the Y51 genome revealed eight open reading frames, including the pceAB genes. Immunoblot analysis revealed that PceA dehalogenase is localized in the periplasm of Y51 cells. Production of PceA dehalogenase was induced upon addition of TCE. Significant growth inhibition of strain Y51 was observed in the presence of cis-DCE, More interestingly, the pce gene cluster was deleted with high frequency when the cells were grown with cis-DCE.     

Characterization of the protein expressed in Escherichia coli by a recombinant plasmid containing the Bacillus megaterium cytochrome P-450BM-3 gene

Summary In two previous reports (Narhi LO, Fulco AJ, J. Biol. Chem. 261: 7160–7169, 1986; Ibid., 262: 6683–6690, 1987) we described the characterization of a catalytically self-sufficient 119000-dalton P-450 cytochrome that was induced by barbiturates in Bacillus megaterium. In the presence of NADPH and O₂, this polypeptide (cytochrome P-450_BM-3) catalyzed the hydroxylation of long-chain fatty acids without the aid of any other protein. The gene encoding this unique monooxygenase was cloned into Escherichia coli and the clone harboring the recombinant plasmid produced a protein that behaved electrophoretically and immunochemically like the B. megaterium enzyme (Wen LP, Fulco AJ, J. Biol. Chem. 262: 6676–6682, 1987). We have now compared authentic P-450_BM-3 from B. megaterium and putative P-450_BM-3 isolated from transformed E. coli and have found them to be indistinguishable with respect to chromatographic and electrophoretic behavior, reaction with specific antibody, prosthetic group (heme, FAD and FMN) analyses, spectra, enzymology, limited trypsin proteolysis and partial amino acid sequencing. We thus conclude that the P-450 cytochrome expressed by the transformed E. coli is essentially identical to native P-450_BM-3 induced by barbiturates in B. megaterium. The evidence furthermore suggests that the primary amino acid sequence of this complex protein is alone sufficient to direct the proper integration of the three prosthetic groups and to specify folding of the polypeptide into the correct tertiary structure.Abbreviations SDS Sodium Dodecylsulfate - PAGE Polyacrylamide Gel Electrophoresis - HPLC High Performance Liquid Chromatography     

Spectroscopic investigations of intermediates in the reaction of cytochrome P450BM3–F87G with surrogate oxygen atom donors

Rapid mixing of substrate-free ferric cytochrome P450_BM3–F87G with m-chloroperoxybenzoic acid (mCPBA) resulted in the sequential formation of two high-valent intermediates. The first was spectrally similar to compound I species reported previously for P450_CAM and CYP 119 using mCPBA as an oxidant, and it featured a low intensity Soret absorption band characterized by shoulder at 370 nm. This is the first direct observation of a P450 compound I intermediate in a type II P450 enzyme. The second intermediate, which was much more stable at pH values below 7.0, was characterized by an intense Soret absorption peak at 406 nm, similar to that seen with P450_CAM [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300–20309]. Double mixing experiments in which NADPH was added to the transient 406 nm-absorbing intermediate resulted in rapid regeneration of the resting ferric state, with the flavins of the flavoprotein domain in their reduced state. EPR results were consistent with this stable intermediate species being a cytochrome c peroxidase compound ES-like species containing a protein-based radical, likely localized on a nearby Trp or Tyr residue in the active site. Iodosobenzene, peracetic acid, and sodium m-periodate also generated the intermediate at 406 nm, but not the 370 nm intermediate, indicating a probable kinetic barrier to accumulating compound I in reactions with these oxidants. The P450 ES intermediate has not been previously reported using iodosobenzene or m-periodate as the oxygen donor.     

Modulation of the reductive metabolism of halothane by microsomal cytochrome b5 in rat liver

To study the modulation of the reductive metabolism of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) by microsomal cytochrome b₅, formation of 2-chloro-1,1,1-trifluoroethane (CTE) and 2-chloro-1,1-difluoroethylene (CDE), major reduced metabolites of halothane, was analyzed in vivo and in vitro. Rats were pretreated with both malotilate (diisopropyl-1,3-dithiol-2-ylidenemalonate) and sodium phenobarbital (malotilate-treated rats) or only with sodium phenobarbital (control rats). The microsomes of malotilate-treated rats had significantly more cytochrome b₅ than the controls, whereas the cytochrome P-450 content was not different between the two groups. At the end of 2-h exposure to 1% halothane in 14% oxygen, the ratio of CDE to CTE in arterial blood was significantly higher in malotilate-treated rats than in the controls. Under anaerobic conditions, the formation of CDE and the ratio of CDE to CTE were significantly greater in microsomal preparations of malotilate-treated rats than those of the controls. In a reconstituted system containing cytochrome P-450_PB purified from rabbit liver, addition of cytochrome b₅ to the system enhanced the formation of CDE and increased the ratio of CDE to CTE. These results suggested that cytochrome b₅ enhances the formation ratio of CDE to CTE by stimulating the supply of a second electron to cytochrome P-450, which might reduce radical reactions in the reductive metabolism of halothane.     

Photoreactions of cytochrome b6 in systems using resolved chloroplast electron-transfer complexes

Photoreactions of cytochrome b₆ have been studied using resolved chloroplast electron-transfer complexes. In the presence of Photosystem (PS) II and the cytochrome b₆-f complex, photoreduction of the cytochrome can be observed. No soluble components are required for this reaction. Cytochrome b₆ photoreduction was found to be inhibited by quinone analogs, which inhibit at the Rieske iron-sulfur center of the cytochrome complex, by the addition of ascorbate and by depletion of the Rieske center and bound plastoquinone from the cytochrome complex. Photoreduction of cytochrome b₆ can also be demonstrated in the presence of the cytochrome complex and PS I. This photoreduction requires plastocyanin and a low-potential electron donor, such as durohydroquinone. Cytochrome b₆ photoreduction in the presence of PS I is inhibited by quinone analogs which interact with the Rieske iron-sulfur center. These results are discussed in terms of a Q-cycle mechanism in which plastosemiquinone serves as the reductant for cytochrome b₆ via an oxidant-induced reductive pathway.     

The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase and cytochrome b(5)

Cytochrome P450 2B4 is a microsomal protein with a multi-step reaction cycle similar to that observed in the majority of other cytochromes P450. The cytochrome P450 2B4-substrate complex is reduced from the ferric to the ferrous form by cytochrome P450 reductase. After binding oxygen, the oxyferrous protein accepts a second electron which is provided by either cytochrome P450 reductase or cytochrome b₅. In both instances, product formation occurs. When the second electron is donated by cytochrome b₅, catalysis (product formation) is ∼10- to 100-fold faster than in the presence of cytochrome P450 reductase. This allows less time for side product formation (hydrogen peroxide and superoxide) and improves by ∼15% the coupling of NADPH consumption to product formation. Cytochrome b₅ has also been shown to compete with cytochrome P450 reductase for a binding site on the proximal surface of cytochrome P450 2B4. These two different effects of cytochrome b₅ on cytochrome P450 2B4 reactivity can explain how cytochrome b₅ is able to stimulate, inhibit, or have no effect on cytochrome P450 2B4 activity. At low molar ratios (<1) of cytochrome b₅ to cytochrome P450 reductase, the more rapid catalysis results in enhanced substrate metabolism. In contrast, at high molar ratios (>1) of cytochrome b₅ to cytochrome P450 reductase, cytochrome b₅ inhibits activity by binding to the proximal surface of cytochrome P450 and preventing the reductase from reducing ferric cytochrome P450 to the ferrous protein, thereby aborting the catalytic reaction cycle. When the stimulatory and inhibitory effects of cytochrome b₅ are equal, it will appear to have no effect on the enzymatic activity. It is hypothesized that cytochrome b₅ stimulates catalysis by causing a conformational change in the active site, which allows the active oxidizing oxyferryl species of cytochrome P450 to be formed more rapidly than in the presence of reductase.     

Cytochrome P-450 mediated reductive dehalogenation of the perhalogenated aromatic compound hexachlorobenzene

Hexachlorobenzene (HCB) elicits concentration-dependent and saturable type 1 binding spectra when added to oxidized (Fe3+) cytochrome P-450 (CYT P-450) in control, phenobarbital- (PB) induced, and beta-naphthoflavone- (BNF) induced male Sprague-Dawley rat liver microsomes. The spectral binding constants (Ks) for HCB in control and PB-induced microsomes are 180 microM and 83 microM, respectively, and correlate inversely with the specific content of CYT P-450 (0.9 and 2.1 nmol/mg) in the two microsomal preparations. BNF-induced microsomes show type 1 interaction only at low HCB concentration. Overall biotransformation of HCB, monitored by loss of [14C]HCB from the reaction medium, is dependent on NADPH and intact microsomes. Dimethyl sulfoxide (Me2SO), a potent hydroxyl radical scavenger and the solvent used for HCB dissolution, does not affect the biotransformation of HCB in aerobic reactions. Pentachlorobenzene (PCB) appears to be the initial and major isolatable CYT P-450 mediated dechlorination product of HCB with NADPH-fortified rat liver microsomes. Trace levels of pentachlorophenol (PCP) and an unidentified metabolite are also observed. PCB formation is enhanced under anaerobic conditions but is inhibited by metyrapone and carbon monoxide. PCB formation is also inhibited with aerobic reaction conditions, while PCP formation is observed. The data indicate that CYT P-450 in hepatic microsomes supports the reductive dechlorination of HCB to PCB.     

Glutathione peroxidase activity of glutathione-s-transferases purified from rat liver.

$\text{Rhizobium}$ hemeproteins P-450$\text{a, b}$, and $\text{c}$ cross react with antibodies to P-450_CAM and P-450_LM-2. Anti P-450_CAM IgG and phenobarbital, each bound to Sepharose 4B, were effective in purification of $\text{Rhizobium}$ P-450$\text{c}$; the latter was more convenient. The amino acid composition of highly purified $\text{Rhizobium}$ P-450$\text{c}$ resembles the compositions of P-450_CAM and P-450_LM-2. These results suggest that P-450 heme proteins of unrelated substrate specificities may nevertheless contain similar structural features.     

A 2.6 kb intron separates the signal peptide coding sequence of an anther-specific protein from the rest of the gene in sunflower

Summary Metabolism of sulfonylurea herbicides by Streptomyces griseolus ATCC 11796 is carried out via two cytochromes P-450, P-450_SU1 and P-450_SU2. Mutants of S. griseolus, selected by their reduced ability to metabolize a fluorescent sulfonylurea, do not synthesize cytochrome P-450_SU1 when grown in the presence of sulfonylureas. Genetic evidence indicated that this phenotype was the result of a deletion of > 15 kb of DNA, including the structural genes for cytochrome P-450_SU1 and an associated ferredoxin Fd-1 (suaC and suaB, respectively). In the absence of this monooxygenase system, the mutants described here respond to the presence of sulfonylureas or phenobarbital in the growth medium with the expression of only the suhC,B gene products (cytochrome P-450_SU2 and Fd-2), previously observed only as minor components in wild-type cells treated with sulfonylurea. These strains have enabled an analysis of sulfonylurea metabolism mediated by cytochrome P-450_SU2 in the absence of P-450_SU1, yielding an in vivo delineation of the roles of the two different cytochrome P-450 systems in herbicide metabolism by S. griseolus.     

Alkaloid metabolism by cytochrome P-450 enzymes in Drosophila melanogaster

The cytochrome P-450 family of enzymes is the primary means of foreign compound detoxification in virtually all organisms. Cytochrome P-450s have been strongly implicated in the metabolism of cactus alkaloids, and consequently, the observed patterns of host plant utilization by cactophilic species of Drosophila in the Sonoran Desert. The current study looked for evidence of alkaloid-metabolizing P-450 enzymes in a non-cactophilic species, D. melanogaster. The results of in vitro metabolism assays indicate the presence of a phenobarbital-inducible P-450 in adult D. melanogaster which is capable of metabolizing alkaloids. P-450 quantification data suggest that the enhanced level of metabolism is not the result of an overall increase in total P-450 content. Results from larval viability and adult longevity studies indicate that D. melanogaster's in vitro activity does not produce an enhanced in vivo tolerance of alkaloids.     

Bioremediation of Hexachlorocyclohexane Isomers,Chlorinated Benzenes,and Chlorinated Ethenes in Soil and Fractured Dolomite

Groundwater at an industrial site is contaminated with α hexachlorocyclohexane (HCH) and γ -HCH (i.e., lindane) (0.3 to 0.5 ppm). Other contaminants in the 1 to 15 ppm range include 1,2,4-trichlorobenezene (TCB), 1,2-dichlorobenzene (DCB), 1,3-DCB, 1,4-DCB, chlorobenzene (CB), benzene, trichloroethene (TCE), and cis-1,2-dichloroethene (cDCE). The aquifer consists of a shallow layer of soil over fractured dolomite, where most of the contaminant mass resides. The objective of this study was to compare (1) anaerobic reductive dechlorination of the polychlorinated contaminants, followed by aerobic biodegradation of the daughter products (mainly DCBs, CB, and benzene); and (2) aerobic biodegradation of α - and γ -HCH, TCB, DCBs, CB, and benzene, followed by anaerobic reduction of TCE and cDCE to ethene. Conventional wisdom suggests that sequential anaerobic and aerobic conditions are desirable for bioremediating sites contaminated by mixtures of polychlorinated organics. The results of this microcosm study suggest that a sequential aerobic and anaerobic approach may be more successful, although implementing this in the field presents some major challenges. In the dolomite microcosms incubated under aerobic conditions first (59 days), α - and γ -HCH were biodegraded close to the maximum contaminant level for lindane; all of the aromatic compounds were consumed; and there was partial removal of TCE and cDCE (presumptively via cometabolism). The subsequent switch to anaerobic conditions (day 101) yielded reductive dechlorination of the remaining TCE; a significant level of ethene was produced, although some cDCE and VC persisted. In contrast, sequential anaerobic (393 days) and aerobic treatment (498 days) for the dolomite microcosms was ineffective in completely removing the aromatic compounds, α -HCH, cDCE, and VC. For the soil microcosms, both treatment sequences were effective, most likely reflecting a greater abundance of the necessary microbes and electron donor in this part of the site.     

Microbial transformation of chlorinated benzoates

Chlorinated benzoates enter the environment through their use as herbicides or as metabolites of other halogenated compounds. Ample evidence is available indicating biodegradation of chlorinated benzoates to CO₂ and chloride in the environment under aerobic as well as anaerobic conditions. Under aerobic conditions, lower chlorinated benzoates can serve as sole electron and carbon sources supporting growth of a large list of taxonomically diverse bacterial strains. These bacteria utilize a variety of pathways ranging from those involving an initial degradative attack by dioxygenases to those initiated by hydrolytic dehalogenases. In addition to monochlorinated benzoates, several bacterial strains have been isolated that can grow on dichloro-, and trichloro- isomers of chlorobenzoates. Some aerobic bacteria are capable of cometabolizing chlorinated benzoates with simple primary substrates such as benzoate. Under anaerobic conditions, chlorinated benzoates are subject to reductive dechlorination when suitable electron-donating substrates are available. Several halorespiring bacteria are known which can use chlorobenzoates as electron acceptors to support growth. For example, Desulfomonile tiedjei catalyzes the reductive dechlorination of 3-chlorobenzoate to benzoate. The benzoate skeleton is mineralized by other microorganisms in the anaerobic environment. Various dichloro- and trichlorobenzoates are also known to be dechlorinated in anaerobic sediments.     

Acetate as a source of reducing equivalents in the reductive dechlorination of 2,5-dichlorobenzoate

Desulfomonile tiedjei is the key dechlorinating organism in a three-tiered bacterial consortium that grows on the methanogenic degradation of 3-chlorobenzoate. 2,5-Dichlorobenzoate, however, is only converted to 2-chlorobenzoate and is not a methanogenic substrate for the consortium. The dechlorinator uses hydrogen produced from benzoate by the benzoate degrading member of consortium as its source of reducing equivalents for the dechlorination reaction. Incubation of 3-chlorobenzoate grown consortium cells with 2,5-dichlorobenzoate resulted in the consumption of acetate concurrent with the formation of 2-chlorobenzoate indicating that acetate can serve as an alternative source of reducing equivalents for reductive dechlorination. This interpretation was confirmed by the finding that the formation of ¹⁴CO₂ from 2-¹⁴C-labeled acetate was stoichiometric. The addition of hydrogen to 2,5-dichlorobenzoate metabolizing cells resulted in (i) an 2.7-fold increase in the rate of dechlorination, and (ii) a drop in the amount of label recovered as CO₂+CH₄ from methyl ¹⁴C-labeled acetate, indicating that hydrogen was the preferred source of reducing equivalents for reductive dechlorination. Benzoate, an indirect source of H₂ in the consortium, also inhibited the oxidation of acetate, while glucose, methanol, and butyrate did not affect labeled gas production and therefore were not suitable electron donors. Concomittant to dechlorination of 2,5-dichlorobenzoate 3- and 4-methoxybenzoate were converted to 3- and 4-hydroxybenzoate respectively. These conversions stimulated the rate of dechlorination 2-fold. Demethylation of 4-methoxybenzoate stimulated, but demethylation of 3-methoxybenzoate inhibited the oxidation of benzoate during the dechlorination of 2,5-dichlorobenzoate, suggesting that these isomers are metabolized through different pathways. Experiments with benzoate, 3-chlorobenzoate and 2,5-dichlorobenzoate metabolizing cells amended with ¹⁴CO₂ showed that actively dechlorinating cells catalyzed an exchange reaction between CO₂ and acetate.