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.     

Biomass, Oleate, and Other Possible Substrates for Chloroethene Reductive Dehalogenation

Comparative studies were conducted with benzoate, propionate, oleate, tetrabutyl orthosilicate (TBOS), and biomass as substrates for dehalogenation of cis-1,2-dichloroethene (cDCE). All five substrates supported dehalogenation. Sufficient calcium was required to precipitate oleate and thus reduce its toxicity to the dehalogenating microorganisms. More cDCE was dehalogenated with TBOS than with benzoate, although TBOS initially had an inhibitory effect. The most efficient dehalogenation was associated with biomass, 20% of which was used for dehalogenation, even higher than the 17% obtained with propionate. The advantages and disadvantages of these organic substances for introduction into an aquifer as electron donors for in situ dehalogenation were examined in terms of efficiency of electron use for reductive dehalogenation, and method and ease of introduction into the aquifer. Benzoate and propionate are useful for recirculation systems, while TBOS, oleate, and biomass are appropriate for more passive approaches.     

Differences in carbohydrate moieties of high molecular weight glycoproteins of human lymphocytes of T and B origins revealed by monoclonal autoantibodies with anti-I and anti-i specificities

NADPH reduced rabbit liver microsomal enzymes catalyzed anaerobic dehalogenation of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) to produce CF₂CHCl and CF₃CH₂Cl. Anaerobic dehalogenation was optimal at pH7.4 and was blocked by either oxygen or carbon monoxide. The degree of inhibition of anaerobic dehalogenation by carbon monoxide was closely correlated to the proportion of carbon monoxide complex of cytochrome P450. Anaerobic dehalogenation was enhanced by pretreatment of the animals with phenobarbital but not with methylcholanthrene.     

Influence of sulfur oxyanions on reductive dehalogenation activities in Desulfomonile tiedjei.

The inhibition of aryl reductive dehalogenation reactions by sulfur oxyanions has been demonstrated in environmental samples, dehalogenating enrichments, and the sulfate-reducing bacterium Desulfomonile tiedjei; however, this phenomenon is not well understood. We examined the effects of sulfate, sulfite, and thiosulfate on reductive dehalogenation in the model microorganism D. tiedjei and found separate mechanisms of inhibition due to these oxyanions under growth versus nongrowth conditions. Dehalogenation activity was greatly reduced in extracts of cells grown in the presence of both 3-chlorobenzoate, the substrate or inducer for the aryl dehalogenation activity, and either sulfate, sulfite, or thiosulfate, indicating that sulfur oxyanions repress the requisite enzymes. In extracts of fully induced cells, thiosulfate and sulfite, but not sulfate, were potent inhibitors of aryl dehalogenation activity even in membrane fractions lacking the cytoplasmically located sulfur oxyanion reductase. These results suggest that under growth conditions, sulfur oxyanions serve as preferred electron acceptors and negatively influence dehalogenation activity in D. tiedjei by regulating the amount of active aryl dehalogenase in cells. Additionally, in vitro inhibition by sulfur oxyanions is due to the interaction of the reactive species with enzymes involved in dehalogenation and need not involve competition between two respiratory processes for reducing equivalents. Sulfur oxyanions also inhibited tetrachloroethylene dehalogenation by the same mechanisms, further indicating that chloroethylenes are fortuitously dehalogenated by the aryl dehalogenase. The commonly observed inhibition of reductive dehalogenation reactions under sulfate-reducing conditions may be due to similar regulation mechanisms in other dehalogenating microorganisms that contain multiple respiratory activities.     

Catalytic reductive dehalogenation of hexachloroethane by molecular variants of cytochrome P450cam (CYP101).

CYP101 (cytochrome P450cam) catalyses the oxidation of camphor but has also been shown to catalyse the reductive dehalogenation of hexachloroethane and pentachloroethane. This reaction has potential applications in the biodegradation of these environmental contaminants. The hexachloroethane dehalogenation activity of CYP101 has been investigated by mutagenesis. The effects of active-site polarity and volume were probed by combinations of active-site mutations. Increasing the active-site hydrophobicity by the Y96A and Y96F mutations strengthened hexachloroethane binding but decreased the rate of reaction. Increasing the polarity with the F87Y mutation drastically weakened hexachloroethane binding but did not affect the rate of reaction. The Y96H mutation had little effect at pH 7.4, but weakened hexachloroethane binding while increasing the rate of dehalogenation by up to 40% at pH 6.5, suggesting that the imidazole side-chain was partially protonated at pH 6.5 but not at pH 7.4. Substitutions by bulkier side-chains at F87, T101 and V247 weakened hexachloroethane binding but increased the dehalogenation rate. The effect of the individual mutations was additive in multiple mutants, and the most active mutant for hexachloroethane reductive dehalogenation at pH 7.4 was F87W-V247L (80 min-1 or 2.5 x the activity of the wild-type). The results suggested that the CYP101 active site shows good match with hexachloroethane, the Y96 side-chain plays an important role in both hexachloroethane binding and dehalogenation, and hexachloroethane binding and dehalogenation places conflicting demands on active-site polarity and compromises were necessary to achieve reasonable values for both.     

Microbial reductive dehalogenation.

A wide variety of compounds can be biodegraded via reductive removal of halogen substituents. This process can degrade toxic pollutants, some of which are not known to be biodegraded by any other means. Reductive dehalogenation of aromatic compounds has been found primarily in undefined, syntrophic anaerobic communities. We discuss ecological and physiological principles which appear to be important in these communities and evaluate how widely applicable these principles are. Anaerobic communities that catalyze reductive dehalogenation appear to differ in many respects. A large number of pure cultures which catalyze reductive dehalogenation of aliphatic compounds are known, in contrast to only a few organisms which catalyze reductive dehalogenation of aromatic compounds. Desulfomonile tiedjei DCB-1 is an anaerobe which dehalogenates aromatic compounds and is physiologically and morphologically unusual in a number of respects, including the ability to exploit reductive dehalogenation for energy metabolism. When possible, we use D. tiedjei as a model to understand dehalogenating organisms in the above-mentioned undefined systems. Aerobes use reductive dehalogenation for substrates which are resistant to known mechanisms of oxidative attack. Reductive dehalogenation, especially of aliphatic compounds, has recently been found in cell-free systems. These systems give us an insight into how and why microorganisms catalyze this activity. In some cases transition metal complexes serve as catalysts, whereas in other cases, particularly with aromatic substrates, the catalysts appear to be enzymes.     

Characterization of the acclimation period before anaerobic dehalogenation of halobenzoates

The acclimation periods prior to detectable dehalogenation of halogenated benzoates in anaerobic lake sediments ranged from 3 weeks to 6 months. These acclimation periods were reproducible over time and among sampling sites and were characteristic of the chemical tested. The lengthy acclimation period appears to represent an induction phase in which little or no aryl dehalogenation is observed, followed by an exponential increase in activity typical of an enrichment response. Continuous growth from the time of the first exposure to the chemical is inconsistent with the extremely low per-cell activities estimated for the early days of the acclimation period and the fact that the dehalogenation yields no carbon to support microbial growth. The finding of a characteristic acclimation time for each chemical argues against nutritional deficiency, inhibition, or predation as an explanation for this phase of metabolism, while the reproducibility of the findings with time and space and among replicates argues against genetic changes as the explanation. The acclimation times did correlate with the eventual dehalogenation rates. This may reflect the general energy limitations in the anaerobic communities and suggests that those chemicals with faster dehalogenation rates provide more energy for the induction and growth phases of the active population.     

Characterization of the acclimation period before anaerobic dehalogenation of halobenzoates.

The acclimation periods prior to detectable dehalogenation of halogenated benzoates in anaerobic lake sediments ranged from 3 weeks to 6 months. These acclimation periods were reproducible over time and among sampling sites and were characteristic of the chemical tested. The lengthy acclimation period appears to represent an induction phase in which little or no aryl dehalogenation is observed, followed by an exponential increase in activity typical of an enrichment response. Continuous growth from the time of the first exposure to the chemical is inconsistent with the extremely low per-cell activities estimated for the early days of the acclimation period and the fact that the dehalogenation yields no carbon to support microbial growth. The finding of a characteristic acclimation time for each chemical argues against nutritional deficiency, inhibition, or predation as an explanation for this phase of metabolism, while the reproducibility of the findings with time and space and among replicates argues against genetic changes as the explanation. The acclimation times did correlate with the eventual dehalogenation rates. This may reflect the general energy limitations in the anaerobic communities and suggests that those chemicals with faster dehalogenation rates provide more energy for the induction and growth phases of the active population.     

Anaerobic Aryl Reductive Dehalogenation of Halobenzoates by Cell Extracts of "Desulfomonile tiedjei"

We studied the transformation of halogenated benzoates by cell extracts of a dehalogenating anaerobe, "Desulfomonile tiedjei." We found that cell extracts possessed aryl reductive dehalogenation activity. The activity was heat labile and dependent on the addition of reduced methyl viologen, but not on that of reduced NAD, NADP, flavin mononucleotide, flavin adenine dinucleotide, desulfoviridin, cytochrome c(3), or benzyl viologen. Dehalogenation activity in extracts was stimulated by formate, CO, or H(2), but not by pyruvate plus coenzyme A or by dithionite. The pH and temperature optima for aryl dehalogenation were 8.2 and 35 degrees C, respectively. The rate of dehalogenation was proportional to the amount of protein in the assay mixture. The substrate specificity of aryl dehalogenation activity for various aromatic compounds in "D. tiedjei" cell extracts was identical to that of whole cells, except differences were observed in the relative rates of halobenzoate transformation. Dehalogenation was 10-fold greater in "D. tiedjei" extracts prepared from cells cultured in the presence of 3-chlorobenzoate, suggesting that the activity was inducible. Aryl reductive dehalogenation in extracts was inhibited by sulfite, sulfide, and thiosulfate, but not sulfate. Experiments with combinations of substrates suggested that cell extracts dehalogenated 3-iodobenzoate more readily than either 3,5-dichlorobenzoate or 3-chlorobenzoate. Dehalogenation activity was found to be membrane associated. This is the first report characterizing aryl dehalogenation activity in cell extracts of an obligate anaerobe.     

Dehalogenation of a mixture of chloroaromatics by immobilized Pseudomonas sp. US1 ex cells

Summary Pseudomonas sp. US1 ex entrapped in calcium alginate could dehalogenate a mixture of isomeric monochlorobenzoates and 2,4-dichlorophenoxyacetic acid. Rates of dehalogenation by the immobilized cells were found to be comparable to those of free cells. Conditions for optimum dehalogenation of chloroaromatics by immobilized cells and their reusability were investigated. Preliminary attempts were made to set up a continuous system for dehalogenation of chloroaromatics using a fluidized bed column reactor. Offprint requests to: V. Modi     

Microbial dehalogenation of trichloroacetic acid

A pure bacterial culture and a two-membered mixed culture were isolated that degraded trichloroacetic acid if a second, readily metabolizable substrate was present in the growth medium. Previous doubts over the microbial dehalogenation of trichloroacetic acid (TCA) may be due to its inability to act as a sole carbon and energy source. TCA dehalogenation was associated with conventional 2-haloalkanoic acid dehalogenases but oxalate, the putative dehalogenase product, was not detected. CO₂ was produced rapidly and concomitantly with Cl^– ion release during dehalogenation of TCA. An alternative mechanism is suggested for TCA dehalogenation via an initial decarboxylation reaction. This mechanism predicts that carbon monoxide is a product of TCA decarboxylation and it was significant that one of the organisms isolated,Pseudomonas carboxydohydrogens, was a carboxytroph and a second was an unidentified facultative methylotroph.     

In Vitro Studies on Reductive Vinyl Chloride Dehalogenation by an Anaerobic Mixed Culture

Reductive dehalogenation of vinyl chloride (VC) was studied in an anaerobic mixed bacterial culture. In growth experiments, ethene formation from VC increased exponentially at a rate of about 0.019 h(sup-1). Reductive VC dehalogenation was measured in vitro by using cell extracts of the mixed culture. The apparent K(infm) for VC was determined to be about 76 (mu)M; the V(infmax) was about 28 nmol (middot) min(sup-1) (middot) mg of protein(sup-1). The VC-dehalogenating activity was membrane associated. Propyl iodide had an inhibitory effect on the VC-dehalogenating activity in the in vitro assay. However, this inhibition could not be reversed by illumination. Cell extracts also catalyzed the reductive dehalogenation of cis-1,2-dichloroethene (cis-DCE) and, at a lower rate, of trichloroethene (TCE). Tetrachloroethene (PCE) was not transformed. The results indicate that the reductive dehalogenation of VC and cis-DCE described here is different from previously reported reductive dehalogenation of PCE and TCE.     

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.     

Bacterial dehalogenases: biochemistry, genetics, and biotechnological applications.

This review is a survey of bacterial dehalogenases that catalyze the cleavage of halogen substituents from haloaromatics, haloalkanes, haloalcohols, and haloalkanoic acids. Concerning the enzymatic cleavage of the carbon-halogen bond, seven mechanisms of dehalogenation are known, namely, reductive, oxygenolytic, hydrolytic, and thiolytic dehalogenation; intramolecular nucleophilic displacement; dehydrohalogenation; and hydration. Spontaneous dehalogenation reactions may occur as a result of chemical decomposition of unstable primary products of an unassociated enzyme reaction, and fortuitous dehalogenation can result from the action of broad-specificity enzymes converting halogenated analogs of their natural substrate. Reductive dehalogenation either is catalyzed by a specific dehalogenase or may be mediated by free or enzyme-bound transition metal cofactors (porphyrins, corrins). Desulfomonile tiedjei DCB-1 couples energy conservation to a reductive dechlorination reaction. The biochemistry and genetics of oxygenolytic and hydrolytic haloaromatic dehalogenases are discussed. Concerning the haloalkanes, oxygenases, glutathione S-transferases, halidohydrolases, and dehydrohalogenases are involved in the dehalogenation of different haloalkane compounds. The epoxide-forming halohydrin hydrogen halide lyases form a distinct class of dehalogenases. The dehalogenation of alpha-halosubstituted alkanoic acids is catalyzed by halidohydrolases, which, according to their substrate and inhibitor specificity and mode of product formation, are placed into distinct mechanistic groups. beta-Halosubstituted alkanoic acids are dehalogenated by halidohydrolases acting on the coenzyme A ester of the beta-haloalkanoic acid. Microbial systems offer a versatile potential for biotechnological applications. Because of their enantiomer selectivity, some dehalogenases are used as industrial biocatalysts for the synthesis of chiral compounds. The application of dehalogenases or bacterial strains in environmental protection technologies is discussed in detail.     

Bacterial dehalogenation

Halogenated organic compounds are produced industrially in large quantities and represent an important class of environmental pollutants. However, an abundance of haloorganic compounds is also produced naturally. Bacteria have evolved several strategies for the enzyme-catalyzed dehalogenation and degradation of both haloaliphatic and haloaromatic compounds: (i) Oxidative dehalogenation is the result of mono- or dioxygenase-catalyzed, co-metabolic or metabolic reactions. (ii) In dehydrohalogenase-catalyzed dehalogenation, halide elimination leads to the formation of a double bond. (iii) Substitutive dehalogenation in most cases is a hydrolytic process, catalyzed by halidohydrolases, but there also is a “thiolytic” mechanism with glutathione as cosubstrate. Dehalogenation by halohydrin hydrogen-halide lyases is the result of an intramolecular substitution reaction. (iv) A distinct dechlorination mechanism involves methyl transfer from chloromethane onto tetrahydrofolate. (v) Reductive dehalogenations are co-metabolic processes, or they are specific reactions involved in substrate utilization (carbon metabolism), or reductive dehalogenation is coupled to energy conservation: some anaerobic bacteria use a specific haloorganic compound as electron acceptor of a respiratory process. This review discusses the mechanisms of enzyme-catalyzed dehalogenation reactions, describes some pathways of the bacterial degradation of haloorganic compounds, and indicates some trends in the biological treatment of organohalogen-polluted air, groundwater, soil, and sediments. Received: 24 June 1998 / Received revision: 1 September 1998 / Accepted: 3 September 1998     

Reductive Dehalogenations of Halobenzoates by Anaerobic Lake Sediment Microorganisms

Methane-producing freshwater lake sediment was found to dehalogenate chloro-, bromo-, and iodobenzoates by a reductive reaction in which the halogen was replaced by a hydrogen atom. The identity of the dehalogenated products was confirmed by mass spectrometry, nuclear magnetic resonance, or cochromatography. Removal of the halogens to produce benzoate was necessary before mineralization to CH₄ + CO₂ could occur. The dehalogenation occurred after a lag period which lasted from 1 week to more than 6 months, depending on the chemical. Dehalogenation was not observed in the absence of CH₄ production, and it was inhibited by the addition of 20% O₂. Once sediment was acclimated to halobenzoate dehalogenation, new additions of the halobenzoate were degraded without lag. Acclimation was observed regardless of whether the parent substrates were eventually mineralized to CH₄ + CO₂. Sediment acclimated to bromo- and chlorobenzoate degradation generally metabolized bromo- and chlorobenzoates, but sediment acclimated to iodobenzoate degradation only metabolized iodobenzoate. Prior acclimation of sediment to benzoate decomposition did not alter the pattern of dehalogenation, and sediment acclimated to dehalogenation was not concurrently acclimated to benzoate degradation. The presence of this apparent specificity, the lag period, and subsequent acclimation, together with our findings of the absence of dehalogenation in sterile sediments and by sediments previously incubated at ≥39°C, suggests that this reaction was biologically catalyzed. Apparently, a pathway for the reductive dehalogenation of aryl halides is present in anaerobic microorganisms of this methanogenic sediment.     

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.     

Anaerobic Biodegradation of 2,4,5-Trichlorophenoxyacetic Acid in Samples from a Methanogenic Aquifer: Stimulation by Short-Chain Organic Acids and Alcohols

The herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was dehalogenated in samples from a methanogenic aquifer to form 2,4- and 2,5-dichlorophenoxyacetic acids as the first detected intermediates. Further incubation of the aquifer slurries resulted in the formation of several intermediates including monochlorophenoxyacetic acids, di- and monochlorophenols, as well as phenol. No transformation of the parent substrate or production of intermediates was detected in autoclaved controls. The pattern of intermediate formation suggested that the anaerobic degradation of 2,4,5-T proceeded by a series of sequential dehalogenation steps with side-chain cleavage reactions occurring at some point before ring cleavage. The addition of short-chain organic acids or alcohols stimulated the onset and rate of 2,4,5-T dehalogenation and decreased the amount of parent substrate still detectable as halogenated intermediates at the end of the experiment. Sulfate addition had the opposite effect on dehalogenation regardless of whether supplemental carbon was added to the aquifer slurries. The inhibitory effect of sulfate on dehalogenation could sometimes be relieved with molybdate, although this effect seemed to be related to the supplemental carbon compound that was used.     

Evidence for a Chemiosmotic Model of Dehalorespiration in Desulfomonile tiedjei DCB-1

Desulfomonile tiedjei DCB-1, a sulfate-reducing bacterium, conserves energy for growth from reductive dehalogenation of 3-chlorobenzoate by an uncharacterized chemiosmotic process. Respiratory electron transport components were examined in D. tiedjei cells grown under conditions for reductive dehalogenation, pyruvate fermentation, and sulfate reduction. Reductive dehalogenation was inhibited by the respiratory quinone inhibitor 2-heptyl-4-hydroxyquinoline N-oxide, suggesting that a respiratory quinoid is a component of the electron transport chain coupled to reductive dehalogenation. Moreover, reductive dehalogenation activity was dependent on 1,4-naphthoquinone, a possible precursor for a respiratory quinoid. However, no ubiquinone or menaquinone could be extracted from D. tiedjei. Rather, a UV-absorbing quinoid which is different from common respiratory quinones in chemical structure according to mass spectrometric and UV absorption spectroscopic analyses was extracted. ATP sulfurylase, adenosine phosphosulfate reductase, and desulfoviridin sulfite reductase, enzymes involved in sulfate reduction, were constitutively expressed in the cytoplasm of D. tiedjei cells grown under all three metabolic conditions. A periplasmic hydrogenase was detected in cells grown under reductive-dehalogenating and pyruvate-fermenting conditions. A membrane-bound, periplasm-oriented formate dehydrogenase was detected only in cells grown with formate as electron donor, while a cytoplasmic formate dehydrogenase was detected in cells grown under reductive-dehalogenating and pyruvate-fermenting conditions. Results from dehalogenation assays with D. tiedjei whole-cell suspensions and cell extracts suggest that the membrane-bound reductive dehalogenase is cytoplasm oriented. The data clearly demonstrate an enzyme topology in D. tiedjei which produces protons directly in the periplasm, generating a proton motive force by a scalar mechanism.     

Dehalogenation of lindane by a variety of porphyrins and corrins

The dehalogenation of lindane by a range of hemoproteins, porphyrins, and corrins has been tested under reducing conditions in the presence of dithiothreitol. In addition, a series of porphyrin-metal ion complexes have been prepared and have also been screened for the capacity to dehalogenate lindane. Hemoglobin, hemin, hematin, and chlorophyll alpha all catalyzed the dehalogenation of lindane, as did all of the corrins tested. The porphyrins which did not contain metal centers--coproporphyrin, hematoporphyrin, protoporphyrin, and uroporphyrin--were inactive. However, when these porphyrins were then complexed with Co, Fe, Mg, Mo, Ni, or V, lindane dehalogenation was observed. In all cases, the reaction proceeded by an initial dechlorination to produce tetrachlorocyclohexene, which was further dehalogenated to yield chlorobenzene as the end product.