Dehalogenation Activity Research Articles

Electron Irradiated p(VDF-TrFE) Copolymers for Use in Naval Transducer Applications

AbstractRecently, it was discovered that the transduction / strain capability of p(VDF-TrFE) copolymers can be enhanced by more than an order of magnitude by irradiating the copolymer with large doses (40–300 Mrads) of β-particles (1–3 MeV electrons). The goal of this project is to understand how irradiation improves the electromechanical properties of p(VDF-TrFE) copolymers while simultaneously identifying the secondary effects of the irradiation process on the material (not critical to the electromechanical properties) and attempting to separate the two contributions. It has been found that β-irradiation affects the material profoundly in several different manners. Reduction in the melt temperature, degree of crystallinity, and the resulting crystal quality have been observed for increasing doses of β-particles. Similar results have been observed for the Curie transition, especially in the energy associated with, and the breadth otf the transition. In addition, thermogravimetric analysis indicates that irradiation causes both chain scission and network polymer formation. Solid-state NMR results are discussed in reference to postulated dehalogenation, dehydrohalogenation, and olefinic bond formation activities.

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.

A homology model for the human theta-class glutathione transferase T1-1.

A manual threading approach is used to model the human glutathione transferase T1-1 based on the coordinates of the related Theta class enzyme T2-2. The low level of sequence identity (about 20%), found in the C-terminal extension in conjunction with a relative deletion of about five residues makes this a challenging modeling problem. The C-terminal extension contributes to the active site of the molecule and is thus of particular interest for understanding the molecular mechanism of the enzyme. Manual docking of known substrates and non-substrates has implicated potential candidates for the T1-1 catalytic residues involved in the dehalogenation and epoxide-ring opening activities. These include the conserved Theta class residues Arg 107, Trp 115, and the conserved GSTT1 subclass residue His 176. Also, the residue at position 234 is implicated in the modulation of T1-1 activity with different substrates between species.

Chloromethane Metabolism by Methylobacterium sp. Strain CM4

Methylobacterium sp. strain CM4 metabolized chloromethane quantitatively with a molar yield of 2.8 g of whole-cell protein/mol of C. This value was similar to that observed after growth with methanol (2.9 g of protein/mol of C) and about three times larger than the yield with formate (0.94 g of protein/mol of C). Chloromethane dehalogenation activity was inducible. MiniTn5 transposon insertion mutants with altered growth characteristics with chloromethane and other C1 compounds were isolated and characterized. Nine of these were unable to grow with chloromethane but were able to grow with methanol, methylamine, or formate. Seventy-three transposon mutants that were defective in the utilization of either methanol, methylamine, methanol plus methylamine, or formate could still grow with chloromethane. Based on the protein yield data and the properties of the transposon mutants, we propose a pathway for chloromethane metabolism that depends on methyltransferase and dehydrogenase activities.

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.

Reductive Dechlorination of 1,2,3,4-Tetrachlorodibenzo-p-dioxin and Its Products by Anaerobic Mixed Cultures from Saale River Sediment

The capability of anaerobic bacterial consortia from different environmental sources including soils, sewage sludges, and sediment of the river Saale (Germany) to dehalogenate chlorinated dioxins was compared using 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD) as the model compound. The inocula were amended with mineral medium and organic acids and spiked with a high concentration (50 μM) of 1,2,3,4-TCDD to stimulate microbial dehalogenating activity. Reductive dechlorination was observed to 1,3-dichlorodibenzo-p-dioxin (1,3-DCDD) as the main product and to minor amounts of the 1,2,4- and 1,2,3-trichlorodibenzo-p-dioxins (TrCDD) using incubations with Saale River sediment. No reaction was observed in the controls and in incubations with soils or sewage sludges. The dechlorination of 1,2,4- and 1,2,3-TrCDD was analyzed in separate subcultures. Reductive dechlorination of 1,2,4-TrCDD was a relative fast process (about 6 μM converted within 58 days) and yielded only one product (1,3-DCDD). 1,2,3-TrCDD was slowly dechlorinated to equal amounts of 1,3- and 2,3-DCDD. These observations suggest that the main dechlorination route of 1,2,3,4-TCDD to 1,3-DCDD proceeds primarily via the removal of a lateral chlorine atom with 1,2,4-TrCDD as the intermediate.

Chlorophenol dechlorination and subsequent degradation in denitrifying microcosms fed low concentrations of nitrate

The potential of using nitrate as a terminal electron acceptor to stimulate anaerobic degradation of mixtures of monochlorophenols (MCPs) or dichlorophenols (DCPs) was evaluated. Contaminated and non-contaminated soils were added to water saturated anaerobic microcosms supplemented with 1 mM or 5 mM nitrate. Denitrification and dechlorination activity were present in three diverse soil types and were maintained upon refeeding both nitrate and the appropriate chlorophenol. However, dechlorination activity could only be serially transferred in enrichments with an added electron donor such as acetate. Dehalogenation activity in enrichments from four of the primary microcosms showed at least five different dechlorination reactions, each mediated by different microbial communities. Three of these are distinct ortho-dechlorinating paths; two are meta-dechlorinating and one is the para-dechlorination of 3,4-DCP. Simultaneous dechlorination and denitrification was observed and both activities could be maintained in microcosms but only in the presence of low nitrate concentrations. Dechlorination and denitrification were mediated by two separate microbial communities; one that dechlorinates without use of nitrate and one that denitrifies while oxidizing the dechlorinated aromatic ring. There was no evidence that dechlorination is mediated by the denitrifying community, however the maintenance of a denitrification potential using low (< 1 mM) nitrate concentrations may be useful for completing the food chain by stimulating the mineralization of phenol and benzoate.

Characteristics of a microbial assay for the detection of halogenated hydrocarbons using cells of an actinomycete‐like organism as a biological component

AbstractCells of an Actinomycete‐like bacterium, strain GJ70, with the ability to degrade several haloalkanes were used as a biological component in a discontinuous microbial bioassay for the detection of 1,3‐dichloropropene and 1,2‐dibromoethane in water. The cells were entrapped in different matrices such as calcium alginate, carrageenan, chitosan, polyacrylamide‐hydrazide and chitosan‐carboxy‐methyl cellulose; the specific dehalogenating activity of the immobilized cells to a stirred sample solution and by the use of an ion selective electrode (ISE) for the quantification of enzymatically released halogen ions, the concentration of halogenated hydrocarbons could be estimated by determining the change of electrode potential within a period of 5 min. The detection limits for 1,3‐dichloropropene and 1,2‐dibromoethane were below 100 μg/l and 25 μg/l, respectively; the relative standard deviation was &lt; 10%. In addition, several chlorinated and brominated hgydrocarbons were converted by the bacterial cells at a reduced rate e.g. 1, 2‐dibromopropane, 1‐bromoethane, 1,5‐dichloropentane, etc. Moreover, temperatures of between 20 and 40%C did not affect the enzymatic activity of the cells, and a pH of between at 5 and 9 had little influence. Several organic substances and non‐metabolizable compounds did not affect the conversion, whereas some heavy metal ions acted as inhibitors.

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.

Dehalogenation of 2-chlorophenol (2-CP) in anaerobic batch cultures

The effects of seed materials from: anaerobically digested sewage sludge; sediment from a bleached kraft mill (BKM) aerated lagoon and an anaerobic bioreactor biomass treating BKM wastewater were investigated as to their potential for dehalogenation of 0.38 mM 2-chlorophenol. In batch cultures using a mineral medium containing 0.1% yeast extract under 20% CO 2 + 80% N 2 or 20% CO 2 + 80% H 2 atmosphere, dehalogenation was observed after extensive lag periods lasting from 97 to 250 days. Methane was detected in the head-space. Phenol was identified as an intermediate which further degraded via the benzoate pathway. The dehalogenation rates increased significantly in all cultures after re-spiking with 2-CP and yeast extract medium. Cultures with digested sewage sludge incubated under CO 2 N 2 head-space exhibited better dehalogenation rates of 2-CP compared to either the lagoon sediment or the bioreactor biomass. Higher dehalogenation rates were also observed over CO 2 N 2 head-space compared to CO 2 H 2 head-space for digested sludge. Digested sludge which was adapted to 2-CP over a one year period converted 0.38 mM 2-CP to phenol immediately. Degradation of 2-CP with various dilutions of adapted sludge indicated that dehalogenation activity did not increase linearly with biomass concentrations. The effect of four different organic electron donors on the dehalogenation of 2-CP by adapted sludge revealed that 0.1% yeast extract was preferred by the consortium compared to 10 mM each of acetate, propionate or ethanol.

Cloning and expression of a haloacid dehalogenase fromPseudomonas sp. strain 19 S

A dehalogenase gene specifying the utilization of a variety of haloacids by Pseudomonas sp. Strain 19S has been cloned and expressed in E. coli. Our cloning strategy employed specific amplification of a fragment homologous to Pseudomonas dehalogenase gene by Polymerase Chain Reaction (PCR). The PCR amplicon successfully acted as a probe to detect the dehalogenase gene in the Southern Blot of the digested Pseudomonas total DNA. Corresponding fragments were cloned into pUC 18 vector and amplified in E. coli MV 1190. One clone with a substantial dehalogenation activity carried a recombinant plasmid containing a 5.5 kb insert.

Monochloroacetate dehalogenase activities of bacterial strains isolated from soil

Seven bacterial strains capable of utilizing monochloroacetate (MCA) at a concentration of 50 mM as the sole carbon source were isolated from soil and displayed MCA dehalogenase activity. Three of them were identified as Pseudomonas spp., and the remaining four strains as Alcaligenes sp., Agrobacterium sp., Arthrobacter sp., and Azotobacter sp. This latter is the first reported example of a bacterium fixing atmospheric nitrogen under aerobic conditions that also uses a chloro-organic compound as sole source of carbon and energy. MCA dehalogenase activity in these strains was found to be inducible under different growth conditions. Crude extracts from all seven isolated strains also displayed dehalogenating activity with a relatively wide range of halogenated organic compounds (aliphatic acids, ketones, alcohols, alkanes, and aromatics), which, depending on the strain, were dehalogenated to different extents. The estimated Kmvalues for MCA were used to classify the dehalogenase activities into three groups: high affinity (30–40 μM) in Alcaligenes and Agrobacterium species, medium affinity (100–180 μM) in Pseudomonas and Azotobacter species, and low affinity (100 mM) in Arthrobacter sp. Both the optimal pH range for MCA dehalogenase activity (between pH 8 and 10) and the pH profile of stability (in the neutral–basic range) were found to be similar in all strains, whereas the thermal stability profiles were variable.Key words: dehalogenase, halohydrolase, monochloroacetate, soil.

Properties of tetrachloroethene and trichloroethene dehalogenase of Dehalospirillum multivorans

Some properties of tetrachloroethene and trichloroethene dehalogenase of the recently isolated, tetrachloroethene-utilizing anaerobe, Dehalospirillum multivorans, were studied with extracts of cells grown on pyruvate plus fumarate. The dehalogenase catalyzed the oxidation of reduced methyl viologen with tetrachloroethene (PCE) or trichloroethene (TCE) as electron acceptor. All other artificial or physiological electron donors tested were ineffective. The PCE and TCE dehalogenase activity was insensitive towards oxygen in crude extracts. When extracts were incubated under anoxic conditions in the presence of titanium citrate as reducing agent, the dehalogenase was rapidly inactivated by propyl iodide (50 μM). Inactivation did not occur in the absence of titanium citrate. The activity of propyl-iodide-treated extracts was restored almost immediately by illumination. The dehalogenase was inhibited by cyanide. The inhibition profile was almost the same under oxic and anoxic conditions independent of the presence or absence of titanium citrate. In addition, N2O, nitrite, and ethylene diamine tetra-acetate (EDTA) were inhibitors of PCE and TCE dehalogenase. Carbon monoxide and azide had no influence on the dehalogenase activity. Trans-1,2-dichloroethene or 1,1-dichloroethene, both of which are isomers of the dechlorination product cis-1,2-dichloroethene, neither inhibited nor inactivated the dehalogenase. PCE and TCE dechlorination appeared to be mediated by the same enzyme since the inhibitors tested had nearly the same effects on the PCE and TCE dehalogenating activity. The data indicated the involvement of a corrinoid and possibly of an additional transition metal in reductive PCE and TCE dechlorination.

Detection of the anaerobic dechlorinating microorganism Desulfomonile tiedjei in environmental matrices by its signature lipopolysacchride branched-long-chain hydroxy fatty acids

Desulfomonile tiedjei is a Gram-negative sulfate-reducing bacterium capable of catalyzing aryl reductive dehalogenation reactions. Since many toxic and persistent contaminants in the subsurface are halogenated aromatic compounds, the detection and enumeration of dehalogenating microorganisms in the environment may be a useful tool for planning and evaluating bioremediation efforts. In this study, we show that D. tiedjei contains unique lipopolysaccharide branched 3-hydroxy fatty acids, unknown as yet in other bacteria, and that it is possible to detect the bacterium in inoculated aquifer sediments based on these signature lipid biomarkers. The detection of D. tiedjei and other dehalogenating microorganisms possessing similar cellular properties in environmental matrices may be possible by this technique. Additionally, the effect of such inoculation on dehalogenation activity is examined.

Detection of the anaerobic dechlorinating microorganism Desulfomonile tiedjei in environmental matrices by its signature lipopolysacchride branched-long-chain hydroxy fatty acids

Desulfomonile tiedjei is a Gram-negative sulfate-reducing bacterium capable of catalyzing aryl reductive dehalogenation reactions. Since many toxic and persistent contaminants in the subsurface are halogenated aromatic compounds, the detection and enumeration of dehalogenating microorganisms in the environment may be a useful tool for planning and evaluating bioremediation efforts. In this study, we show that D. tiedjei contains unique lipopolysaccharide branched 3-hydroxy fatty acids, unknown as yet in other bacteria, and that it is possible to detect the bacterium in inoculated aquifer sediments based on these signature lipid biomarkers. The detection of D. tiedjei and other dehalogenating microorganisms possessing similar cellular properties in environmental matrices may be possible by this technique. Additionally, the effect of such inoculation on dehalogenation activity is examined.

Reductive dehalogenation as a respiratory process.

Anaerobic bacteria can reductively dehalogenate aliphatic and aromatic halogenated compounds in a respiratory process. Only a few of these bacteria have been isolated in pure cultures. However, long acclimation periods, substrate specificity, high dehalogenation rates, and the possibility to enrich for the dehalogenation activity by subcultivation in media containing an electron donor indicate that many of the reductive dehalogenations in the environment are catalyzed by specific bacteria. Molecular hydrogen or formate appear to be good electron donors for the enrichment of such organisms. Furthermore, systems have to be employed which supply the cultures with the halogenated compounds beyond their toxicity level. All bacteria that are presently available in pure culture and grow with a halogenated compound as electron acceptor are members of new genera. Based on experimental results with the membrane-impermeable electron mediator methyl viologen, a model of the respiration system of Dehalobacter restrictus, a tetrachloroethene-dechlorinating bacterium, is presented. Further studies of the biochemistry and energetics of respiratory-dehalogenating strains will help to understand the mechanisms involved and perhaps reveal the evolutionary origin of the dehalogenating enzyme systems.

Stimulation of Reductive Dechlorination of Tetrachloroethene in Anaerobic Aquifer Microcosms by Addition of Short-Chain Organic Acids or Alcohols

The effect of the addition of common fermentation products on the dehalogenation of tetrachloroethene was studied in methanogenic slurries made with aquifer solids. Lactate, propionate, crotonate, butyrate, and ethanol stimulated dehalogenation activity, while acetate, methanol, and isopropanol did not.

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°C only about 50% of the initial activity was left. Treatment at 55°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 Mg 2+. 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.

Separate cloning and expression analysis of two protein components of 4-chlorobenzoate dehalogenase from Pseudomonas sp. C8S3

The two protein components, II and III, of the 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 were cloned separately into Escherichia coli. Component II was obtained on plasmid pCBSII, containing a 3.0 kbp HindIII fragment, and component III on plasmid pCBSIIIb, containing a 1.3 kbp SalI/PstI fragment. The identities of the two components were confirmed by comparison with the authentic components from Pseudomonas sp. CBS3. Both components were expressed constitutively in E. coli. Neither component alone showed dehalogenating activity. Only in the mixture of crude extracts from both clones was 4-chlorobenzoate dehalogenase detectable. The specific activities in E. coli crude extracts were 2.9 mU (mg protein)-1 for component II and 3.5 mU (mg protein)-1 for component III. Expression analysis by minicell experiments revealed a single polypeptide chain of 29 kDa for component III and of 31 kDa for component III.

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.