Reductive dechlorination of tetrachloroethene to ethene by a two-component enzyme pathway

Two membrane-bound, reductive dehalogenases that constitute a novel pathway for complete dechlorination of tetrachloroethene (perchloroethylene [PCE]) to ethene were partially purified from an anaerobic microbial enrichment culture containing Dehalococcoides ethenogenes 195. When titanium (III) citrate and methyl viologen were used as reductants, PCE-reductive dehalogenase (PCE-RDase) (51 kDa) dechlorinated PCE to trichloroethene (TCE) at a rate of 20 micromol/min/mg of protein. TCE-reductive dehalogenase (TCE-RDase) (61 kDa) dechlorinated TCE to ethene. TCE, cis-1,2-dichloroethene, and 1,1-dichloroethene were dechlorinated at similar rates, 8 to 12 micromol/min/mg of protein. Vinyl chloride and trans-1,2-dichloroethene were degraded at rates which were approximately 2 orders of magnitude lower. The light-reversible inhibition of TCE-RDase by iodopropane and the light-reversible inhibition of PCE-RDase by iodoethane suggest that both of these dehalogenases contain Co(I) corrinoid cofactors. Isolation and characterization of these novel bacterial enzymes provided further insight into the catalytic mechanisms of biological reductive dehalogenation.     

Trichloroethene Reductive Dehalogenase from Dehalococcoides ethenogenes: Sequence of tceA and Substrate Range Characterization

The anaerobic bacterium Dehalococcoides ethenogenes is the only known organism that can completely dechlorinate tetrachloroethene or trichloroethene (TCE) to ethene via dehalorespiration. One of two corrinoid-containing enzymes responsible for this pathway, TCE reductive dehalogenase (TCE-RDase) catalyzes the dechlorination of TCE to ethene. TCE-RDase dehalogenated 1,2-dichloroethane and 1,2-dibromoethane to ethene at rates of 7.5 and 30 μmol/min/mg, respectively, similar to the rates for TCE, cis-dichloroethene (DCE), and 1,1-DCE. A variety of other haloalkanes and haloalkenes containing three to five carbon atoms were dehalogenated at lower rates. The gene encoding TCE-RDase, tceA, was cloned and sequenced via an inverse PCR approach. Sequence comparisons of tceA to proteins in the public databases revealed weak sequence similarity confined to the C-terminal region, which contains the eight-iron ferredoxin cluster binding motif, (CXXCXXCXXXCP)₂. Direct N-terminal sequencing of the mature enzyme indicated that the first 42 amino acids constitute a signal sequence containing the twin-arginine motif, RRXFXK, associated with the Sec-independent membrane translocation system. This information coupled with membrane localization studies indicated that TCE-RDase is located on the exterior of the cytoplasmic membrane. Like the case for the two other RDases that have been cloned and sequenced, a small open reading frame, tceB, is proposed to be involved with membrane association of TCE-RDase and is predicted to be cotranscribed with tceA.     

Quantitative PCR Confirms Purity of Strain GT, a Novel Trichloroethene-to-Ethene-Respiring Dehalococcoides Isolate

A novel Dehalococcoides isolate capable of metabolic trichloroethene (TCE)-to-ethene reductive dechlorination was obtained from contaminated aquifer material. Growth studies and 16S rRNA gene-targeted analyses suggested culture purity; however, the careful quantitative analysis of Dehalococcoides 16S rRNA gene and chloroethene reductive dehalogenase gene (i.e., vcrA, tceA, and bvcA) copy numbers revealed that the culture consisted of multiple, distinct Dehalococcoides organisms. Subsequent transfers, along with quantitative PCR monitoring, yielded isolate GT, possessing only vcrA. These findings suggest that commonly used qualitative 16S rRNA gene-based procedures are insufficient to verify purity of Dehalococcoides cultures. Phylogenetic analysis revealed that strain GT is affiliated with the Pinellas group of the Dehalococcoides cluster and shares 100% 16S rRNA gene sequence identity with two other Dehalococcoides isolates, strain FL2 and strain CBDB1. The new isolate is distinct, as it respires the priority pollutants TCE, cis-1,2-dichloroethene (cis-DCE), 1,1-dichloroethene (1,1-DCE), and vinyl chloride (VC), thereby producing innocuous ethene and inorganic chloride. Strain GT dechlorinated TCE, cis-DCE, 1,1-DCE, and VC to ethene at rates up to 40, 41, 62, and 127 μmol liter⁻¹ day⁻¹, respectively, but failed to dechlorinate PCE. Hydrogen was the required electron donor, which was depleted to a consumption threshold concentration of 0.76 ± 0.13 nM with VC as the electron acceptor. In contrast to the known TCE dechlorinating isolates, strain GT dechlorinated TCE to ethene with very little formation of chlorinated intermediates, suggesting that this type of organism avoids the commonly observed accumulation of cis-DCE and VC during TCE-to-ethene dechlorination.     

Characterization of the corrinoid iron-sulfur protein tetrachloroethene reductive dehalogenase of Dehalobacter restrictus

The membrane-bound tetrachloroethene reductive dehalogenase (PCE-RDase) (PceA; EC 1.97.1.8), the terminal component of the respiratory chain of Dehalobacter restrictus, was purified 25-fold to apparent electrophoretic homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 60 +/- 1 kDa, whereas the native molecular mass was 71 +/- 8 kDa according to size exclusion chromatography in the presence of the detergent octyl-beta-D-glucopyranoside. The monomeric enzyme contained (per mol of the 60-kDa subunit) 1.0 +/- 0.1 mol of cobalamin, 0.6 +/- 0.02 mol of cobalt, 7.1 +/- 0.6 mol of iron, and 5.8 +/- 0.5 mol of acid-labile sulfur. Purified PceA catalyzed the reductive dechlorination of tetrachloroethene and trichloroethene to cis-1,2-dichloroethene with a specific activity of 250 +/- 12 nkat/mg of protein. In addition, several chloroethanes and tetrachloromethane caused methyl viologen oxidation in the presence of PceA. The K(m) values for tetrachloroethene, trichloroethene, and methyl viologen were 20.4 +/- 3.2, 23.7 +/- 5.2, and 47 +/- 10 micro M, respectively. The PceA exhibited the highest activity at pH 8.1 and was oxygen sensitive, with a half-life of activity of 280 min upon exposure to air. Based on the almost identical N-terminal amino acid sequences of PceA of Dehalobacter restrictus, Desulfitobacterium hafniense strain TCE1 (formerly Desulfitobacterium frappieri strain TCE1), and Desulfitobacterium hafniense strain PCE-S (formerly Desulfitobacterium frappieri strain PCE-S), the pceA genes of the first two organisms were cloned and sequenced. Together with the pceA genes of Desulfitobacterium hafniense strains PCE-S and Y51, the pceA genes of Desulfitobacterium hafniense strain TCE1 and Dehalobacter restrictus form a coherent group of reductive dehalogenases with almost 100% sequence identity. Also, the pceB genes, which may code for a membrane anchor protein of PceA, and the intergenic regions of Dehalobacter restrictus and the three desulfitobacteria had identical sequences. Whereas the cprB (chlorophenol reductive dehalogenase) genes of chlorophenol-dehalorespiring bacteria are always located upstream of cprA, all pceB genes known so far are located downstream of pceA. The possible consequences of this feature for the annotation of putative reductive dehalogenase genes are discussed, as are the sequence around the iron-sulfur cluster binding motifs and the type of iron-sulfur clusters of the reductive dehalogenases of Dehalobacter restrictus and Desulfitobacterium dehalogenans identified by electron paramagnetic resonance spectroscopy.     

Purification, Cloning, and Sequencing of a 3,5-Dichlorophenol Reductive Dehalogenase from Desulfitobacterium frappieri PCP-1

A membrane-associated 3,5-dichlorophenol reductive dehalogenase was isolated from Desulfitobacterium frappieri PCP-1. The highest dehalogenase activity was observed with the biomass cultured at 22°C, compared to 30 and 37°C, where the cell suspensions were 2.2 and 9.6 times less active, respectively. The reductive dehalogenase was purified 12.7-fold to apparent homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 57 kDa. Its dechlorinating activity was not inhibited by sulfate and nitrate but was completely inhibited by 2.5 mM sulfite and 10 mM KCN. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activities, suggesting the involvement of a corrinoid cofactor. Several polychlorophenols were dechlorinated at the meta and para positions. The apparent K_m for 3,5-dicholorophenol was 49.3 ± 3.1 μM at a methyl viologen concentration of 2 mM. Six internal tryptic peptides were sequenced by mass spectrometry. One open reading frame (ORF) was found in the Desulfitobacterium hafniense genome containing these peptide sequences. This ORF corresponds to a gene coding for a CprA-type reductive dehalogenase. The corresponding ORF (named cprA5) in D. frappieri PCP-1 was cloned and sequenced. The cprA5 gene codes for a 548-amino-acid protein that contains a twin-arginine-type signal for secretion. The gene product has a cobalamin binding site motif and two iron-sulfur binding motifs and shows 66% identity (76 to 77% similarity) with some tetrachloroethene reductive dehalogenases. This is the first CprA-type reductive dehalogenase that can dechlorinate chlorophenols at the meta and para positions.     

Quantitative PCR confirms purity of strain GT, a novel trichloroethene-to-ethene-respiring Dehalococcoides isolate

A novel Dehalococcoides isolate capable of metabolic trichloroethene (TCE)-to-ethene reductive dechlorination was obtained from contaminated aquifer material. Growth studies and 16S rRNA gene-targeted analyses suggested culture purity; however, the careful quantitative analysis of Dehalococcoides 16S rRNA gene and chloroethene reductive dehalogenase gene (i.e., vcrA, tceA, and bvcA) copy numbers revealed that the culture consisted of multiple, distinct Dehalococcoides organisms. Subsequent transfers, along with quantitative PCR monitoring, yielded isolate GT, possessing only vcrA. These findings suggest that commonly used qualitative 16S rRNA gene-based procedures are insufficient to verify purity of Dehalococcoides cultures. Phylogenetic analysis revealed that strain GT is affiliated with the Pinellas group of the Dehalococcoides cluster and shares 100% 16S rRNA gene sequence identity with two other Dehalococcoides isolates, strain FL2 and strain CBDB1. The new isolate is distinct, as it respires the priority pollutants TCE, cis-1,2-dichloroethene (cis-DCE), 1,1-dichloroethene (1,1-DCE), and vinyl chloride (VC), thereby producing innocuous ethene and inorganic chloride. Strain GT dechlorinated TCE, cis-DCE, 1,1-DCE, and VC to ethene at rates up to 40, 41, 62, and 127 micromol liter-1 day-1, respectively, but failed to dechlorinate PCE. Hydrogen was the required electron donor, which was depleted to a consumption threshold concentration of 0.76+/-0.13 nM with VC as the electron acceptor. In contrast to the known TCE dechlorinating isolates, strain GT dechlorinated TCE to ethene with very little formation of chlorinated intermediates, suggesting that this type of organism avoids the commonly observed accumulation of cis-DCE and VC during TCE-to-ethene dechlorination.     

Trichloroethene reductive dehalogenase from Dehalococcoides ethenogenes: sequence of tceA and substrate range characterization

The anaerobic bacterium Dehalococcoides ethenogenes is the only known organism that can completely dechlorinate tetrachloroethene or trichloroethene (TCE) to ethene via dehalorespiration. One of two corrinoid-containing enzymes responsible for this pathway, TCE reductive dehalogenase (TCE-RDase) catalyzes the dechlorination of TCE to ethene. TCE-RDase dehalogenated 1,2-dichloroethane and 1, 2-dibromoethane to ethene at rates of 7.5 and 30 micromol/min/mg, respectively, similar to the rates for TCE, cis-dichloroethene (DCE), and 1,1-DCE. A variety of other haloalkanes and haloalkenes containing three to five carbon atoms were dehalogenated at lower rates. The gene encoding TCE-RDase, tceA, was cloned and sequenced via an inverse PCR approach. Sequence comparisons of tceA to proteins in the public databases revealed weak sequence similarity confined to the C-terminal region, which contains the eight-iron ferredoxin cluster binding motif, (CXXCXXCXXXCP)(2). Direct N-terminal sequencing of the mature enzyme indicated that the first 42 amino acids constitute a signal sequence containing the twin-arginine motif, RRXFXK, associated with the Sec-independent membrane translocation system. This information coupled with membrane localization studies indicated that TCE-RDase is located on the exterior of the cytoplasmic membrane. Like the case for the two other RDases that have been cloned and sequenced, a small open reading frame, tceB, is proposed to be involved with membrane association of TCE-RDase and is predicted to be cotranscribed with tceA.     

Characterization of a Highly Enriched Dehalococcoides-Containing Culture That Grows on Vinyl Chloride and Trichloroethene

A highly enriched culture that reductively dechlorinates trichloroethene (TCE), cis-1,2-dichloroethene (cDCE), and vinyl chloride (VC) to ethene without methanogenesis is described. The Dehalococcoides strain in this enrichment culture had a yield of (5.6 ± 1.4) × 10⁸ 16S rRNA gene copies/μmol of Cl⁻ when grown on VC and hydrogen. Unlike the other VC-degrading cultures described in the literature, strains VS and BAV1, this culture maintained the ability to grow on TCE with a yield of (3.6 ± 1.3) × 10⁸ 16S rRNA gene copies/μmol of Cl⁻. The yields on an electron-equivalent basis measured for the culture grown on TCE and on VC were not significantly different, indicating that both substrates supported growth equally well. PCR followed by denaturing gradient gel electrophoresis, cloning, and phylogenetic analyses revealed that this culture contained one Dehalococcoides 16S rRNA gene sequence, designated KB-1/VC, that was identical (over 1,386 bp) to the sequences of previously described organisms FL2 and CBDB1. A second Dehalococcoides sequence found in separate KB-1 enrichment cultures maintained on cDCE, TCE, and tetrachloroethene was no longer present in the VC-H₂ enrichment culture. This second Dehalococcoides sequence was identical to that of BAV1. As neither FL2 nor CBDB1 can dechlorinate VC to ethene in a growth-related fashion, it is clear that current 16S rRNA gene-based analyses do not provide sufficient information to distinguish between metabolically diverse members of the Dehalococcoides group.     

Reductive dechlorination of tetrachloroethene by a stepwise catalysis of different organohalide respiring bacteria and reductive dehalogenases

The enrichment culture SL2 dechlorinating tetrachloroethene (PCE) to ethene with strong trichloroethene (TCE) accumulation prior to cis-1,2-dichloroethene (cis-DCE) formation was analyzed for the presence of organohalide respiring bacteria and reductive dehalogenase genes (rdhA). Sulfurospirillum-affiliated bacteria were identified to be involved in PCE dechlorination to cis-DCE whereas “Dehalococcoides”-affiliated bacteria mainly dechlorinated cis-DCE to ethene. Two rdhA genes highly similar to tetrachloroethene reductive dehalogenase genes (pceA) of S. multivorans and S. halorespirans were present as well as an rdhA gene very similar to the trichloroethene reductive dehalogenase gene (tceA) of “Dehalococcoides ethenogenes” strain 195. A single strand conformation polymorphism (SSCP) method was developed allowing the simultaneous detection of the three rdhA genes and the estimation of their abundance. SSCP analysis of different SL2 cultures showed that one pceA gene was expressed during PCE dechlorination whereas the second was expressed during TCE dechlorination. The tceA gene was involved in cis-DCE dechlorination to ethene. Analysis of the internal transcribed spacer region between the 16S and 23S rRNA genes revealed two distinct sequences originating from Sulfurospirillum suggesting that two Sulfurospirillum populations were present in SL2. Whether each Sulfurospirillum population was catalyzing a different dechlorination step could however not be elucidated.     

Reductive Dechlorination of Chlorinated Ethenes and 1,2-Dichloroethane by “Dehalococcoides ethenogenes” 195

“Dehalococcoides ethenogenes” 195 can reductively dechlorinate tetrachloroethene (PCE) completely to ethene (ETH). When PCE-grown strain 195 was transferred (2% [vol/vol] inoculum) into growth medium amended with trichloroethene (TCE), cis-dichloroethene (DCE), 1,1-DCE, or 1,2-dichloroethane (DCA) as an electron acceptor, these chlorinated compounds were consumed at increasing rates over time, which indicated that growth occurred. Moreover, the number of cells increased when TCE, 1,1-DCE, or DCA was present. PCE, TCE, 1,1-DCE, and cis-DCE were converted mainly to vinyl chloride (VC) and then to ETH, while DCA was converted to ca. 99% ETH and 1% VC. cis-DCE was used at lower rates than PCE, TCE, 1,1-DCE, or DCA was used. When PCE-grown cultures were transferred to media containing VC or trans-DCE, products accumulated slowly, and there was no increase in the rate, which indicated that these two compounds did not support growth. When the intermediates in PCE dechlorination by strain 195 were monitored, TCE was detected first, followed by cis-DCE. After a lag, VC, 1,1-DCE, and trans-DCE accumulated, which is consistent with the hypothesis that cis-DCE is the precursor of these compounds. Both cis-DCE and 1,1-DCE were eventually consumed, and both of these compounds could be considered intermediates in PCE dechlorination, whereas the small amount of trans-DCE that was produced persisted. Cultures grown on TCE, 1,1-DCE, or DCA could immediately dechlorinate PCE, which indicated that PCE reductive dehalogenase activity was constitutive when these electron acceptors were used.     

Complete Detoxification of Vinyl Chloride by an Anaerobic Enrichment Culture and Identification of the Reductively Dechlorinating Population as a Dehalococcoides Species

A major obstacle in the implementation of the reductive dechlorination process at chloroethene-contaminated sites is the accumulation of the intermediate vinyl chloride (VC), a proven human carcinogen. To shed light on the microbiology involved in the final critical dechlorination step, a sediment-free, nonmethanogenic, VC-dechlorinating enrichment culture was derived from tetrachloroethene (PCE)-to-ethene-dechlorinating microcosms established with material from the chloroethene-contaminated Bachman Road site aquifer in Oscoda, Mich. After 40 consecutive transfers in defined, reduced mineral salts medium amended with VC, the culture lost the ability to use PCE and trichloroethene (TCE) as metabolic electron acceptors. PCE and TCE dechlorination occurred in the presence of VC, presumably in a cometabolic process. Enrichment cultures supplied with lactate or pyruvate as electron donor dechlorinated VC to ethene at rates up to 54 μmol liter⁻¹day⁻¹, and dichloroethenes (DCEs) were dechlorinated at about 50% of this rate. The half-saturation constant (K_S) for VC was 5.8 μM, which was about one-third lower than the concentrations determined for cis-DCE and trans-DCE. Similar VC dechlorination rates were observed at temperatures between 22 and 30°C, and negligible dechlorination occurred at 4 and 35°C. Reductive dechlorination in medium amended with ampicillin was strictly dependent on H₂ as electron donor. VC-dechlorinating cultures consumed H₂ to threshold concentrations of 0.12 ppm by volume. 16S rRNA gene-based tools identified a Dehalococcoides population, and Dehalococcoides-targeted quantitative real-time PCR confirmed VC-dependent growth of this population. These findings demonstrate that Dehalococcoides populations exist that use DCEs and VC but not PCE or TCE as metabolic electron acceptors.     

Anaerobic dechlorination of trichloroethene,tetrachloroethene and 1,2-dichloroethane by an acetogenic mixed culture in a fixed-bed reactor

An anaerobic enrichment culture with glucose as the sole source of carbon and energy plus trichloroethene (TCE) as a potential electron acceptor was inoculated with material from a full size anaerobic charcoal reactor that biologically eliminated dichloromethane from contaminated groundwater (Stromeyer et al. 1991). In subcultures of this enrichment complete sequential transformation of 10 µM TCE viacis-dichloroethene and chloroethene to ethene was reproducibly observed. Maintenance of this activity on subcultivation required the presence of TCE in the medium. The enrichment culture was used to inoculate an anaerobic fixed-bed reactor containing sintered glass Raschig elements as support material. The reactor had a total volume of 1780 ml and was operated at 20 °C in an up-flow mode with a flow rate of 50 ml/h. It was fed continuously with 2 mM glucose and 55 µM TCE. Glucose was converted to acetate as the major product and to a minor amount of methane; TCE was quantitatively dehalogenated to ethene. When, in addition to TCE, tetrachloroethene or 1,2-dichloroethane were added to the system, these compounds were also dehalogenated to ethene. In contrast, 1,1,1-trichloroethane was not dehalogenated, but at 40 µM severely inhibited acetogenesis and methanogenesis. When the concentration of TCE in the feed was raised to 220 µM, chloroethene transiently accumulated, but after an adaptation period ethene was again the only volatile product detected in the effluent. The volumetric degradation rate at this stage amounted to 6.2 µmol/l/h. Since complete transformation of TCE occurred in the first sixth of the reactor volume, the degradation capacity of the system is estimated to exceed this value by factor of about ten.Abbreviations CA chloroethane - 1,1-DCA 1,1-dichloroethane - 1,2-DCA 1,2-dichloroethane - 1,1-DCE 1,1-dichloroethene - c-DCE cis-1,2-dichloroethene - t-DCE trans-1,2-dichloroethene - PCE tetrachloroethene, perchloroethene - 1,1,1-TCA 1,1,1-trichloroethane - TCE trichloroethene - VC chloroethene, vinyl chloride     

Purification, cloning, and sequencing of a 3,5-dichlorophenol reductive dehalogenase from Desulfitobacterium frappieri PCP-1

A membrane-associated 3,5-dichlorophenol reductive dehalogenase was isolated from Desulfitobacterium frappieri PCP-1. The highest dehalogenase activity was observed with the biomass cultured at 22 degrees C, compared to 30 and 37 degrees C, where the cell suspensions were 2.2 and 9.6 times less active, respectively. The reductive dehalogenase was purified 12.7-fold to apparent homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 57 kDa. Its dechlorinating activity was not inhibited by sulfate and nitrate but was completely inhibited by 2.5 mM sulfite and 10 mM KCN. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activities, suggesting the involvement of a corrinoid cofactor. Several polychlorophenols were dechlorinated at the meta and para positions. The apparent K(m) for 3,5-dicholorophenol was 49.3 +/- 3.1 microM at a methyl viologen concentration of 2 mM. Six internal tryptic peptides were sequenced by mass spectrometry. One open reading frame (ORF) was found in the Desulfitobacterium hafniense genome containing these peptide sequences. This ORF corresponds to a gene coding for a CprA-type reductive dehalogenase. The corresponding ORF (named cprA5) in D. frappieri PCP-1 was cloned and sequenced. The cprA5 gene codes for a 548-amino-acid protein that contains a twin-arginine-type signal for secretion. The gene product has a cobalamin binding site motif and two iron-sulfur binding motifs and shows 66% identity (76 to 77% similarity) with some tetrachloroethene reductive dehalogenases. This is the first CprA-type reductive dehalogenase that can dechlorinate chlorophenols at the meta and para positions.     

Identity and Substrate Specificity of Reductive Dehalogenases Expressed in Dehalococcoides-Containing Enrichment Cultures Maintained on Different Chlorinated Ethenes

Many reductive dehalogenases (RDases) have been identified in organohalide-respiring microorganisms, and yet their substrates, specific activities, and conditions for expression are not well understood. We tested whether RDase expression varied depending on the substrate-exposure history of reductive dechlorinating communities. For this purpose, we used the enrichment culture KB-1 maintained on trichloroethene (TCE), as well as subcultures maintained on the intermediates cis-dichloroethene (cDCE) and vinyl chloride (VC). KB-1 contains a TCE-to-cDCE dechlorinating Geobacter and several Dehalococcoides strains that together harbor many of the known chloroethene reductases. Expressed RDases were identified using blue native polyacrylamide gel electrophoresis, enzyme assays in gel slices, and peptide sequencing. As anticipated but never previously quantified, the RDase from Geobacter was only detected transiently at the beginning of TCE dechlorination. The Dehalococcoides RDase VcrA and smaller amounts of TceA were expressed in the parent KB-1 culture during complete dechlorination of TCE to ethene regardless of time point or amended substrate. The Dehalococcoides RDase BvcA was only detected in enrichments maintained on cDCE as growth substrates, in roughly equal abundance to VcrA. Only VcrA was detected in subcultures enriched on VC. Enzyme assays revealed that 1,1-DCE, a substrate not used for culture enrichment, afforded the highest specific activity. trans-DCE was substantially dechlorinated only by extracts from cDCE enrichments expressing BvcA. RDase gene distribution indicated enrichment of different strains of Dehalococcoides as a function of electron acceptor TCE, cDCE, or VC. Each chloroethene reductase has distinct substrate preferences leading to strain selection in mixed communities.     

Biodegradation of cis-Dichloroethene as the Sole Carbon Source by a β-Proteobacterium

An aerobic bacterium capable of growth on cis-dichloroethene (cDCE) as a sole carbon and energy source was isolated by enrichment culture. The 16S ribosomal DNA sequence of the isolate (strain JS666) had 97.9% identity to the sequence from Polaromonas vacuolata, indicating that the isolate was a β-proteobacterium. At 20°C, strain JS666 grew on cDCE with a minimum doubling time of 73 ± 7 h and a growth yield of 6.1 g of protein/mol of cDCE. Chloride analysis indicated that complete dechlorination of cDCE occurred during growth. The half-velocity constant for cDCE transformation was 1.6 ± 0.2 μM, and the maximum specific substrate utilization rate ranged from 12.6 to 16.8 nmol/min/mg of protein. Resting cells grown on cDCE could transform cDCE, ethene, vinyl chloride, trans-dichloroethene, trichloroethene, and 1,2-dichloroethane. Epoxyethane was produced from ethene by cDCE-grown cells, suggesting that an epoxidation reaction is the first step in cDCE degradation.     

Purification and characterization of the tetrachloroethene reductive dehalogenase of strain PCE-S

The membrane-associated tetrachloroethene reductive dehalogenase from the tetrachloroethene-reducing anaerobe, strain PCE-S, was purified 165-fold to apparent homogeneity in the presence of the detergent Triton X-100. The purified dehalogenase catalyzed the reductive dechlorination of tetrachloroethene to trichloroethene and of trichloroethene to cis-1,2-dichloroethene with reduced methyl viologen as the electron donor, showing a specific activity of 650 nkat/mg protein. The apparent K _m values of the enzyme for tetrachloroethene, trichloroethene, and methyl viologen were 10 μM, 4 μM, and 0.3 mM, respectively. SDS-PAGE revealed a single protein band with an apparent molecular mass of 65 kDa. The apparent molecular mass of the native enzyme was 200 kDa as determined by gel filtration. Tetrachloroethene dehalogenase contained 0.7 ± 0.3 mol corrinoid, 1.0 ± 0.3 mol cobalt, 7.8 ± 0.5 mol iron, and 10.3 ± 2.0 mol acid-labile sulfur per mol subunit. The pH optimum was approximately 7.2, and the temperature optimum was approximately 50 °C. The dehalogenase was oxygen-sensitive with a half-life of approximately 50 min. The N-terminal amino acid sequence of the enzyme was determined, and no significant similarity was found to any part of the amino acid sequence of the tetrachloroethene (PCE) reductive dehalogenase from Dehalospirillum multivorans. Received: 4 December 1997 / Accepted: 10 February 1998     

Complete reductive dechlorination of tetrachloroethene to ethene by anaerobic microbial enrichment culture developed from sediment

A mixed, anaerobic microbial enrichment culture, AMEC-4P, was developed that uses lactate as the electron donor for the reductive dechlorination of tetrachloroethene (PCE) to ethene. AMEC-4P consistently and completely converted 2 mM PCE to cis-1,2-dichloroethene (cis-DCE) within 13 days, and the intermediate, cis-DCE, was then completely dechlorinated to ethene after 130 days. Dechlorination rates for PCE to cis-DCE, cis-DCE to VC, and VC to ethene were 243, 27, and 41 μmol/l/day, respectively. Geobacter lovleyi and a Dehalococcoides sp. were identified from their 16S rRNA sequences to be the dominant phylotypes in AMEC-4P.     

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 M). 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.Abbreviations PCE Tetrachloroethene - TCE Trichloroethene - DCE Dichloroethene - EDTA Ethylene diamine tetra-acetate - MV Methyl viologen - BV Benzyl viologen - PI Propyl iodide, 1-iodopropane - TC Titanium(III) citrate     

Dehalococcoides mccartyi Strain DCMB5 Respires a Broad Spectrum of Chlorinated Aromatic Compounds

Polyhalogenated aromatic compounds are harmful environmental contaminants and tend to persist in anoxic soils and sediments. Dehalococcoides mccartyi strain DCMB5, a strain originating from dioxin-polluted river sediment, was examined for its capacity to dehalogenate diverse chloroaromatic compounds. Strain DCMB5 used hexachlorobenzenes, pentachlorobenzenes, all three tetrachlorobenzenes, and 1,2,3-trichlorobenzene as well as 1,2,3,4-tetra- and 1,2,4-trichlorodibenzo-p-dioxin as electron acceptors for organohalide respiration. In addition, 1,2,3-trichlorodibenzo-p-dioxin and 1,3-, 1,2-, and 1,4-dichlorodibenzo-p-dioxin were dechlorinated, the latter to the nonchlorinated congener with a remarkably short lag phase of 1 to 4 days following transfer. Strain DCMB5 also dechlorinated pentachlorophenol and almost all tetra- and trichlorophenols. Tetrachloroethene was dechlorinated to trichloroethene and served as an electron acceptor for growth. To relate selected dechlorination activities to the expression of specific reductive dehalogenase genes, the proteomes of 1,2,3-trichlorobenzene-, pentachlorobenzene-, and tetrachloroethene-dechlorinating cultures were analyzed. Dcmb_86, an ortholog of the chlorobenzene reductive dehalogenase CbrA, was the most abundant reductive dehalogenase during growth with each electron acceptor, suggesting its pivotal role in organohalide respiration of strain DCMB5. Dcmb_1041 was specifically induced, however, by both chlorobenzenes, whereas 3 putative reductive dehalogenases, Dcmb_1434, Dcmb_1339, and Dcmb_1383, were detected only in tetrachloroethene-grown cells. The proteomes also harbored a type IV pilus protein and the components for its assembly, disassembly, and secretion. In addition, transmission electron microscopy of DCMB5 revealed an irregular mode of cell division as well as the presence of pili, indicating that pilus formation is a feature of D. mccartyi during organohalide respiration.