Factors controlling the carbon isotope fractionation of tetra- and trichloroethene during reductive dechlorination by Sulfurospirillum ssp. and Desulfitobacterium sp. strain PCE-S

Carbon stable isotope fractionation of tetrachloroethene (PCE) and trichloroethene (TCE) was investigated during reductive dechlorination. Growing cells of Sulfurospirillum multivorans, Sulfurospirillum halorespirans, or Desulfitobacterium sp. strain PCE-S, the respective crude extracts and the abiotic reaction with cyanocobalamin (vitamin B(12)) were used. Fractionation of TCE (alphaC=1.0132-1.0187) by S. multivorans was more than one order of magnitude higher than values previously observed for tetrachloroethene (PCE) (alphaC=1.00042-1.0017). Similar differences in fractionation were observed during reductive dehalogenation by the close relative S. halorespirans with alphaC=1.0046-1.032 and alphaC=1.0187-1.0229 for PCE and TCE respectively. TCE carbon isotope fractionation (alphaC=1.0150) by the purified PCE-reductive dehalogenase from S. multivorans was more than one order of magnitude higher than fractionation of PCE (alphaC=1.0017). Carbon isotope fractionation of TCE by Desulfitobacterium sp. strain PCE-S (alphaC=1.0109-1.0122) as well as during the abiotic reaction with cyanocobalamin (alphaC=1.0154) was in a similar range to previously reported values for fractionation by mixed microbial cultures. In contrast with previous results with PCE, no effects due to rate limitations, uptake or transport of the substrate to the reactive site could be observed during TCE dechlorination. Our results show that prior to a mechanistic interpretation of stable isotope fractionation factors it has to be carefully verified how other factors such as uptake or transport affect the isotope fractionation during degradation experiments with microbial cultures.     

Stable Isotope Fractionation of Tetrachloroethene during Reductive Dechlorination by Sulfurospirillum multivorans and Desulfitobacterium sp. Strain PCE-S and Abiotic Reactions with Cyanocobalamin

Carbon stable isotope fractionation of tetrachloroethene (PCE) during reductive dechlorination by whole cells and crude extracts of Sulfurospirillum multivorans and Desulfitobacterium sp. strain PCE-S and the abiotic reaction with cyanocobalamin (vitamin B₁₂) was studied. Fractionation was largest during the reaction with cyanocobalamin with αC = 1.0132. Stable isotope fractionation was lower but still in a similar order of magnitude for Desulfitobacterium sp. PCE-S (αC = 1.0052 to 1.0098). The isotope fractionation of PCE during dehalogenation by S. multivorans was lower by 1 order of magnitude (αC = 1.00042 to 1.0017). Additionally, an increase in isotope fractionation was observed with a decrease in cell integrity for both strains. For Desulfitobacterium sp. strain PCE-S, the carbon stable isotope fractionation factors were 1.0052 and 1.0089 for growing cells and crude extracts, respectively. For S. multivorans, αC values were 1.00042, 1.00097, and 1.0017 for growing cells, crude extracts, and the purified PCE reductive dehalogenase, respectively. For the field application of stable isotope fractionation, care is needed as fractionation may vary by more than an order of magnitude depending on the bacteria present, responsible for degradation.     

Development of primers for amplifying genes encoding CprA- and PceA-like reductive dehalogenases in anaerobic microbial consortia, dechlorinating trichlorobenzene and 1,2-dichloropropane

Gene sequence alignments of the reductive dehalogenases PceA (Dehalospirillum multivorans) and CprA (Desulfitobacterium dehalogenans) were used to develop specific PCR primers binding to conserved regions of these sequences. These primers enabled us to amplify and subsequently sequence cprA-like gene fragments from the chlororespiring species Dehalobacter restrictus, Desulfitobacterium sp. strain PCE1, and D. hafniense. No specific amplicons were obtained from the chlororespiring species D. frappieri, D. chlororespirans, and Desulfomonile tiedjei. Furthermore, we were able to amplify and sequence cprA/pceA-like gene fragments from both trichlorobenzene (TCB)- and 1,2-dichloropropane (DCP)-dechlorinating microbial consortia using the novel primers. Subsequent sequence analysis of the fragments obtained from the microbial consortia revealed a group of four clusters (I-IV). Of these, clusters I and II showed the highest similarities to the cprA-like gene of Dehalobacter restrictus (79.0 and 96.2%, respectively). Cluster III comprised cprA-like sequences found in both the TCB- and the DCP-dechlorinating consortia, whereas sequences of cluster IV were most similar to the pceA gene of Dehalospirillum multivorans (97.8%). Our detection of genes encoding reductive dehalogenases, the key enzymes of chlororespiration, supports the hypothesis that reductive dechlorination of TCB and DCP occurs via a respiratory pathway.     

Isolation and characterization of Tn-Dha1, a transposon containing the tetrachloroethene reductive dehalogenase of Desulfitobacterium hafniense strain TCE1

A new 9.9 kb catabolic transposon, Tn-Dha1, containing the gene responsible for tetrachloroethene (PCE) reductive dechlorination activity, was isolated from Desulfitobacterium hafniense strain TCE1. Two fully identical copies of the insertion sequence ISDha1, a new member of the IS256 family, surround the gene cluster pceABCT, a truncated gene for another transposase and a short open reading frame with homology to a member of the twin-arginine transport system (tatA). Evidence was obtained by Southern blot for an alternative form of the transposon element as a circular molecule containing only one copy of ISDha1. This latter structure most probably represents a dead-end product of the transposition of Tn-Dha1. Strong indications for the transposition activity of ISDha1 were given by polymerase chain reaction (PCR) amplification and sequencing of the intervening sequence located between both inverted repeats (IR) of ISDha1 (IR junction). A stable genomic ISDha1 tandem was excluded by quantitative real-time PCR. Promoter mapping of the pceA gene, encoding the reductive dehalogenase, revealed the presence of a strong promoter partially encoded in the right inverted repeat of ISDha1. A sequence comparison with pce gene clusters from Desulfitobacterium sp. strains PCE-S and Y51 and from Dehalobacter restrictus, all of which show 100% identity for the pceAB genes, indicated that both Desulfitobacterium strains seem to possess the same transposon structure, whereas only the pceABCT gene cluster is conserved in D. restrictus.     

Development of degenerate and specific PCR primers for the detection and isolation of known and putative chloroethene reductive dehalogenase genes

Degenerate and specific PCR primers were designed for the detection of chloroethene reductive dehalogenases (CE-RDase), the key enzymes of chloroethene dehalorespiration, based on sequence information of three CE-RDases and three chlorophenol (CP) RDases. For the design of the degenerate primers, seven conserved amino-acid blocks identified with different bioinformatic tools were used. For one block degenerate, primers containing a 5'-consensus clamp region specific for CE-RDases and a 3'-end degenerate core region specific for RDases in general were designed using the Consensus-Degenerate Hybrid Oligonucleotide Primer (CDHOP) design method. Applying the degenerate primers to genomic DNA of Sulfurospirillum multivorans strain K, Dehalobacter restrictus strain PER-K23, and Desulfitobacterium sp. strain PCE1 led to the isolation of the known CE-RDase genes and three new genes encoding putative reductive dehalogenases that cluster with CE-RDases and not with CP-RDases. In addition, primers designed to be specific for the three known CE-RDase genes, namely pceA of S. multivorans, pceA of D. restrictus, and tceA of Dehalococcoides ethenogenes were successfully tested on genomic DNA of different chloroethene-dehalorespiring bacteria. Nested PCR using degenerate primers followed by a PCR with specific primers allowed a sensitive detection of only 10(2) copies per reaction.     

Two distinct enzyme systems are responsible for tetrachloroethene and chlorophenol reductive dehalogenation in Desulfitobacterium strain PCE1

Desulfitobacterium strain PCE1 is able to use tetrachloroethene and chloroaromatics as terminal electron acceptors for growth. Cell extracts of Desulfitobacterium strain PCE1 grown with tetrachloroethene as electron acceptor showed no dehalogenase activity with 3-chloro-4-hydroxyphenylacetate (Cl-OH-phenylacetate) and other ortho-chlorophenolic compounds in an in vitro assay. Extracts of cells that were grown with Cl-OH-phenylacetate as electron acceptor dechlorinated tetrachloroethene at 10% of the dechlorination rate of Cl-OH-phenylacetate. In both cell extracts dechlorination was inhibited by the addition of 1-iodopropane and dinitrogen oxide, inhibitors of cobalamin-containing enzymes. The enzymes responsible for tetrachloroethene and Cl-OH-phenylacetate dechlorination were partially purified. A 100-fold enriched fraction of chlorophenol reductive dehalogenase was obtained that mainly contained a protein with a subunit size of 48 kDa. The characteristics of this enzyme are similar to that of the chlorophenol reductive dehalogenase of D. dehalogenans. After partial purification of the tetrachloroethene reductive dehalogenase, a fraction was obtained that also contained a 48-kDa protein, but the N-terminal sequence showed no similarity with that of the chlorophenol reductive dehalogenase sequence or with the N-terminal amino acid sequence of tetra- and trichloroethene reductive dehalogenase of Desulfitobacterium strain TCE1. These results provide strong evidence that two different enzymes are responsible for tetrachloroethene and chlorophenol dechlorination in Desulfitobacterium strain PCE1. Furthermore, the characterization of partially purified tetrachloroethene reductive dehalogenase indicated that this enzyme is a novel type of reductive dehalogenase.     

Tetrachloroethene reductive dehalogenase of Dehalospirillum multivorans: substrate specificity of the native enzyme and its corrinoid cofactor

The substrate specificity of the tetrachloroethene reductive dehalogenase of Dehalospirillum multivoransand its corrinoid cofactor were studied. Besides reduced methyl viologen, titanium(III) citrate could serve as electron donor for reductive dehalogenation of tetrachloroethene (PCE) and trichloroethene to cis-1,2-dichloroethene. In addition to chlorinated ethenes, chlorinated propenes were reductively dechlorinated solely by the native enzyme. trans-1,3-Dichloropropene, 1,1,3-trichloropropene and 2,3-dichloropropene were reduced to a mixture of mono-chloropropenes, 1,1-dichloropropene, and 2-chloropropene, respectively. Other halogenated compounds that were rapidly reduced by the enzyme were also dehalogenated abiotically by the heat-inactivated enzyme and by commercially available cyanocobalamin. The rate of this abiotic reaction was dependent on the number and type of halogen substituents and on the type of catalyst. The corrinoid cofactor purified from the tetrachloroethene dehalogenase of D. multivorans exhibited an activity about 50-fold higher than that of cyanocobalamin (vitamin B(12)) with trichloroacetate as electron acceptor, indicating that the corrinoid cofactor of the PCE dehalogenase is not cyanocobalamin. Corrinoids catalyzed the rapid dehalogenation of trichloroacetic acid. The rate was proportional to the amount of, e.g. cyanocobalamin; therefore, the reductive dehalogenation assay can be used for the sensitive and rapid quantification of this cofactor.     

Resolution of culture Clostridium bifermentans DPH-1 into two populations, a Clostridium sp. and tetrachloroethene-dechlorinating Desulfitobacterium hafniense strain JH1

Clostridium bifermentans strain DPH-1 reportedly dechlorinates tetrachloroethene (PCE) to cis-1,2-dichloroethene. Cultivation-based approaches resolved the DPH-1 culture into two populations: a nondechlorinating Clostridium sp. and PCE-dechlorinating Desulfitobacterium hafniense strain JH1. Strain JH1 carries pceA, encoding a PCE reductive dehalogenase, and shares other characteristics with Desulfitobacterium hafniense strain Y51.     

Molecular characterization of the PceA reductive dehalogenase of desulfitobacterium sp. strain Y51

The tetrachloroethene (PCE) reductive dehalogenase (encoded by the pceA gene and designated PceA dehalogenase) of Desulfitobacterium sp. strain Y51 was purified and characterized. The expression of the enzyme was highly induced in the presence of PCE and trichloroethene (TCE). The purified enzyme catalyzed the reductive dehalogenation of PCE via TCE to cis-1,2-dichloroethene at a specific activity of 113.6 nmol x min(-1) x mg of protein(-1). The apparent K(m) values for PCE and TCE were 105.7 and 535.3 microM, respectively. Chlorinated ethenes other than PCE and TCE were not dehalogenated. However, the enzyme exhibited dehalogenation activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane, and 1,1,2,2-tetrachloroethane. The pceA gene of Desulfitobacterium sp. strain Y51 was identified in a 2.8-kb DNA fragment and used to express the protein in Escherichia coli for the preparation of antibodies. Immunoblot analyses located PceA in the periplasm of the cell.     

Phenyl methyl ethers: novel electron donors for respiratory growth of<Emphasis Type="Italic"> Desulfitobacterium hafniense</Emphasis> and<Emphasis Type="Italic"> Desulfitobacterium</Emphasis> sp. strain PCE-S

Desulfitobacterium hafniense and Desulfitobacterium sp. strain PCE-S grew under anoxic conditions with a variety of phenyl methyl ethers as electron donors in combination with fumarate as electron acceptor. The phenyl methyl ethers were O-demethylated to the corresponding phenol compounds. O-demethylation was strictly dependent on the presence of fumarate; no O-demethylation occurred with CO₂ as electron acceptor. One mol phenyl methyl ether R-O-CH₃ was O-demethylated to R-OH per 3 mol fumarate reduced to succinate. The growth yields with vanillate or syringate plus fumarate were approximately 15 g cells (dry weight) per mol methyl moiety converted. D. hafniense utilized vanillate or syringate as an electron donor for reductive dehalogenation of 3-Cl-4-hydroxyphenylacetate, whereas strain PCE-S was not able to dechlorinate tetrachloroethene with phenyl methyl ethers. Crude extracts of both organisms showed O-demethylase activity in the O-demethylase assay with vanillate or syringate as substrates when the organism was grown on syringate plus fumarate. Besides the homoacetogenic bacteria, only growing cells of Desulfitobacterium frappieri PCP-1 have thus far been reported to be capable of phenyl methyl ether O-demethylation. This present study is the first report of Desulfitobacteria utilizing phenyl methyl ethers as electron donors for fumarate reduction and for growth.Abbreviations PCE Tetrachloroethene - TCE Trichloroethene - DCE cis-1,2-Dichloroethene - ClOHPA 3-Cl-4-Hydroxyphenylacetate - OHPA 4-Hydroxyphenylacetate - FH₄ Tetrahydrofolate     

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.     

A non-dechlorinating strain of Dehalospirillum multivorans: evidence for a key role of the corrinoid cofactor in the synthesis of an active tetrachloroethene dehalogenase

A strain of Dehalosprillum multivorans, designated strain N, was isolated from the same source as the formerly described tetrachloroethene (PCE)-dechlorinating D. multivorans, herein after referred to as strain K. Neither growing cells nor cell extracts of strain N were able to dechlorinate PCE. The pceA and pceB genes encoding for the PCE-reductive dehalogenase were detected in cells of strain N; and they were 100% homologous to the corresponding genes of strain K. Since the PCE dehalogenase of D. multivorans strain K contains a corrinoid cofactor, the corrinoids of strain N cells were extracted. Analysis of the corrinoids revealed the absence of the specific corrinoid, which is the cofactor of the PCE dehalogenase of strain K cells. RT-PCR of mRNA indicated that the pceA gene was transcribed in strain N cells to a far lower extent than the pceA gene of strain K under the same experimental conditions. Western blot analysis of crude extracts of strain N showed that, if at all, an insignificant amount of the apoprotein of the PCE dehalogenase was present. The results indicate that the inability of strain N to dechlorinate is due to the absence of the corrinoid cofactor of the enzyme mediating PCE dechlorination.     

Influence of different electron donors and acceptors on dehalorespiration of tetrachloroethene by Desulfitobacterium frappieri TCE1

Strain TCE1, a strictly anaerobic bacterium that can grow by reductive dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE), was isolated by selective enrichment from a PCE-dechlorinating chemostat mixed culture. Strain TCE1 is a gram-positive, motile, curved rod-shaped organism that is 2 to 4 by 0.6 to 0.8 microm and has approximately six lateral flagella. The pH and temperature optima for growth are 7.2 and 35 degrees C, respectively. On the basis of a comparative 16S rRNA sequence analysis, this bacterium was identified as a new strain of Desulfitobacterium frappieri, because it exhibited 99.7% relatedness to the D. frappieri type strain, strain PCP-1. Growth with H(2), formate, L-lactate, butyrate, crotonate, or ethanol as the electron donor depends on the availability of an external electron acceptor. Pyruvate and serine can also be used fermentatively. Electron donors (except formate and H(2)) are oxidized to acetate and CO(2). When L-lactate is the growth substrate, strain TCE1 can use the following electron acceptors: PCE and TCE (to produce cis-1,2-dichloroethene), sulfite and thiosulfate (to produce sulfide), nitrate (to produce nitrite), and fumarate (to produce succinate). Strain TCE1 is not able to reductively dechlorinate 3-chloro-4-hydroxyphenylacetate. The growth yields of the newly isolated bacterium when PCE is the electron acceptor are similar to those obtained for other dehalorespiring anaerobes (e.g., Desulfitobacterium sp. strain PCE1 and Desulfitobacterium hafniense) and the maximum specific reductive dechlorination rates are 4 to 16 times higher (up to 1.4 micromol of chloride released. min(-1). mg of protein(-1)). Dechlorination of PCE and TCE is an inducible process. In PCE-limited chemostat cultures of strain TCE1, dechlorination is strongly inhibited by sulfite but not by other alternative electron acceptors, such as fumarate or nitrate.     

Sulfonates as terminal electron acceptors for growth of sulfite-reducing bacteria (Desulfitobacterium spp.) and sulfate-reducing bacteria: effects of inhibitors of sulfidogenesis.

This study demonstrates the ability of Desulfitobacterium spp. to utilize aliphatic sulfonates as terminal electron acceptors (TEA) for growth. Isethionate (2-hydroxyethanesulfonate) reduction by Desulfitobacterium hafniense resulted in acetate as well as sulfide accumulation in accordance with the expectation that the carbon portion of isethionate was oxidized to acetate and the sulfur was reduced to sulfide. The presence of a polypeptide, approximately 97 kDa, was evident in isethionate-grown cells of Desulfitobacterium hafniense, Desulfitobacterium sp. strain PCE 1, and the two sulfate-reducing bacteria (SRB)-Desulfovibrio desulfuricans IC1 (T. J. Lie, J. R. Leadbetter, and E. R. Leadbetter, Geomicrobiol. J. 15:135-149, 1998) and Desulfomicrobium norvegicum; this polypeptide was not detected when these bacteria were grown on TEA other than isethionate, suggesting involvement in its metabolism. The sulfate analogs molybdate and tungstate, effective in inhibiting sulfate reduction by SRB, were examined for their effects on sulfonate reduction. Molybdate effectively inhibited sulfonate reduction by strain IC1 and selectively inhibited isethionate (but not cysteate) reduction by Desulfitobacterium dehalogenans and Desulfitobacterium sp. strain PCE 1. Desulfitobacterium hafniense, however, grew with both isethionate and cysteate in the presence of molybdate. In contrast, tungstate only partially inhibited sulfonate reduction by both SRB and Desulfitobacterium spp. Similarly, another inhibitor of sulfate reduction, 1,8-dihydroxyanthraquinone, effectively inhibited sulfate reduction by SRB but only partially inhibited sulfonate reduction by both SRB and Desulfitobacterium hafniense.     

Enzymes involved in the anoxic utilization of phenyl methyl ethers by <Emphasis Type="Italic">Desulfitobacterium hafniense</Emphasis> DCB2 and <Emphasis Type="Italic">Desulfitobacterium hafniense</Emphasis> PCE-S

Phenyl methyl ethers are utilized by Desulfitobacterium hafniense DCB2 and Desulfitobacterium hafniense PCE-S; the methyl group derived from the O-demethylation of these substrates can be used as electron donor for anaerobic fumarate respiration or dehalorespiration. The activity of all enzymes involved in the oxidation of the methyl group to carbon dioxide via the acetyl-CoA pathway was detected in cell extracts of both strains. In addition, a carbon monoxide dehydrogenase activity could be detected. Activity staining of this enzyme indicated that the enzyme is a bifunctional CO dehydrogenase/acetyl-CoA synthase.     

Stable carbon isotope fractionation during degradation of dichloromethane by methylotrophic bacteria

Stable carbon isotope fractionation during dichloromethane (DCM) degradation by methylotrophic bacteria was investigated under aerobic and nitrate-reducing conditions. The strains studied comprise several Hyphomicrobium strains, Methylobacterium, Methylopila, Methylophilus and Methylorhabdus spp. that are considered to degrade DCM by a glutathione (GSH)-dependent dehalogenase enzyme system in the initial step. The stable carbon isotope fractionation factors (alphaC) of the strains varied under aerobic conditions between 1.043 and 1.071 and under nitrate-reducing conditions between 1.048 and 1.065. Comparison of isotope fractionation under aerobic and nitrate-reducing conditions by individual strains revealed only minor to no differences. The variability in isotope fractionation among strains was found to be related to the polymorphism of the functional genes encoding the DCM dehalogenase.     

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.     

Physiological adaptation of Desulfitobacterium hafniense strain TCE1 to tetrachloroethene respiration

Desulfitobacterium spp. are ubiquitous organisms with a broad metabolic versatility, and some isolates have the ability to use tetrachloroethene (PCE) as terminal electron acceptor. In order to identify proteins involved in this organohalide respiration process, a comparative proteomic analysis was performed. Soluble and membrane-associated proteins obtained from cells of Desulfitobacterium hafniense strain TCE1 that were growing on different combinations of the electron donors lactate and hydrogen and the electron acceptors PCE and fumarate were analyzed. Among proteins increasingly expressed in the presence of PCE compared to fumarate as electron acceptor, a total of 57 proteins were identified by mass spectrometry analysis, revealing proteins involved in stress response and associated regulation pathways, such as PspA, GroEL, and CodY, and also proteins potentially participating in carbon and energy metabolism, such as proteins of the Wood-Ljungdahl pathway and electron transfer flavoproteins. These proteomic results suggest that D. hafniense strain TCE1 adapts its physiology to face the relative unfavorable growth conditions during an apparent opportunistic organohalide respiration.     

Functional Heterologous Production of Reductive Dehalogenases from Desulfitobacterium hafniense Strains

The anaerobic dehalogenation of organohalides is catalyzed by the reductive dehalogenase (RdhA) enzymes produced in phylogenetically diverse bacteria. These enzymes contain a cobamide cofactor at the active site and two iron-sulfur clusters. In this study, the tetrachloroethene (PCE) reductive dehalogenase (PceA) of the Gram-positive Desulfitobacterium hafniense strain Y51 was produced in a catalytically active form in the nondechlorinating, cobamide-producing bacterium Shimwellia blattae (ATCC 33430), a Gram-negative gammaproteobacterium. The formation of recombinant catalytically active PceA enzyme was significantly enhanced when its dedicated PceT chaperone was coproduced and when 5,6-dimethylbenzimidazole and hydroxocobalamin were added to the S. blattae cultures. The experiments were extended to D. hafniense DCB-2, a reductively dehalogenating bacterium harboring multiple rdhA genes. To elucidate the substrate spectrum of the rdhA3 gene product of this organism, the recombinant enzyme was tested for the conversion of different dichlorophenols (DCP) in crude extracts of an RdhA3-producing S. blattae strain. 3,5-DCP, 2,3-DCP, and 2,4-DCP, but not 2,6-DCP and 3,4-DCP, were reductively dechlorinated by the recombinant RdhA3. In addition, this enzyme dechlorinated PCE to trichloroethene at low rates.