Purification and properties of the formate dehydrogenase and characterization of the<Emphasis Type="Italic"> fdhA</Emphasis> gene of<Emphasis Type="Italic"> Sulfurospirillum multivorans</Emphasis>

The soluble periplasmic subunit of the formate dehydrogenase FdhA of the tetrachloroethene-reducing anaerobe Sulfurospirillum multivorans was purified to apparent homogeneity and the gene (fdhA) was identified and sequenced. The purified enzyme catalyzed the oxidation of formate with oxidized methyl viologen as electron acceptor at a specific activity of 1683 nkat/mg protein. The apparent molecular mass of the native enzyme was determined by gel filtration to be about 100 kDa, which was confirmed by the fdhA nucleotide sequence. fdhA encodes for a pre-protein that differs from the truncated mature protein by an N-terminal 35-amino-acid signal peptide containing a twin arginine motif. The amino acid sequence of FdhA revealed high sequence similarities to the larger subunits of the formate dehydrogenases of Campylobacter jejuni, Wolinella succinogenes, Escherichia coli (FdhN, FdhH, FdhO), and Methanobacterium formicicum. According to the nucleotide sequence, FdhA harbors one Fe₄/S₄ cluster and a selenocysteine residue as well as conserved amino acids thought to be involved in the binding of a molybdopterin guanidine dinucleotide cofactor.Abbreviations Fdh Formate dehydrogenase - PCE Tetrachloroethene     

Growth substrate dependent localization of tetrachloroethene reductive dehalogenase in Sulfurospirillum multivorans

Sulfurospirillum multivorans is a dehalorespiring organism, which is able to utilize tetrachloroethene as terminal electron acceptor in an anaerobic respiratory chain. The localization of the tetrachloroethene reductive dehalogenase in dependence on different growth substrates was studied using the freeze-fracture replica immunogold labeling technique. When the cells were grown with pyruvate plus fumarate, a major part of the enzyme was either localized in the cytoplasm or membrane associated facing the cytoplasm. In cells grown on pyruvate or formate as electron donors and tetrachloroethene as electron acceptor, most of the enzyme was detected at the periplasmic side of the cytoplasmic membrane. These results were confirmed by immunoblots of the enzyme with and without the twin arginine leader peptide. Trichloroethene exhibited the same effect on the enzyme localization as tetrachloroethene. The data indicated that the localization of the enzyme was dependent on the electron acceptor utilized.     

Isolation and characterization of Dehalospirillum multivorans gen. nov., sp. nov., a tetrachloroethene-utilizing,strictly anaerobic bacterium

A strictly anaerobic bacterium dechlorinating tetrachloroethene (perchloroethylene, PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (DCE) was isolated from activated sludge with pyruvate plus PCE as energy substrates. The organism, called Dehalospirillum multivorans, is a gram-negative spirillum that does not form spores. The G+C content of the DNA was 41.5 mol%. According to 16S rRNA gene sequence analysis, D. multivorans represents a new genus and a new species belonging to the epsilon subdivision of Proteobacteria. Quinones, cytochromes b and c, and corrinoids were extracted from the cells. D. multivorans grew in defined medium with PCE and H₂ as sole energy sources and acetate as carbon source; the growth yield under these conditions was 1.4g of cell protein per mol chloride released. Alternatively to PCE, fumarate and nitrate could serve as electron acceptors; sulfate could not replace fumarate, nitrate, or PCE in this respect. In addition to H₂, the organism utilized a variety of electron donors for dechlorination (pyruvate, lactate, ethanol, formate, glycerol). Upon growth on pyruvate plus PCE, the main fermentation products formed were acetatc, lactate, DCE, and H₂. At optimal pH (7.3–7.6) and temperature (30°C), and in the presence of pyruvate (20mM) and PCE (160M), a dechlorination rate of about 50 nmol min^-1 (mg cell protein)^-1 and a doubling time of about 2.5h were obtained with growing cultures. The ability to reduce PCE to DCE appears to be constitutive under the experimental conditions applied since cultures growing in the absence of PCE for several generations immediately started dechlorination when transferred to a medium containing PCE. The organism may be useful for bioremediation of environments polluted with tetrachloroethene.Abbreviations PCE Perchloroethylene, tetrachloroethene - TCE Trichloroethene - DCE cis-1,2-Dichloroethene - CHC Chlorinated hydrocarbon     

Impact of Chlorofluorocarbon 113 on Chlorinated Ethene Biodegradation

The inhibition of tetrachloroethene (PCE) degradation in anaerobic, ethanol-fed PCE-enrichment cultures by chlorofluorocarbon 113 (CFC113) was a function of the initial CFC113 concentration. Typically, aqueous CFC113 concentrations up to 1 mg/L slowed, but did not stop PCE-degradation, but cis-1,2-dichloroethene (cDCE) degradation was inhibited by 0.2 mg/L CFC113. In some cultures, however, PCE degradation was stopped by as little as 0.15 mg/L CFC113. CFC113 also slowed the consumption of hydrogen and the concurrent methane production. CFC113 slowly degraded in PCE-enrichment cultures to hydrochlorofluorocarbon 123a (HCFC123a). Chlorotrifluoroethene was also detected. Although relatively non-toxic, CFC113 may nevertheless pose remediation challenges when present at sites that also contain PCE.     

Functional characterization of the trigger factor protein PceT of tetrachloroethene-dechlorinating Desulfitobacterium hafniense Y51

Desulfitobacterium hafniense strain Y51 dechlorinates tetrachloroethene to cis-1,2-dichloroethene (cis-DCE) via trichloroethene by the action of the PceA reductive dehalogenase encoded by pceA. The pceA gene constitutes a gene cluster with pceB, pceC, and pceT. However, the gene components, except for pceA, still remained to be characterized. In the present study, we characterized the function of PceT. PceT of strain Y51 showed a sequence homology with trigger factor proteins, although it is evolutionally distant from the well-characterized trigger factor protein of Escherichia coli. The PceT protein tagged with 6x histidine was expressed as a soluble form in E. coli. The recombinant PceT fusion protein exhibited peptidyl-proryl cis–trans isomerase activity toward the chromogenic peptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The PceT fusion protein also exhibited chaperon activity towards the chemically denatured citrate synthase. Immunoprecipitation analysis using antibodies raised against PceA and PceT demonstrated that PceT specifically binds to the precursor form of PceA with an N-terminal twin-arginine translocation (TAT) signal sequence. On the other hand, PceT failed to bind the mature form of PceA that lost the TAT signal sequence. This is the first report in dehalorespiring bacteria, indicating that PceT is responsible for the correct folding of the precursor PceA.     

Continuous dechlorination of tetrachloroethene in an upflow anaerobic sludge blanket reactor

Influences of hydraulic retention time (HRT) on dechlorination of tetrachloroethene (PCE) were investigated in an upflow anaerobic sludge blanket (UASB) reactor inoculated with anaerobic granular sludge non-pre-exposed to chlorinated compounds. PCE was introduced into the reactor at a loading rate of 3 mg/l d. PCE removal increased from 51 +/- 5% to 87 +/- 3% when HRT increased from 1 to 4 d, corresponding to an increase in the PCE biotransformation rate from 10.5 +/- 2.3 to 21.3 +/- 3.7 mumol/d. A higher ethene production rate, 0.9 +/- 0.2 mumol/d, was attained without accumulation of dichloroethenes at the HRT of 4 d. Dehalococcoides-like species were detected in sludge granules by fluorescence in situ hybridization, with signal strength in proportion to the extent of PCE dechlorination.     

Effect of chloroethene concentrations and granular activated carbon on reductive dechlorination rates and growth of Dehalococcoides spp

This study focused on the investigation of (i) the tetrachloroethene (PCE) toxicity threshold of a reductively dechlorinating mixed culture containing Dehalococcoides spp., (ii) the adsorption of PCE on different types of granular activated carbon (GAC), and (iii) the bioavailability and reductive dechlorination in the presence of GAC. The abundance of Dehalococcoides spp. detected by quantitative real-time polymerase chain reaction (qPCR) was found to increase by 2-4 orders of magnitude during degradation of PCE. No degradation occurred at dissolved concentrations beyond 420 μM (70 mg/L). Different adsorption isotherms were determined for thermally and chemically activated carbons. The addition of GAC to biological assays reduced the dissolved PCE concentration below the toxicity threshold. The combination of microbial reductive dechlorination with GAC adsorption proved to be a promising method for remediation of groundwater contaminated by high concentrations of chloroethenes.     

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     

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