Reductive biodegradation of 1,2-dichloroethane by methanogenic granular sludge: perspectives for in situ remediation.

Granular methanogenic sludge was able to dechlorinate 1,2-dichloroethane (1,2-DCA) to ethene in UASB reactors. Ethanol served as the sole carbon and energy source. The average dechlorination rate measured on the basis of ethene production varied between 1.7 and 2.1 micromol 1,2-DCA/(h.gVSS) (46.7 and 57.4 mg/L.d). In order to elucidate the microbial origin of this bioconversion, enrichment cultures of the methanogenic sludge were prepared with different carbon and electron sources: pyruvate, lactate, H2/CO2, ethanol and formate. Dithiothreitol (DTT) was the strong reductant in order to increase the negative redox potential in the media. A homo-acetogenic gram-positive strain could be isolated in the presence of formate. 16S rRNA of the isolated strain showed that the bacterium was closely related (99.7%) to Acetobacterium wieringae. The strain also grew on pyruvate, lactate, H2/CO2 and ethanol, although dechlorination rates of 1,2-DCA were at least 5 times higher when formate was the (only) electron source. Average conversion rates reached 3 micromol/(h.g(dry cells)) and appeared t relate to cometabolic biocatalysis on the corrinoid centers of the homo-acetogenic strain. Some perspectives of anaerobic in situ bioremediation of groundwater polluted with chloroethanes are presented.

Isolation and characterization of a veratrol:corrinoid protein methyl transferase from Acetobacterium dehalogenans.

From 3-methoxyphenol-grown cells of Acetobacterium dehalogenans, an inducible enzyme was purified that mediated the transfer of the methyl groups of veratrol (1,2-dimethoxybenzene) to a corrinoid protein enriched from the same cells. In this reaction, veratrol was converted via 2-methoxyphenol to 1,2-dihydroxybenzene. The veratrol:corrinoid protein methyl transferase, designated MTIver, had an apparent molecular mass of about 32 kDa. With respect to the N-terminal amino acid sequence and other characteristics, MTIver is different from the vanillate:corrinoid protein methyl transferase (MTIvan) isolated earlier from the same bacterium. For the methyl transfer from veratrol to tetrahydrofolate, two additional protein fractions were required, one of which contained a corrinoid protein. This protein was not identical with the corrinoid protein of the vanillate O-demethylase system. However, the latter corrinoid protein could also serve as methyl acceptor for the veratrol:corrinoid protein methyl transferase. MTIver catalyzed the demethylation of veratrol, 3,4-dimethoxybenzoate, 2-methoxyphenol, and 3-methoxyphenol. Vanillate (3-methoxy-4-hydroxybenzoate), 2-methoxybenzoate, or 4-methoxybenzoate could not serve as substrates.

O-demethylase from Acetobacterium dehalogenans--cloning, sequencing, and active expression of the gene encoding the corrinoid protein.

The ether-cleaving O-demethylase from the strictly anaerobic homoacetogen Acetobacterium dehalogenans catalyses the methyltransfer from 4-hydroxy-3-methoxy-benzoate (vanillate) to tetrahydrofolate. In the first step a vanillate :corrinoid protein methyltransferase (methyltransferase I) mediates the methylation of a 25-kDa corrinoid protein with the cofactor reduced to cob(I)alamin. The methyl group is then transferred to tetrahydrofolate by the action of a methylcorrinoid protein:tetrahydrofolate methyltransferase (methyltransferase II). Using primers derived from the amino-terminal sequences of the corrinoid protein and the vanillate:corrinoid protein methyltransferase (methyltransferase I), a 723-bp fragment was amplified by PCR, which contained the gene odmA encoding the corrinoid protein of O-demethylase. Downstream of odmA, part of the odmB gene encoding methyltransferase I was identified. The amino acid sequence deduced from odmA showed about 60% similarity to the cobalamin-binding domain of methionine synthase from Escherichia coli (MetH) and to corrinoid proteins of methyltransferase systems involved in methanogenesis from methanol and methylamines. The sequence contained the DXHXXG consensus sequence typical for displacement of the dimethylbenzimidazole base of the corrinoid cofactor by a histidine from the protein. Heterologous expression of odmA in E. coli yielded a colourless, oxygen-insensitive apoprotein, which was able to bind one mol cobalamin or methylcobalamin/mol protein. Both of these reconstituted forms of the protein were active in the overall O-demethylation reaction. OdmA reconstituted with hydroxocobalamin and reduced by titanium(III) citrate to the cob(I)alamin form was methylated with vanillate by methyltransferase I in an irreversible reaction. Methylcobalamin carrying OdmA served as methyl group donor for the methylation of tetrahydrofolate by methyltransferase II. This reaction was found to be reversible, since methyltranSferase II also catalysed the methylation of cob(I)alamin containing OdmA with methyltetrahydrofolate.

Purification and characterization of the 3-chloro-4-hydroxy-phenylacetate reductive dehalogenase of Desulfitobacterium hafniense.

The membrane-bound 3-chloro-4-hydroxyphenylacetate (Cl-OHPA) reductive dehalogenase from the chlorophenol-reducing anaerobe Desulfitobacterium hafniense was purified 11.3-fold to apparent homogeneity in the presence of the detergent CHAPS. The purified dehalogenase catalyzed the reductive dechlorination of Cl-OHPA to 4-hydroxyphenylacetate with reduced methyl viologen as the electron donor at a specific activity of 103.2 nkat/mg protein. SDS-PAGE revealed a single protein band with an apparent molecular mass of 46.5 kDa. The enzyme contained 0.68 +/- 0.2 mol corrinoid, 12.0 +/- 0.7 mol iron, and 13.0 +/- 0.7 mol acid-labile sulfur per mol subunit. The N-terminal amino acid sequence of the enzyme was determined and no significant similarity was found to any protein present in the gene bank.

Tetrachloroethene dehalogenase from Dehalospirillum multivorans: cloning, sequencing of the encoding genes, and expression of the pceA gene in Escherichia coli.

The genes encoding tetrachloroethene reductive dehalogenase, a corrinoid-Fe/S protein, of Dehalospirillum multivorans were cloned and sequenced. The pceA gene is upstream of pceB and overlaps it by 4 bp. The presence of a sigma70-like promoter sequence upstream of pceA and of a rho-independent terminator downstream of pceB indicated that both genes are cotranscribed. This assumption is supported by reverse transcriptase PCR data. The pceA and pceB genes encode putative 501- and 74-amino-acid proteins, respectively, with calculated molecular masses of 55,887 and 8,354 Da, respectively. Four peptides obtained after trypsin treatment of tetrachloroethene (PCE) dehalogenase were found in the deduced amino acid sequence of pceA. The N-terminal amino acid sequence of the PCE dehalogenase isolated from D. multivorans was found 30 amino acids downstream of the N terminus of the deduced pceA product. The pceA gene contained a nucleotide stretch highly similar to binding motifs for two Fe4S4 clusters or for one Fe4S4 cluster and one Fe3S4 cluster. A consensus sequence for the binding of a corrinoid was not found in pceA. No significant similarities to genes in the databases were detected in sequence comparisons. The pceB gene contained two membrane-spanning helices as indicated by two hydrophobic stretches in the hydropathic plot. Sequence comparisons of pceB revealed no sequence similarities to genes present in the databases. Only in the presence of pUBS 520 supplying the recombinant bacteria with high levels of the rare Escherichia coli tRNA4Arg was pceA expressed, albeit nonfunctionally, in recombinant E. coli BL21 (DE3).

O-demethylase from Acetobacterium dehalogenans--substrate specificity and function of the participating proteins.

The ether-cleaving O-demethylase isolated from syringate-grown cells of Acetobacterium dehalogenans (formerly named strain MC) consists of four proteins, components A, B, C and D. The enzyme system converts only phenyl methyl ethers with a hydroxyl group in the ortho position to the methoxyl moiety. The presence of a carboxyl group in the aromatic compound was not required for O-demethylase reaction. Component B mediated the conversion of vanillate to 3,4-dihydroxybenzoate in the presence of the Ti(III)-reduced corrinoid-containing component A. After addition of component D and tetrahydrofolate, methyl tetrahydrofolate was formed from vanillate in stoichiometric amounts. Titanium(III) citrate as a reductant could be replaced by H2, methyl viologen or ferredoxin, partially purified hydrogenase, purified component C obtained from A. dehalogenans, and ATP. From these findings, it was deduced that component B serves as vanillate:corrinoid protein methyltransferase (methyltransferase I) mediating the methyl transfer from vanillate to the reduced corrinoid protein component A. Component D functions as methylcorrinoid protein:tetrahydrofolate transferase (methyltransferase II). The role of component C is probably that of an activating protein reversing accidental oxidation of the protein-bound cob(I)alamin to cob(II)alamin in the presence of ATP and reducing equivalents supplied by the enzymatic oxidation of hydrogen.

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 Km values of the enzyme for tetrachloroethene, trichloroethene, and methyl viologen were 10 microM, 4 microM, 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 degrees 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.

Isolation of O-demethylase, an ether-cleaving enzyme system of the homoacetogenic strain MC.

The O-demethylase of the methylotrophic homoacetogenic bacterium strain MC was purified to apparent homogeneity. The enzyme system consisted of four different components that were designated A, B, C, and D according to their elution sequence from the anionic-exchange chromatography column. All four components were essentially required for catalysis of the transfer of the methyl group from phenyl methyl ethers to tetrahydrofolate. According to gel filtration and SDS-PAGE, components A and B were monomers with apparent molecular masses of approximately 26 kDa (subunit 25 kDa) and 36 (subunit 41 kDa), respectively; component C appeared to be a trimeric protein (195 kDa, subunit 67 kDa); and component D was probably a dimer (64 kDa, subunit 30 kDa). Component A contained one corrinoid per monomer. In crude extracts, component D appeared to be the rate-limiting protein for the complete methyl transfer reaction. Additional requirements for the reaction were ATP and low-potential reducing equivalents supplied by either titanium(III) citrate or H2 plus hydrogenase purified from strain MC.

Anaerobic dehalogenases.

Several anaerobic bacteria are able to reductively dehalogenate chlorinated hydrocarbons and to couple this reaction to the synthesis of ATP via a chemiosmotic mechanism (dehalorespiration). A few reductive dehalogenases have recently been purified and characterized. Preliminary investigations have been performed to elucidate the mechanism of dehalorespiration.

Comparative studies on tetrachloroethene reductive dechlorination mediated by Desulfitobacterium sp. strain PCE-S.

Tetrachloroethene reductive dechlorination was studied with cell extracts of a newly isolated, tetrachloroethene-utilizing bacterium, Desulfitobacterium sp. strain PCE-S. Tetrachloroethene dehalogenase mediated the reductive dechlorination of tetrachloroethene and trichloroethene to cis-1,2-dichloroethene with artificial electron donors such as methyl viologen. The chlorinated aromatic compounds tested so far were not reduced. A low-potential electron donor (E0' < -0.4 V) was required for tetrachloroethene reduction. The enzyme in its reduced state was inactivated by propyl iodide and reactivated by light, indicating the involvement of a corrinoid in reductive tetrachloroethene dechlorination.

Studies on tetrachloroethene respiration in Dehalospirillum multivorans.

Tetrachloroethene (PCE) respiration was studied in the tetrachloroethene-utilizing anaerobe, Dehalospirillum multivorans, with respect to localization of the catabolic enzymes, the electron carriers potentially involved in electron transport, and the response to ionophores and specific inhibitors. Hydrogenase and formate dehydrogenase were recovered in the periplasmic cell fraction and were membrane-associated. Electron-accepting tetrachloroethene dehalogenase was found in the cytoplasmic fraction. In the PCE dehalogenase assay, only artificial electron donors with a standard redox potential of D. multivorans (Eo' = -445 mV) could serve as electron donor for PCE reduction. However, the reaction rate with ferredoxin was only 1% of that with methyl viologen, whereas the pyruvate-ferredoxin oxidoreductase exhibited almost the same reaction rates with methyl viologen and ferredoxin as electron acceptors for pyruvate oxidation. Reduced menadione (2-methyl-1, 4-naphthoquinone) did not serve as electron donor in the PCE dehalogenase reaction. 2-Heptyl-4-hydroxyquinoline-N-oxide (HOQNO) had no significant effect on PCE dechlorination in cell suspensions and in crude extracts. Whole cells catalyzed the reductive dechlorination of PCE with H2 or formate as electron donors. The dechlorination in cell suspensions rather than in cell extracts was inhibited by the ionophores carbonylcyanide-p-(trifluoromethoxy)phenylhydrazone (FCCP) and tetrachlorosalicylanilide (TCS), indicating that a membrane potential and/or a pH gradient may be required for the reaction in vivo.

Purification and characterization of tetrachloroethene reductive dehalogenase from Dehalospirillum multivorans.

Tetrachloroethene reductive dehalogenase from the tetrachloroethene-utilizing anaerobe, Dehalospirillum multivorans, was purified approximately 100-fold to apparent homogeneity. The purified dehalogenase catalyzed the reductive dechlorination of tetrachloroethene (PCE) to trichloroethene and of trichloroethene to cis-1,2-dichloroethene with reduced methyl viologen as the electron donor at a specific activity of 2.6 microkatal/mg. The apparent Km values for tetrachloroethene and trichloroethene were 0.20 and 0.24 mM, respectively. The apparent molecular mass of the native enzyme was determined by gel filtration to be 58 kDa. Sodium dodecyl sulfate-gel electrophoresis revealed a single protein band with a molecular mass of 57 kDa. One mol of dehalogenase contained 1.0 mol of corrinoid, 9.8 mol of iron, and 8.0 mol of acid-labile sulfur. The pH optimum was about 8.0. The enzyme had a temperature optimum of 42 degrees C. It was slightly oxygen-sensitive and was thermolabile above 50 degrees C. The dechlorination of PCE was stimulated by ammonium ions. Chlorinated methanes severely inhibited PCE dehalogenase activity.

Tetrachloroethene metabolism of Dehalospirillum multivorans.

Dehalospirillum multivorans is a strictly anaerobic bacterium that is able to dechlorinate tetrachloroethene (perchloroethylene; PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (DCE) as part of its energy metabolism. The present communication describes some features of the dechlorination reaction in growing cultures, cell suspensions, and cell extracts of D. multivorans. Cell suspensions catalyzed the reductive dechlorination of PCE with pyruvate as electron donor at specific rates of up to 150 nmol (chloride released) min-1 (mg cell protein)-1 (300 microM PCE initially, pH 7.5, 25 degrees C). The rate of dechlorination depended on the PCE concentration; concentrations higher than 300 microM inhibited dehalogenation. The temperature optimum was between 25 and 30 degrees C; the pH optimum at about 7.5. Dehalogenation was sensitive to potential alternative electron acceptors such as fumarate or sulfur; nitrate or sulfate had no significant effect on PCE reduction. Propyl iodide (50 microM) almost completely inhibited the dehalogenation of PCE in cell suspensions. Cell extracts mediated the dehalogenation of PCE and of TCE with reduced methyl viologen as the electron donor at specific rates of up to 0.5 mumol (chloride released) min-1 (mg protein).-1 An abiotic reductive dehalogenation could be excluded since cell extracts heated for 10 min at 95 degrees C were inactive. The PCE dehalogenase was recovered in the soluble cell fraction after ultracentrifugation. The enzyme was not inactivated by oxygen.

Metabolism of homocetogens.

Homoacetogenic bacteria are strictly anaerobic microorganisms that catalyze the formation of acetate from C1 units in their energy metabolism. Most of these organisms are able to grow at the expense of hydrogen plus CO2 as the sole energy source. Hydrogen then serves as the electron donor for CO2 reduction to acetate. The methyl group of acetate is formed from CO2 via formate and reduced C1 intermediates bound to tetrahydrofolate. The carboxyl group is derived from carbon monoxide, which is synthesized from CO2 by carbon monoxide dehydrogenase. The latter enzyme also catalyzes the formation of acetyl-CoA from the methyl group plus CO. Acetyl-CoA is then converted either to acetate in the catabolism or to cell carbon in the anabolism of the bacteria. The homoacetogens are very versatile anaerobes, which convert a variety of different substrates to acetate as the major end product.

Anaerobic transformation of 2,4,6-trinitrotoluene (TNT).

A sulfate-reducing bacterium using trinitrotoluene (TNT) as the sole nitrogen source was isolated with pyruvate and sulfate as the energy sources. The organism was able to reduce TNT to triaminotoluene (TAT) in growing cultures and cell suspensions and to further transform TAT to still unknown products. Pyruvate, H2, or carbon monoxide served as the electron donors for the reduction of TNT. The limiting step in TNT conversion to TAT was the reduction of 2,4-diamino-6-nitrotoluene (2,4-DANT) to triaminotoluene. The reduction proceeded via 2,4-diamino-6-hydroxylaminotoluene (DAHAT) as an intermediate. The intermediary formation of DAHAT was only observed in the presence of carbon monoxide or hydroxylamine, respectively. The reduction of DAHAT to triaminotoluene was inhibited by both CO and NH2OH. The inhibitors as well as DANT and DAHAT significantly inhibited sulfide formation from sulfite. The data were taken as evidence for the involvement of dissimilatory sulfite reductase in the reduction of DANT and/or DAHAT to triaminotoluene. Hydrogenase purified from Clostridium pasteurianum and carbon monoxide dehydrogenase partially purified from Clostridium thermoaceticum also catalyzed the reduction of DANT in the presence of methyl viologen or ferredoxin, however, as the main reduction product DAHAT rather than triaminotoluene was formed. The findings could explain the function of CO as an electron donor for the DANT reduction (to DAHAT) and the concomitant inhibitory effect of CO on triaminotoluene formation (from DAHAT) by the inhibition of sulfite reductase.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of NADP(+)-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Peptostreptococcus productus marburg.

The 5,10-methylenetetrahydrofolate dehydrogenase of heterotrophically grown Peptostreptococcus productus Marburg was purified to apparent homogeneity. The purified enzyme catalyzed the reversible oxidation of methylenetetrahydrofolate with NADP+ as the electron acceptor at a specific activity of 627 U/mg of protein. The Km values for methylenetetrahydrofolate and for NADP+ were 27 and 113 microM, respectively. The enzyme, which lacked 5,10-methenyltetrahydrofolate cyclohydrolase activity, was insensitive to oxygen and was thermolabile at temperatures above 40 degrees C. The apparent molecular mass of the enzyme was estimated by gel filtration to be 66 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a single subunit of 34 kDa, accounting for a dimeric alpha 2 structure of the enzyme. Kinetic studies on the initial reaction velocities with different concentrations of both substrates in the absence and presence of NADPH as the reaction product were interpreted to indicate that the enzyme followed a sequential reaction mechanism. After gentle ultracentrifugation of crude extracts, the enzyme was recovered to greater than 95% in the soluble (supernatant) fraction. Sodium (10 microM to 10 mM) had no effect on enzymatic activity. The data were taken to indicate that the enzyme was similar to the methylenetetrahydrofolate dehydrogenases of other homoacetogenic bacteria and that the enzyme is not involved in energy conservation of P. productus.

Purification and properties of a NADH-dependent 5,10-methylenetetrahydrofolate reductase from Peptostreptococcus productus.

The methylenetetrahydrofolate reductase from the carbon-monoxide-utilizing homoacetogen Peptostreptococcus productus (strain Marburg) has been purified to apparent homogeneity. The purified enzyme catalyzed the oxidation of NADH with methylenetetrahydrofolate as the electron acceptor at a specific activity of 380 mumols.min-1 mg protein-1 (37 degrees C; pH 5.5). The apparent Km for NADH was near 10 microM. The apparent molecular mass of the enzyme was determined by gel filtration to be approximately 250.0 kDa. The enzyme consists of eight identical subunits with a molecular mass of 32 kDa. It contains 4 FAD/mol octamer which were reduced by the enzyme with NADH as the electron donor; iron could not be detected. Oxygen had no effect on the enzyme. Ultracentrifugation of cell extracts revealed that about 40% of the enzyme activity was recovered in the particulate fraction, suggesting that the enzyme is associated with the membrane. The enzyme also catalyzed the methylenetetrahydrofolate reduction with methylene blue as an artificial electron donor. The oxidation of methyltetrahydrofolate was mediated with methylene blue as the electron acceptor; neither NAD+ nor viologen dyes could replace methylene blue in this reaction. NADP(H) or FAD(H2) were not used to substrates for the reaction in either direction. The activity of the purified enzyme, which was proposed to be involved in sodium translocation across the cytoplasmic membrane, was not affected by the absence or presence of added sodium. The properties of the enzyme differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase of the homoacetogen Clostridium formicoaceticum and of the NADP(+)-dependent reductase of eucaryotes investigated so far.

Sodium dependent acetate formation from CO2 in Peptostreptococcus products (strain Marburg).

Washed cells of Peptostreptococcus products (strain Marburg), which were incubated in the presence of CO/CO2/N2 (50%/17%/33%; 200 kPa) catalyzed the synthesis of acetate from carbon monoxide. The rate of acetate formation from CO was stimulated more than threefold by the addition of sodium (10 mM); potassium did not effect acetate synthesis. The degree of stimulation was dependent on the sodium concentration; the dependence followed simple Michaelis-Menten kinetics. The apparent Km for sodium was determined to be about 2 mmol/l. Sodium also stimulated acetate synthesis from H2 plus CO2. In the absence of added sodium the formation of formate as an intermediate in methyl group synthesis was stimulated. It is suggested that the sodium dependent reaction(s) is one (or more) of the reactions involved in methyl group synthesis from CO2.

Evidence for a nickel-containing carbon monoxide dehydrogenase in Methanobrevibacter arboriphilicus.

In growing cultures of Methanobrevibacter arboriphilicus (Methanobrevibacter arboriphilus), the synthesis of active carbon monoxide dehydrogenase required nickel. The 21-fold-purified enzyme from 63Ni-labeled cells of M. arboriphilicus comigrated with 63Ni during gel filtration. These results provide evidence that the carbon monoxide dehydrogenase of methanogens is a nickel protein.