Jun 25, 1993 - obtained by addition of HC1 to a final concentration of 0.2 M to a solution of ..... thanopterm: coenzyme M methyltransferase of Methanosarcina.
Arch M~crobiol (1993) 160:383-387
Archives of
Hicrebiolegy 9 SpringerVerlag 1993
Methyl chloride metabolism of the strictly anaerobic, methyl chloride-utilizing homoacetogen strain MC Michael Meflmer. Gert Wohlfarth, Gabriele Diekert Institut ffir Mikrobiologie, Universitfit Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany Received: 27 April 1993/Accepted: 25 June 1993
Abstract. The methyl chloride metabolism of the homoacetogenie, methyl chloride-utilizing strain MC was investigated with cell extracts and cell suspensions of the organism. Cell extracts were found to contain all enzyme activities required for the conversion of methyl chloride or of H 2 plus COz to acetate. They catalyzed the dechlorination of methyl chloride with tetrahydrofolate as the methyl acceptor at a rate of ~ 20 nmol/min x mg of cell protein. Also, the O-demethylation of vanillate with tetrahydrofolate could be measured at a rate of 40 nmol/min x mg. Different enzyme systems appeared to be responsible for the dehalogenation of CH3C1 and for the O-demethylation of methoxylated aromatic compounds, since cells grown with methoxylated aromatic compounds exhibited a significantly lower activity of CH3C1 conversion than methyl chloride grown cells and vice versa. In addition, ammonium thiocyanate (5 mM) completely inhibited CH3C1 dechlorination, whereas the consumption of vanillate was not affected significantly. The data were taken to indicate, that the methyl chloride dehalogenation is catalyzed by a specific, inducible enzyme present in strain MC, and that tetrahydrofolate rather than the corrinoid-protein involved in acetate formation is the primary acceptor of the methyl group in the dechlorination reaction. Key words: Homoacetogenic bacteria - Acetate formation from methyl chloride - Strain MC - Tetrahydrofolate enzymes - Methyl tetrahydrofolate formation - Anaerobic dechlorination - O-demethylation of methoxylated aromatics
The acetate formation from C1 units catalyzed by homoacetogenic bacteria (Diekert 1992) involves the formation of a methyl group and a carboxyl group. Most of the homoacetogens are able to grow with H 2 plus CO 2 as the sole energy sources. The methyl group of acetate is
Correspondence to" G. Diekert
formed from CO2 involving methyl tetrahydrofalate as the intermediate (for a recent review see Diekert 1992). The methyl group is transferred to a corrinoid protein by the methyl transferase. The carboxyl group is formed via reduction of CO 2 to a bound carbon monoxide. The bound carbonyl and the corrinoid-bound methyl group are then converted to acetyl CoA, from which acetate is formed. When reduced C 1 units at the redox state of methanol are converted to acetate, the methyl precursor has to be oxidized to CO2 in a pathway reverse to the methyl group formation. The reducing equivalents derived from the oxidation of the methyl group are required for the reduction of CO2 to carbon monoxide. In the presence of CO, the oxidation of the methyl group is, at least in some cases, dispensable, since the carboxyl group of acetate can be directly formed from CO (Ma et al. 1987). Strain MC is a strictly anaerobic, methylotrophic homoacetogen, which is able to grow at the expense of methyl chloride as the sole energy source (Traunecker et al. 1991). The organism converts CH3C1 to acetate in its energy metabolism. The enzymes involved in acetate formation from methyl chloride have been investigated. Until now, it is not known how methyl chloride is fed into the homoacetogenic pathway. Here we report for the first time on methyl chloride conversion to methyl tetrahydrofolate in cell extracts of the organism.
Materials and methods Source of materials N 2 (grade 4.6), CO2 (grade 4.5), CO (grade 4.7), and CH3C1 (grade 2.8) were from Messer Griesheim (Dfisseldorf, Germany). Cellosyl was a gift from Dr. Br/iu (Hoechst, Frankfurt, Germany). All chemicals and biochemacalsused were of the highest available purity and were purchased from Merck (Darmstadt, Germany), Boehringer (Mannheim, Germany), Sigma (Mfinchen, Germany), and Biomol (Hamburg, Germany). Bio-Rad reagent was from Bio-Rad Laboratories (M/inchen, Germany). Strain MC was isolated from sewage sludge using CHaC1 as the sole energy source (Traunecker et al. 1991). The organism was grown on CHaC1 as described earlier (Traunecker et al. 1991). Cells were harvested anaerobically in the late logarithmic growth phase by centrifugatlon at 4500 x g and 4 ~ for 15 rain.
384
Analytical procedures CH3C1 and CO were determined gas chromatographically (Traunecker et al. 1991). Methyl tetrahydrofolate and methoxylated aromatic compounds were measured by high-performance liquid chromatography on a Cs-reversed phase column with 0.175% H3PO4 in HzO containing 10.5% methanol as the eluent. The compounds were detected photometrically at 210 nm.
Preparation of cell extracts from strain MC Strain MC was grown and harvested as described above. Crude extracts were obtained by incubation of cells in 50 mM Tns-HC1 pH 7.5 containing 5 mM dithiothreitol, 1 mM MgC12, 1 mM phenylmethylsulfonylfluoride, 1 mg deoxyribonuclease I, and 10 mg cellosyl per ml at 37 ~ for 30 rain. The cells were then disrupted in a French pressure cell at 137000 kPa. Cell debris was removed by anaerobic centrifugation at 100000 • g and 4 ~ for 30 rain. The protein concentration was determined according to Bradford (1976) using the Bio-Rad reagent.
Determination of enzyme activities The enzymatic activaties were tested under anaerobic conditions in rubber-stoppered glass cuvettes at 30 ~ w~th Nz as the gas phase unless otherwise mentioned. The assays were started by the addition of cell extracts (10-30rag protein per ml). Carbon monoxide dehydrogenase was measured spectrophotometrically following the reductmn of methyl viologen at 578 nm. The assay was conducted in 100 mM Tris-HC1 pH 7.5 containing 10 mM methyl viologen and CO (100 kPa) as the gas phase (Diekert and Thauer 1978). Formate dehydrogenase was determined by the reduction of methyl viologen at 578 nm (Ljungdahl and Andreesen 1978). Sodium formate (40 raM) was added to 100 mM Tris-HC1 pH 7.5 containing 10 mM methyl viologen. Formyl tetrahydrofolate synthetase catalyzed the formation of formyl tetrahydrofolate from 40 mM sodium formate, 2raM tetrahydrofolate, and 5 mM ATP in 100raM Tris-HC1 pH 7.5 contaimng 50raM 2-mercaptoethanol, 2 mM NH4C1, and 10 mM MgC12 (Rabinowitz and Pricer 1962). Formyl tetrahydrofolate was converted to methenyl tetrahydrofolate by acidification. The increase of the absorption at 350 nm was measured photometrically. Methenyl tetrahydrofolate cyclohydrolase was assayed spectrophotometrically at 350 nm by the decrease ofmethenyl tetrahydrofolate (Clark et al. 1982). Methenyl tetrahydrofolate was obtained by addition of HC1 to a final concentration of 0.2 M to a solution of 1.7 mM 5-formyl tetrahydrofolate in 0.1 mM maleate buffer pH 7.5. The solution was diluted 10-fold with 0.1 M maleate buffer pH 7.5. The reaction was started by the addition of cell extract. Methylene tetrahydrofolate dehydrogenase was assayed by the photometric determination of the reduction of 0.3 mM NAD + with 0.5 mM methylene tetrahydrofolate in 100 mM potassium phosphate buffer pH 7.5 containing 50 mM 2-mercaptoethanol. Methylene tetrahydrofolate was formed non-enzymatically from 0.5 mM tetrahydrofolate and 10 mM formaldehyde in the reaction vessel (Uyeda and Rabmowitz 1967). Methylene tetrahydrofolate reductase was measured spectrophotometrically by following the oxidation of methyl viologen at 578 nm. The assay was conducted in 100 mM Tris-HC1 pH 7.5 containing 10 mM methyl viologen, 0.5raM tetrahydrofolate, 10raM formaldehyde, and 50raM 2-mercaptoethanol. Methyl viologen was reduced with 50 mM sodium dithionite in 100 mM Tris-HC1 pH 7.5. In the phosphate acetyltransferase reaction coenzyme A was released from acetyt CoA under formataon of acetyl phosphate. 3-carboxy-4-nitrothiophenol was formed non-enzymaticallyin the reaction of coenzyme A with 5,5'-dithio-bis-(2-nitro-benzoate) and measured spectrophotometrically at 405 nm. The reaction was conducted aerobacaIly in 100 mM potassium phosphate buffer pH 7.2 containing 0.2 mM acetyl CoA, 1 mM 5,5'-dithio-bis-(2-nitro-benzoate), and 25 mM (NH~),SO,~ pH 7.5 (Wohlfarth and Buckel 1985). Acetate kinase was determined in a coupled enzymatic test system involving the
following reactions: i) the formation of acetyl phosphate plus ADP from acetate plus ATP, ~i) conversion of phosphoenolpyruvate plus ADP to pyruvate plus ATP, and iii) reduction of pyruvate with NADH to lactate. The oxidation of NADH was followed spectrophotometrically at 365 nm (Bergmeyer et al. 1983). The assay was conducted aerobically in 100 mM Tris-HC1 pH 8.0 containing 250 mM acetate, 2.5 mM phosphoenolpyruvate, 0.5 mM NADH, 5 mM ATP, 100 mM MgC12, 5 mM dithlothreitol, 10 U lactate dehydrogenase, and 10 U pyruvate kinase. Hydrogenasewas assayed spectrophotometrically following the reduction of 10 mM methyl viologen in 100 mM Tris-HC1 pH 7.5 with H2 (100 kPa) as the gas phase at 578 nm (Ragsdale and Ljungdahl 1984).
CHaCI consumption and vanillate demethylation in cell extracts of strain MC Assays were conducted at 25 ~ in 25-ml rubber-stoppered glass bottles containing 4.7 ml 100raM Mops buffer pH 7.2, 5 mM potassium phosphate buffer, 6.5 mM MgC12, 2.5 mM dithiothreJtol, 10 ~tM resazurin, and N 2 (165 kPa) as the gas phase. CH3C1, tetrahydrofolate, vanillate, and ATP were added as indicated in the figures and figure legends. Where indicated, ammonium thiocyanate (5 raM) was added. The reactions were started by the addition of 0.3 ml crude extract
Methyl chloride consumption in cell suspensions Strain MC was grown eather with CH3C1 or with syringate (Traunecker et al. 1991) and harvested as described above. Cells were washed in test buffer (100 mM Mops buffer pH 7.2 containing 10 gM resazurin, and 0.75 mM cysteine) and resuspended in the same buffer. Assays were conducted in 40-ml rubber-stoppered glass bottles at 25 ~ The experiments were started by the addition of 0.45 ml cell suspension to 4.55 ml 100 mM Mops buffer pH 7.5 containing 120mM cysteine and Nz (165 kPa) contaimng 2% CH3C1 as the gas phase. Where indicated, the CH3C1 was replaced by 2 mM vanillate. At the times indicated, samples were taken from the gas or liquid phase and analyzed for CH3C1 or vanillate.
Results
Enzymes involved in acetate synthesis from C 1 units Cell extracts of strain M C were o b t a i n e d from m e t h y l chloride g r o w n cells ( T r a u n e c k e r et al. i991). The e n z y m e activities were d e t e r m i n e d as described in the Materials a n d m e t h o d s section. The following enzymes were detected (Table 1). Carbon monoxide dehydrogenase oxidized C O with m e t h y l v i o l o g e n as the electron acceptor. T h e enzyme, which was oxygen labile, was slightly inhibited by cyanide. P y r i d i n e nucleotides could n o t serve as electron acceptors for C O oxidation. The a p p a r e n t K m values for C O a n d m e t h y l v i o l o g e n were n e a r 0.1 m M (CO) a n d 4.4 m M (MV). T h e extracts also catalyzed the r e d u c t i o n of Co In h y d r o x o c o b a l a m i n to the c o r r e s p o n d i n g Co I corrinoid, which was s p o n t a n e o u s l y c o n v e r t e d abiotically to m e t h y l c o b a l a m i n in the presence of m e t h y l chloride (data n o t shown). Formate dehydrogenase m e d i a t e d the r e d u c t i o n of m e t h y l viologen with f o r m a t e as the electron d o n o r . The e n z y m e was severely affected by oxygen. P y r i d i n e nucleotides were n o t reduced. Formyl tetrahydrofolate synthetase catalyzed the f o r m a t i o n o f formyl t e t r a h y d r o folate f r o m f o r m a t e a n d t e t r a h y d r o f o l a t e . Methenyl tetrahydrofolate cyclohydrolase a n d methylene tetrahy-
385 Table 1. Enzyme activities determined m cell extracts of strain MC. FHa, tetrahydrofolate; MV, methyl viologen. For enzyme assays see 'Materials and methods' Enzyme
Reaction
Specific activity (gmol/min x rag)
CO dehydrogenase Formate dehydrogenase Formyl-FH4 synthetase Methenyl-FH4 cyclohydrolase Methylene-FHr dehydrogenase Methylene-FH4 reductase Phosphate acetyltransferase Acetate kmase Hydrogenase CH3C1 dehalogenase Vanillate demethylase
CO + H20 + 2 MVox --+ CO2 + 2 H + + 2 MVrea H C O O - + 2 MVox ~ CO2 + H + + 2 MV~,d H C O O - + FH4 + ATP ~ O H C - F H 4 + A D P + P1 CH =- F H 4 + H 2 0 --+ O H C - F H 4 C H 2 = F H 4 + NAD § -~ C H - F H ~ + N A D H + H § C H / = F H ~ + 2 MVrcd -~ C H 3 - F H 4 + 2 MVox Acetyl CoA + P, ~ acetyl phosphate + CoA Acetate + ATP ~ acetyl phosphate + ADP H2 + 2 MVo~ ~ 2 H + + 2 MV~a CH3C1 + F H 4 --~ C H 3 - F H a + CI + H + Vanillate + FH4 --, C H 3 - F H 4 + 3,4-dihydroxybenzoate
23.50 1.10 1.10 0.05 5.10 9.53 0.34 14.80 3.30 0.02 0.04
drofolate dehydrogenase activities were also detected. Methylene tetrahydrofolate reductase mediated the reduction of methylene tetrahydrofolate with reduced methyl viologen as the artificial electron donor; methyl viologen could not be replaced by N A D H or N A D P H . The enzyme appeared to be extremely oxygen sensitive. Phosphate acetyltransferase and acetate kinase were oxygen insensitive. The apparent K m values for acetate and for ATP in the latter reaction were 26.1 and 0.47 mM, respectively. Since strain MC is able to grow with H 2 plus CO 2 as the sole energy sources, the presence of a hydrogenase was not surprising. The data on the presence of the enzyme activities are summarized in Table 1. All enzymes required for the acetate formation from C1 units were measured in activities sufficiently high to account for growth of the bacteria with methyl chloride at a growth rate of # = 0.023 h - 1 and a growth yield with methyl chloride of 7.9 g cells (dry weight) per mol of methyl chloride (Traunecker et al. 1991). The data indicate, that methyl chloride as a novel substrate for homoacetogenic bacteria is metabolized via the "classical" homoacetogenic pathway, which is also operative in homoacetogens growing with other methylotrophic substrates. For growth with C 1 substrates at the redox level of methanol enzymes are required, which introduce the methyl group into the homoacetogenic pathway. The methyl group could either be transferred to tetrahydrofolate or to the corrinoid enzyme, which supplies the methyl group for the acetyl CoA synthesis. We tested the formation of methyl tetrahydrofolate from tetrahydrofolate plus vanillate (3-methoxy-4-hydroxybenzoate) or methyl chloride as the substrates. For the experiment with vanillate syringate-grown cells were used (syringate = 3,5-dimethoxy-4-hydroxybenzoate). Cell extracts of strain MC catalyzed the formation of methyl tetrahydrofolate from both substrates (Table 1, Fig. 1). This indicated that tetrahydrofolate rather than the corrinoid protein was the acceptor for the methyl group. The rate of O-demethylation of vanillate was nearly in the same order as the rate expected for the growth rates and growth yields of the bacterium. The O-demethylating activity was bound to the soluble cell fraction. The methyl chloride dehalogenase activity depended on the experimental conditions applied. Usually, the
activity was between 5 and 25 nmol per min and mg of cell protein in the presence of ATP. This activity is too low by a factor of ~ 5 to explain growth with methyl chloride at the growth rate and growth yield given above. However, it should be considered, that for technical reasons the methyl chloride concentration was only near 0.5% in the gas phase under the experimental conditions used, whereas 2.5% methyl chloride are applied to the gas phases of growing cultures of the organism. In addition, the dehalogenase activity appeared to be extremely labile. When concentrated cell extracts were incubated anaerobically at 0 ~ the activity decreased more than 50% within a few hours. Frozen extracts exhibited no dehalogenase activity after thawing under the experimental conditions applied. The sensitivity of the enzyme could be another reason for the relatively low activity. In addition, the enzyme seemed to be very oxygensensitive. The kinetics of methyl chloride consumption and the formation of methyl tetrahydrofolate is shown in Fig. 1. The CH3C1 dehalogenase was found in the soluble cell fraction after ultracentrifugation of the cell extracts. The dehalogenation of CH3C1 in cell extracts was slightly stimulated by ATP (Fig. 2). The extent of stimula-
5 I
0 o
'
1 Time (h)
'
2
Fig. 1. Kinetics of methyl chloride conversion to methyl tetrahydrofolate in cell extracts of strain MC. The assay buffer contained 3 mM ATP and 3 mM tetrahydrofolate initially; the cell protein concentration was 8.3 rag/5 ml. At the times indicated samples were taken from the tiqmd and the gas phases and analyzed for CH3C1 and methyl tetrahydrofolate. For further details on the experimental conditions see 'Materials and methods'
386
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30
02
-5 20 E =;L
A
10 8
~
6
o
:t
{11
::s lO
0.I
c
o
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+ATP oo
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0
I
30
60
0
I
90
Time (rnln) Fig. 2. Effect of ATP (3 mM initially) on methyl chloride conversion to methyl tetrahydrofolate in cell extracts of strain MC. The tetrahydrofolate concentration was 3 mM at t = 0 h. The protein concentration was 8.3 rag/5 ml. For further details on the experimental conditions see 'Materials and methods' tion appeared to depend on the preparation of the crude extracts. The same effect of ATP was observed for the O-demethylation of vanillate (data not shown) and has been reported before for the O-demethylation of methoxylated aromatic compounds in Acetobacterium woodii (Berman and Frazer 1992). Whereas in the latter report the O-demethylation was stimulated by a factor of ,-, 5, the effect was in any case by far less significant with strain MC. The methyl chloride dehalogenase was severely inhibited by ammonium thiocyanate (5 mM), which is known to be an inhibitor of methyl chloride formation and methylation reactions with CH3C1 in wood-rotting fungi (Harper and Kennedy 1986). The O-demethylating activity was not significantly influenced by ammonium thiocyanate (Fig. 3 A, B).
1
2
3
4
5 0 1 2 3 4- 5 T,rne (h) Fig. 4. Kinetics of CH3C1 dehalogenation and vanillate demethytation in suspensions of CH3Cl-grown (A) and in syringate-grown (B) cells. The cell protein concentration was 3.8 mg/5 ml (A) or 3.0 mg/5 ml (B),respectively.The gas phase was N2/CO2. For details on the experimental conditions see 'Materials and methods' reactions. Therefore we tested the dehalogenating and the O-demethylating activity in suspensions of methyl chloride- and syringate-grown cells. Vanillate was chosen as a substrate for the demethylating activity, since this compound contains just one methoxy group, and therefore the rate of demethylation can be calculated more easily. The kinetics of methyl chloride and vanillate consumption in methyl chloride- and syringate-grown cells are shown in Fig. 4A, B. The maximum specific activity related to the cell protein was 110 nmol/min x mg for methyl chloride consumption and near 11 nmol/ m i n x mg for vanillate demethylation in CH3Cl-grown cells. In syringate-grown cells the rates were about 7 nmol/min x mg for methyl chloride dehalogenation and near 31 nmol per min and mg for vanillate demethylation. Similar results were obtained with cell extracts of the organism. The results indicated that the methyl chloride dehalogenase is inducible.
Inducibility of the methyl chloride dehalogenating activity Since both the CH3C1 dehalogenase as well as the O-demethylating activity form methyl tetrahydrofolate from a methyl precursor at the redox level of methanol, it could not be excluded, that one enzyme catalyzes both 5 A
n
o
o
12
B
10 O
E 3 =L -r C3
:J_
6
2
4 =r > 2
-SCN-
I
~
0
i o
~SCN_ i
i
I
2
2
Time (h)
Fig. 3. IAIcct ol ammonium thiocyanate (5 mM) on methyl chloride consumpuon 111extracts of methyl chloride-grown cells (A) and on vanillate consumption in extracts of syringate-grown cells (B). The tetrahydrofolate and ATP concentrations were 3 mM initially, each. The protein concentration was 6 mg/5 ml (A) or 4 rag/5 ml (B). For further details on the experimental conditions see 'Materials and methods'
Discussion
In this communication some characteristics of the methyl chloride metabolism in cell extracts and cell suspensions are described. The enzymes involved in acetate formation from methyl chloride and from methoxylated aromatic compounds could all be measured with the exception of methyl transferase, which cannot be assayed easily, since the reaction product is enzyme-bound. The acetate formation from methyl chloride or from methoxylated aromatic compounds is summarized in Fig. 5. Although from the morphology and the G + C content strain MC resembles the carbon monoxide-utilizing homoacetogen Peptostreptococcus productus (strain Marburg) (Traunecker etal. 1991), the substrate specificity and other characteristics of the enzymes involved in the homoacetogenic pathway of both organisms are different. In addition, P. productus is not able to grow with methyl chloride as the energy substrate. All attempts to demonstrate the formation of acetate from methyl chloride in cell extracts failed so far. However, cell extracts were found to convert methyl chloride or methoxylated aromatic compounds to methyl
387
CH3-O~ R
CH3Cl
k / CHa-FH4 31 ~
3 CH3C151
CH2=FH4 ~ 3 C02 3 CH3-FH, 41, ---~-~60"t] --~,I,8 CH-FH,~ ] 3[C0~ ~11 51 / 8 ~3 CH3-//~-E OHC-FH4 / 3 acetyl CoA
81/
HCOO- / CO~
19
3 acetyl~P 3 acetate
Fig. 5. Scheme of acetate synthesis from CH3C1 or from methoxylated aromatic compounds in strain MC. The enzymes measured in cell extracts (see Table 1) are indicated by the thick arrows. FH4 = tetrahydrofolate: C H 3 - C o - E = corrinoid enzyme. The enzymes are the following: 1 methyl chloride dehalogenase; 2 O-demethylating activity; 3 CH2=FH4 reductase; 4 CH2=FH4 dehydrogenase; 5 CH-=FH~ cyclohydrolase; 6 formyl-FH4 synthetase; 7 formate dehydrogenase; 8 carbon monoxide dehydrogenase; 9 phosphotransacetylase; 10 acetate kinase; 11 methyl transferase tetrahydrofolate. Two possible pathways of methyl tetrahydrofolate formation from methyl chloride can be envisaged: i) The methyl group is transferred directly to tetrahydrofolate as the primary acceptor. This would imply the involvement of the methyl transferase reaction in the formation of acetate from CH3C1. ii) The primary methyl group acceptor is the corrinoid-protein. This would require the involvement of methyl transferase in methyl tetrahydrofolate synthesis from CH3C1 rather than in acetate formation. The involvement of methyl transferase in methyl tetrahydrofolate formation, however, appears unlikely. If the methyl transfer from tetrahydrofolate to the corrinoid protein - in analogy to the methyl transferase reaction in methanogens (Becher et at. 1992) - is coupled to a sodium extrusion (Heise et al. 1989), the reverse reaction should be driven by a sodium gradient, which would require intact cells or vesicles with a sodium gradient across the membrane. In addition, the methyl transferase should be b o u n d to the particulate rather than to the soluble cell fraction. Moreover, the formation of acetate from methyl chloride plus C O in cell suspensions was stimulated by sodium (data not shown), indicating, that the methyl transferase is required for the formation of the methyl group of acetate from methyl chloride. F o r these reasons we believe, that the methyl group is directly transferred to methyl tetrahydrofolate rather than via the corrinoidprotein. The experiments on methyl chloride conversion to acetate or methyl tetrahydrofolate in suspensions or extracts, respectively, of methyl chloride- and syringate-grown cells point to the inducibility of the CH3C1 dehalogenase and suggest, that the dehalogenation and the O-demethylation are catalyzed by two different enzyme systems. The finding, that the CH3C1 dehalogenase rather than
the vanillate demethylase is inhibited by a m m o n i u m thiocyanate further confirms this assumption. The inducibility of the dehalogenase clearly indicates, that the dechlorination in strain M C is a biotic rather than an abiotic process. The dehalogenation therefore must be considered to be a novel enzyme activity specific for this organism rather than a side activity of an enzyme involved in the homoacetogenic pathway. This is supported by the finding, that e.g. the homoacetogen Peptostreptococcus productus was not able to grow at the expense of methyl chloride or to dechlorinate methyl chloride (data not shown). Acknowledgements. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Bonn-Bad Godesberg, Germany) and by the Fonds der Chemischen Industrie.
References Becher B. Miiller V, Gottschalk G (1992) NS-methyl-tetrahydromethanopterm: coenzyme M methyltransferase of Methanosarcina strain G61 is an Na+-translocating membrane protein. J Bacteriol 174:7656 7660 Bergmeyer HU, Gral31M, Walter HE (1983) Acetate kinase. In: Bergmeyer HU (ed) Methods of enzymatic analysis, vol II, 3rd edn. Verlag Chemic, Weinheim, pp 127-128 Berman MH, FrazerAC (1992) Importance of tetrahydrofolate and ATP in the anaerobic O-demethylation reaction for phenylmethylethers. Appl Environ Microbiol 58:925-931 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248 254 Clark JE, Ragsdale SW, Ljungdahl LG, Wiegel J (1982) Levels of enzymes involved in the synthesis of acetate from CO2 in Clostridium thermoautotrophicum. J Bacteriol 151:507-509 Diekert G (1992) The acetogenic bacteria. In: Balows A, Triiper HG, Dworkin M, Harder W, Schleifer KH (eds) The prokaryotes, vol I. Springer, New York Berlin Heidelberg, pp 517 to 533 Daekert GB, Thauer RK (1978) Carbon monoxide oxidation by Clostridium thermoaceticum and Clostridmm formzcoacetieum. J Bacteriol 136:597-606 Harper DB. Kennedy JT (1986) Effect of growth conditions on halomethane production by PhelIinus species: biological and environmental implications. J Gen Microbiol 132:1231-1246 Heise R, MiillerV. Gottschalk G (1989) Sodium dependence of acetate formation by the acetogenic bacterium Acetobacterium woodii. J Bacteriol 171:5473-5478 Ljungdahl LG, Andreesen JR (1978) Formate dehydrogenase, a selenium-tungsten enzyme from Clostridium thermoaceticum. Methods Enzymol 53? 360-372 Ma K, Siemon S, Diekert G (1987) Carbon monoxide metabolism in cell suspensions of Peptostreptococcus productus strain Marburg. FEMS Microbiol Lett 43:367-371 Rabinowitz JC, Pricer WE (1962) Formyltetrahydrofolate synthetase: Isolation and crystallization of the enzyme. J Biol Chem 237:2898 2902 Ragsdale SW, Ljungdahl LG (1984) Hydrogenase from Acetobacterium woodii. Arch Microbiol 139:361-365 Traunecker J, Preul3 A, Diekert G (1991) Isolation and characterization of a methyl chloride utilizing, strictly anaerobic bacterium. Arch Microbiol 156:416-421 Uyeda K, Rabinowitz JC (1967) Enzymes of clostridial purine fermentation. Methylene tetrahydrofolate dehydrogenase. J Biol Chem 242:4378-4385 Wohlfarth G, Buckel W (1985) A sodium ion gradient as energy source for Peptostreptococcus asaccharolyticus. Arch Microbiol 142:128-135
