Viologen dye inhibition of methane formation by Methanobacillus omelianskii

Wolin, E. A. (University of Illinois, Urbana), R. S. Wolfe, and M. J. Wolin. Viologen dye inhibition of methane formation by Methanobacillus omelianskii. J. Bacteriol. 87:993-998. 1964.-Low concentrations of methyl or benzyl viologen inhibit the formation of CH(4) from ethanol and CO(2) by washed cells of Methanobacillus omelianskii. Hydrogen, which is normally formed from ethanol, accumulates in greater quantities when CH(4) formation is inhibited by viologens. The viologens do not stimulate H(2) formation from ethanol in the absence of CO(2). Inhibition of CH(4) formation by the viologens is not reversed by H(2). A variety of other dyes and possible electron acceptors were tested for inhibition, and none was inhibitory in the same low-concentration range at which the viologens were effective.     

Ferredoxin-Dependent Conversion of Acetaldehyde to Acetate and H2 in Extracts of S Organism

S organism, an anaerobic gram-negative rod, which is one of two bacterial species isolated from the culture known as "Methanobacillus omelianskii," ferments ethanol to acetate and H(2). The present study shows that extracts of this organism contain ferredoxin and produce acetate from acetaldehyde via aldehyde: ferredoxin oxidoreductase activity. Electrons generated in the reaction are given off as H(2) by a previously demonstrated ferredoxin-linked hydrogenase system. Extracts were shown to contain good phosphotransacetylase and acetokinase activities, but no mechanism of adenosine triphosphate generation during acetaldehyde conversion to acetate could be detected. No evidence could be obtained for coenzyme A (CoASH) or phosphate requirement or for formation of acetyl CoA or acetyl phosphate.     

Hydrogen-oxidizing Methane Bacteria I. Cultivation and Methanogenesis

A method for the mass culture of hydrogen-oxidizing methane bacteria has been developed; yields of 50 to 60 g (wet weight) of cells per 12 to 14 liters of culture medium were obtained. The methanogenic organism from the culture of Methanobacillus omelianskii was grown in a complex medium which was aerated with a gas mixture of hydrogen and carbon dioxide. Extracts prepared from hydrogen-grown cells formed methane from methyl cobalamin, 5-methyl tetrahydrofolate, serine, pyruvate, and carbon dioxide; these substrates have been shown to be precursors of methane in extracts of the ethyl alcohol-grown culture of M. omelianskii.     

Thymidine Phosphorylation in Arachis hypogaea by Combined Activities of Adenosine Triphosphate Phosphohydrolase and Nucleoside Phosphotransferase

Extracts from hypocotyls of germinating peanuts (Arachis hypogaea L.) stimulated the formation of thymidine monophosphate from thymidine and adenosine triphosphate in the presence of magnesium ions. Such extracts were incapable of incorporating isotopic phosphorus from gamma-labelled adenosine triphosphate into thymidine during the synthesis of thymidine monophosphate but were competent in transferring phosphorus from alpha-labelled adenosine triphosphate to thymidine. The apparent thymidine kinase activity thus appeared to result from the combined activities of an adenosine triphosphatase (adenosine triphosphate pyrophosphohydrolase, E. C. 3.6.1.3) and a nucleoside phosphotransferase (E. C. 2.7.1.77). The latter two enzymes could be largely separated by using DEAE-Sephadex chromatography.     

Anaerobic Simulated Mixed Culture System

A compartmented, autoclavable culture vessel has been developed for the purpose of studying interactive associations of microorganisms which are essential to the anaerobic decomposition of sewage sludge. The unit employs sterile filter membranes to subdivide the interior culture space into individual compartments. Bacteria cultured in one compartment are denied access to adjacent compartments, even though rapid interchange of nutrients and metabolic waste products occurs throughout the unit. The obligate methane-forming anaerobe, Methanobacillus omelianskii has been successfully grown and concentrated in this system by use of a synthetic medium reduced with sodium sulfide. The feasibility of using this system to study microbial interactions was, in part, demonstrated by growing M. omelianskii in a thoroughly aerated medium which had been biologically reduced by Escherichia coli prior to inoculation with the anaerobe. Under this condition of simulated mixed culture growth, the cell yield of both microorganisms, as well as specific metabolic activities ascribed to each organism, was readily monitored.     

Cobalamin-dependent methionine synthase: probing the role of the axial base in catalysis of methyl transfer between methyltetrahydrofolate and exogenous cob(I)alamin or cob(I)inamide

Cobalamin-dependent methionine synthase (MetH) catalyzes the transfer of methyl groups between methyltetrahydrofolate (CH(3)-H(4)folate) and homocysteine, with the enzyme-bound cobalamin serving as an intermediary in the methyl transfers. An MetH fragment comprising residues 2-649 contains modules that bind and activate CH(3)-H(4)folate and homocysteine and catalyze methyl transfers to and from exogenous cobalamin. Comparison of the rates of reaction of cobalamin, which contains a dimethylbenzimidazole nucleotide coordinated to the cobalt in the lower axial position, and cobinamide, which lacks the dimethylbenzimidazole nucleotide, allows assessment of the degree of stabilization the dimethylbenzimidazole base provides for methyl transfer between CH(3)-H(4)folate bound to MetH(2-649) and exogenous cob(I)alamin. When the reactions of cob(I)alamin or cob(I)inamide with CH(3)-H(4)folate are compared, the observed second-order rate constants are 2.7-fold faster for cob(I)alamin; in the reverse direction, methylcobinamide reacts 35-fold faster than methylcobalamin with enzyme-bound tetrahydrofolate. These measurements can be used to estimate the influence of the dimethylbenzimidazole ligand on both the thermodynamics and kinetics of methyl transfer between methyltetrahydrofolate and cob(I)alamin or cob(I)inamide. The free energy change for methyl transfer from CH(3)-H(4)folate to cob(I)alamin is 2.8 kcal more favorable than that for methyl transfer to cob(I)inamide. Dimethylbenzimidazole contributes approximately 0.6 kcal/mol of stabilization for the forward reaction and approximately 2.2 kcal/mol of destabilization for the reverse reaction. Binding of methylcobalamin to full-length methionine synthase is accompanied by ligand substitution, and switching between "base-on" and "base-off" states of the cofactor has been demonstrated [Bandarian, V., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 8156-8163]. The present results disfavor a major role for such switching in catalysis of methyl transfer, and are consistent with the hypothesis that the primary role of the ligand triad in methionine synthase is controlling the distribution of enzyme conformations during catalysis.     

Effects of Alkaline Phosphatase Activity on Nucleotide Measurements in Aquatic Microbial Communities

Alkaline phosphatase (APase) activity was detected in aquatic microbial assemblages from the subtropics to Antarctica. The occurrence of APase in environmental nucleotide extracts was shown to significantly affect the measured concentrations of cellular nucleotides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, guanosine triphosphate, uridine triphosphate, and cytidine triphosphate), adenylate energy charge, and guanosine triphosphate/adenosine triphosphate ratios, when conventional methods of nucleotide extraction were employed. Under the reaction conditions specified in this report, the initial rate of hydrolysis of adenosine triphosphate was directly proportional to the activity of APase in the sample extracts and consequently can be used as a sensitive measure of APase activity. A method was devised for obtaining reliable nucleotide measurements in naturally occurring microbial populations containing elevated levels of APase activity. The metabolic significance of APase activity in microbial cells is discussed, and it is concluded that the occurrence and regulation of APase in nature is dependent upon microscale inorganic phosphate limitation of the autochthonous microbial communities.     

Deoxynucleoside Kinases of Bacillus megaterium KM

Dialyzed extracts of Bacillus megaterium KM contain thymidine, deoxyadenosine, and deoxyguanosine kinase activities. Thymidine kinase activity is best with deoxyadenosine triphosphate or deoxyguanosine triphosphate (dGTP) as the phosphoryl donor, whereas the best deoxyadenosine kinase activity is obtained with dGTP or adenosine triphosphate. Deoxyguanosine kinase activity functions optimally with deoxycytidine triphosphate as the donor. Although the thymidine kinase activity of crude extracts does not have a demonstrable divalent cation requirement, the addition of Mg(2+) or Mn(2+) is necessary for the formation of thymidine di- and triphosphates. The synthesis of thymidine kinase appears to be partially derepressed by thymine starvation. Incubation of extracts with deoxyadenosine and dGTP results in the substantial accumulation of deoxyadenosine di- and triphosphates. Extracts deaminate deoxycytidine to deoxyuridine, presumably as a consequence of the action of deoxycytidine deaminase, and then convert deoxyuridine to deoxyuridylic acid. B. megaterium extracts do not contain any detectable deoxycytidine kinase activity.     

Methane fermentation of rubber (Hevea brasiliensis) latex effluent.

Four species of bacteria capable of CH4 fermentation of rubber latex effluent were isolated and identified as a Methanococcus, a strain of M. vannielii, a Methanobacterium and a strain of M. omelianskii. Auxanographic tests using the four strains showed growth and CH4 formation on a basal medium containing mineral salts or added H2 and Co2. Varied response was obtained when the basal medium was added to formate, acetate, butyrate, methanol, ethanol, and glucose. Previous work has established acid fermentation of Hevea latex arising from bacterial contamination and decomposition of the non-rubber constituents which consist of N-compounds, 2% quebrachitol, and smaller concentration of carbohydrates. This suggests that reduction of CO2 and fermentation of acids formed during metabolism of Hevea latex are possible pathways of CH4 production.     

Regulation of phosphatidylcholine synthesis in rat liver endoplasmic reticulum.

The biosynthesis of phosphatidylcholine in rat liver microsomal preparations catalysed by CDP-choline-1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) was inhibited by a combination of ATP and CoA or ATP and pantetheine. ATP alone at high concentrations (20 mM) inhibits phosphatidylcholine formation to the extent of 70%. In the presence of 0.1 mM-CoA, ATP (2 mM) inhibits to the extent of 80% and in the presence of 1 mM-pantetheine to the extent of 90%. ADP and other nucleotide triphosphates in combination with either CoA or pantetheine are only 10-30% as effective in inhibiting phosphatidylcholine synthesis. AMP(CH2)PP [adenosine 5'-(alphabeta-methylene)triphosphate] together with CoA inhibits to the extent of 59% and with pantetheine by 48%. AMP-P(CH2)P [adenosine 5'-(betagamma-methylene)triphosphate] together with either CoA or pantetheine had no significant effect on phosphatidylcholine formation. Other closely related derivatives of pantothenic acid were without effect either alone or in the presence of ATP, as were thiol compounds such as cysteine, homocysteine, cysteamine, dithiothreitol and glutathione. Several mechanisms by which this inhibition might take place were ruled out and it is concluded that ATP together with either CoA or pantetheine interacts reversibly with phosphatidylcholine synthetase to cause temporarily the inhibition of phosphatidylcholine formation.     

Role of phosphoenolpyruvate carboxylation in Acetobacter xylinum

Glucose-grown cells of Acetobacter xylinum oxidized acetate only when the reaction mixture was supplemented with catalytic quantities of glucose or intermediates of the citrate cycle. Extracts, prepared by sonic treatment, catalyzed the formation of oxalacetate when incubated with phosphoenolpyruvate (PEP) and bicarbonate. Oxalacetate was not formed in the presence of pyruvate plus adenosine triphosphate. The ability to promote carboxylation of PEP was lower in succinate-grown cells than in glucose-grown cells. PEP carboxylase, partially purified from extracts by ammonium sulfate fractionation, catalyzed the stoichiometric formation of oxalacetate and inorganic phosphate from PEP and bicarbonate. The enzyme was not affected by acetyl-coenzyme A or inorganic phosphate. It was inhibited by adenosine diphosphate in a manner competitive with PEP (K(1) = 1.3 mm) and by dicarboxylic acids of the citrate cycle; of these, succinate was the most potent inhibitor. It is suggested that the physiological role of PEP carboxylase in A. xylinum is to affect the net formation of C(4) acids from C(3) precursors, which are essential for the maintainance of the citrate cycle during growth on glucose. The relationship of PEP carboxylase to other enzyme systems metabolizing PEP and oxalacetate in A. xylinum is discussed.     

Adenosine Triphosphate Pools in Methanobacterium

Certain aspects of adenosine triphosphate (ATP) metabolism in the strict anaerobe Methanobacterium strain M.o.H. have been investigated. Results of growth yield studies suggest that ATP conservation is very inefficient (0.06 mole of ATP per mole of hydrogen) under the conditions used to grow the bacterium in a fermentor. Experiments designed to demonstrate net ATP formation in cell-free extracts were negative. In whole-cell studies, substances which decreased ATP pool levels and increased adenosine monophosphate (AMP) pool levels were air, chloroform, 2,4-dinitrophenol, carbonylcyanide-m-chlorophenylhydrazone, and pentachlorophenol. The results suggest that the latter compounds act either as inhibitors of electron transport or as uncouplers of an energy-linked process. All the above compounds also inhibit methane formation in cell-free extracts, an ATP-requiring process. Methods are described for estimation of ATP, adenosine diphosphate (ADP), and AMP in whole cells, with a sensitivity in the range of 10 to 200 pmoles. An apparatus for quick sampling from an anaerobic suspension of whole cells also is described.     

In vitro methane and methyl coenzyme M formation from acetate: evidence that acetyl-CoA is the required intermediate activated form of acetate

Buffer-soluble extracts of acetate-grown Methanosarcina barkeri catalyzed methanogenesis from acetate in the presence of hydrogen and ATP. The rates of methane formation from either acetate plus ATP, or acetylphosphate without ATP added, were approximately doubled by the addition of coenzyme A (CoA). In vitro methyl group transfer from [2-14C]acetate to form [14CH3]methyl coenzyme M (14CH3S-CoM) was monitored by causing the accumulation of 14CH3S-CoM (14CH3-SCH2CH2SO3-) in the presence of 2-bromoethanesulfonate. The rate of 14CH3S-CoM formation was increased 2.5-fold by 0.2 mM CoA.     

A stereochemical study of the mechanism of activation of donor oligonucleotides by RNA ligase from bacteriophage T4 infected Escherichia coli

RNA ligase from bacteriophage T4 infected Escherichia coli catalyzes the activation of adenosine 3',5'-bisphosphate (representing the donor oligonucleotide) by adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate with retention of configuration at P alpha. Since single-step enzyme-catalyzed nucleotidyl transfer reactions proceed with inversion, this stereochemical result provides support for a double displacement mechanism involving an adenylyl-enzyme intermediate as proposed previously from isotope exchange experiments.     

Characteristics of S Organism Isolated from Methanobacillus omelianskii

Previous work showed that Methanobacillus omelianskii was a mixed culture of an ethanol-oxidizing organism called S organism and a hydrogen-utilizing methane bacterium, strain MOH. S organism grows poorly on ethanol unless a hydrogen-utilizing methanogenic bacterium is included to utilize the H(2) produced during growth. Further studies have shown that, among many substrates tested, only ethanol, n-propanol, n-butanol, isobutanol, n-pentanol, acetaldehyde, oxalacetate, and pyruvate are fermented by S organism, either alone or in combination with Methanobacterium ruminantium. It grew better in pure culture with pyruvate than with alcohols. H(2) gas phase inhibited growth on pyruvate as well as on alcohol. When grown alone on pyruvate, S organism produced mainly acetate, ethanol, and CO(2), in addition to a small amount of H(2). When combined with M. ruminantium, no H(2) and very little ethanol were produced and acetate production was increased. When M. ruminantium was present, electrons from pyruvate oxidation by S organism were channeled almost entirely to H(2) and hence to methane formation rather than ethanol. Also, S organism utilized more pyruvate when grown with M. ruminantium. Attempts to obtain better growth of S organism on ethanol by addition of many possible electron acceptors were unsuccessful. It grew best between 32 and 45 C, had a per cent guanine plus cytosine content of deoxyribonucleic acid bases of 47.27 +/- 0.1, contained no cytochrome, and could be grown on a defined medium with pyruvate as the energy and carbon source and with (NH(4))(2)SO(4) as the main nitrogen source. These and other results suggest that S organism belongs in a new genus, but assignment of a definite taxonomic status should await isolation and characterization of more strains.     

An assessment of growth yields and energy coupling inDesulfovibrio

The yield coefficients forDesulfovibrio vulgaris andD. gigas varied with the electron donoracceptor combinations and with the bacterial strain. The only evidence for electron transport coupled formation of adenosine triphosphate (ATP) was with sulfate as the electron acceptor. WithD. vulgaris the ATP formation coupling to electron flow with pyruvate oxidation was 1:4 electrons and with lactate oxidation was 1:8 electrons. WithD. gigas these ratios were 1:8 electrons and 1:16 electrons for the oxidation of pyruvate and lactate. The clearest resolution of energy coupling was withD. vulgaris growing on formatesulfate medium where 2 ATP appear to be formed with the transfer of electrons from formate to adenosine phosphosulfate and one ATP with the transfer of electrons from formate to sulfite.     

Mechanism of activation of phenylalanine and synthesis of P1, P4-bis(5'-adenosyl) tetraphosphate by yeast phenylalanyl-tRNA synthetase

The activation of L-phenylalanine by yeast phenylalanyl-tRNA synthetase using adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate is shown to proceed with inversion of configuration at P alpha of ATP. This observation taken together with the lack of positional isotope exchange when adenosine 5'-[beta,beta-18O2]triphosphate is incubated with the enzyme in the absence of phenylalanine and in the presence of the competitive inhibitor phenylalaninol indicates that activation of phenylalanine occurs by a direct "in-line" adenylyl-transfer reaction. In the presence of Zn2+, yeast phenylalanyl-tRNA synthetase also catalyzes the phenylalanine-dependent hydrolysis of ATP to AMP and the synthesis of P1,P4-bis(5'-adenosyl) tetraphosphate (Ap4A). With adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate, the formation of AMP and Ap4A is shown to occur with inversion and retention of configuration, respectively. It is concluded that phenylalanyl adenylate is an intermediate in both processes, Zn2+ promoting AMP formation by hydrolytic cleavage of the C-O bond and Ap4A formation by displacement at phosphorus of phenylalanine by ATP.     

Some properties of adenosine kinase from Ehrlich ascites-tumour cells

1. Adenosine kinase was measured in dialysed extracts from Ehrlich ascites-tumour cells by a chromatographic procedure. 2. In the absence of added Mg(2+) the K(m) values for ATP and adenosine were 0.22mm and 2.8mum respectively. 3. The maximum velocity of adenosine kinase with free ATP was about three times that with the Mg(2+)-ATP complex. Free Mg(2+) was a non-competitive inhibitor of the reaction. A small amount of added Mg(2+), Mn(2+) or Ca(2+) was required for maximum adenosine kinase activity after cation bound to the enzyme had been released by treatment with p-chloromercuribenzoate and then removed by dialysis. 4. GTP, ITP, deoxy-ATP, deoxy-GTP, CTP, xanthosine triphosphate, UTP and thymidine triphosphate could partially or completely replace ATP as a phosphate donor. 5. The reaction of ATP with adenosine kinase was competitively inhibited by AMP, GMP, IMP, ADP, deoxy-ADP and IDP (K(i) 0.2, 1.1, 5.9, 1.2, 0.5 and 0.78mm respectively). Enzymic activity was markedly affected by the relative concentrations of AMP, ADP and ATP in assay mixtures. 6. The results are discussed in terms of possible mechanisms regulating the rate of adenosine kinase in vivo.     

Effect of inorganic sulfide on the growth and metabolism of Methanosarcina barkeri strain DM

Minimal growth of Methanosarcina barkeri strain DM occurred when sulfide was omitted fromthe growth medium, and addition of either sodium sulfate or coenzyme M to sulfide-depleted media failed to restore growth. Optimal growth occurred in the presence of 1.25 mM added sulfide, giving a molar growth yield (YCH4) of 4.4 mg (dry weight) of cells per mmol of CH4 produced. Increasing sulfide to 12.5 mM led to decrease in YCH4 (1.9 mg [dry weight]/mmol of CH4), in the specific growth rate and in be intracellular levels of adenosine triphosphate. However, the specific rate of methane production increased. The data suggested that at elevated sulfide levels (12.5 mM) the decrease in YCH4 might be a result of an increase in the relative energy needed for maintnenace and of uncoupling of growth from energy production.     

Effect of inorganic sulfide on the growth and metabolism of Methanosarcina barkeri strain DM.

Minimal growth of Methanosarcina barkeri strain DM occurred when sulfide was omitted fromthe growth medium, and addition of either sodium sulfate or coenzyme M to sulfide-depleted media failed to restore growth. Optimal growth occurred in the presence of 1.25 mM added sulfide, giving a molar growth yield (YCH4) of 4.4 mg (dry weight) of cells per mmol of CH4 produced. Increasing sulfide to 12.5 mM led to decrease in YCH4 (1.9 mg [dry weight]/mmol of CH4), in the specific growth rate and in be intracellular levels of adenosine triphosphate. However, the specific rate of methane production increased. The data suggested that at elevated sulfide levels (12.5 mM) the decrease in YCH4 might be a result of an increase in the relative energy needed for maintnenace and of uncoupling of growth from energy production.