Acetate catabolism by Methanosarcina barkeri: evidence for involvement of carbon monoxide dehydrogenase, methyl coenzyme M, and methylreductase.

The pathway of acetate catabolism in Methanosarcina barkeri strain MS was studied by using a recently developed assay for methanogenesis from acetate by soluble enzymes in cell extracts. Extracts incubated with [2-14C]acetate, hydrogen, and ATP formed 14CH4 and [14C]methyl coenzyme M as products. The apparent Km for acetate conversion to methane was 5 mM. In the presence of excess acetate, both the rate and duration of methane production was dependent on ATP. Acetyl phosphate replaced the cell extract methanogenic requirement for both acetate and ATP (the Km for ATP was 2 mM). Low concentrations of bromoethanesulfonic acid and cyanide, inhibitors of methylreductase and carbon monoxide dehydrogenase, respectively, greatly reduced the rate of methanogenesis. Precipitation of CO dehydrogenase in cell extracts by antibodies raised to 95% purified enzyme inhibited both CO dehydrogenase and acetate-to-methane conversion activity. The data are consistent with a model of acetate catabolism in which methylreductase, methyl coenzyme M, CO dehydrogenase, and acetate-activating enzymes are components. These results are discussed in relation to acetate uptake and rate-limiting transformation mechanisms in methane formation.     

Oxidation of trimethylamine to the level of formaldehyde by Methanosarcina barkeri is dependent on the protonmotive force

The conversion of trimethylamine to methane, carbon dioxide and ammonia as catalyzed by cell suspensions of Methanosarcina barkeri was coupled to the generation of a protonmotive force and to the synthesis of ATP. Methanogenesis as well as ATP formation and protonmotive force generation was abolished by the uncoupler tetrachloro-salicylanilide (TCS). Inhibition of methane formation was reversed by addition of formaldehyde, which was predominantly oxidized to carbon dioxide, whereas trimethylamine was predominantly reduced to methane and ammonia under these conditions. Cell extracts of M. barkeri were unable to convert trimethylamine to methane, carbon dioxide and ammonia independent from the presence or absence of ATP.     

An ancient pathway combining carbon dioxide fixation with the generation and utilization of a sodium ion gradient for ATP synthesis

Synthesis of acetate from carbon dioxide and molecular hydrogen is considered to be the first carbon assimilation pathway on earth. It combines carbon dioxide fixation into acetyl-CoA with the production of ATP via an energized cell membrane. How the pathway is coupled with the net synthesis of ATP has been an enigma. The anaerobic, acetogenic bacterium Acetobacterium woodii uses an ancient version of this pathway without cytochromes and quinones. It generates a sodium ion potential across the cell membrane by the sodium-motive ferredoxin:NAD oxidoreductase (Rnf). The genome sequence of A. woodii solves the enigma: it uncovers Rnf as the only ion-motive enzyme coupled to the pathway and unravels a metabolism designed to produce reduced ferredoxin and overcome energetic barriers by virtue of electron-bifurcating, soluble enzymes.     

Lactate Metabolism by Veillonella parvula

A strain of Veillonella parvula M4, which grows readily in lactate broth without a requirement for carbon dioxide, has been isolated from the oral cavity. Anaerobic, washed cells of this organism fermented sodium lactate to the following products (moles/100 moles of lactate): propionate, 66; acetate, 40; carbon dioxide, 40; and hydrogen, 14. Cells grew readily in tryptone-yeast extract broth with pyruvate, oxaloacetate, malate, and fumarate, but poorly with succinate. The fermentation of pyruvate, oxaloacetate, or lactate plus oxaloacetate by washed cells resulted in the formation of propionate and acetate in ratios significantly lower than those observed with lactate as the sole carbon source. This was primarily due to increased acetate production. Cell-free extracts were unable to degrade lactate but metabolized lactate in the presence of oxaloacetate, indicating the presence of malic-lactic transhydrogenase in this organism. Lactic dehydrogenase activity was not observed. Evidence is presented for oxaloacetate decarboxylase and malic dehydrogenase activities in extracts.     

Methanogenic cleavage of acetate by lysates of Methanosarcina barkeri.

Cell lysates of acetate-grown Methanosarcina barkeri 227 were found to cleave acetate to CH4 and CO2. The aceticlastic reaction was identified by using radioactive methyl-labeled acetate. Cell lysates decarboxylated acetate in a nitrogen atmosphere, conserving the methyl group in methane. The rate of methanogenesis from acetate in the cell lysates was comparable to that observed with whole cells. Aceticlastic activity was found in the particulate fraction seperate from methylcoenzyme M methylreductase activity, which occurs in the soluble fraction. Pronase treatment eliminated methylcoenzyme M methylreductase activity in lysates and stimulated aceticlastic activity, indicating the aceticlastic activity was not derived from unbroken cells, which are unaffected by proteolytic treatment.     

Acetic acid from H2 and CO2

Cell extracts of a nonsporeforming strictly anaerobic bacterium, Acetobacterium woodii produced acetate in N-tris(Hydroxymethyl)methyl-2-aminoethane sulfonic acid or phosphate buffers from hydrogen and carbon dioxide. The formation of acetate was not dependent on the presence of ATP in the reaction mixture; ADP also did not influence the acetate production. Since acetic acid is the main fermentation product during growth of A. woodii with H₂ and CO₂, ATP must be synthesized in the course of acetate formation. The possible sites of ATP synthesis are discussed.     

Bioenergetics of methanogenesis from acetate by Methanosarcina barkeri.

Methane formation from acetate by resting cells of Methanosarcina barkeri was accompanied by an increase in the intracellular ATP content from 0.9 to 4.0 nmol/mg of protein. Correspondingly, the proton motive force increased to a steady-state level of -120 mV. The transmembrane pH gradient however, was reversed under these conditions and amounted to +20 mV. The addition of the protonophore 3,5,3',4'-tetrachlorosalicylanilide led to a drastic decrease in the proton motive force and in the intracellular ATP content and to an inhibition of methane formation. The ATPase inhibitor N,N'-dicyclohexylcarbodiimide stopped methanogenesis, and the intracellular ATP content decreased. The proton motive force decreased also under these conditions, indicating that the proton motive force could not be generated from acetate without ATP. The overall process of methane formation from acetate was dependent on the presence of sodium ions; upon addition of acetate to cell suspensions of M. barkeri, a transmembrane Na+ gradient in the range of 4:1 (Na+ out/Na+ in) was established. Possible sites of involvement of the Na+ gradient in the conversion of acetate to methane and carbon dioxide are discussed. Na+ is not involved in the CO dehydrogenase reaction.     

Methanogenesis from acetate by cell-free extracts of the thermophilic acetotrophic methanogen Methanothrix thermophila CALS-1

High rates of methanogenesis from acetate and ATP were observed from cell-free extracts of the thermophilic acetotrophic methanogen Methanothrix (Methanosaeta) thermophila strain CALS-1 when cultures were grown in a pH auxostat fed with acetic acid. Specific methanogenic activities ranged from 50–300 nmol min^–1 (mg protein)^–1, which was comparable to those for whole cells. In contrast to results with Methanosarcina spp., the reaction did not require high levels of H₂ in the headspace. CO was inhibitory to methanogenesis from acetate. The inhibition by CO and the lack of effect of H₂ on methanogenesis from acetate resemble previous results with whole cells of CALS-1. Protein concentrations in extracts > 5 mg/ml were required for good activity, and the optimum temperature for the methanogenesis was near 65° C. ATP was required in substrate quantities and was converted mainly to AMP. The maximum CH₄/ATP stoichiometry obtained was near 1.0, consistent with acetate activation using an acetyl-CoA synthetase mechanism that converts ATP to AMP and pyrophosphate. Methanogenesis in extracts was inhibited by bromoethane sulfonate and cyanide, indicating the involvement of methylcoenzyme M methylreductase and a carbon monoxide dehydrogenase complex with methanogenesis from acetate. These results are consistent with acetyl-coenzyme A (CoA) as the form of activated acetate involved in methanogenesis from acetate in strain CALS-1, but no activity could be obtained from extracts using acetyl-CoA as a substrate. Received: 18 March 1996 / Accepted: 14 June 1996     

A methyl-CoM methylreductase system from methanogenic bacterium strain G? 1 not requiring ATP for activity

Crude inside-out vesicles from the methanogenic strain G?1 were prepared via protoplasts. These vesicles catalyzed methane formation from methyl-CoM and H2 at a maximal rate of 35 nmol/min.mg protein. Methane formation by the vesicles did not depend on the addition of ATP. This was in contrast to conventionally prepared crude extracts from the same organism or from Methanosarcina barkeri which exhibited strict ATP dependence of methanogenesis. ATP analogues inhibited methanogenesis by extracts to a much higher extent than that by vesicles. Both, particulate and soluble components prepared from the crude vesicles by ultracentrifugation were necessary for ATP-independent methane formation from methyl-CoM and H2. Hydrogenase activity was mainly associated with the particulate fraction whereas methyl-CoM methylreductase could be assigned to the soluble fraction. The detergent sulfobetaine inhibited methane formation from methyl-CoM without affecting hydrogenase or titanium citrate-dependent methylreductase activities, indicating that an additional membraneous component is involved in methanogenesis for methyl-CoM and H2.     

Utilization of acetate by Methanomonas emthanooxidans.

Methanomonas methanooxidans incorporates both carbon atoms of acetate into the glutamate and aspartate families of amino acids during growth on methane; carbon dioxide is also evolved from both carbon atoms of acetate. The distribution of carboxyl-labeled acetate incorporated into convalently bound glutamate is consistent with the operation of the tricarboxylic acid cycle in this species, and the presence of alpha-ketoglutarate dehydrogenase was demonstrated in cell-free extracts.     

Metabolism of Dichloromethane by the Strict Anaerobe Dehalobacterium formicoaceticum

The metabolism of dichloromethane by Dehalobacterium formicoaceticum in cell suspensions and crude cell extracts was investigated. The organism is a strictly anaerobic gram-positive bacterium that utilizes exclusively dichloromethane as a growth substrate and ferments this compound to formate and acetate in a molar ratio of 2:1. When [¹³C]dichloromethane was degraded by cell suspensions, formate, the methyl group of acetate, and minor amounts of methanol were labeled, but there was no nuclear magnetic resonance signal corresponding to the carboxyl group of acetate. This finding and previously established carbon and electron balances suggested that dichloromethane was converted to methylene tetrahydrofolate, of which two-thirds was oxidized to formate while one-third gave rise to acetate by incorporation of CO₂ from the medium in the acetyl coenzyme A synthase reaction. When crude desalted extracts were incubated in the presence of dichloromethane, tetrahydrofolate, ATP, methyl viologen, and molecular hydrogen, dichloromethane and tetrahydrofolate were consumed, with the concomitant formation of stoichiometric amounts of methylene tetrahydrofolate. The in vitro transfer of the methylene group of dichloromethane onto tetrahydrofolate required substoichiometric amounts of ATP. The reaction was inhibited in a light-reversible fashion by 20 μM propyl iodide, thus suggesting involvement of a Co(I) corrinoid in the anoxic dehalogenation of dichloromethane. D. formicoaceticum exhibited normal growth with 0.8 mM sodium in the medium, and crude extracts contained ATPase activity that was partially inhibited by N,N′-dicyclohexylcarbodiimide and azide. During growth with dichloromethane, the organism thus may conserve energy not only by substrate-level phosphorylation but also by a chemiosmotic mechanism involving a sodium-independent F₀F₁-type ATP synthase.     

Inhibition of growth by erythritol catabolism in Brucella abortus.

The growth of Brucella abortus (US-19) in a complex tryptose-yeast extract medium containing D-glucose is inhibited by 10 mM erythritol. The enzymes of the erythritol pathway, except for D-erythrulose 1-phosphate dehydrogenase (D-glycero-2-tetrulose 1-phosphate:nicotinamide adenine dinucleotide (NAD+) 4-oxidoreductase) were detected in the soluble and membrane fractions of cell extracts. Glucose catabolism by cell extracts was inhibited by erythritol, whereas, phosphorylated intermediates of the hexose monophosphate pathway were converted to pyruvic acid with oxygen consumption. Erythritol kinase (EC 2.7.1.27; adenosine 5'-triphosphate (ATP): erythritol 1-phosphotransferase) was found to be eightfold higher in activity than the hexokinase in cell extracts. In vivo, ATP is apparently consumed with the accumulation of D-erythrulose 1-phosphate (D-glycero-2-tetrulose 1-phosphate) and no substrate level phosphorylation. ATP levels dropped 10-fold in 30 min after addition of erythritol to log phase cells in tryptose-yeast extract medium with D-glucose as the carbon source. These data suggest bacteriostasis in the presence of erythritol results from the ATP drain caused by erythritol kinase.     

The assimilation of carbon by Chloropseudomonas ethylicum

1. The enzymes in ultrasonically prepared extracts of Chloropseudomonas ethylicum were studied to elucidate how this organism assimilates acetate and carbon dioxide and why it cannot grow with either of these two compounds alone. 2. Such extracts can (i) convert acetate and oxaloacetate into alpha-oxoglutarate, (ii) convert oxaloacetate into succinyl-CoA, (iii) convert phosphopyruvate into 3-phosphoglyceraldehyde and (iv) interconvert phosphopyruvate and pyruvate via oxaloacetate. 3. Pyruvate kinase, alpha-oxoglutarate dehydrogenase, ribulose diphosphate carboxylase, isocitrate lyase and malate synthase were not detected. 4. It is difficult to detect aconitate hydratase, fumarate hydratase and citrate synthase in extracts of the organism ultrasonically treated in tris buffer; to demonstrate these enzymes extracts should be prepared in phosphate buffer containing 2-mercaptoethanol. 5. Provided that this organism can synthesize pyruvate from acetate and carbon dioxide, the enzymes detected are sufficient to account for the nutritional requirements of this organism.     

Isolation and characterization of acetyl-coenzyme A synthetase from Methanothrix soehngenii.

In Methanothrix soehngenii, acetate is activated to acetyl-coenzyme A (acetyl-CoA) by an acetyl-CoA synthetase. Cell extracts contained high activities of adenylate kinase and pyrophosphatase, but no activities of a pyrophosphate:AMP and pyrophosphate:ADP phosphotransferase, indicating that the activation of 1 acetate in Methanothrix requires 2 ATP. Acetyl-CoA synthetase was purified 22-fold in four steps to apparent homogeneity. The native molecular mass of the enzyme from M. soehngenii estimated by gel filtration was 148 kilodaltons (kDa). The enzyme was composed of two subunits with a molecular mass of 73 kDa in an alpha 2 oligomeric structure. The acetyl-CoA synthetase constituted up to 4% of the soluble cell protein. At the optimum pH of 8.5, the Vmax was 55 mumol of acetyl-CoA formed per min per mg of protein. Analysis of enzyme kinetic properties revealed a Km of 0.86 mM for acetate and 48 microM for coenzyme A. With varying amounts of ATP, weak sigmoidal kinetic was observed. The Hill plot gave a slope of 1.58 +/- 0.12, suggesting two interacting substrate sites for the ATP. The kinetic properties of the acetyl-CoA synthetase can explain the high affinity for acetate of Methanothrix soehngenii.     

The biosynthesis of valine from isobutyrate by Peptostreptococcus elsdenii and Bacteroides ruminicola

1. Growing cultures of Peptostreptococcus elsdenii and Bacteroides ruminicola incorporate (14)C from [1-(14)C]isobutyrate into the valine of cell protein. With P. elsdenii some of the (14)C is also incorporated into leucine. 2. Crude cell-free extracts of both organisms in the presence of glutamine, carbon dioxide and suitable sources of energy and electrons incorporate (14)C from [1-(14)C]isobutyrate into valine but not into leucine. 3. With extracts of P. elsdenii treated with DEAE-cellulose the reaction is dependent on ATP, CoA, thiamin pyrophosphate, molecular hydrogen and a low-potential electron carrier (ferredoxin, flavodoxin or benzyl viologen). 4. The same extracts incorporate (14)C from NaH(14)CO(3) into valine in the presence of isobutyrate plus ATP, CoA, glutamine and ferredoxin; isobutyryl-CoA or isobutyryl phosphate plus CoA will replace the isobutyrate plus CoA and ATP. With acetyl phosphate in place of isobutyryl phosphate, (14)C is incorporated into alanine. With isovalerate or 2-methylbutyrate in place of isobutyrate, (14)C is incorporated into leucine and isoleucine respectively. 5. When carrier 2-oxoisovalerate is added to the carboxylating system (14)C from [1-(14)C]isobutyrate passes into the oxo acid fraction. 6. It is concluded that these two organisms form valine from isobutyrate by the sequence isobutyrate-->isobutyryl-CoA-->2-oxoisovalerate-->valine and that the reductive carboxylation of isobutyrate is catalysed by a system similar to the pyruvate synthetase of clostridia and photosynthetic bacteria.     

The incorporation of acetate by the chemoautotroph Thiobacillus neapolitanus strain C

Summary Cultures of Thiobacillus neapolitanus strain C assimilate ¹⁴C-labelled acetate and aspartate. Both carbon atoms of acetate are incorporated, and 25% of the cell carbon can arise from acetate. Aspartate-¹⁴C contributes 4–5% of the cell carbon, and is found in pyrimidines and in protein as aspartate and its related amino acids. Acetate-¹⁴C contributes to lipid, glutamate, arginine, proline and leucine, but not to aspartate. Acetate assimilation by washed organisms requires carbon dioxide and energy from thiosulphate oxidation. Degradation of ¹⁴C-glutamic acid from acetate-¹⁴C-labelled bacteria; the accumulation of ¹⁴C-citrate in the presence of fluoroacetate and [¹⁴C] acetate; short-term kinetic experiments on acetate-¹⁴C turnover; and the demonstration of citrate synthesis by cell-free extracts all indicate glutamate synthesis from -ketoglutarate formed by reactions of the tricarboxylic acid cycle. The cycle is believed to be incomplete, probably not proceeding further than -ketoglutarate, and functions as a glutamate-synthesising system, using oxaloacetate derived solely from carbon dioxide fixation. Malate synthase (and the glyoxylate cycle) appear to be insignificant in the metabolism, but extracts did form citramalate from acetate and pyruvate.     

Decomposition of the water-soluble fraction of sudangrass residue in soil

Summary Ground sudangrass forage was extracted at room temperature with demineralized water. The insoluble residue, the water-soluble residue, unextracted sudangrass and the recombined soluble and insoluble fractions were incubated in remoistened, air-dried soil to measure the influence of the water-soluble fraction on net carbon dioxide evolution. The maximum rate of carbon dioxide evolution from the check soil and from any treatment containing the water-soluble fraction of sudangrass occurred about 14 hours after initiation of incubation. Maximum rate of carbon dioxide evolution from the soil treated with the insoluble residue occurred about 39 hours after initiation of the incubation. The rate of carbon dioxide evolution from the soil which received the water-soluble residue was not significantly different from the rate from the check soil after 44 hours of incubation.Contribution from the Department of Agronomy. Approved as Journal Paper No. J-4955, Project 1378. Financial assistance of Phillips Petroleum Company is acknowledged.     

The Kinetics of Isocitrate Lyase Formation in Chlorella: Evidence for the Promotion of Enzyme Synthesis by Photophosphorylation

The addition of acetate to aerobic Chlorella pyrenoidosa indarkness was followed by the formations of isocitrate lyaseactiity. After a lag period of 40 minutes the formation proceededat a constant rate. By use of actylamide gel electrophoresisit was shown that the increase in enzyme activity was accompaniedby the formation of a new protein which, after separation byelectrophoresis, contained isocitrate lyase activity. The formationof isocitrate lyase was repressed by glucose; it was repressedby light in the presence of carbon dioxide, but not when DCMUwas added. In light, plus DCMU, isocitrate lyase was formedanaerobically and the capacity for photo-formation of isocitratelyase was saturated at 500 ergs/cm²/sec. In this respect theprocess resembled the photo-conversion of glucose to polysaccharidebut differed from the photo-assimilation of carbon dioxide whichbecame saturated at a heigher light intensity. Monochromaticlight of 706 mµ wavelength supported both isocitrate layseformation and the conversion of glucose to polysaccharide butnot carbon dioxide fixation. It is concluded that ATP generatedby cyclic photophosphorylatin can provide the energy for isocitratelyase synthesis in Chlorella.     

ATP-stimulated degradation of endogenous proteins in cell-free extracts of BHK 21/C13 fibroblasts. A key role for the proteinase, macropain, in the ubiquitin-dependent degradation of short-lived proteins.

Baby hamster kidney (BHK) 21/C13 cell proteins, labeled with [35S]methionine, [14C]leucine or [3H]leucine in intact cells, were degraded in soluble, cell-free extracts by an ATP-stimulated process. The stimulatory effect of ATP appeared to require ATP hydrolysis and was mediated to a large extent by ubiquitin. Although the cell extracts contained endogenous ubiquitin, supplementation with exogenous ubiquitin increased ATP-dependent proteolysis by up to 2-fold. Furthermore, antibodies against the E1 ubiquitin conjugating enzyme specifically inhibited both conjugation of [125I]ubiquitin to endogenous proteins and ATP/ubiquitin-dependent proteolysis. Addition of purified E1 to antibody-treated extracts restored conjugation and proteolysis. Proteins containing the amino acid analogues canavanine and azatryptophan were also degraded in vitro by an ATP/ubiquitin-dependent process but at a rate up to 2-fold faster than normal proteins. These results indicate that soluble, cell-free extracts of BHK cells can selectively degrade proteins whose rates of degradation are increased in intact cells. Treatment of cell-free extracts with antibodies against the high molecular weight proteinase, macropain, also greatly inhibited the ATP/ubiquitin-dependent degradation of endogenous proteins. Proteolysis was specifically restored when purified macropain L was added to the antibody-treated extracts. Treatment of cell extracts with both anti-macropain and anti-E1 antibodies reduced ATP/ubiquitin-dependent proteolysis to the same extent as treatment with either antibody alone. Furthermore, proteolysis could be restored to the double antibody treated extracts only after addition of both purified E1 and macropain. These results provide strong evidence for an important role for macropain in the ATP/ubiquitin-dependent degradation of endogenous proteins in BHK cell extracts.     

Effect of Vitreoscilla hemoglobin dosage on microaerobic Escherichia coli carbon and energy metabolism

The amount of Vitreoscilla hemoglobin (VHb) expression was modulated over a broad range with an isopropyl-beta-D-thiogalactopyranoside- (IPTG-) inducible plasmid, and the consequences on microaerobic Escherichia coli physiology were examined in glucose fed-batch cultivations. The effect of IPTG induction on growth under oxygen-limited conditions was most visible during late fed-batch phase where the final cell density increased initially linearly with increasing VHb concentrations, ultimately saturating at a 2.7-fold increase over the VHb-negative (Vhb(-)) control. During the same growth phase, the specific excretions of fermentation by-products, acetate, ethanol, formate, lactate, and succinate from the culture expressing the highest amount of VHb were reduced by 25%, 49%, 68%, 72%, and 50%, respectively, relative to the VHb(-) control. During the exponential growth phase, VHb exerted a positive but smaller control on growth rate, growth yield, and respiration. Varying the amount of VHb from 0 to 3.8 mumol/g dry cell weight (DCW) increased the specific growth rate, the growth yield, and the oxygen consumption rate by 33%, 35%, and 60%, respectively. Increasing VHb concentration to 3.8 mumol/g DCW suppressed the rate of carbon dioxide evolution in the exponential phase by 30%. A metabolic flux distribution analysis incorporating data from these cultivations discloses that VHb(+) cells direct a larger fraction of glucose toward the pentose phosphate pathway and a smaller fraction of carbon through the tricarboxylic acid cycle from acetyl coenzyme A. The overall nicotinamide adenine dinucleotide [NAD(P)H] flux balance indicates that VHb-expressing cells generate a net NADH flux by the NADH/NADPH transhydrogenase while the VHb(-) cells yield a net NADPH flux under the same growth conditions. Flux distribution analysis also reveals that VHb(+) cells have a smaller adenosine triphosphate (ATP) synthesis rate from substrate-level phosphorylation but a larger overall ATP production rate under microaerobic conditions. The thermodynamic efficiency of growth, based on reducing equivalents generated per unit of biomass produced, is greater for VHb(+) cells. (c) 1996 John Wiley & Sons, Inc.