Metabolism of One-Carbon Compounds by the Ruminal Acetogen Syntrophococcus sucromutans

Syntrophococcus sucromutans is the predominant species capable of O demethylation of methoxylated lignin monoaromatic derivatives in the rumen. The enzymatic characterization of this acetogen indicated that it uses the acetyl coenzyme A (Wood) pathway. Cell extracts possess all the enzymes of the tetrahydrofolate pathway, as well as carbon monoxide dehydrogenase, at levels similar to those of other acetogens using this pathway. However, formate dehydrogenase could not be detected in cell extracts, whether formate or a methoxyaromatic was used as electron acceptor for growth of the cells on cellobiose. Labeled bicarbonate, formate, [1-¹⁴C] pyruvate, and chemically synthesized O-[methyl-¹⁴C]vanillate were used to further investigate the catabolism of one-carbon (C₁) compounds by using washed-cell preparations. The results were consistent with little or no contribution of formate dehydrogenase and pointed out some unique features. Conversion of formate to CO₂ was detected, but labeled formate predominantly labeled the methyl group of acetate. Labeled CO₂ readily exchanged with the carboxyl group of pyruvate but not with formate, and both labeled CO₂ and pyruvate predominantly labeled the carboxyl group of acetate. No CO₂ was formed from O demethylation of vanillate, and the acetate produced was position labeled in the methyl group. The fermentation pattern and specific activities of products indicated a complete synthesis of acetate from pyruvate and the methoxyl group of vanillate.     

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.     

One-carbon metabolism in methanogens: evidence for synthesis of a two-carbon cellular intermediate and unification of catabolism and anabolism in Methanosarcina barkeri

One-carbon metabolic transformations associated with cell carbon synthesis and methanogenesis were analyzed by long- and short-term ¹⁴CH₃OH or ¹⁴CO₂ incorporation studies during growth and by cell suspensions. ¹⁴CH₃OH and ¹⁴CO₂ were equivalently incorporated into the major cellular components (i.e., lipids, proteins, and nucleic acids) during growth on H₂-CO₂-methanol. ¹⁴CH₃OH was selectively incorporated into the C-3 of alanine with decreased amounts fixed in the C-1 and C-2 positions, whereas ¹⁴CO₂ was selectively incorporated into the C₁ moiety with decreasing amounts assimilated into the C-2 and C-3 atoms. Notably, ¹⁴CH₄ and [3-¹⁴C]alanine synthesized from ¹⁴CH₃OH during growth shared a common specific activity distinct from that of CO₂ or methanol. Cell suspensions synthesized acetate and alanine from ¹⁴CO₂. The addition of iodopropane inhibited acetate synthesis but did not decrease the amount of ¹⁴CH₃OH or ¹⁴CO₂ fixed into one-carbon carriers (i.e., methyl coenzyme M or carboxydihydromethanopterin). Carboxydihydromethanopterin was only labeled from ¹⁴CH₃OH in the absence of hydrogen. Cell extracts catalyzed the synthesis of acetate from ¹⁴CO (~1 nmol/min per mg of protein) and an isotopic exchange between CO₂ or CO and the C-1 of pyruvate. Acetate synthesis from ¹⁴CO was stimulated by methyl B₁₂ but not by methyl tetrahydrofolate or methyl coenzyme M. Methyl coenzyme M and coenzyme M were inhibitory to acetate synthesis. Cell extracts contained high levels of phosphotransacetylase (>6 μmol/min per mg of protein) and acetate kinase (>0.14 μmol/min per mg of protein). It was not possible to distinguish between acetate and acetyl coenzyme A as the immediate product of two-carbon synthesis with the methods employed.     

Metabolism of homoacetogens

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.     

Carbon assimilation pathways in sulfate reducing bacteria. Formate,carbon dioxide,carbon monoxide,and acetate assimilation by Desulfovibrio baarsii

Desulfovibrio baarsii is a sulfate reducing bacterium, which can grown on formate plus sulfate as sole energy source and formate and CO₂ as sole carbon sources. It is shown by ¹⁴C labelling studies that more than 60% of the cell carbon is derived from CO₂ and the rest from formate. The cells thus grow autotrophically. Labelling studies with [¹⁴C]acetate, ¹⁴CO and [¹⁴C]formate indicate that CO₂ fixation does not proceed via the Calvin cycle. The labelling patterns of alanine, aspartate, glutamate, and glucosamine indicate that acetate (or activated acetic acid) is an early intermediate in formate and CO₂ assimilation; the methyl group of acetate is derived from formate, and the carboxyl group from CO₂ via CO; pyruvate is formed from acetyl-CoA by reductive carboxylation. The capacity to synthesize an acetate unit from two C₁-compounds obviously distinguishes D. baarsii from those Desulfovibrio species, which require acetate as a carbon source in addition to CO₂.     

Isolation and characterization of Dehalobacterium formicoaceticum gen. nov. sp. nov., a strictly anaerobic bacterium utilizing dichloromethane as source of carbon and energy

A strictly anaerobic, dichloromethane-utilizing bacterium was isolated from a previously described dichloromethane-fermenting, two-component mixed culture. In a mineral medium with vitamins, the organism converted 5 mM dichloromethane within 7 days to formate plus acetate in a molar ratio of 2:1 and to biomass and traces of pyruvate. Of 50 potential substrates and combinations of substrates tested, only dichloromethane supported growth. The organism had a DNA G+C content of 42.7 mol%. From its phylogenetic position deduced from 16S rDNA analysis and from its unique substrate range, we conclude that the organism represents a new genus and a new species within the phylum of the gram-positive bacteria for which we propose the name Dehalobacterium formicoaceticum. Cell extracts were found to contain carbon monoxide dehydrogenase, methylene tetrahydrofolate dehydrogenase, formyl tetrahydrofolate synthetase, and hydrogenase activities, whereas activities of methenyl tetrahydrofolate cyclohydrolase and methylene tetrahydrofolate reductase were not detectable. Activity for dehalogenation of dichloromethane was lost on preparation of cell extracts, but was maintained in cell suspensions. Oxygen and reagents that react with thiol groups caused irreversible inhibition, and propyl iodide caused reversible inhibition of dehalogenation. Our observations suggest: 1) conversion of dichloromethane to methylene tetrahydrofolate, which gives rise to both formate and the methyl group of acetate, or 2) conversion of two molecules of dichloromethane to methylene tetrahydrofolate (which is oxidized to formate) and parallel reductive dehalogenation of one dichloromethane to the methyl group of the corrinoid-protein involved in acetate formation. Received: 11 March 1996 / Accepted 3 May 1996     

Acetate synthesis from 2 CO2 in acetogenic bacteria: is carbon monoxide an intermediate?

Cultures of Acetobacterium woodii and Clostridium thermoaceticum growing on fructose or glucose, respectively, were found to produce small, but significant amounts of carbon monoxide. In the gas phase of the cultures up to 53 ppm CO were determined. The carbon monoxide production was completely inhibited by 1 mM cyanide. Cultures and cell suspensions of both acetogens incorporated ¹⁴CO specifically into the carboxyl group of acetate. This CO fixation into C₁ of acetate was unaffected by cyanide (1 mM). The findings are taken to indicate that CO (in a bound form) is the physiological precursor of the C₁ of acetate in acetate synthesis from CO₂. The cyanide inhibition experiments support the hypothesis that the cyanide-sensitive carbon monoxide dehydrogenase may serve to reduce CO₂ to CO rather than to incorporate the carbonyl into C₁ of acetate.     

Maltose fermentation to acetate,CO2 and H2 in the anaerobic hyperthermophilic archaeon Pyrococcus furiosus: evidence for the operation of a novel sugar fermentation pathway

The hyperthermophilic anaerobe Pyrococcus furiosus was grown on maltose as energy and carbon source. During growth 1 mol maltose was fermented to 3–4 mol acetate, 6–7 mol H₂ and 3–4 mol CO₂. The presence of the following enzyme activities in cell extracts of maltose-grown P. furiosus indicate that the sugar is degraded to pyruvate and H₂ by a modified non-phosphorylated Entner-Doudoroff-pathway (the values given in brackets are specific enzyme activities at 100 °C): Glucose: methyl viologen oxidoreductase (0.03 U/mg); 2-keto-3-deoxy-gluconate aldolase (0.03 U/mg); glyceraldehyde: benzyl viologen oxidoreductase (2.6 U/mg), glycerate kinase (2-phosphoglycerate forming) (0.48 U/mg), enolase (10.4 U/mg), pyruvate kinase (1.4 U/mg). Hexokinase, glucose-6-phosphate dehydrogenase, 2-keto-3-deoxy-6-phosphogluconate aldolase and phosphofructokinase could not be detected. Further conversion of pyruvate to acetate, CO₂ and H₂ involves pyruvate: ferredoxin oxidoreductase (0.4 U/mg; T=60°C with Clostridium pasteurianum ferredoxin as electron acceptor), hydrogen: methyl viologen ixodoreductase (3.4 U/mg) and ADP-dependent acetyl-CoA synthetase (1.9 U/mg). Phosphate acetyl transferase and acetate kinase could not be detected. The ADP-dependent acetyl-CoA synthetase catalyzes ATP synthesis via the mechanism of substrate level phosphorylation and apparently constitutes the only ATP conserving site during maltose catabolism in P. furiosus.This novel pathway of maltose fermentation to acetate, CO₂ and H₂ in the anaerobic archaeon P. furiosus may represent a phylogenetically ancient pathway of sugar fermentation.Non-standard abbreviations DTE dithioerythritol - MV methyl viologen - BV benzyl viologen - CHES cyclohexylamino-ethane sulfonic acid - ABTS 2,2-Azino-di-(3-ethylbenzthiazoliumsulfonate)     

Pyruvate metabolism of the hyperthermophilic archaebacterium Pyrococcus furiosus

The hyperthermophilic anaerobe Pyrococcus furiosus was found to grow on pyruvate as energy and carbon source. Growth was dependent on yeast extract (0.1%). The organism grew with doublings times of about 1 h up to cell densities of 1–2×10⁸ cells/ml. During growth 0.6–0.8 mol acetate and 1.2–1.5 mol CO₂ and 0.8 mol H₂ were formed per mol of pyruvate consumed. The molar growth yield was 10–11 g cells(dry weight)/mol pyruvate. Cell suspensions catalyzed the conversion of 1 mol of pyruvate to 0.6–0.8 mol acetate, 1.2–1.5 mol CO₂, 1.2 mol H₂ and 0.03 mol acetoin. After fermentation of [3-¹⁴C]pyruvate the specific radioactivities of pyruvate, CO₂ and acetate were equal to 1:0.01:1. Cellfree extracts contained the following enzymatic activities: pyruvate: ferredoxin (methyl viologen) oxidoreductase (0.2 U mg^-1, T=60°C, with Clostridium pasteurianum ferredoxin as electron acceptor; 1.4 U mg^-1 at 90°C, with methyl viologen as electron acceptor); acetyl-CoA synthetase (ADP forming) [acetyl-CoA+ADP+Piacetate+ATP+CoA] (0.34 U mg^-1, T=90°C), and hydrogen: methyl viologen oxidoreductase (1.75 U mg^-1). Phosphate acetyl-transferase activity, acetate kinase activity, and carbon monoxide:methyl viologen oxidoreductase activity could not be detected. These findings indicate that the archaebacterium P. furiosus ferments pyruvate to acetate, CO₂ and H₂ involving only three enzymes, a pyruvate:ferredoxin oxidoreductase, a hydrogenase and an acetyl-CoA synthetase (ADP forming).Non-standard abbreviations DTE dithioerythritol - MV methyl viologen - MOPS morpholinopropane sulfonic acid - Tricine N-tris(hydroxymethyl)-methylglycine Part of the work was performed at the Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Karlvon-Frisch-Strasse, W-3550 Marburg/Lahn, Federal Republic of Germany     

Operation of the CO dehydrogenase/acetyl coenzyme A pathway in both acetate oxidation and acetate formation by the syntrophically acetate-oxidizing bacterium Thermacetogenium phaeum

Thermacetogenium phaeum is a homoacetogenic bacterium that can grow on various substrates, such as pyruvate, methanol, or H2/CO2. It can also grow on acetate if cocultured with the hydrogen-consuming methanogenic partner Methanothermobacter thermautotrophicus. Enzyme activities of the CO dehydrogenase/acetyl coenzyme A (CoA) pathway (CO dehydrogenase, formate dehydrogenase, formyl tetrahydrofolate synthase, methylene tetrahydrofolate dehydrogenase) were detected in cell extracts of pure cultures and of syntrophic cocultures. Mixed cell suspensions of T. phaeum and M. thermautotrophicus oxidized acetate rapidly and produced acetate after addition of H2/CO2 after a short time lag. CO dehydrogenase activity staining after native polyacrylamide gel electrophoresis exhibited three oxygen-labile bands which were identical in pure culture and coculture. Protein profiles of T. phaeum cells after sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the strain exhibited basically the same protein patterns in both pure and syntrophic culture. These results indicate that T. phaeum operates the CO dehydrogenase/acetyl-CoA pathway reversibly both in acetate oxidation and in reductive acetogenesis by using the same biochemical apparatus, although it has to couple this pathway to ATP synthesis in different ways.     

The effect of ferrous ions,tungstate and selenite on the level of formate dehydrogenase inClostridium formicoaceticum and formate synthesis from CO2 during pyruvate fermentation

In a medium containing a trace element solution and 10^-4 M ferrous ions the growth yield ofClostridium formicoaceticum on fructose was 5.5 g of weight per l; in the absence of metal ion solution it was 1 g per l. The specific activity of methyl viologen dependent formate dehydrogenase under both conditions was 0.28 and 0.03 units per mg of protein, respectively. It could be increased to 9.75 units when the growth medium contained 10^-4 M tungstate and 10^-5 M selenite in addition. Molybdate was only about 40% as effective as tungstate. Tungstate or molybdate could not be replaced by vanadate, selenite not by sulfide. The formate dehydrogenase catalyzed also the reduction of CO₂ to formate. The highest rate of formate synthesis was observed when pyruvate served as the reductant. No pyruvate: formate exchange but rapid pyruvate: CO₂ exchange could be observed with cell-free extracts ofC. formicoaceticum. Pyruvate is fermented byC. formicoaceticum to yield up to 1.16 mole acetate per mole of pyruvate. Resting cells accumulated some formate in addition to acetate.     

Anaerobic acetate oxidation to CO2 by Desulfotomaculum acetoxidans

Desulfotomaculum acetoxidans has been proposed to oxidize acetate to CO₂ via an oxidative acetyl-CoA/carbon monoxide dehydrogenase pathway rather than via the citric acid cycle. We report here the presence of the enzyme activities required for the operation of the novel pathway. In cell extracts the following activities were found (values in brackets=specific activities and apparent K _m; 1 U·mg^-1=1 mol·min^-1·mg protein^-1 at 37°C): Acetate kinase (6.3 U·mg^-1; 2 mM acetate; 2.4 mM ATP); phosphate acetyltransferase (60 U·mg^-1, 0.4 mM acetylphosphate; 0.1 mM CoA); carbon monoxide dehydrogenase (29 U·mg^-1; 13% carbon monoxide; 1.3 mM methyl viologen); 5,10-methylenetetrahydrofolate reductase (3 U·mg^-1, 0.06 mM CH₃–FH₄); methylenetetrahydrofolate dehydrogenase (3.6 U·mg^-1, 0.9 mM NAD, 0.1 mM CH₂=FH₄); methenyltetrahydrofolate cyclohydrolase (0.3 U·mg^-1); formyltetrahydrofolate synthetase (3 U·mg^-1, 1.4 mM FH₄, 0.4 mM ATP, 13 mM formate); and formate dehydrogenase (10 U·mg^-1, 0.4 mM formate, 0.5 mM NAD). The specific activities are sufficient to account for the in vivo acetate oxidation rate of 0.26 U·mg^-1.Non-standard abbreviations FH₄ Tetrahydrofolate - CHO-FH₄ N¹⁰-formyltetrahydrofolate - CHFH₄ N⁵,N¹⁰-methenyltetrahydrofolate - CH₂=FH₄ N⁵,N¹⁰-methylenetetrahydrofolate - CH₃–FH₄ N⁵-methyltetrahydrofolate - MOPS morpholinopropane sulfonic acid - DTT d,l-1,4-dithiothreitol - TRIS tris-(hydroxymethyl)-aminomethane - Ap₅A p¹,P⁵-di(adenosine-5)pentaphosphate - MV methyl viologen     

Anaerobic acetate oxidation to CO2 by Desulfotomaculum acetoxidans

Desulfotomaculum acetoxidans oxidizes acetate to CO₂ with sulfate. This organism metabolizes acetate via a pathway in which C₁ units rather than tri- and dicarboxylic acids are intermediates. We report here that cell extracts of D. acetoxidans catalyzed an exchange between CO₂ and the carboxyl group of acetate at a rate of 90 nmol · min^-1 · mg^-1 protein which is sufficient to account for the in vivo acetate oxidation rate of 250 nmol · min^-1 · mg^-1 protein. The reaction was strictly dependent on both ATP and coenzyme A. The extracts contain high activities of acetate kinase (6.3 U · mg^-1 protein) and phosphotransacetylase (60 U · mg^-1 protein). These findings indicate that acetyl-CoA rather than acetyl-phosphate or acetate is the substrate of the carbon-carbon cleavage activity. Exchange was only observed in the presence of strong reducing agents such as Ti³⁺. Interestingly, the cell extracts also catalyzed the reduction of CO₂ to CO with Ti³⁺ as electron donor (120 nmol · min^-1 · mg^-1 protein). Carbon monoxide dehydrogenase and other oxidoreductases involved in acetate oxidation were found to be partially associated with the membrane fraction suggesting a membrane localization of these enzymes.Abbreviations MOPS Morpholinopropane sulfonic acid - Tricine N-tris(hydroxymethyl)-methylglycine - DTT d,l-1,4-Dithiothreitol - DMN 2,3-Dimethyl-1,4-naphthoquinone - MV_OX Methyl viologen, oxidized - APS Adenosinephosphosulfate - SRB Sulfate reducing bacteria - U mol product formed per min     

Comparison of unitrophic and mixotrophic substrate metabolism by acetate-adapted strain of Methanosarcina barkeri

We examined the unitrophic metabolism of acetate and methanol individually and the mixotrophic utilization of these compounds by using detailed ¹⁴C-labeled tracer studies in a strain of Methanosarcina barkeri adapted to grow on acetate as the sole carbon and energy source. The substrate consumption rate and methane production rate were significantly lower on acetate alone than during the unitrophic or mixotrophic metabolism of methanol. Cell yields (in grams per mole of substrate) were identical during exponential growth on acetate and exponential growth on methanol. During unitrophic metabolism of acetate, the methyl moiety accounted for the majority of the CH₄ produced, but 14% of the CO₂ generated originated from the methyl moiety. This correlated with the concurrent reduction of equivalent amounts of the C-1 of acetate to CH₄. ¹⁴CH₄ was also produced from added ¹⁴CO₂, although to a lesser extent than from reduction of the C-1 of acetate. During mixotrophic metabolism, methanol and acetate were catabolized simultaneously. The rates of ¹⁴CH₄ and ¹⁴CO₂ generation from [2-¹⁴C]acetate were logarithmic and higher in mixotrophic than in unitrophic cultures at substrate concentrations of 50 mM. A comparison of the oxidoreductase activities in cell extracts of the acetate-adapted strain grown on acetate and of strain MS grown on methanol or on H₂ plus CO₂ indicated that the pyruvate, α-ketoglutarate, and isocitrate dehydrogenase activities remained constant, whereas the CO dehydrogenase activity was significantly higher (5,000 nmol/min per mg of protein) in the acetate-adapted strain. These results suggested that a significant intramolecular redox pathway is possible for the generation of CH₄ from acetate, that energy metabolism from acetate by M. barkeri is not catabolite repressed by methanol, and that the acetate-adapted strain is a metabolic mutant with derepressed CO dehydrogenase activity.     

Autotrophic synthesis of activated acetic acid from two CO2 in Methanobacterium thermoautotrophicum

In a previous study with Methanobacterium thermoautotrophicum evidence was presented that methanogenesis and autotrophic synthesis of activated acetic acid from CO₂ are linked processes. In this study one-carbon metabolism was investigated with growing cultures and in vitro.Serine was shown to be converted into glycine and activated formaldehyde, but only traces of label from [¹⁴C-3] of serine appeared in biosynthetic one-carbon positions. This seeming discrepancy could be explained if the same activated formaldehyde is an intermediate in biosynthesis and in methanogenesis from CO₂. This hypothesis was supported by demonstrating that [¹⁴C-3] of serine and [¹⁴C] formaldehyde were rapidly converted into methane, but a small portion of the label was also specifically incorporated into the methyl group of acetate. Methane and acetate synthesis in vitro were similarly stimulated by various compounds. These experiments indicate that the methyl of acetate and methane share common one-carbon precursor(s), i.e. methylene tetrahydromethanopterin, which can also be formed enzymatically from C-3 of serine or chemically from formaldehyde.Propyl iodide 20–40 M) and methyl iodide (1–3 M) completely inhibited growth in the dark. This effect was abolished by light. Methane formation was hardly affected. When ¹⁴CH₃I was applied at an only slightly inhibitory concentration, ¹⁴C was incorporated into the methyl of acetate. In vitro, similar effects on [¹⁴C] acetate formation from ¹⁴CO₂ or from [¹⁴C-3] of serine were observed, except that methyl iodide did not inhibit, but even stimulated acetate synthesis. These experiments indicate that a corrinoid is involved in acetate synthesis and probably not in methanogenesis from CO₂; the metal is light-reversibly alkylated and functions in methyl transfer to the acetate methyl.     

Autotrophic growth and CO2 fixation of Chloroflexus aurantiacus

Chlorofluexus aurantiacus OK-70 fl was grown photoautotrophically with hydrogen as the electron source. The lowest doubling time observed was 26 h.The mechanism of CO₂ fixation in autotrophically grown cells was studied. The presence of ribulose-1,5-bis-phosphate carboxylase and phosphoribulokinase could not be demonstrated. Carbon isotope fractionation (¹³C) was small, and alanine and aspartate but not 3-phosphoglycerate were the major labelled compounds in short term ¹⁴CO₂ labelling. Thus CO₂ is not fixed by the Calvin cycle.Fluoroacetate (FAc) completely inhibited protein synthesis in cultures and caused a slight citrate accumulation. However, CO₂ fixation continued and increased polyglucose formation occurred. Under these conditions added acetate was metabolized to polyglucose, as were glycine, serine, glyoxylate and succinate, but to a lesser extent; little or no formate or CO was utilised.Glyoxylate inhibited CO₂ fixation in vivo, indicating that pyruvate is formed from acetyl-CoA and CO₂ by pyruvate synthase. Two key enzymes of the reductive TCA cycle, citrate lyase and -ketoglutarate synthase were not detected in cell free extracts, but pyruvate synthase and phosphoenolpyruvate carboxylase were demonstrated. It is concluded that acetyl-CoA is a central intermediate in the CO₂ fixation process, but the mechanism of its synthesis is not clear.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase - TCA cycle tricarboxylic acid cycle - FAc monofluoroacetate - PEP phosphoenolpyruvate - MV methyl viologen - TTC triphenyltetrazolium chloride - PMS phenazine methosulfate     

Growth phase-dependant enzyme profile of pyruvate catabolism and end-product formation in Clostridium thermocellum ATCC 27405

End-product synthesis and enzyme activities involved in pyruvate catabolism, H₂ synthesis, and ethanol production in mid-log (OD₆₀₀  0.25), early stationary (OD₆₀₀  0.5), and stationary phase (OD₆₀₀  0.7) cell extracts were determined in Clostridium thermocellum ATCC 27405 grown in batch cultures on cellobiose. Carbon dioxide, hydrogen, ethanol, acetate and formate were major end-products and their production paralleled growth and cellobiose consumption. Lactate dehydrogenase, pyruvate:formate lyase, pyruvate:ferredoxin oxidoreductase, methyl viologen-dependant hydrogenase, ferredoxin-dependant hydrogenase, NADH-dependant hydrogenase, NADPH-dependant hydrogenase, NADH-dependant acetaldehyde dehydrogenase, NADH-dependant alcohol dehydogenase, and NADPH-dependant alcohol dehydrogenase activities were detected in all extracts, while pyruate dehydrogenase and formate dehydrogenase activities were not detected. All hydrogenase activities decreased (2–12-fold) as growth progressed from early exponential to stationary phase. Alcohol dehydrogenase activities fluctuated only marginally (<45%), while lactate dehydrogenase, pyruvate:formate lyase, and pyruvate:ferredoxin oxidoreductase remained constant in all cell extracts. We have proposed a pathway involved in pyruvate catabolism and end-product formation based on enzyme activity profiles in conjunction with bioinformatics analysis.     

Function of methanofuran,tetrahydromethanopterin, and coenzyme F420 in Archaeoglobus fulgidus

Archaeoglobus fulgidus is an extremely thermophilic archaebacterium that can grow at the expense of lactate oxidation with sulfate to CO₂ and H₂S. The organism contains coenzyme F₄₂₀, tetrahydromethanopterin, and methanofuran which are coenzymes previously thought to be unique for methanogenic bacteria. We report here that the bacterium contains methylenetetrahydromethanopterin: F₄₂₀ oxidoreductase (20 U/mg), methenyltetrahydromethanopterin cyclohydrolase (0.9 U/mg), formyltetrahydromethanopterin: methanofuran formyltransferase (4.4 U/mg), and formylmethanofuran: benzyl viologen oxidoreductase (35 mU/mg). Besides these enzymes carbon monoxide: methyl viologen oxidoreductase (5 U/mg), pyruvate: methyl viologen oxidoreductase (0.7 U/mg), and membranebound lactate: dimethylnaphthoquinone oxidoreductase (0.1 U/mg) were found. 2-Oxoglutarate dehydrogenase, which is a key enzyme of the citric acid cycle, was not detectable. From the enzyme outfit it is concluded that in A. fulgidus lactate is oxidized to CO₂ via a modified acetyl-CoA/carbon monoxide dehydrogenase pathway involving C₁-intermediates otherwise only used by methanogenic bacteria.Non-standard abbreviations APS adenosine 5-phosphosulfate - BV benzyl viologen - DCPIP 2,6-dichlorophenolindophenol - DMN 2,3-dimethyl-1,4-naphthoquinone - DTT DL-1,4-dithiothreitol - H₄F tetrahydrofolate - H₄MPT tetrahydromethanopterin - CH₂ H₄MPT, methylene-H₄MPT - CH H₄MPT, methenyl-H₄MPT - Mes morpholinoethane sulfonic acid - MFR methanofuran - Mops morpholinopropane sulfonic acid - MV methyl viologen - Tricine N-tris(hydroxymethyl)-methylglycine - U mol product formed per min     

Acetate oxidation to CO2 in anaerobic bacteria via a novel pathway not involving reactions of the citric acid cycle

In several sulfate-reducing bacteria capable of complete oxidation of acetate (or acetyl CoA), the citric acid cycle is not operative. No 2-oxoglutarate dehydrogenase activity was found in these organisms, and the labelling pattern of oxaloacetate excludes its synthesis via 2-oxo-glutarate. These sulfate-reducers contained, however, high activities of the enzymes carbon monoxide dehydrogenase and formate dehydrogenase and catalyzed an isotope exchange between CO₂ and the carboxyl group of acetate (or acetyl CoA), showing a direct C-C-cleavage of activated acetic acid. These findings suggest that in the investigated sulfate-reducers acetate is oxidized to CO₂ via C₁ intermediates. The proposed pathway provides a possible explanation for the reported different fluoroacetate sensitivity of acetate oxidation by anaerobic bacteria, for mini-methane formation, as well as for the postulated anaerobic methane oxidation by special sulfate-reducers.     

Carbon monoxide pathway enzyme activities in a thermophilic anaerobic bacterium grown acetogenically and in a syntrophic acetate-oxidizing coculture

We recently isolated an acetate-oxidizing rodshaped eubacterium (AOR) which was capable of oxidizing acetate to CO₂ when grown in coculture with the hydrogenotrophic methanogen Methanobacterium sp. strain THF. The AOR was also capable of growing axenically on H₂CO₂ which it converted to acetate. Previous results for the acetate oxidizing coculture showed isotopic exchange between acetate and CO₂, suggesting that the AOR was using a pathway for acetate oxidation resembling a reveral of the acetogenic (carbon monoxide) pathway. In this study, it was found that production of ¹⁴CO₂ from ¹⁴CH₃COO^- by the coculture was inhibited by 200 M cyanide, while methanogenesis from H₂–CO₂ was unaffected, implying the involvement of carbon monoxide dehydrogenase (CODH) in acetate oxidation. CODH was present at 0.055 mol methyl viologen reduced min^-1 mg^-1 protein in extracts of Methanobacterium sp. strain THF, but was present in higher levels in the acetate oxidizing coculture and in the AOR grown axenically and on H₂–CO₂ (2.0 and 6.4 mol min^-1 mg^-1 protein respectively). Anaerobic activity stains for CODH in native polyacrylamide gels from the AOR coculture showed components co-migrating with bands from both organisms, as well as an additional band in extracts of the coculture. Formate dehydrogenase (FDH) was present in both the AOR coculture and monoculture but not in extracts of H₂–CO₂ grown cells of Methanobacterium sp. strain THF. Formyltetrahydrofolate (FTHF) synthetase was not detectable in extracts of the AOR monoculture or coculture, although it was found in high amounts in extracts of H₂–CO₂ grown cells of the thermophilic acetogen Acetogenium kivui. Extracts of H₂–CO₂ grown cells of the AOR showed a fluorescence spectrum typical of pterin derivatives. Bioassay for folates showed levels to be at anabolic rather than catabolic levels. It is possible that the AOR uses pterins distinct from folate for catabolism. Isocitrate dehydrogenase, a citric acid cycle enzyme, was also present in the AOR, but at anabolic levels and -ketoglutarate dehydrogenase was not detectable.Abbreviations (AOR) acetate-oxidizing rod - (CODH) carbon monoxide dehydrogenase - (FDH) formate dehydrogenase - (FTHF) formyltetrahydrofolate