Pathway of propionate formation in Desulfobulbus propionicus

Whole cells of Desulfobulbus propionicus fermented [1-¹³C]ethanol to [2-¹³C] and [3-¹³C]propionate and [1-¹³C]-acetate, which indicates the involvement of a randomizing pathway in the formation of propionate. Cell-free extracts prepared from cells grown on lactate (without sulfate) contained high activities of methylmalonyl-CoA: pyruvate transacetylase, acetase kinase and reasonably high activities of NAD(P)-independent L(+)-lactate dehydrogenase NAD(P)-independent pyruvate dehydrogenase, phosphotransacetylase, acetate kinase and reasonably high activity of NAD(P)-independent L(+)-lactate dehydrogenase, fumarate reductase and succinate dehydrogenase. Cell-free extracts catalyzed the conversion of succinate to propionate in the presence of pyruvate, CoA and ATP and the oxaloacetate-dependent conversion of propionate to succinate. After growth on lactate or propionate in the presence of sulfate similar enzyme levels were found except for fumarate reductase which was considerably lower. Fermentative growth on lactate led to higher cytochrome b contents than growth with sulfate as electron acceptor.The labeling studies and the enzyme measurements demonstrate that in Desulfobulbus propionate is formed via a succinate pathway involving a transcarboxylase like in Propionibacterium. The same pathway may be used for the degradation of propionate to acetate in the presence of sulfate.Abbreviations DCPIP 2,6-dichlorophenolindophenol - PEP phosphoenolpyruvate     

Acetate assimilation pathway of Methanosarcina barkeri.

The pathway of acetate assimilation in Methanosarcina barkeri was determined from analysis of the position of label in alanine, aspartate, and glutamate formed in cells grown in the presence of [14C]acetate and by measurement of enzyme activities in cell extracts. The specific radioactivity of glutamate from cells grown on [1-14C]- or [2-14C]acetate was approximately twice that of aspartate. The methyl and carboxyl carbons of acetate were incorporated into aspartate and glutamate to similar extents. Degradation studies revealed that acetate was not significantly incorporated into the C1 of alanine, C1 or C4 of aspartate, or C1 of glutamate. The C5 of glutamate, however, was partially derived from the carboxyl carbon of acetate. Cell extracts were found to contain the following enzyme activities, in nanomoles per minute per milligram of protein at 37 degrees C: F420-linked pyruvate synthase, 170; citrate synthase, 0.7; aconitase, 55; oxidized nicotinamide adenine dinucleotide phosphate-linked isocitrate dehydrogenase, 75; and oxidized nicotinamide adenine dinucleotide-linked malate dehydrogenase, 76. The results indicate that M. barkeri assimilates acetate into alanine and aspartate via pyruvate and oxaloacetate and into glutamate via citrate, isocitrate, and alpha-ketoglutarate. The data reveal differences in the metabolism of M. barkeri and Methanobacterium thermoautotrophicum and similarities in the assimilation of acetate between M. barkeri and other anaerobic bacteria, such as Clostridium kluyveri.     

Microbial metabolism of the pyridine ring. Metabolic pathways of pyridine biodegradation by soil bacteria.

1. Two bacteria, a Bacillus sp. and a Nocardia sp. (strain Z1) were isolated from soil by enrichment with 0.1 percent (v/v) pyridine and grew rapidly on this compound as sole C, N and energy source. The monohydroxypyridines, tetrahydropyridine, piperidine and some other analogues were not utilized for growth or oxidized by washed suspensions of either bacterium. 2. Cell-free extracts were unable to metabolize pyridine even after supplementation with a variety of cofactors or protecting agents. Treatment of cells with toluene led to rapid loss of the ability to oxidize pyridine. 3. In the presence of 10mM-semicarbazide at pH 6.0, Nocardia Z1 accumulated a semialdehyde idenditied as its 2,4-dinitrophenylhydrazone by chromatography, mixed melting point, mass spectrometry and isotope trapping from [2,6(-14)C]pyridine as glutarate semialdehyde. 4. Extracts of this bacterium prepared from cells grown with pyridine or exposed to the gratuitous inducer 2-picoline, contained high activities of a specific glutarate semialdehyde dehydrogenase. 5. Cells grown with pyridine or glutarate also contained a glutaric dialdehyde dehydrogenase, an acyl-CoA synthetase and elevated amounts of isocitrate lyase but no glutaryl-CoA dehydrogenase. 6. Bacillus 4 accumulated in the presence of 10mM-semicarbazide several acidic carbonyl compounds from pyridine among which was succinate semialdehyde. Extracts of this bacillus after growth of the cells with pyridine contained an inducible succinate semialdehyde dehydrogenase in amounts at least 50-fold over those found in succinate-grown cells. 7. Two mutants of this bacillus, selected for their inability to grow on pyridine were deficient in succinate semialdehyde dehydrogenase. 8. In the presence of 0.2mM-KCN, washed suspensions of Bacillus 4 accumulated formate and possibly formamide from pyridine. The use of [14C]pyridine showed that formate was derived from C-2 of the pyridine ring. 9. The organism had a specific formamide amidohydrolase cleaving formamide quantitatively to formate and NH3. 10. Formate was further oxidized by the particle fraction. There was no soluble formate dehydrogenase in extracts.     

Enzymatic preparation of radiolabeled succinic semialdehyde

[U-14C]Succinic semialdehyde was prepared with yields of 30-40% by oxidation of purified [U-14C]4-aminobutyric acid with commercially available bovine plasma monoamine oxidase. [U-14C]Succinic semialdehyde was purified by cation-exchange chromatography and quantified as the oxime and methoxime derivatives using liquid partition chromatography on silicic acid. The availability of [U-14C]succinic semialdehyde permits the reliable assay of succinic semialdehyde dehydrogenase in crude cell extracts of lymphocytes isolated from human blood, cultured human lymphoblasts, and other tissues where 4-aminobutyric acid metabolism is known to occur.     

Interactions between carbon and nitrogen metabolism in Fibrobacter succinogenes S85: a 1H and 13C nuclear magnetic resonance and enzymatic study

The effect of the presence of ammonia on [1-13C]glucose metabolism in the rumen fibrolytic bacterium Fibrobacter succinogenes S85 was studied by 13C and 1H nuclear magnetic resonance (NMR). Ammonia halved the level of glycogen storage and increased the rate of glucose conversion into acetate and succinate 2.2-fold and 1.4-fold, respectively, reducing the succinate-to-acetate ratio. The 13C enrichment of succinate and acetate was precisely quantified by 13C-filtered spin-echo difference 1H-NMR spectroscopy. The presence of ammonia did not modify the 13C enrichment of succinate C-2 (without ammonia, 20.8%, and with ammonia, 21.6%), indicating that the isotopic dilution of metabolites due to utilization of endogenous glycogen was not affected. In contrast, the presence of ammonia markedly decreased the 13C enrichment of acetate C-2 (from 40 to 31%), reflecting enhanced reversal of the succinate synthesis pathway. The reversal of glycolysis was unaffected by the presence of ammonia as shown by 13C-NMR analysis. Study of cell extracts showed that the main pathways of ammonia assimilation in F. succinogenes were glutamate dehydrogenase and alanine dehydrogenase. Glutamine synthetase activity was not detected. Glutamate dehydrogenase was active with both NAD and NADP as cofactors and was not repressed under ammonia limitation in the culture. Glutamate-pyruvate and glutamate-oxaloacetate transaminase activities were evidenced by spectrophotometry and 1H NMR. When cells were incubated in vivo with [1-13C]glucose, only 13C-labeled aspartate, glutamate, alanine, and valine were detected. Their labelings were consistent with the proposed amino acid synthesis pathway and with the reversal of the succinate synthesis pathway.     

Clostridium neopropionicum sp. nov., a strict anaerobic bacterium fermenting ethanol to propionate through acrylate pathway

Strain X4 was isolated several years ago from an anaerobic mesophilic plant treating vegetable cannery waste waters. It was the first example of propionic fermentation from ethanol. Morphologic and physiologic characterizations of the strain are presented here. This strain is described as type strain of a new species, Clostridium neopropionicum sp. nov. Whole cells of strain X4 ferment [1-¹³C]ethanol and CO₂ to [2-¹³C]propionate, [1-¹³C]acetate and [2-¹³C]propanol, suggesting the absence of a randomizing pathway during the propionate formation. Enzymes involved in this fermentation were assayed in cell-free extracts of cells grown with ethanol as sole substrate. Alcohol dehydrogenase, aldehyde dehydrogenase, phosphate acetyl transferase, acetate kinase, pyruvate synthase, lactate dehydrogenases, and the enzymes of the acrylate pathway were detected at activities sufficient to be involved in ethanol fermentation. The same pathway may be used for the degradation of lactate or acrylate to acetate.     

Catabolism of alpha-ketoglutarate by a sucA mutant of Bradyrhizobium japonicum: evidence for an alternative tricarboxylic acid cycle

A complete tricarboxylic acid (TCA) cycle is generally considered necessary for energy production from the dicarboxylic acid substrates malate, succinate, and fumarate. However, a Bradyrhizobium japonicum sucA mutant that is missing alpha-ketoglutarate dehydrogenase is able to grow on malate as its sole source of carbon. This mutant also fixes nitrogen in symbiosis with soybean, where dicarboxylic acids are its principal carbon substrate. Using a flow chamber system to make direct measurements of oxygen consumption and ammonium excretion, we confirmed that bacteroids formed by the sucA mutant displayed wild-type rates of respiration and nitrogen fixation. Despite the absence of alpha-ketoglutarate dehydrogenase activity, whole cells of the mutant were able to decarboxylate alpha-[U-(14)C]ketoglutarate and [U-(14)C]glutamate at rates similar to those of wild-type B. japonicum, indicating that there was an alternative route for alpha-ketoglutarate catabolism. Because cell extracts from B. japonicum decarboxylated [U-(14)C]glutamate very slowly, the gamma-aminobutyrate shunt is unlikely to be the pathway responsible for alpha-ketoglutarate catabolism in the mutant. In contrast, cell extracts from both the wild type and mutant showed a coenzyme A (CoA)-independent alpha-ketoglutarate decarboxylation activity. This activity was independent of pyridine nucleotides and was stimulated by thiamine PP(i). Thin-layer chromatography showed that the product of alpha-ketoglutarate decarboxylation was succinic semialdehyde. The CoA-independent alpha-ketoglutarate decarboxylase, along with succinate semialdehyde dehydrogenase, may form an alternative pathway for alpha-ketoglutarate catabolism, and this pathway may enhance TCA cycle function during symbiotic nitrogen fixation.     

Acetate oxidation through a modified citric acid cycle in Propionibacterium freudenreichii

Propionibacterium freudenreichii strain DSM 20271 was grown in a mineral medium containing 0.1% (w/v) yeast extract. Acetate was oxidized by growing cells with potassium hexacyanoferrate as electron acceptor, which was oxidized by a three-electrode poised-potential system at a redox potential of +510 mV. Growth with acetate under these conditions followed linear rather than expenential kinetics, whereas growth with other substrates such as lactate under the same conditions was exponential. Cell-free extracts of P. freudenreichii cells grown with acetate contained all enzymes of the classical citric acid cycle except 2-oxoglutarate-oxidizing activity. No activity of anaplerotic reactions such as isocitrate lyase or malate synthase was found. Instead, moderate activities of glutamate decarboxylase, 4-aminobutyrate:2-oxoglutarate aminotransferase, and succinate semialdehyde dehydrogenase were detected. In short-term radiolabeling experiments with U-¹⁴C-acetate, 4-aminobutyrate was identified as a major early intermediate in acetate oxidation by these cells. These findings allow the construction of a modified citric acid cycle that compensates the lack of 2-oxoglutarate dehydrogenase by a subcycle through glutamate, 4-aminobutyrate, and succinate semialdehyde. Lack of anaplerotic reactions explains subexponential growth kinetics during growth with acetate.     

Fermentation of 4-aminobutyrate by Clostridium aminobutyricum: cloning of two genes involved in the formation and dehydration of 4-hydroxybutyryl-CoA

Clostridium aminobutyricum ferments 4-aminobutyrate via succinic semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl-CoA and crotonyl-CoA to acetate and butyrate. The genes coding for the enzymes that catalyse the interconversion of these intermediates are arranged in the order abfD (4-hydroxybutyryl-CoA dehydratase), abfT (4-hydroxybutyrate CoA-transferase), and abfH (NAD-dependent 4-hydroxybutyrate dehydrogenase). The genes abfD and abfT were cloned, sequenced and expressed as active enzymes in Escherichia coli. Hence the insertion of the [4Fe-4S]clusters and FAD into the dehydratase required no additional specific protein from C. aminobutyricum. The amino acid sequences of the dehydratase and the CoA-transferase revealed close relationships to proteins deduced from the genomes of Clostridium difficile, Porphyromonas gingivalis and Archaeoglobus fulgidus. In addition the N-terminal part of the dehydratase is related to those of a family of FAD-containing mono-oxygenases from bacteria. The putative assignment in the databank of Cat2 (OrfZ) from Clostridium kluyveri as 4-hydroxybutyrate CoA-transferase, which is thought to be involved in the reductive pathway from succinate to butyrate, was confirmed by sequence comparison with AbfT (57% identity). Furthermore, an acetyl-CoA:4-hydroxybutyrate CoA-transferase activity could be detected in cell-free extracts of C. kluyveri. In contrast to glutaconate CoA-transferase from Acidaminococcus fermentans, mutation studies suggested that the glutamate residue of the motive EXG, which is conserved in many homologues of AbfT, does not form a CoA-ester during catalysis.     

Restricted oxygen supply and excretion of metabolites

Summary The effect of restricted oxygen supply on the excretion of metabolites was studied in Pseudomonas acidovorans (DSM 39), P. delafieldii (DSM 64) and a mutant strain of Paracoccus denitrificans unable to accumulate poly-3-hydroxybutanoic acid. Different metabolites were produced at distinct submaximum respiration rates by these strains. These metabolites were, in order of decreasing respiration rates; 2-oxoglutarate, 2-oxo-3-methylbutanoate, cisaconitate, 3-hydroxybutanoate, succinate, hydrogen gas, formate, acetate, butanoate, acetoin, meso- and D,L-2,3-butanediol, and ethanol. Poly-3-hydroxy-butanoic acid (PHB) accumulated intracellularly at almost the same respiration rates at which the excretion of 3-hydroxybutanoate occurred.The production of ethanol, 2,3-butanediol, butanoate, formate, and hydrogen gas indicate the function of enzymes such as ethanol and butanediol dehydrogenases, pyruvate formate lyase, formate hydrogen lyase, and butanoyl-CoA dehydrogenase. These enzymes are not expected to be present in strict aerobes at different degrees of restricted oxygen supply.Excreted metabolites are indicators of the degree to which the oxygen demand of cells is met. On the other hand, a fermentation process designed for the production of a distinct metabolite can be controlled by maintaining the appropriate oxygen supply.     

13C-NMR study of autotrophic CO2 fixation pathways in the sulfur-reducing Archaebacterium Thermoproteus neutrophilus and in the phototrophic Eubacterium Chloroflexus aurantiacus.

The unresolved autotrophic CO2 fixation pathways in the sulfur-reducing Archaebacterium Thermoproteus neutrophilus and in the phototrophic Eubacterium Chloroflexus aurantiacus have been investigated. Autotrophically growing cultures were labelled with [1,4-13C1]succinate, and the 13C pattern in cell constituents was determined by 1H- and 13C-NMR spectroscopy of purified amino acids and other cell constituents. In both organisms succinate contributed to less than 10% of cell carbon, the major part of carbon originated from CO2. All cell constituents became 13C-labelled, but different patterns were observed in the two organisms. This proves that two different cyclic CO2 fixation pathways are operating in autotrophic carbon assimilation in both of which succinate is an intermediate. The 13C-labelling pattern in T. neutrophilus is consistent with the operation of a reductive citric acid cycle and rules out any other known autotrophic CO2 fixation pathway. Surprisingly, the proffered [1,4-13C1]succinate was partially converted to double-labelled [3,4-13C2]glutamate, but not to double-labelled aspartate. These findings suggest that the conversion of citrate to 2-oxoglutarate is readily reversible under the growth conditions used, and a reversible citrate cleavage reaction is proposed. The 13C-labelling pattern in C. aurantiacus disagrees with any of the established CO2 fixation pathways; it therefore demands a novel autotrophic CO2 fixation cycle in which 3-hydroxypropionate and succinate are likely intermediates. The bacterium excreted substantial amounts of 3-hydroxypropionate (5 mM) and succinate (0.5 mM) at the end of autotrophic growth. Autotrophically grown Chloroflexus cells contained acetyl-CoA carboxylase and propionyl-CoA carboxylase activity. These enzymes are proposed to be the main CO2-fixing enzymes resulting in malonyl-CoA and methylmalonyl-CoA formation; from these carboxylation products 3-hydroxypropionate and succinate, respectively, can be formed.     

Growth of Paracoccus denitrificans on [2,3-13C]succinate and [1,4-13C]succinate. II. Isoleucine biosynthesis

Paracoccus denitrificans was grown on either [2,3-13C]succinate or [1,4-13C]succinate, and extracts were analysed by using gas chromatography-mass spectrometry. The distribution of label in isoleucine indicated that the 2-ketobutyrate required for isoleucine biosynthesis was mainly produced from pyruvate by 2-keto-acid chain elongation (i.e. the 'pyruvate elongation pathway'). Approximately 10% of isoleucine was produced by a second pathway involving propionyl CoA. Threonine and glutamate were not utilized by P. denitrificans as a source of 2-ketobutyrate production for isoleucine biosynthesis under the growth conditions used.     

13C NMR studies of butyric fermentation in Clostridium kluyveri

The fermentation of 13C-labeled ethanol and acetate into butyrate and caproate by Clostridium kluyveri has been studied by using 13C NMR. The pathway involves the conversion of both ethanol and acetate into acetyl coenzymes A, two of which condense to form CoA-linked precursors of butyrate. If butyryl-CoA is involved in the condensation, caproate is the ultimate product. ATP is produced from acetyl-CoA via the reactions catalyzed by phosphotransacetylase and acetate kinase with acetate, a required carbon source, as a co-product. In spectra of whole cells incubated with the labeled carbon sources, label from ethanol appears rapidly in acetate, which then reaches a lower, steady-state concentration due to its re-entry into the pathway. The rapid initial production of acetate indicates equally rapid production of ATP. Label from acetate appears in ethanol only if ethanol is already present, indicating that this process is one of isotopic equilibration rather than net synthesis of ethanol from acetate. The ratio of butyrate to caproate produced depends strongly on the initial ratio of ethanol to acetate in the medium. The relative rates of utilization of ethanol and acetate vary as the fermentation proceeds. 13C-13C coupling in the butyrate and caproate produced from [1-13C]ethanol and [2-13C]acetate can be used to determine if the acetyl-CoA molecules arising from ethanol and acetate enter the same pool or if they remain separated. The data are consistent with random mixing of the acetyl-CoA produced from the two carbon sources.     

Glycolysis and Entner-Doudoroff pathways in Halobacterium halobium: some new observations based on 13C NMR spectroscopy

13C NMR was used to study glucose metabolism in intact cells of Halobacterium halobium. Spectra of glucose grown cells incubated with [1-13C] glucose indicate the presence of gluconate as the initial product. The existence of glycolytic pathway is also indicated. In the extracts of these cells an NADP dependent glucose dehydrogenase was detected. Galactose grown cells failed to metabolise glucose but exhibited glucose dehydrogenase activity although about 20-50% less than that for glucose grown cells. Possible explanations of these experiments are discussed.     

The tricarboxylic acid cycle of Helicobacter pylori.

The composition and properties of the tricarboxylic acid cycle of the microaerophilic human pathogen Helicobacter pylori were investigated in situ and in cell extracts using [1H]- and [13C]-NMR spectroscopy and spectrophotometry. NMR spectroscopy assays enabled highly specific measurements of some enzyme activities, previously not possible using spectrophotometry, in in situ studies with H. pylori, thus providing the first accurate picture of the complete tricarboxylic acid cycle of the bacterium. The presence, cellular location and kinetic parameters of citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate oxidase, fumarate reductase, fumarase, malate dehydrogenase, and malate synthase activities in H. pylori are described. The absence of other enzyme activities of the cycle, including alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase, and succinate dehydrogenase also are shown. The H. pylori tricarboxylic acid cycle appears to be a noncyclic, branched pathway, characteristic of anaerobic metabolism, directed towards the production of succinate in the reductive dicarboxylic acid branch and alpha-ketoglutarate in the oxidative tricarboxylic acid branch. Both branches were metabolically linked by the presence of alpha-ketoglutarate oxidase activity. Under the growth conditions employed, H. pylori did not possess an operational glyoxylate bypass, owing to the absence of isocitrate lyase activity; nor a gamma-aminobutyrate shunt, owing to the absence of both gamma-aminobutyrate transaminase and succinic semialdehyde dehydrogenase activities. The catalytic and regulatory properties of the H. pylori tricarboxylic acid cycle enzymes are discussed by comparing their amino acid sequences with those of other, more extensively studied enzymes.     

Excretion of metabolites by hydrogen bacteria I. Autotrophic and heterotrophic fermentations

Summary The excretion of metabolites by 48 wild-type and mutant strains belonging to various species and genera of aerobic hydrogen-oxidizing bacteria was studied. The cells were grown autotrophically and heterotrophically, and samples were analyzed by gas chromatrographic techniques. The following metabolites were identified and quantitatively determined: acetate, ethanol, malate, citrate, lactate, succinate, 2-propanol, 2-methylpropanoate, 3-methylbutanoate, cis-aconitate, acetone, 2-oxoglutarate, isocitrate, butanoate, and methanol. The excretion of the metabolites started when ammonia and oxygen became limiting. The concentrations reached a maximum, whereupon the excreted products were reconsumed.The total concentration of the metabolites identified reached 5 g/l. Maximum concentrations were measured when mutants of Alcaligenes eutrophus lacking the ability to accumulate poly-3-hydroxybutanoate were grown on fructose, gluconate, or lactate in the fermenter under conditions of ammonia limitation and when the carbon source was present in excess.     

Altered cerebral glucose and acetate metabolism in succinic semialdehyde dehydrogenase-deficient mice: evidence for glial dysfunction and reduced glutamate/glutamine cycling

Succinic semialdehyde dehydrogenase (SSADH) catalyzes the NADP-dependent oxidation of succinic semialdehyde to succinate, the final step of the GABA shunt pathway. SSADH deficiency in humans is associated with excessive elevation of GABA and γ-hydroxybutyrate (GHB). Recent studies of SSADH-null mice show that elevated GABA and GHB are accompanied by reduced glutamine, a known precursor of the neurotransmitters glutamate and GABA. In this study, cerebral metabolism was investigated in urethane-anesthetized SSADH-null and wild-type 17-day-old mice by intraperitoneal infusion of [1,6-¹³C₂]glucose or [2-¹³C]acetate for different periods. Cortical extracts were prepared and measured using high-resolution ¹H-[¹³C] NMR spectroscopy. Compared with wild-type, levels of GABA, GHB, aspartate, and alanine were significantly higher in SSADH-null cortex, whereas glutamate, glutamine, and taurine were lower. ¹³C Labeling from [1,6-¹³C₂]glucose, which is metabolized in neurons and glia, was significantly lower (expressed as μmol of ¹³C incorporated per gram of brain tissue) for glutamate-(C4,C3), glutamine-C4, succinate-(C3/2), and aspartate-C3 in SSADH-null cortex, whereas Ala-C3 was higher and GABA-C2 unchanged. ¹³C Labeling from [2-¹³C]acetate, a glial substrate, was lower mainly in glutamine-C4 and glutamate-(C4,C3). GHB was labeled by both substrates in SSADH-null mice consistent with GABA as precursor. Our findings indicate that SSADH deficiency is associated with major alterations in glutamate and glutamine metabolism in glia and neurons with surprisingly lesser effects on GABA synthesis.     

The inhibition of acetate oxidation by ethanol in Acetobacter aceti

Ethanol grown Acetobacter aceti differed from acetate grown. In ethanol grown cells, acetate uptake, caused by the oxidation of acetate, was completely inhibited by ethanol, in acetate grown cells only to 20%. This was correlated with a 65-fold higher specific activity of the membrane bound NAD(P)-independent alcohol dehydrogenase in ethanol grown than in acetate grown cells. In comparison with ethanol grown cells, acetate grown cells showed a 3-fold higher acetate respiration rate and 3-fold higher specific activities of some tricarboxylic acid cycle enzymes tested. Both adaptations were due to induction by the homologous and not to repression by the heterologous growth substrate. A. aceti showed a membrane bound NAD(P)-independent malate dehydrogenase and no activity of a soluble NAD(P)-dependent one, as was known before from A. xylinum. A hypothesis was proposed explaining the observed inhibition of malate dehydrogenase and of functioning of the tricarboxylic acid cycle in the presence of ethanol or butanol or glucose by a competition of two electron currents for a common link in the convergent electron transport chains. The electrons coming from the quinoproteins, alcohol dehydrogenase and glucose dehydrogenase on the one side and those coming from the flavoproteins, malate dehydrogenase and succinate dehydrogenase via ubiquinonecytochrome c reductase on the other side are meeting at cytochrome c. Here the quinoproteins may be favoured by higher affinity and so inhibit the flavoproteins. Inhibition could be alleviated in the cell free system by increasing the oxygen supply.Dedicated to Professor Carl Martius on the occasion of his 80th birthday, March 1st 1986     

2,3-Cyclopyrophosphoglycerate in methanogens: evidence by 13C NMR spectroscopy for a role in carbohydrate metabolism

The novel compound 2,3-cyclopyrophosphoglycerate (CPP) is the major small molecule carbon pool in Methanobacterium thermoautotrophicum. High-field 13C NMR 13CO2 pulse/unenriched CO2 chase experiments have shown that the labeled CPP rapidly loses its 13C to an insoluble pool, while the CPP steady-state concentration is maintained (as monitored by 31P NMR spectroscopy). The biosynthesis of CPP from CO2, acetyl coenzyme A, and pyruvate as precursors has been established by a 13C NMR study of ethanol extracts of Mb. thermoautotrophicum fed with 13CO2, [1-13C]- and [2-13C]acetate, and [1-13C]pyruvate. That CPP is a post-phosphoenolpyruvate metabolite has been confirmed by in vitro experiments with cell extracts. A role for CPP in carbohydrate metabolism was established when [1-13C]glucose fed to cells resulted in the formation of [3-13C]CPP exclusively. Possible functions of CPP within the cell are discussed.     

Acetate Utilization in Lactococcus lactis Deficient in Lactate Dehydrogenase: a Rescue Pathway for Maintaining Redox Balance

Acetate was shown to improve glucose fermentation in Lactococcus lactis deficient in lactate dehydrogenase. 13C and 1H nuclear magnetic resonance studies using [2-13C]glucose and [2-(13)C]acetate as substrates demonstrated that acetate was exclusively converted to ethanol. This novel pathway provides an alternative route for NAD+ regeneration in the absence of lactate dehydrogenase.