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     

The influence of acetate on the oxidation of sulfide by Rhodopseudomonas capsulata

The capacity to oxidize sulfide and the influence of the simultaneous presence of acetate in heterotrophically (acetate) and autotrophically (sulfide/CO₂) grown Rhodopseudomonas capsulata was investigated.Sulfide oxidation of acetate-limited cultures was found inversely related to the specific growth rate. Upon acetate deprevation (metering pump stopped) increased rates of sulfide oxidation were observed. This points to the existence of a constitutive acceptor for the electrons from sulfide. It is suggested that a carrier functional in the light-induced cyclic electron flow operates as such. The rate of sulfide oxidation, however, is low when compared to autotrophically-grown cells. This is probably due to the low levels of Calvin cycle enzymes present in the acetate-grown cells.In cells growing on sulfide/CO₂, the addition of acetate resulted in less sulfide being oxidized. Upon depletion of the acetate, the rate of sulfide oxidation again increased, however, insufficiently to maintain the accelerated growth rate. This indicates that under mixotrophic conditions the enzymes of the Calvin cycle are being synthesized to a far lesser extent.Non-Standurd Abbreviations PHB poly--hydroxybutyric acid - D dilution rate - TCA Tri carboxylic acid cycle - RubPcase ribulose 1,5-bisphosphate carboxylase - RP reducing power     

Microbial manganese(III) reduction fuelled by anaerobic acetate oxidation

Soluble manganese in the intermediate +III oxidation state (Mn³⁺) is a newly identified oxidant in anoxic environments, whereas acetate is a naturally abundant substrate that fuels microbial activity. Microbial populations coupling anaerobic acetate oxidation to Mn³⁺ reduction, however, have yet to be identified. We isolated a Shewanella strain capable of oxidizing acetate anaerobically with Mn³⁺ as the electron acceptor, and confirmed this phenotype in other strains. This metabolic connection between acetate and soluble Mn³⁺ represents a new biogeochemical link between carbon and manganese cycles. Genomic analyses uncovered four distinct genes that allow for pathway variations in the complete dehydrogenase‐driven TCA cycle that could support anaerobic acetate oxidation coupled to metal reduction in Shewanella and other Gammaproteobacteria. An oxygen‐tolerant TCA cycle supporting anaerobic manganese reduction is thus a new connection in the manganese‐driven carbon cycle, and a new variable for models that use manganese as a proxy to infer oxygenation events on early Earth.     

Mechanism of acetate oxidation to CO2 with elemental sulfur in Desulfuromonas acetoxidans

The strict anaerobe Desulfuromonas acetoxidans can oxidize acetate to CO₂ with elemental sulfur as electron acceptor. ¹⁴C-labelling experiments and enzyme studies are described revealing that acetate oxidation proceeds via the citric acid cycle with the synthesis of oxaloacetate from acetate and 2 CO₂ via pyruvate as anaplerotic reaction. An oxidation of acetate via one carbon unit intermediates as proposed for anaerobic bacteria fermenting acetate to 2 CO₂ and 4 H₂ was excluded.Dedicated to Professor Dr. Gerhart Drews on the occasion of his 60th birthday     

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.     

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.     

Partial characterization of the pathway from propionate to acetate in the cabbage looper,Trichoplusia ni

Experiments were performed to characterize the metabolism of propionate to acetate in the cabbage looper Trichoplusia ni and correlate the results with vitamin B₁₂ levels. Fourth and fifth instar larvae contain 2–4 pg vitamin B₁₂/mg dry wt whereas pupae and adults do not contain detectable amounts. In vivo studies as a function of time in larvae, pupae and adults gave evidence that [2-¹⁴C]propionate was converted to 3-hydroxypropionate and then to acetate, which subsequently labeled Krebs cycle intermediates. Radioactivity from [1-¹⁴C]propionate was recovered only in the propionate and 3-hydroxypropionate fractions, and not in acetate or Krebs cycle intermediates, suggesting that carbon 1 of propionate was lost as carbon dioxide and that carbons 2 and 3 of propionate were retained during conversion to acetate. The enzymes of this pathway were located entirely in the mitochondrial fraction. Cyanide inhibited the metabolism of propionate to 3-hydroxypropionate and acetate in mitochondrial preparations, whereas carbon monoxide did not. [2,3-¹⁴C]Acrylic acid was metabolized to 3-hydroxypropionate, which is consistent with a dehydrogenase converting propionate to acrylate which is then hydrated to 3-hydroxypropionate and then oxidized and decarboxylated to acetate.     

Metabolic fluxes in Schizosaccharomyces pombe grown on glucose and mixtures of glycerol and acetate

Growth on glycerol has already been a topic of research for several yeast species, and recent publications deal with the regulatory mechanisms of glycerol assimilation by the fission yeast Schizosaccharomyces pombe. We investigated glycerol metabolism of S. pombe from a physiological point of view, characterizing growth and metabolism on a mixture of glycerol and acetate and comparing it to growth on glucose under respirative growth conditions in chemostat experiments. On glycerol/acetate mixtures, the cells grew with a maximum specific growth rate of 0.11 h^?1 where 46 % of the carbon was channeled into biomass and the key fermentation product ethanol was not detectable. ¹³C-assisted metabolic flux analysis resolved substrate distributions through central carbon metabolism, proving that glycerol is used as a precursor for glycolysis, gluconeogenesis, and the pentose phosphate pathway, while acetate enters the tricarboxylic acid cycle via acetyl-CoA. Considering compartmentalization between cytosol and mitochondria in the metabolic model, we found compartmentalization of biosynthesis for the amino acids aspartate and leucine. Balancing of redox cofactors revealed an abundant production of cytosolic NADPH that must be finally regenerated via the respiratory chain shown by the simulated and measured CO₂ production and oxygen consumption rates which were in good agreement.     

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     

Photoassimilation of acetate and metabolism of carbohydrate in Chlorobium thiosulfatophilum

1. Washed cell suspensions of Chlorobium thiosulfatophilum form large amounts of a polyglucose in the light. Addition of acetate to the cells increases the formation of polysaccharide considerably. During incubation in the dark, polysaccharide decreases with time, and organic acids such as succinic and propionic acid are excreted into the medium. 2. Glucose isolated from cells which had photoassimilated 1-, 2-, and U-¹⁴C-acetate had a specific activity which lay between 1 and 2 times that of the acetate substrates. 3. To analyse the distribution of radioactivity in the glucose units formed during photoassimilation of ¹⁴C-acetate, 2 microbial degradations, with bakers' yeast and Zymomonas mobilis respectively, were used. The results show that acetate gives rise to carbon atoms 1+2 and 5+6 of glucose, whereas carbon atomes 3+4 are not labelled. Further, the results indicate that glucose is not formed via the reductive pentose phosphate cycle when acetate is present.     

Redox responses in yeast to acetate as the carbon source

Following a shift to medium with acetate as the carbon source, a parental yeast strain exhibited a transient moderate 20% reduction in total cellular [NAD⁺ + NADH] but showed a ∼10-fold increase in the ratio of [NAD⁺]:[NADH] after 36 h. A mutant strain (idhΔ) lacking the tricarboxylic acid cycle enzyme isocitrate dehydrogenase had 50% higher cellular levels of [NAD⁺ + NADH] relative to the parental strain but exhibited similar changes in cofactor concentrations following a shift to acetate medium, despite an inability to grow on that carbon source; essentially all of the cofactor was in the oxidized form within 36 h. The salvage pathway for NAD(H) biosynthesis was found to be particularly important for viability during early transition of the parental strain to stationary phase in acetate medium. However, oxygen consumption was not affected, suggesting that the NAD(H) produced during this time may support other cellular functions. The idhΔ mutant exhibited increased flux through the salvage pathway in acetate medium but was dependent on the de novo pathway for viability. Long-term chronological lifespans of the parental and idhΔ strains were similar, but viability of the mutant strain was dependent on both pathways for NAD(H) biosynthesis.     

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.     

13C Metabolic Flux Analysis of acetate conversion to lipids by Yarrowia lipolytica

Volatile fatty acids (VFAs) are an inexpensive and renewable carbon source that can be generated from gas fermentation and anaerobic digestion of fermentable wastes. The oleaginous yeast Yarrowia lipolytica is a promising biocatalyst that can utilize VFAs and convert them into triacylglycerides (TAGs). However, currently there is limited knowledge on the metabolism of Y. lipolytica when cultured on VFAs. To develop a better understanding, we used acetate as the sole carbon source to culture two strains, a control strain and a previously engineered strain for lipid overaccumulation. For both strains, metabolism during the growth phase and lipid production phase were investigated by metabolic flux analysis using two parallel sodium acetate tracers. The resolved flux distributions demonstrate that the glyoxylate shunt pathway is constantly active and the flux through gluconeogenesis varies depending on strain and phase. In particular, by regulating the activities of malate transport and pyruvate kinase, the cells divert only a portion of the glyoxylate shunt flux required to satisfy the needs for anaplerotic reactions and NADPH production through gluconeogenesis and the oxidative pentose phosphate pathway (PPP). Excess flux flows back to the tricarboxylic acid (TCA) cycle for energy production. As with the case of glucose as the substrate, the primary source for lipogenic NADPH is derived from the oxidative PPP.     

Studies on the mechanism of acetate oxidation by bacteria. V. evidence for the participation of fumarate,malate, and oxalacetate in the oxidation of acetic acid by Escherichia coli

1. Simultaneous oxidation of C¹⁴-methyl-labeled acetate, and unlabeled malate or fumarate and α-ketoglutarate results in entrapment of labeled carbon in the C₄-dicarboxylic acids, but not in α-ketoglutarate, although all substrates are utilized at comparable rates. 2. A large endogenous reduction of all C₄-dicarboxylic acids (fumarate, oxalacetate, and malate) to succinate is observed under aerobic conditions, and when vigorous oxidation is proceeding. This effect occurs with both freshly harvested young (18 hour) cells and stored (2 week) cells. 3. This reduction can be considerably minimized under high oxygen tensions. 4. The quantitative concordance of these results with a Thunberg-Knoop cyclic mechanism for acetate oxidation is shown. Possible alternative C₄ products formed prior to succinate are not completely excluded, but it appears that the cells can utilize the succinate condensation as a major pathway in acetate oxidation.     

Identification of in vivo enzyme activities in the cometabolism of glucose and acetate by Saccharomyces cerevisiae by using 13C-labeled substrates

A detailed characterization of the central metabolic network of Saccharomyces cerevisiae CEN.PK 113-7D was carried out during cometabolism of different mixtures of glucose and acetate, using aerobic C-limited chemostats in which one of these two substrates was labeled with ¹³C. To confirm the role of malic enzyme, an isogenic strain with the corresponding gene deleted was grown under the same conditions. The labeling patterns of proteinogenic amino acids were analyzed and used to estimate metabolic fluxes and/or make inferences about the in vivo activities of enzymes of the central carbon metabolism and amino acid biosynthesis. Malic enzyme flux increased linearly with increasing acetate fraction. During growth on a very-high-acetate fraction, the activity of malic enzyme satisfied the biosynthetic needs of pyruvate in the mitochondria, while in the cytosol pyruvate was supplied via pyruvate kinase. In several cases enzyme activities were unexpectedly detected, e.g., the glyoxylate shunt for a very-low-acetate fraction, phosphoenolpyruvate carboxykinase for an acetate fraction of 0.46 C-mol of acetate/C-mol of substrate, and glucose catabolism to CO₂ via the tricarboxylic acid cycle for a very-high-acetate fraction. Cytoplasmic alanine aminotransferase activity was detected, and evidence was found that α-isopropylmalate synthase has two active forms in vivo, one mitochondrial and the other a short cytoplasmic form.     

Heterotrophic nitrification by Arthrobacter sp. (strain 9006) as influenced by different cultural conditions,growth state and acetate metabolism

An Arthrobacter sp. (strain 9006), isolated from lake water, accumulated nitrite up to about 15 mg N/l, but no nitrate. In a mineral medium supplemented with tryptone, yeast extract, acetate and ammonium, the cells released nitrite into the medium parallel to growth or when growth had virtually ceased. The nitrite formed was proportional to the initial acetate concentration, indicating an involvement of acetate metabolism with nitrification. The organism grew with a wide variety of organic carbon sources, but washed cells formed nitrite from ammonium only in the presence of citrate, malate, acetate or ethanol. Magnesium ions were required for nitrification of ammonium and could not be replaced by other divalent metal ions. Analysis of the glyoxylate cycle key enzymes in washed suspensions incubated in a minimal medium revealed that isocitrate lyase and malate synthase were most active during the nitrification phase. Nitrite accumulation but not growth was inhibited by glucose, tryptone and yeast extract. A possible explanation for the different nitrification patterns during growth is based on the regulatory properties of glyoxylate cycle enzymes.Abbreviations IL Isocitrate lyase [threo-D_s-isocitrate glyoxylate-lase, E.C. 4.1.3.1.] - MS malate synthase [l-malate glyoxylate-lyase (CoA-acetylating), E.C. 4.1.3.2.]     

C nuclear magnetic resonance study of acetate incorporation into malate during ca-uptake by isolated leaf tissues

¹³C Nuclear magnetic resonance spectroscopy of leaflets of Gleditsia triacanthos and Albizia julibrisin was used to determine the fate of acetate taken up during the absorption of calcium from ¹³C-labeled Ca-acetate solution. Small amounts of acetate accumulated temporarily in the leaf tissues, but the bulk of acetate was incorporated into malate. The initial rate of malate synthesis was very low, but increased rapidly during acetate treatment and reached its maximum after 8 hours; the enzymes involved in malate synthesis thus appear to be substrate induced. Use of acetate-2-¹³C yielded malate labeled in C-3, indicating that vacuolar malate accumulating during Ca-uptake might be synthesized via malate synthase from acetate and glyoxalate. However, a source of glyoxalate condensing with acetate during malate synthesis could not be identified. Glycolate produced in photorespiration is an unlikely source, because glycolate-2-¹³C was absorbed and metabolized by the leaf tissues into products of the glycolate pathway, but was not a major precursor in malate synthesis. Malate synthesis via the glyoxalate cycle is also unlikely, because no evidence for the recycling of a ¹³C-labeled 4-carbon organic acid was found. Malate synthesis in the leaflets of Gleditsia and Albizia thus appears to involve the inducible condensation of acetate with a 2-carbon compound of unidentified nature and origin.     

Organic Acid Metabolism by Isolated Rhizobium japonicum Bacteroids

Rhizobium japonicum bacteroids isolated from soybean (Glycine max L.) nodules oxidized ¹⁴C-labeled succinate, pyruvate, and acetate in a manner consistent with operation of the tricarboxylic acid cycle and a partial glyoxylate cycle. Substrate carbon was incorporated into all major cellular components (cell wall + membrane, nucleic acids, and protein).