Metabolism of acetylene by Nocardia rhodochrous.

A Nocardia rhodochrous strain capable of utilizing acetylene as its sole source of carbon and energy exhibited slow growth on low concentrations of acetaldehyde. Resting cells incubated with acetylene formed a product identified as acetaldehyde, but attempts to demonstrate acetylene hydrase activity in cell-free extracts were unsuccessful. Acetaldehyde dehydrogenase in N. rhodochrous was found to be NAD+ linked and nonacylating, converting acetaldehyde to acetate. Specific activities of acetaldehyde dehydrogenase, acetothiokinase, and isocitrate lyase were enhanced in cells grown on acetylene and ethanol as compared with cells grown on alternate substrates. These results suggest that acetylene is catabolized via acetaldehyde to acetate and eventually to acetyl coenzyme A. Acetylene oxidation in N. rhodochrous appears to be constitutive and is not inhibited in the presence of either ethylene, nitrous oxide, or methane.     

New types of acetate-oxidizing,sulfate-reducing Desulfobacter species,D. hydrogenophilus sp. nov., D. latus sp. nov., and D. curvatus sp. nov.

Sulfate-reducing bacteria with oval to rod-shaped cells (strains AcRS1, AcRS2) and vibrio-shaped cells (strains AcRM3, AcRM4, AcRM5) differing by size were isolated from anaerobic marine sediment with acetate as the only electron donor. A vibrio-shaped type (strain AcKo) was also isolated from freshwater sediment. Two strains (AcRS1, AcRM3) used ethanol and pyruvate in addition to acetate, and one strain (AcRS1) grew autotrophically with H₂, sulfate and CO₂. Higher fatty acids or lactate were never utilized. All isolates were able to grow in ammonia-free medium in the presence of N₂. Nitrogenase activity under such conditions was demonstrated by the acetylene reduction test. The facultatively lithoautotrophic strain (AcRS1), a strain (AcRS2) with unusually large cells (2×5 m), and a vibrio-shaped strain (AcRM3) are described as new Desulfobacter species, D. hydrogenophilus, D. latus, and D. curvatus, respectively.     

Escherichia coli mutants with altered control of alcohol dehydrogenase and nitrate reductase.

Mutants of Escherichia coli which overproduce alcohol dehydrogenase were obtained by selection for the ability to use ethanol as an acetate source in a strain auxotrophic for acetate. A mutant having a 20-fold overproduction of alcohol dehydrogenase was able to use ethanol only to fulfill its acetate requirement, whereas two mutants with a 60-fold overproduction were able to use ethanol as a sole carbon source. The latter two mutants produced only 25% of the wild-type level of nitrate reductase, when grown under anaerobic conditions. Alcohol dehydrogenase production was largely unaffected by catabolite repression but was repressed by nitrate under both aerobic and anaerobic conditions. The genetic locus responsible for alcohol dehydrogenase overproduction was located at min 27 on the E. coli genetic map; the gene order, as determined by transduction, was trp tonB adh chlC hemA. The possible relationship of alcohol dehydrogenase to anaerobic redox systems such as formate-nitrate reductase is discussed.     

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     

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     

Fermentation of methoxyacetate to glycolate and acetate by newly isolated strains of Acetobacterium sp.

Three strains of new mesophilic homoacetogenic bacteria were enriched and isolated from sewage sludge and from marine sediment samples with methoxyacetate as sole organic substrate in a carbonate-buffered medium under anoxic conditions. Two freshwater isolates were motile, Gram-positive, non-sporeforming rods. The marine strain was an immotile, Gram-positive rod with a slime capsula. All strains utilized only the methyl residue of methoxyacetate and released glycolic acid. They also fermented methyl groups of methoxylated aromatic compounds and of betaine to acetate with growth yields of 6–10 g dry matter per mol methyl group. H₂/CO₂, formate, methanol, hexamethylene tetramine, as well as fructose, numerous organic acids, glycerol, ethylene glycol, and glycol ethers were fermented to acetate as well. High activities of carbon monoxide dehydrogenase (0.4–2.2 U x mg protein^–1) were detected in all three isolates. The guanine-plus-cytosine-content of the DNA of the freshwater isolates was 42.7 and 44.4 mol %, with the marine isolate it was 47.7 mol %. The freshwater strains were assigned to the genus Acetobacterium as new strains of the species A. carbinolicum. One freshwater isolate, strain KoMac1, was deposited with the Deutsche Sammlung von Mikroorganismen GmbH, Braunschweig, under the number DSM 5193.     

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.     

Investigations of the anaerobic metabolism of the polychaete wormNereis diversicolor M.

Summary The anaerobic metabolism ofNereis diversicolor M. was studied during various periods of experimental anaerobiosis.The degradation of glycogen is shown to be the main source of anaerobic energy production. During first hours of anaerobiosis, aspartate, in addition to glycogen, is metabolized in considerable quantities.Five acids were found to accumulate as end-products: alanine, D-lactate, succinate, acetate and propionate (Table 2).Alanine is accumulated only during the first hours of anaerobiosis. The increase in alanine is correlated with a decrease in aspartate.D-Lactate is the main end-product during the first 24 h of anaerobiosis, and continues to be produced even during prolonged anaerobiosis. In accordance with lactate production,Nereis diversicolor possesses a high glycolytic capacity (Table 4).The major end-products of long term fermentation are propionate and acetate. In contrast to other end-products, these acids are excreted in substantial amounts.Abbreviations GAPDH glyceraldehydephosphate dehydrogenase, EC 1.2.1.12 - LDH lactate dehydrogenase, EC 1.1.1.27 - GOT aspartate aminotransferase, EC 2.6.1.1 - GPT alanine aminotransferase, EC 2.6.1.2 - MDH malate dehydrogenase, EC 1.1.1.37 Supported by Deutsche Forschungsgemeinschaft (Gr 456/5 and Gr 456/6)     

Relationship between porewater organic carbon content, sulphate reduction and nitrogen fixation (acetylene reduction) in the rhizosphere of Zostera noltii

Depth profiles of nitrogen fixation (acetylene reduction), sulphate reduction, NH ₄ ⁺ concentration and porewater volatile fatty acids concentrations were measured in Zostera noltii colonised sediments in the Bassin d'Arcachon, France in March 1994. Acetylene reduction activity (ARA) was detectable throughout sediment profiles. Addition of sodium molybdate (20 mmol l^–1) a specific inhibitor of sulphate reduction to slurries inhibited ARA by >75% inferring that sulphate-reducing bacteria (SRB) were the dominant component of the nitrogen fixing microflora. The peak of ARA was coincident with that of sulphate reduction and a relatively constant relationship of 40 mole sulphate reduced per mole acetylene reduced was recorded throughout the profiles. From this ratio it was calculated that at least 17% of the ATP yield from sulphate reduction would be required to support the measured rates of nitrogen fixation (acetylene reduction).Acetate was the dominant constituent of the porewater volatile fatty acids pool, accounting for >90% of the total pool as measured by HPLC. Concentrations of porewater acetate recorded by HPLC were compared with those measured using an enzymatic technique and these data indicate that approximately 10% of the total porewater acetate pool was not available to microbial metabolism. Profiles of porewater acetate concentrations measured by both techniques were similar to those recorded for both ARA and sulphate reduction and thus acetate oxidation may fuel these activities.     

A new 3-hydroxybutyrate fermenting anaerobe,Ilyobacter polytropus,gen. nov. sp. nov., possessing various fermentation pathways

From marine anoxic mud, a new strictly anaerobic, Gram-negative, non-sporeforming bacterium was isolated with 3-hydroxybutyrate as substrate. 3-Hydroxybutyrate and crotonate were fermented to acetate and butyrate. Glycerol was fermented to 1,3-propanediol and 3-hydroxypropionate. Acetate and formate were the only products of pyruvate or citrate fermentation. Glucose and fructose were fermented to acetate, formate and ethanol. Malate and fumarate were fermented to acetate, formate and propionate. Neither sulfate, sulfur, nor nitrate was reduced. The DNA base ratio was 32.2±0.5 mol% guanine plus cytosine. Strain CuHbu1 is described as type strain of a new genus and species, Ilyobacter polytropus gen. nov. sp. nov., in the family Bacteroidaceae.     

不同浓度外源乙酸钠对耐乙酸大肠埃希菌DA19代谢和关键酶酶活的影响

为研究外源乙酸钠对大肠埃希菌DA19生长代谢的影响,将该菌株在氮源限制基本培养基及添加不同浓度乙酸钠的氮源限制基本培养基中连续培养,测定稳态时生长代谢参数和胞内关键酶酶活。与MN培养基相比,葡萄糖比消耗速率和延胡索酸比生成速率随外源乙酸钠质量浓度增加而逐渐下降,丙酮酸比生成速率则随外源乙酸钠质量浓度增加而明显增加,而乙酸比生成速率则明显降低(除9 g/L乙酸钠外)。磷酸果糖激酶、异柠檬酸脱氢酶、异柠檬酸裂解酶、苹果酸脱氢酶、磷酸烯醇式丙酮酸羧化酶和乙酸激酶酶活随外源乙酸钠质量浓度增加而呈先下降后上升的趋势,而6-磷酸葡萄糖脱氢酶则随着外源乙酸钠质量浓度增加而逐渐降低。为了应对外源乙酸钠压力,大肠埃希菌DA19的生长代谢和中心代谢途径酶活都发生了明显改变。     

Peculiarities of Ethanol Metabolism in an Acinetobacter sp. Mutant Strain Defective in Exopolysaccharide Synthesis

Activities of the key enzymes of ethanol metabolism were assayed in ethanol-grown cells of an Acinetobacter sp. mutant strain unable to synthesize exopolysaccharides (EPS). The original EPS-producing strain could not be used for enzyme analysis because its cells could not to be separated from the extremely viscous EPS with a high molecular weight. In Acinetobacter sp., ethanol oxidation to acetaldehyde proved to be catalyzed by the NAD⁺-dependent alcohol dehydrogenase (EC 1.1.1.1.). Both NAD⁺ and NADP⁺ could be electron accepters in the acetaldehyde dehydrogenase reaction. Acetate is implicated in the Acinetobacter sp. metabolism via the reaction catalyzed by acetyl-CoA-synthetase (EC 6.2.1.1.). Isocitrate lyase (EC 4.1.3.1.) activity was also detected, indicating that the glyoxylate cycle is the anaplerotic mechanism that replenishes the pool of C₄-dicarboxylic acids in Acinetobacter sp. cells. In ethanol metabolism by Acinetobacter sp., the reactions involving acetate are the bottleneck, as evidenced by the inhibitory effect of sodium ions on both acetate oxidation in the intact cells and on acetyl-CoA-synthetase activity in the cell-free extracts, as well as by the limitation of the C₂-metabolism by coenzyme A. The results obtained may be helpful in developing a new biotechnological procedure for obtaining ethanol-derived exopolysaccharide ethapolan.     

Inorganic nitrogen metabolism in two cellulose-degrading clostridia

Inorganic nitrogen metabolism in two cellulose degrading clostridia, the mesophile Clostridium cellobioparum and the thermophile Clostridium thermocellum was investigated. Both strains show acetylene reduction (i.e. possibly nitrogenase activity), contain glutamine synthetase, glutamate dehydrogenase and glutamate-dependent transaminases. C. cellobioparum additionally contains a NADH-dependent glutamate synthase and a NH ₄ ⁺ -repressible glycine dehydrogenase (NADPH). Remarkably, acetylene reduction in C. thermocellum is not repressed by ammonium, casting doubt whether this activity is due to nitrogenase. The results are compared with the data from other saccharolytic clostridia.Abbreviation GOGAT glutamine-oxoglutarate amidotransferase (glutamate synthase)     

Fermentation of trihydroxybenzenes by Pelobacter acidigallici gen. nov. sp. nov., a new strictly anaerobic,non-sporeforming bacterium

Five strains of rod-shaped, Gram-negative, non-sporing, strictly anaerobic bacteria were isolated from limnic and marine mud samples with gallic acid or phloroglucinol as sole substrate. All strains grew in defined mineral media without any growth factors; marine isolates required salt concentrations higher than 1% for growth, two freshwater strains only thrived in freshwater medium. Gallic acid, pyrogallol, 2,4,6-trihydroxybenzoic acid, and phloroglucinol were the only substrates utilized and were fermented stoichiometrically to 3 mol acetate (and 1 mol CO₂) per mol with a growth yield of 10g cell dry weight per mol of substrate. Neither sulfate, sulfur, nor nitrate were reduced. The DNA base ratio was 51.8% guanine plus cytosine. A marine isolate, Ma Gal 2, is described as type strain of a new genus and species, Pelobacter acidigallici gen. nov. sp. nov., in the family Bacteroidaceae. In coculture with Acetobacterium woodii, the new isolates converted also syringic acid completely to acetate. Cocultures with Methanosarcina barkeri converted the respective substrates completely to methane and carbon dioxide.     

Enzymatic regulation of glucose catabolism by Lactobacillus plantarum in response to pH shifts in a chemostat

Summary The influence of medium pH on the regulation of glucose catabolism by Lactobacillus plantarum 8014 was examined in anaerobic chemostat cultures. When L. plantarum was grown in a chemostat at pH 5.5, and the pH shifted to pH 7.5, acetate was produced in addition to lactate and acetoin. After the shift, acetate kinase and NAD-dependent lactate dehydrogenase activities increased while the acetoin dehydrogenase and alpha-acetolactate synthase activities decreased. The high acetate kinase activity together with low acetoin dehydrogenase and alpha-acetolactate synthase activities may explain why L. plantarum made more acetate at the expense of acetoin in response to alkaline conditions.Offprint requests to: T.J. Montville     

Oxygen dependent lactate utilization by Lactobacillus plantarum

Lactobacillus plantarum P5 grew aerobically in rich media at the expense of lactate; no growth was observed in the absence of aeration. The oxygen-dependent growth was accompanied by the conversion of lactate to acetate which accumulated in the growth medium. Utilization of oxygen with lactate as substrate was observed in buffered suspensions of washed whole cells and in cell-free extracts. A pathway which accounts for the generation of adenosine triphosphate during aerobic metabolism of lactate to acetate via pyruvate and acetyl phosphate is proposed. Each of the enzyme activities involved, nicotinamide adenine dinucleotide independent lactic dehydrogenase, nicotinamide adenine dinucleotide dependent lactic dehydrogenase, pyruvate oxidase, acetate kinase and NADH oxidase were demonstrated in cell-free extracts. The production of pyruvate, acetyl phosphate and acetate was demonstrated using cell-free extracts and cofactors for the enzymes of the proposed pathway.Abbreviations MRS Man, Rogosa and Sharpe (1960) medium modified as in Materials and methods - TY Tryptone Yeast Extract broth - OUL Oxygen uptake with lactate as substrate - DCPIP 2,6-Dichlorophenolindophenol - LDH Lactic dehydrogenase     

Comparative proteomics of Geobacter sulfurreducens PCAT in response to acetate,formate and/or hydrogen as electron donor

Geobacter sulfurreducens is a model bacterium to study the degradation of organic compounds coupled to the reduction of Fe(III). The response of G. sulfurreducens to the electron donors acetate, formate, hydrogen and a mixture of all three with Fe(III) citrate as electron acceptor was studied using comparative physiological and proteomic approaches. Variations in the supplied electron donors resulted in differential abundance of proteins involved in the citric acid cycle (CAC), gluconeogenesis, electron transport, and hydrogenases and formate dehydrogenase. Our results provided new insights into the electron donor metabolism of G. sulfurreducens. Remarkably, formate was the preferred electron donor compared to acetate, hydrogen, or acetate plus hydrogen. When hydrogen was the electron donor, formate was formed, which was associated with a high abundance of formate dehydrogenase. Notably, abundant proteins of two CO₂ fixation pathways (acetyl-CoA pathway and the reversed oxidative CAC) corroborated chemolithoautotrophic growth of G. sulfurreducens with formate or hydrogen and CO₂, and provided novel insight into chemolithoautotrophic growth of G. sulfurreducens.     

化能自养硫氧化细菌Halothiobacillus sp.LS2介导的以乙炔为电子受体的硫氧化反应

【目的】探究化能自养硫氧化细菌Halothiobacillus sp. LS2介导的以乙炔为电子受体的厌氧硫氧化反应。【方法】稀释涂布法测定细胞生长情况,离子色谱仪测试硫氧化动力学中SO_4~(2–)和S_2O_3~(2–)以及基于相对荧光定量法的基因表达分析。【结果】尽管菌株LS2在以氧气为电子受体时的最大反应速率V_(max)更高,但在厌氧条件下且以乙炔为电子受体时,菌株LS2的生长量是氧气为电子受体时的2倍,且硫氧化酶基因soxB的表达量显著高于氧气作为电子受体时。【结论】菌株LS2不仅可以以乙炔为电子受体完成厌氧硫氧化反应,且这一代谢过程的产能效率较有氧硫氧化过程更高。本研究首次发现了微生物介导的以乙炔为电子受体的厌氧硫氧化反应,对丰富硫的生物地球化学循环理论有积极意义。