Carrier-mediated acetate transport in Acetobacterium woodii

Intracellular and extracellular acetate concentrations of Acetobacterium woodii DSM 1030 were determined during growth or incubation of resting cell suspensions. The internal concentrations during growth decreased from initially 350 mM to 145 mM at the end of the experiment. The intracellular pH was lowered from 7.5 to 6.6 and the pH was enlarged from 0.2 to 0.6 units. Both, growing and resting cells of A. woodii showed no equilibrium between internal and external acetate concentrations during glucose consumption; the internal concentrations were always higher than expected assuming equal concentrations of the free acid inside and outside the cells. From counterflow experiments it is suggested that acetate does not only leave A. woodii cells by passive diffusion but also by carrier-mediated transport.     

CO Metabolism in the Acetogen Acetobacterium woodii

The Wood-Ljungdahl pathway allows acetogenic bacteria to grow on a number of one-carbon substrates, such as carbon dioxide, formate, methyl groups, or even carbon monoxide. Since carbon monoxide alone or in combination with hydrogen and carbon dioxide (synthesis gas) is an increasingly important feedstock for third-generation biotechnology, we studied CO metabolism in the model acetogen Acetobacterium woodii. When cells grew on H₂-CO₂, addition of 5 to 15% CO led to higher final optical densities, indicating the utilization of CO as a cosubstrate. However, the growth rate was decreased by the presence of small amounts of CO, which correlated with an inhibition of H₂ consumption. Experiments with resting cells revealed that the degree of inhibition of H₂ consumption was a function of the CO concentration. Since the hydrogen-dependent CO₂ reductase (HDCR) of A. woodii is known to be very sensitive to CO, we speculated that cells may be more tolerant toward CO when growing on formate, the product of the HDCR reaction. Indeed, addition of up to 25% CO did not influence growth rates on formate, while the final optical densities and the production of acetate increased. Higher concentrations (75 and 100%) led to a slight inhibition of growth and to decreasing rates of formate and CO consumption. Experiments with resting cells revealed that the HDCR is a site of CO inhibition. In contrast, A. woodii was not able to grow on CO as a sole carbon and energy source, and growth on fructose-CO or methanol-CO was not observed.     

Carbonic anhydrase in Acetobacterium woodii and other acetogenic bacteria.

Acetobacterium woodii, Acetohalobium arabaticum, Clostridium formicoaceticum, and Sporomusa silvacetica were found to contain carbonic anhydrase (CA). Minimal to no CA activity was detected in Moorella thermoautotrophica, Moorella thermoacetica subsp. "pratumsolum," Sporomusa termitida, and Thermoanaerobacter kivui. Of the acetogens tested, A. woodii had the highest CA specific activity, approximately 14 U mg of protein(-1), in extracts of either glucose- or H2-CO2-cultivated cells. CA of A. woodii was cytoplasmic and was purified approximately 300-fold to a specific activity of 5,236 U mg of protein(-1). Intracellular acetate concentrations inhibited CA activity of A. woodii by 50 to 85%, indicating that intracellular acetate may affect in situ CA activity.     

Autotrophic CO2 fixation in Acetobacterium woodii

Earlier labeling experiments have shown that autotrophically grown Acetobacterium woodii assimilates cell carbon via direct acetyl CoA formation from 2 CO₂, rather than via the Calvin cycle. Cell extracts contained the enzymes required for biosynthesis starting from acetyl CoA and CO₂. Notably, pyruvate synthase, pyruvate phosphate dikinase, and phosphoenolpyruvate carboxytransphosphorylase were present in sufficiently high activities. Ribulose-1,5-bisphosphate carboxylase activity could not be detected. The observed enzyme pattern was consistent with the postulated biosynthetic pathway as deduced from ¹⁴C-labeling experiments.     

Additional characteristics of one-carbon-compound utilization by Eubacterium limosum and Acetobacterium woodii.

Growth characteristics of Eubacterium limosum and Acetobacterium woodii during one-carbon-compound utilization were investigated. E. limosum RF grew with formate as the sole energy source. Formate also replaced a requirement for CO2 during growth with methanol. Growth with methanol required either rumen fluid, yeast extract, or acetate, but their effects were not additive. Cultures were adapted to grow in concentrations of methanol of up to 494 mM. Growth occurred with methanol in the presence of elevated levels of Na+ (576 mM). The pH optima for growth with methanol, H2-CO2, and carbon monoxide were similar (7.0 to 7.2). Growth occurred with glucose at a pH of 4.7, but not at 4.0. The apparent Km values for methanol and hydrogen were 2.7 and 0.34 mM, respectively. The apparent Vmax values for methanol and hydrogen were 1.7 and 0.11 mumol/mg of protein X min-1, respectively. The Ks value for CO was estimated to be less than 75 microM. Cellular growth yields were 70.5, 7.1, 3.38, and 0.84 g (dry weight) per mol utilized for glucose, methanol, CO, and hydrogen (in H2-CO2), respectively. E. limosum was also able to grow with methoxylated aromatic compounds as energy sources. Glucose apparently repressed the ability of E. limosum to use methanol, hydrogen, or isoleucine but not CO. Growth with mixtures of methanol, H2, CO, or isoleucine was not diauxic. The results, especially the relatively high apparent Km values for H2 and methanol, may indicate why E. limosum does not usually compete with rumen methanogens for these energy sources.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonacetogenic Growth of the Acetogen Acetobacterium woodii on 1,2-Propanediol

Acetogenic bacteria can grow by the oxidation of various substrates coupled to the reduction of CO₂ in the Wood-Ljungdahl pathway. Here, we show that growth of the acetogen Acetobacterium woodii on 1,2-propanediol (1,2-PD) as the sole carbon and energy source is independent of acetogenesis. Enzymatic measurements and metabolite analysis revealed that 1,2-PD is dehydrated to propionaldehyde, which is further oxidized to propionyl coenzyme A (propionyl-CoA) with concomitant reduction of NAD. NADH is reoxidized by reducing propionaldehyde to propanol. The potential gene cluster coding for the responsible enzymes includes genes coding for shell proteins of bacterial microcompartments. Electron microscopy revealed the presence of microcompartments as well as storage granules in cells grown on 1,2-PD. Gene clusters coding for the 1,2-PD pathway can be found in other acetogens as well, but the distribution shows no relation to the phylogeny of the organisms.     

Transformation of tetrachloromethane to dichloromethane and carbon dioxide by Acetobacterium woodii.

Five anaerobic bacteria were tested for their abilities to transform tetrachloromethane so that information about enzymes involved in reductive dehalogenations of polychloromethanes could be obtained. Cultures of the sulfate reducer Desulfobacterium autotrophicum transformed some 80 microM tetrachloromethane to trichloromethane and a small amount of dichloromethane in 18 days under conditions of heterotrophic growth. The acetogens Acetobacterium woodii and Clostridium thermoaceticum in fructose-salts and glucose-salts media, respectively, degraded some 80 microM tetrachloromethane completely within 3 days. Trichloromethane accumulated as a transient intermediate, but the only chlorinated methanes recovered at the end of the incubation were 8 microM dichloromethane and traces of chloromethane. Desulfobacter hydrogenophilus and an autotrophic, nitrate-reducing bacterium were unable to transform tetrachloromethane. Reduction of chlorinated methanes was thus observed only in the organisms with the acetyl-coenzyme A pathway. Experiments with [14C]tetrachloromethane were done to determine the fate of this compound in the acetogen A. woodii. Radioactivity in an 11-day heterotrophic culture was largely (67%) recovered in CO2, acetate, pyruvate, and cell material. In experiments with cell suspensions to which [14C]tetrachloromethane was added, 14CO2 appeared within 20 s as the major transformation product. A. woodii thus catalyzes reductive dechlorinations and transforms tetrachloromethane to CO2 by a series of unknown reactions.     

Sodium dependence of acetate formation by the acetogenic bacterium Acetobacterium woodii.

Growth of Acetobacterium woodii on fructose was stimulated by Na+; this stimulation was paralleled by a shift of the acetate-fructose ratio from 2.1 to 2.7. Growth on H2-CO2 or on methanol plus CO2 was strictly dependent on the presence of sodium ions in the medium. Acetate formation from formaldehyde plus H2-CO by resting cells required Na+, but from methanol plus H2-CO did not. This is analogous to H2-CO2 reduction to methane by Methanosarcina barkeri, which involves a sodium pump (V. Müller, C. Winner, and G. Gottschalk, Eur. J. Biochem. 178:519-525, 1988). This suggests that the reduction of methylenetetrahydrofolate to methyltetrahydrofolate is the Na+-requiring reaction. A sodium gradient (Na+ out/Na+ in = 32, delta pNa = -91 mV) was built up when resting cells of A. woodii were incubated under H2-CO2. Acetogenesis was inhibited when the delta pNa was dissipated by monensin.     

Production and Utilization of Ethanol by the Homoacetogen Acetobacterium woodii

Acetobacterium woodii formed ethanol as a fermentation product in addition to acetate when the phosphate concentration of the medium was between 0.2 and 8.4 mM. Considerable amounts of alanine were also found (2 to 11 mM). Supplementation with phosphate caused a shift to acetate as the only end product. Ethanol could also serve as a substrate for A. woodii. The fermentation yielded predominantly acetate and was strictly dependent on high bicarbonate concentrations. 1-Propanol, 1-butanol, and 1-pentanol were converted to the corresponding fatty acids but allowed only marginal growth. A. wieringae and A. carbinolicum grown under identical conditions were also able to form ethanol, and A. wieringae could use ethanol as a substrate, too. Alcohol dehydrogenase and acetaldehyde dehydrogenase activities were determined in A. woodii. Activity stains of polyacrylamide gels with crude extracts allowed the detection of acetaldehyde dehydrogenase but not of alcohol dehydrogenase. Trace amounts of methane were detected during growth of A. woodii on glucose and ethanol.     

Regulation of Caffeate Respiration in the Acetogenic Bacterium Acetobacterium woodii

The anaerobic acetogenic bacterium Acetobacterium woodii can conserve energy by oxidation of various substrates coupled to either carbonate or caffeate respiration. We used a cell suspension system to study the regulation and kinetics of induction of caffeate respiration. After addition of caffeate to suspensions of fructose-grown cells, there was a lag phase of about 90 min before caffeate reduction commenced. However, in the presence of tetracycline caffeate was not reduced, indicating that de novo protein synthesis is required for the ability to respire caffeate. Induction also took place in the presence of CO₂, and once a culture was induced, caffeate and CO₂ were used simultaneously as electron acceptors. Induction of caffeate reduction was also observed with H₂ plus CO₂ as the substrate, but the lag phase was much longer. Again, caffeate and CO₂ were used simultaneously as electron acceptors. In contrast, during oxidation of methyl groups derived from methanol or betaine, acetogenesis was the preferred energy-conserving pathway, and caffeate reduction started only after acetogenesis was completed. The differential flow of reductants was also observed with suspensions of resting cells in which caffeate reduction was induced prior to harvest of the cells. These cell suspensions utilized caffeate and CO₂ simultaneously with fructose or hydrogen as electron donors, but CO₂ was preferred over caffeate during methyl group oxidation. Caffeate-induced resting cells could reduce caffeate and also p-coumarate or ferulate with hydrogen as the electron donor. p-Coumarate or ferulate also served as an inducer for caffeate reduction. Interestingly, caffeate-induced cells reduced ferulate in the absence of an external reductant, indicating that caffeate also induces the enzymes required for oxidation of the methyl group of ferulate.     

Regulation of caffeate respiration in the acetogenic bacterium Acetobacterium woodii

The anaerobic acetogenic bacterium Acetobacterium woodii can conserve energy by oxidation of various substrates coupled to either carbonate or caffeate respiration. We used a cell suspension system to study the regulation and kinetics of induction of caffeate respiration. After addition of caffeate to suspensions of fructose-grown cells, there was a lag phase of about 90 min before caffeate reduction commenced. However, in the presence of tetracycline caffeate was not reduced, indicating that de novo protein synthesis is required for the ability to respire caffeate. Induction also took place in the presence of CO(2), and once a culture was induced, caffeate and CO(2) were used simultaneously as electron acceptors. Induction of caffeate reduction was also observed with H(2) plus CO(2) as the substrate, but the lag phase was much longer. Again, caffeate and CO(2) were used simultaneously as electron acceptors. In contrast, during oxidation of methyl groups derived from methanol or betaine, acetogenesis was the preferred energy-conserving pathway, and caffeate reduction started only after acetogenesis was completed. The differential flow of reductants was also observed with suspensions of resting cells in which caffeate reduction was induced prior to harvest of the cells. These cell suspensions utilized caffeate and CO(2) simultaneously with fructose or hydrogen as electron donors, but CO(2) was preferred over caffeate during methyl group oxidation. Caffeate-induced resting cells could reduce caffeate and also p-coumarate or ferulate with hydrogen as the electron donor. p-Coumarate or ferulate also served as an inducer for caffeate reduction. Interestingly, caffeate-induced cells reduced ferulate in the absence of an external reductant, indicating that caffeate also induces the enzymes required for oxidation of the methyl group of ferulate.     

Growth yield increase linked to caffeate reduction in Acetobacterium woodii

Growth yields were determined with Acetobacterium woodii strain NZva 16 on hydrogen and CO₂, formate, methanol, vanillate, ferulate and fructose in mineral medium in the absence and presence of 0.05% yeast extract. Yeast extract was not essential for growth but enhanced growth yields by 25–100% depending on the substrate fermented. Comparison of yields on formate or methanol allowed calculation of an energy yield in the range of 1.5–2 mol ATP per mol acetate formed during homoacetate fermentation of A. woodii. In the presence of 6 mM caffeate, growth yields were determined with the substrates formate or methanol. Caffeate was reduced to hydrocaffeate and increased growth yields were obtained. An ATP yield of about 1 mol per mol of caffeate reduced was calculated. Cytochromes were not detectable in cell free extracts or membrane preparations.     

Transformation of Tetrachloromethane to Dichloromethane and Carbon Dioxide by Acetobacterium woodii

[This corrects the article on p. 2822 in vol. 54.].     

Transformation of tetrachloromethane to dichloromethane and carbon dioxide by Acetobacterium woodii

Five anaerobic bacteria were tested for their abilities to transform tetrachloromethane so that information about enzymes involved in reductive dehalogenations of polychloromethanes could be obtained. Cultures of the sulfate reducer Desulfobacterium autotrophicum transformed some 80 microM tetrachloromethane to trichloromethane and a small amount of dichloromethane in 18 days under conditions of heterotrophic growth. The acetogens Acetobacterium woodii and Clostridium thermoaceticum in fructose-salts and glucose-salts media, respectively, degraded some 80 microM tetrachloromethane completely within 3 days. Trichloromethane accumulated as a transient intermediate, but the only chlorinated methanes recovered at the end of the incubation were 8 microM dichloromethane and traces of chloromethane. Desulfobacter hydrogenophilus and an autotrophic, nitrate-reducing bacterium were unable to transform tetrachloromethane. Reduction of chlorinated methanes was thus observed only in the organisms with the acetyl-coenzyme A pathway. Experiments with [14C]tetrachloromethane were done to determine the fate of this compound in the acetogen A. woodii. Radioactivity in an 11-day heterotrophic culture was largely (67%) recovered in CO2, acetate, pyruvate, and cell material. In experiments with cell suspensions to which [14C]tetrachloromethane was added, 14CO2 appeared within 20 s as the major transformation product. A. woodii thus catalyzes reductive dechlorinations and transforms tetrachloromethane to CO2 by a series of unknown reactions.     

Additional characteristics of one-carbon-compound utilization by Eubacterium limosum and Acetobacterium woodii

Growth characteristics of Eubacterium limosum and Acetobacterium woodii during one-carbon-compound utilization were investigated. E. limosum RF grew with formate as the sole energy source. Formate also replaced a requirement for CO2 during growth with methanol. Growth with methanol required either rumen fluid, yeast extract, or acetate, but their effects were not additive. Cultures were adapted to grow in concentrations of methanol of up to 494 mM. Growth occurred with methanol in the presence of elevated levels of Na+ (576 mM). The pH optima for growth with methanol, H2-CO2, and carbon monoxide were similar (7.0 to 7.2). Growth occurred with glucose at a pH of 4.7, but not at 4.0. The apparent Km values for methanol and hydrogen were 2.7 and 0.34 mM, respectively. The apparent Vmax values for methanol and hydrogen were 1.7 and 0.11 mumol/mg of protein X min-1, respectively. The Ks value for CO was estimated to be less than 75 microM. Cellular growth yields were 70.5, 7.1, 3.38, and 0.84 g (dry weight) per mol utilized for glucose, methanol, CO, and hydrogen (in H2-CO2), respectively. E. limosum was also able to grow with methoxylated aromatic compounds as energy sources. Glucose apparently repressed the ability of E. limosum to use methanol, hydrogen, or isoleucine but not CO. Growth with mixtures of methanol, H2, CO, or isoleucine was not diauxic. The results, especially the relatively high apparent Km values for H2 and methanol, may indicate why E. limosum does not usually compete with rumen methanogens for these energy sources.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Na+-translocating pyrophosphatase in the acetogenic bacterium Acetobacterium woodii

The anaerobic acetogenic bacterium Acetobacterium woodii employs a novel type of Na(+)-motive anaerobic respiration, caffeate respiration. However, this respiration is at the thermodynamic limit of energy conservation, and even worse, in the first step, caffeate is activated by caffeyl-CoA synthetase, which hydrolyzes ATP to AMP and pyrophosphate. Here, we have addressed whether or not the energy stored in the anhydride bond of pyrophosphate is conserved by A. woodii. Inverted membrane vesicles of A. woodii have a membrane-bound pyrophosphatase that catalyzes pyrophosphate hydrolysis at a rate of 70-120 milliunits/mg of protein. Pyrophosphatase activity was dependent on the divalent cation Mg(2+). In addition, activity was strictly dependent on Na(+) with a K(m) of 1.1 mM. Hydrolysis of pyrophosphate was accompanied by (22)Na(+) transport into the lumen of the inverted membrane vesicles. Inhibitor studies revealed that (22)Na(+) transport was primary and electrogenic. Next to the Na(+)-motive ferredoxin:NAD(+) oxidoreductase (Fno or Rnf), the Na(+)-pyrophosphatase is the second primary Na(+)-translocating enzyme in A. woodii.     

Electron microscopic investigation of the hydrogen-oxidizing acetate-forming anaerobic bacterium Acetobacterium woodii

Acetobacterium woodii is a Gram-positive anaerobic nonsporeforming bacterium able to grow on H₂ and CO₂ as sole sources of energy. The product of fermentation is acetic acid. Fine structural analysis showed rod-shaped flagellated cells, and coccoid cells without flagella arranged predominantly in pairs and chains. The cell wall was found to be composed of three layers. The cell surface exhibited a periodic array of particles consisting of subunits. The cytoplasmic membrane showed particles either either in random distribution or in a hexagonal pattern. Intracytoplasmic membranes were rarely observed, whereas inclusion bodies of varying shapes, predominantly in an uncommon disc-shape, could frequently be observed. Their content was dissolved in ultrathin sections indicating hydrophobic nature.     

Improvement of the long-term survival of Acetobacterium woodii by limiting acetate production

Summary The viable count in fructose-grown cultures of Acetobacterium woodii fell to undetectable levels 186 h after the stationary phase was reached. The loss of viability was due to accumulation of undissociated acetic acid, since loss of viability could be induced artificially in cultures grown at pH 6.8 by adjusting the pH to 5.0 or by adding high concentrations of acetate. Survival was greatly improved when cultures were grown at low initial fructose concentrations ( 2 g/l) to limit acetate production, since viable cells could still be detected after 73d.