Differential effects of sodium on hydrogen- and glucose-dependent growth of the acetogenic bacterium Acetogenium kivui

Acetogenium kivui could not be revived or maintained in a sodium-deficient medium (0.2 mM sodium) under H2-dependent conditions, and neither lithium nor potassium replaced the sodium requirement of H2-cultivated cells. Conversely, the revival and maintenance of glucose-cultivated cells did not display a dependency on supplemental sodium. In the absence of growth, formate became a major end product in both sodium-deficient and metabolically impaired H2-grown cultures of A. kivui. Harmaline, a putative inhibitor of Na+/H+ antiporters, uncoupled acetogenesis from H2-dependent growth but was less effective when growth was at the expense of glucose. Significantly, carbon monoxide (CO) stimulated H2-dependent growth of A. kivui but inhibited glucose-dependent growth. Collectively, these findings demonstrate that sodium plays a critical role in the H2-dependent bioenergetics of A. kivui and indicate that autotrophic and heterotrophic cells may utilize dissimilar mechanisms of energy conservation. In contrast to the growth of A. kivui, supplemental sodium was not required for the glucose-, H2-, and CO-dependent growth of Clostridium thermoaceticum.     

Acetogenium kivui,a new thermophilic hydrogen-oxidizing acetogenic bacterium

Hydrogen-oxidizing acetogenic bacteria in pure culture are presently represented by the two mesophilic species, Acetobacterium woodii and Clostridium aceticum. From Lake Kivu we have isolated a Gram negative, chemolithotrophic, thermophilic anaerobe (LKT-1) that oxidizes hydrogen and reduces carbon dioxide to acetic acid. It is a non-motile, non-sporeforming rod, about 0.7m in width and 2–7.5m in length, often occuring in pairs or chains. The cell wall has a banded appearance; the surface layer contains a regular array of particles with six-fold rotational symmetry. No outer membrane is present. The temperature optimum for growth is 66°C, and the pH optimum is 6.4. Organic growth substrates include glucose, mannose, fructose, pyruvate, and formate; acetate is the principal product. The doubling time for growth on hydrogen and carbon dioxide is about 2h. Vitamins are neither required nor stimulatory. Yeast extract and Trypticase enhance the final yield but do not affect the growth rate. Cysteine or sulfide are required and cannot be replaced by thioglycolate or dithiothreitol. LKT-1 was mass cultured on hydrogen and carbon dioxide in a 24.1 fermentor with a yield of 34g (wet weight) of cells. The DNA base composition as determined by buoyant density is 38 mol % guanine plus cytosine. LKT-1 appears only distantly related to physiologically similar bacteria. A new genus Acetogenium is proposed, and the species is Acetogenium kivui.     

Nickel transport by the thermophilic acetogen Acetogenium kivui.

Exogenous 63Ni was incorporated into carbon monoxide dehydrogenase when Acetogenium kivui ATCC 33488 was cultivated in the presence of 63NiCl2. The capacity for nickel (63NiCl2) transport was greatest with cells harvested from the mid- to late exponential phases of growth. Nickel transport was linear during the transport assay period and displayed saturation kinetics. The apparent Km and Vmax for nickel transport by H2-cultivated cells approximated 2.3 microM Ni and 670 pmol of Ni transported per min per mg (dry weight) of cells, respectively. The nickel transport system was not appreciably affected by the other divalent cations that were tested, and transported nickel was not readily exchangeable with exogenous nickel. Nickel transport was stimulated by glucose or H2 and was decreased by various metabolic inhibitors; however, nickel uptake by glucose- and H2-cultivated cells displayed differential sensitivities to ATPase inhibitors.     

A thermostable mannitol-1-phosphate dehydrogenase is required in mannitol metabolism of the thermophilic acetogenic bacterium Thermoanaerobacter kivui

Acetogenic bacteria recently attracted attention because they reduce carbon dioxide (CO₂) with hydrogen (H₂) to acetate or to other products such as ethanol. Besides gases, acetogens use a broad range of substrates, but conversion of the sugar alcohol mannitol has rarely been reported. We found that the thermophilic acetogenic bacterium Thermoanaerobacter kivui grew on mannitol with a specific growth rate of 0.33 h⁻¹ to a final optical density (OD₆₀₀) of 2.2. Acetate was the major product formed. A lag phase was observed only in cultures pre-grown on glucose, not in those pre-grown on mannitol, indicating that mannitol metabolism is regulated. Mannitol-1-phosphate dehydrogenase (MtlD) activity was observed in cell-free extracts of cells grown on mannitol only. A gene cluster (TKV_c02830–TKV_c02860) for mannitol uptake and conversion was identified in the T. kivui genome, and its involvement was confirmed by deleting the mtlD gene (TKV_c02860) encoding the key enzyme MtlD. Finally, we overexpressed mtlD, and the recombinant MtlD carried out the reduction of fructose-6-phosphate with NADH, at a high V_MAX of 1235 U mg⁻¹ at 65°C. The enzyme was thermostable for 40 min at 75°C, thereby representing the first characterized MtlD from a thermophile.     

Domain structure of the Acetogenium kivui surface layer revealed by electron crystallography and sequence analysis.

The three-dimensional structure of the Acetogenium kivui surface layer (S-layer) has been determined to a resolution of 1.7 nm by electron crystallographic techniques. Two independent reconstructions were made from layers negatively stained with uranyl acetate and Na-phosphotungstate. The S-layer has p6 symmetry with a center-to-center spacing of approximately 19 nm. Within the layer, six monomers combine to form a ring-shaped core surrounded by a fenestrated rim and six spokes that point towards the axis of threefold symmetry and provide lateral connectivity to other hexamers in the layer. The structure of the A. kivui S-layer protein is very similar to that of the Bacillus brevis middle wall protein, with which it shares an N-terminal domain of homology. This domain is found in several other extracellular proteins, including the S-layer proteins from Bacillus sphaericus and Thermus thermophilus, Omp alpha from Thermotoga maritima, an alkaline cellulase from Bacillus strain KSM-635, and xylanases from Clostridium thermocellum and Thermoanaerobacter saccharolyticum, and may serve to anchor these proteins to the peptidoglycan. To our knowledge, this is the first example of a domain conserved in several S-layer proteins.     

Homoacetogenic Fermentation of Cellulose by a Coculture of Clostridium thermocellum and Acetogenium kivui

Interrelationships between methanogens and fermentative or hydrolytic bacteria are well documented; however, such cocultures do not allow a complete fermentation shift to a peculiar metabolite. We describe here a new stable association between Clostridium thermocellum and Acetogenium kivui which converts 1 mol of cellulose (anhydroglucose equivalent) into 2.7 mol of acetate.     

S-layer protein gene of Acetogenium kivui: cloning and expression in Escherichia coli and determination of the nucleotide sequence.

Acetogenium kivui is anaerobically growing thermophilic bacterium with a gram-positive type of cell wall structure. The outer surface is covered with a hexagonally packed surface (S) layer. The gene coding for the S-layer polypeptide was cloned in Escherichia coli on two overlapping fragments by using the plasmid pUC18 as the vector. It was expressed under control of a cloned Acetogenium promoter or the lacZ gene. We determined the complete sequence of the structural gene. The mature polypeptide comprises 736 amino acids and is preceded by a typical procaryotic signal sequence of 26 amino acids. It i weakly acidic, weakly hydrophilic, and contains a relatively high proportion of hydroxyamino acids, including two clusters of serine and threonine residues. An N-terminal region of about 200 residues is homologous to the N-terminal part of the middle wall protein, one of the two S-layer proteins of Bacillus brevis, and there is also an internal homology within the N-terminal region of the A. kivui polypeptide.     

Characterization of the H2- and CO-dependent chemolithotrophic potentials of the acetogens Clostridium thermoaceticum and Acetogenium kivui.

Strains of Clostridium thermoaceticum were tested for H2- and CO-dependent growth in a defined medium containing metals, minerals, vitamins, cysteine-sulfide, CO2-bicarbonate, and H2 or CO. Ten of the thirteen strains tested grew at the expense of H2 and CO, and C. thermoaceticum ATCC 39073 was chosen for further study. The doubling times for H2- and CO-dependent growth under chemolithotrophic conditions (the defined medium with nicotinic acid as sole essential vitamin and sulfide as sole reducer) were 25 and 10 h, respectively. Product stiochiometries for chemolithotrophic cultures approximated: 4.1H2 + 2.4CO2----CH3COOH + 0.1 cell C + 0.3 unrecovered C and 6.8CO----CH3COOH + 3.5CO2 + 0.4 cell C + 0.9 unrecovered C. H2-dependent growth produced significantly higher acetate concentrations per unit of biomass synthesized than did CO- or glucose-dependent growth. In contrast, the doubling time for H2-dependent growth under chemolithotrophic conditions (the defined medium without vitamins and sulfide as sole reducer) by Acetogenium kivui ATCC 33488 was 2.7 h; as a sole energy source, CO was not growth supportive for A. kivui. The YH2 values for A. kivui and C. thermoaceticum were 0.91 and 0.46 g of cell dry weight per mol of H2 consumed, respectively; the YCO value for C. thermoaceticum was 1.28 g of cell dry weight per mol of CO consumed. The specific activities of hydrogenase and CO dehydrogenase in both acetogens were influenced by the energy source utilized for growth and were significantly lower in C. thermoaceticum than in A. kivui. With extracts of H2-cultivated cells and benzyl viologen as electron acceptor, the Vmax values for hydrogenase from C. thermoaceticum and A. kivui were 155.7 and 1,670 micromoles of H2 oxidized per min mg of protein, respectively; the Vmax values for CO dehydrogenase from C. thermoaceticum and A. kivui were 90.6 and 2,973 micromoles of CO oxidized per min per mg of protein, respectively.     

The regular surface layer of Acetogenium kivui: some structural, developmental and evolutionary aspects

Abstract The structure of the regular surface layer of Acetogenium kivui has been investigated by electron microscopy in conjunction with image processing techniques. From the averaged unit cell structure a tentative model of the subunit organization is derived. The occurrence of mono- and polycrystalline S-layer sheets is interpreted in terms of transitions related to the growth cycle of cell. The question is raised as to whether the striking structural similarity of S-layers, even from evolutionary rather distant species, is due to divergent or convergent evolution.     

A sodium-stimulated ATP synthase in the acetogenic bacterium Acetobacterium woodii.

Experiments with resting cells of Acetobacterium woodii were performed to elucidate the coupling ion used by the ATP synthase. A. woodii synthesized ATP in response to an artificial delta pH, indicating the presence of a proton-translocating ATPase. On the other hand, a delta pNa, as well as a proton diffusion potential, could serve as a driving force for ATP synthesis with the latter strictly dependent on Na+. These results are indicative for the presence of a Na(+)-translocating ATP synthase in A. woodii.     

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.     

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.     

Electron carriers involved in autotrophic and heterotrophic acetogenesis in the thermophilic bacterium Thermoanaerobacter kivui

Extremophiles - Thermoanaerobacter kivui is an acetogenic model organism that reduces CO2 with electrons derived from H2 or CO, or from organic substrates in the Wood–Ljugdahl pathway (WLP)....     

Differential effects of sodium on two types of opiate binding sites.

Effects of Na⁺ on saturable naloxone binding were studied $\text{in vitro}$ using particulate fractions obtained from rat brain homogenates. Na⁺ stimulated the saturable naloxone binding in thalamus-hypothalamus regions, but inhibited it in cerebellum. These findings strongly support the hypothesis that two types of naloxone binding sites exist in brain tissues.     

Role of carbon monoxide dehydrogenase in acetate synthesis by the acetogenic bacterium, Acetobacterium woodii

Carbon monoxide dehydrogenase (CODH) plays a key role in acetate synthesis by the acetogenic bacterium, Clostridium thermoaceticum. Acetobacterium woodii, like C. thermoaceticum contains high levels of CODH. In this work we show that crude extracts of A. woodii synthesize acetate from methyl tetrahydrofolate or methyl iodide, carbon monoxide and coenzyme A (CoA). The purified CODH from A. woodii catalyzes an exchange reaction between CO and the carbonyl group of acetyl-CoA even faster than the C. thermoaceticum enzyme, indicating the CODH of A. woodii, like that of C. thermoaceticum is an acetyl-CoA synthetase. Fluorescence and EPR studies further support this postulate by demonstrating that CODH binds CoA near the CO binding site involving a tryptophan residue. The UV absorption spectra and the amino acid compositions of A. woodii and C. thermoaceticum CODHs are very similar. Evidence is presented using purified enzymes from A. woodii that the synthesis of acetyl-CoA occurs by a pathway similar to that utilized by C. thermoaceticum.     

Some physiological effects of near-maximum growth temperatures on an obligately psychrophilic marine bacterium.

The heat inactivation of the obligately psychrophilic marine bacterium Ant-300 was investigated in terms of glucose uptake, the oxidation of glucose to CO2, and permeability control. At 13C, the maximum temperature for growth, and at slightly higher temperatures, CO2 evolution decreased with time during the oxidation of exogenously supplied glucose. The decrease in CO2 evolution appeared to be a result of heat-induced restrictions on glucose uptake. Leakage of intracellular metabolites apparently contributed to the cells decreased ability to take up glucose at elevated temperatures. A consequence of these heat-induced changes seemed to be the acceleration of cell starvation.     

The Rnf complex from the acetogenic bacterium Acetobacterium woodii: Purification and characterization of RnfC and RnfB

rnf genes are widespread in anaerobic bacteria and hypothesized to encode a respiratory enzyme that couples exergonic reduction of NAD with reduced ferredoxin as a reductant to vectorial ion (Na⁺, H⁺) translocation across the cytoplasmic membrane. However, despite its importance for the physiology of these bacteria, little is known about the subunit composition and the function of subunits. Here, we have purified the entire Rnf complex from the acetogen Acetobacterium woodii or after its production in Escherichia coli. These studies revealed covalently bound flavin in RnfB and RnfD. Unfortunately, the complex did not catalyze electron transfer from reduced ferredoxin to NAD. We, therefore, concentrated on the two cytosolic subunits RnfC and RnfB. RnfC was produced in E. coli, purified and shown to have 8.3 mol iron and 8.6 mol sulfur per mol of the subunit, consistent with the presence of two [4Fe-4S] centers, which were verified by EPR analysis. Flavins could not be detected, but RnfC catalyzed NADH-dependent FMN reduction. These data confirm RnfC as NADH-binding subunit and FMN as an intermediate in the electron transport chain. RnfB could only be produced as a fusion to the maltose-binding protein. It contained 25 mol iron and 26 mol sulfur, consistent with the predicted six [4Fe4S] centers. The FeS centers in RnfB were reduced with reduced ferredoxin as reductant. These data are consistent with RnfB as the ferredoxin-binding subunit of the complex.