Rolf Schauder

Acetate oxidation to CO2 in anaerobic bacteria via a novel pathway not involving reactions of the citric acid cycle

In several sulfate-reducing bacteria capable of complete oxidation of acetate (or acetyl CoA), the citric acid cycle is not operative. No 2-oxoglutarate dehydrogenase activity was found in these organisms, and the labelling pattern of oxaloacetate excludes its synthesis via 2-oxo-glutarate. These sulfate-reducers contained, however, high activities of the enzymes carbon monoxide dehydrogenase and formate dehydrogenase and catalyzed an isotope exchange between CO2 and the carboxyl group of acetate (or acetyl CoA), showing a direct C-C-cleavage of activated acetic acid. These findings suggest that in the investigated sulfate-reducers acetate is oxidized to CO2 via C1 intermediates. The proposed pathway provides a possible explanation for the reported different fluoroacetate sensitivity of acetate oxidation by anaerobic bacteria, for mini-methane formation, as well as for the postulated anaerobic methane oxidation by special sulfate-reducers.

Carbon assimilation pathways in sulfate-reducing bacteria. II: Enzymes of a reductive citric acid cycle in the autotrophic Desulfobacter hydrogenophilus

The strict anaerobe Desulfobacter hydrogenophilus is able to grow autotrophically with CO2, H2, and sulfate as sole carbon and energy sources. The generation time at 30°C under autotrophic conditions in a pure mineral medium was 15 h, the growth yield was 8 g cell dry mass per mol sulfate reduced to H2S. Enzymes of the autotrophic CO2 assimilation pathway were investigated. Key enzymes of the Calvin cycle and of the acetyl CoA pathway could not be found. All enzymes of a reductive citric acid cycle were present at specific activities sufficient to account for the observed growth rate. Notably, an ATP-citrate lyase (1.3 μmol · min-1 · mg cell protein-1) was present both in autotrophically and in heterotrophically grown cells, which was rapidly inactivated in the absence of ATP. The data indicate that in D. hydrogenophilus a reductive citric acid cycle is operating in autotrophic CO2 fixation. Since other autotrophic sulfate reducers possess an acetyl CoA pathway for CO2 fixation, two different autotrophic pathways occur in the same physiological group.

Oxidative and reductive acetyl CoA/carbon monoxide dehydrogenase pathway in Desulfobacterium autotrophicum

It has been proposed that in some anaerobic facultatively autotrophic bacteria the acetyl CoA/CO dehydrogenase pathway is operating both in the reductive and in the oxidative direction, depending on the growth conditions. One of these anaerobes, the Gram-negative sulfate-reducing cubacterium Desulfobacterium autotrophicum, was examined for enzymes of the proposed pathway. All the required enzyme activities were present in sufficient amounts both in autotrophically and in heterotrophically grown cells, provided that the cellular tetrahydropterin rather than tetrahydrofolate was used as cosubstrate in some of the enzyme assays. The question arises whether two sets of enzymes are operating in the reductive and oxidative direction, respectively. The key enzyme of this pathway, CO dehydrogenase, which was reasonably oxygen stable, was analysed by native polyacrylamide gel electrophoresis and anaerobic activity staining. Extracts from heterotrophically grown cells exhibited five enzyme activity bands. Extracts from autotrophically grown cells showed the same pattern but an additional activity band appeared.

Bacterial sulphur respiration

The reduction of elemental sulphur to HSis coupled to the phosphorylation of ADP with inorganic phosphate in the catabolism of certain anaerobic bacteria. This process is called sulphur respiration. Since the discovery of sulphur respiration with the isolation of Desulfuromonas acetoxidans (Pfennig and Biebl 1976), many other bacteria have been found that catalyze the reduction of elemental sulphur (for review see Widdel 1988; Stetter et al. 1990: Adams 1990; Fauque et al. 1991). Most of these organisms belong to the group of extremely thermophilic archaebacteria. Also some species of Thermotoga, the earliest branch of the eubacterial domain, as well as the methanogens catalyze sulphur reduction. These organisms are thought to be more closely related to the common ancestor than any other living organism. Therefore, sulphur respiration may represent one of the first means of biological energy conservation in evolution (Stetter and Gaag 1983). For bacteria that grow with H 2 or formate as electron donors and sulphur as acceptor it is evident from growth that sulphur reduction is coupled to phosphorylation, since the growth reactions do not allow substrate level phosphorylation. In contrast, the metabolic significance of sulphur reduction is uncertain in other bacteria. After a brief discussion of the biology of sulphur reducers, this review will focus on the chemistry, enzymology and bioenergetics of catabolic sulphur reduction. Sulphur reduction serving anabolic purposes will not be discussed here (for review see Le Faou et al. 1990).

Polysulfide as a possible substrate for sulfur-reducing bacteria

Because of its low solubility it is unlikely that elemental sulfur serves as the direct substrate for sulfur-reducing bacteria. To test the hypothesis that polysulfide may represent a soluble intermediate of sulfur reduction, the maximal polysulfide concentrations formed from elemental sulfur in aqueous sulfide solutions were measured at near neutral pH and at temperatures up to 90°C. The saturation concentrations decreased by two orders of magnitude when the pH was lowered from 7 to 6 at a given temperature, and increased about tenfold when the temperature was raised from 37°C to 90°C at a given pH. The dissolution of 0.1 mM zerovalent sulfur in 1 mM sulfide (H2S+HS−) required a pH of 7.5 at 20°C and of only 6.1 at 100°C. A comparison with the growth optima of sulfur-reducers suggests that polysulfide is present at sufficient concentration at the growth conditions of the Bacteria and the moderately acidophilic Archaea. Polysulfide is apparently not available at the growth conditions of the extremely acidophilic Archaea. Alternative mechanisms for the sulfur utilization under these conditions are discussed.

Acetate oxidation to CO2 via a citric acid cycle involving an ATP-citrate lyase: a mechanism for the synthesis of ATP via substrate level phosphorylation in Desulfobacter postgatei growing on acetate and sulfate

Desulfobacter postgatei is an acetate-oxidizing, sulfate-reducing bacterium that metabolizes acetate via the citric acid cycle. The organism has been reported to contain a si-citrate synthase (EC 4.1.3.7) which is activated by AMP and inorganic phosphate. It is show now, that the enzyme mediating citrate formation is an ATP-citrate lyase (EC 4.1.3.8) rather than a citrate synthase. Cell extracts (160,000xg supernatant) catalyzed the conversion of oxaloacetate (apparent Km=0.2 mM), acetyl-CoA (app. Km=0.1 mM), ADP (app. Km=0.06 mM) and phosphate (app. Km=0.7 mM) to citrate, CoA and ATP with a specific activity of 0.3 μmol·min-1·mg-1 protein. Per mol citrate formed 1 mol of ATP was generated. Cleavage of citrate (app. Km=0.05 mM; Vmax=1.2 μmol · min-1 · mg-1 protein) was dependent on ATP (app. Km=0.4 mM) and CoA (app. Km=0.05 mM) and yielded oxaloacetate, acetyl-CoA, ADP, and phosphate as products in a stoichiometry of citrate:CoA:oxaloacetate:ADP=1:1:1:1. The use of an ATP-citrate lyase in the citric acid cycle enables D. postgatei to couple the oxidation of acetate to 2 CO2 with the net synthesis of ATP via substrate level phosphorylation.

Anaerovibrio glycerini sp. nov., an anaerobic bacterium fermenting glycerol to propionate, cell matter, and hydrogen

A strictly anaerobic, Gram-negative bacterium was isolated in continuous culture from black freshwater sediment with glycerol as sole source of carbon and energy. It was present in such sediments at 108 cells per ml. The isolate was highly specialized and used only glycerol and the glycerol residue of diolein as substrate, and fermented it quantitatively to propionate. During growth in mineral medium, small amounts of hydrogen were produced which corresponded exactly to the calculated amount of electrons released in cell matter formation from glycerol. Yeast extract enhanced cell yields with glycerol, but did not support growth itself. In cell-free extracts, benzylviologen-dependent hydrogenase activity as well as a b-type cytochrome and some of the enzymes of the methylmalonylCoA pathway were found. The guanine-plus-cytosine content of the DNA was 34.3±1.0 mol% and corresponded well with that of Anaerovibrio lipolytica which was found to be 31.4 mol%. The consequences of the electron balance of this glycerol fermentation are discussed with respect to glycerol fermentation by other propionic acid-forming bacteria.

Carbon Isotope Fractionation by Autotrophic Bacteria with Three Different C02 Fixation Pathways

Abstract Carbon isotope fractionation during autotrophic growth o f different bacteria which possess different autotrophic CO2 fixation pathways has been studied. 13C /12C -Ratios in the cell carbon of the following bacteria were determined (CO2 fixation pathway suggested or proven in parentheses): Alkaligenes eutrophus (reductive pentose phosphate cycle), Desulfobacterium autotrophicum and Acetobacterium woodii (reductive acetyl-CoA pathway), Desulfobacter hydrogenophilus and Thermoproteus neutrophilus (reductive citric acid cycle). The Δδ13C values, which indicate the per mille deviation of the 13C content of cell carbon from that of the CO : used as the sole carbon source, range from - 10%° (reductive citric acid cycle) over - 26%° (reductive pentose phosphate cycle) to -36%° (reductive acetyl-CoA pathway). Acetate formed via the acetyl-CoA pathway by the acetogenic Acetobacterium woodii showed a Δδ13C = -40%°. These data are discussed in view of the different CO2 fixation reactions used by the bacteria and especially with regard to the isotopic composition of sedimentary carbon through time.

Growth of Wolinella succinogenes with elemental sulfur in the absence of polysulfide

Wolinella succinogenes grows by anaerobic respiration with formate and polysulfide. Polysulfide forms spontaneously from sulfur and sulfide. Here we report that this eubacterium also grows with formate and elemental sulfur under conditions that do not allow polysulfide formation. With the appropriate amount of Fe2+ added to the medium, the concentration of polysulfide was calculated to be 0.4 nM, which is 1/400th of the concentration that of dissolved elemental sulfur. At commensurable growth rates, the growth yield with sulfur was one quarter of that with polysulfide as electron acceptor. The same low growth yield either with sulfur or with polysulfide as electron acceptor was measured for a Δpsr mutant that lacks the genes encoding polysulfide reductase (Psr).

Autotrophic CO2 Fixation in Chemotrophic Anaerobic Bacteria

An increasing number of strictly anaerobic bacteria are being found which are able to grow autotrophically with CO2 as the only or the main source of cell carbon. They belong to different systematic groups [1], including methanogenic archaebacteria [2], gram-positive and gram-negative sulfate reducing eubacteria [3], sulfur-dependent archaebacteria [4], gram-positive and gram-negative acetogenic eubacteria [5–7], and possibly others. These anaerobes have in common that they do not use the reactions of the reductive pentose phosphate cycle (Calvin cycle) for CO2 assimilation. Rather, two alternative pathways were found. Reviews on this subject have recently appeared [5–10].