Georg Fuchs

Acetate assimilation and the synthesis of alanine, aspartate and glutamate inMethanobacterium thermoautotrophicum

Cultures of the autotrophic bacteriumMethanobacterium thermoautotrophicum were shown to assimilate acetate when grown on CO2 and H2 in the presence of acetate. At 1 mM acetate 10% of the cell carbon came from acetate, the rest from CO2. At higher concentrations the percentage increased to reach a maximum of 65%at acetate concentrations higher than 20 mM. The data suggest that acetate may be an important carbon source under physiological conditions.The incorporation of acetate into alanine, aspartate and glutamate was studied in more detail. The cells were grown on CO2 and H2 in the presence of 1 mM U-14C-acetate. The three amino acids were isolated from the labelled cells by a simplified procedure. Alanine, aspartate and glutamate were found to have the same specific radioactivity. Degradation studies showed that C1 of alanine C1 and C4 of aspartate, and C1 and C5 of glutamate were exclusively derived from CO2, whereas C2 and C3 alamine and aspartate, and C3 and C4 of glutamate were partially derived from acetate. These findings and the presence of pyruvate synthase, phosphoenolpyruvate carboxylase and α-ketoglutarate synthase inM. thermoautotrophicum indicate that CO2 is assimilated into the three amino acids via acetyl CoA carboxylation to pyruvate, phosphoenolpyruvate carboxylation to oxaloacetate, and succinyl CoA carboxylation to α-ketoglutarate.

Autotrophic CO2 fixation in Chlorobium limicola. Evidence for the operation of a reductive tricarboxylic acid cycle in growing cells

Chlorobium limicola was grown on a mineral salts medium with CO2 as the main carbon source supplemented with specifically labeled 14C propionate and the incorporation of 14C into alanine (≙ intracellular pyruvate), aspartate (≙ oxaloacetate), and glutamate (≙ α-ketoglutarate) was studied in long term labeling experiments. During growth in presence of propionate 30% of the cell carbon were derived from propionate and 70% from CO2. Propionate was not oxidized to CO2.All three amino acids were found to be labeled. The labeling patterns indicate that propionate was assimilated via propionyl CoA, methylmalonyl CoA and succinyl CoA. When 1-14C propionate was the labeled precursor no radioactivity was found in the carboxyl group(s) of alanine, aspartate and glutamate, excluding the incorporation of propionate into the amino acids via succinate oxidation to fumarate. With 1-14C propionate preferentially aspartate (C-3) and glutamate (C-2) became labeled, with 2-14C propionate alanine (C-3) and glutamate (C-4). These findings indicate that propionate was incorporated into the amino acids via succinyl CoA, α-ketoglutarate, isocitrate, and citrate, followed by a si-type cleavage of citrate to oxaloacetate and acetyl CoA (or acetate). Similar experiments with U-14C acetate confirm these conclusions. Thus, all reactions of the proposed reductive tricarboxylic acid cycle could be demonstrated in autotrophically growing cells.

Carbon isotope fractionation by Methanobacterium thermoautotrophicum

The fractionation of carbon isotopes by Methanobacterium thermoautotrophicum was studied during growth of the bacterium on H2 plus CO2 as sole carbon and energy sources. A 80% H2/20% CO2 gas mixture was continuously bubbled through the culture. At high gassing rates, in the absence of a “closed system effect”, cells and methane were found to be depleted in 13C relative to CO2 in the gas mixture by 2.4% and 3.4%, respectively. At low gassing rates, when more than 90% of the CO2 was converted to methane, the cells were enriched in 13C by 1.3% and methane was depleted in 13C by 0.5%; residual CO2 was enriched in 13C by 3.4%. The magnitude of isotope fractionation suggests that CO2 rather than bicarbonate is the active species of CO2 mainly utilized in both CO2 assimilation and CO2 reduction to methane. The apparent positive 13C-discrimination in cell carbon synthesis, which was observed at low gassing rates, indicates that most of the CO2 assimilated into cell material is not incorporated via reactions involved in CO2 reduction to methane.

Acetyl CoA, a central intermediate of autotrophic CO2 fixation in Methanobacterium thermoautotrophicum

AbstractThe pathway of autotrophic CO2 fixation in Methanobacterium thermoautotrophicum has been investigated by long term labelling of the organism with isotopic acetate and pyruvate while exponentially growing on H2 plus CO2. Maximally 2% of the cell carbon were derived from exogeneous tracer, 98% were synthesized from CO2. Since growth was obviously autotrophic the labelled compounds functioned as tracers of the cellular acetyl CoA and pyruvate pool during cell carbon synthesis from CO2.nM. thermoautotrophicum growing in presence of U-14C acetate incorporated 14C into cell compounds derived from acetyl CoA (N-acetyl groups) as well as into compounds derived from pyruvate (alanine), oxaloacetate (aspartate), α-ketoglutarate (glutamate), hexosephosphates (galactosamine), and pentosephosphates (ribose). The specific radioactities of N-acetylgroups and of the three amino acids were identical. The hexosamine exhibited a two times higher specific radioactivity, and the pentose a 1.6 times higher specific radioactivity than e.g. alanine.nM. thermoautotrophicum growing in presence of 3-14C pyruvate, however, did not incorporate 14C into cell compounds directly derived from acetyl CoA. Those compounds derived from pyruvate, dicarboxylic acids and hexosephosphates became labelled. The specific radioactivities of alanine, aspartate and glutamate were identical; the hexosamine had a specific radioactivity twice as high as e.g. alanine.The finding that pyruvate was not incorporated into compounds derived from acetyl CoA, whereas acetate was incorporated into derivatives of acetyl CoA and pyruvate in a 1:1 ratio demonstrates that pyruvate is synthesized by reductive carboxylation of acetyl CoA. The data further provide evidence that in this autotrophic CO2 fixation pathway hexosephosphates and pentosephosphates are synthesized from CO2 via acetyl CoA and pyruvate.

Evidence for an incomplete reductive carboxylic acid cycle in Methanobacterium thermoautotrophicum

The involvement of reactions of the tricarboxylic acid cycle in autotrophic CO2 fixation in Methanobacterium thermoautotrophicum was investigated. The incorporation of succinate into glutamate (=α-ketoglutarate), aspartate (=oxaloacetate) and alanine (=pyruvate) was studied. The organism was grown on H2 plus CO2 at pH 6.5 in the presence of 1 mM [U-14C-]succinate. Significant amounts of the dicarboxylic acid were incorporated into cellular material under these conditions. Alanine, aspartate, and glutamate were isolated and their specific radioactivities were determined. Only glutamate was found to be labelled. Degradation of glutamate revealed that C-1 of glutamate was derived from CO2 and C-2-C-5 from succinate indicating that in M. thermoautotrophicum α-ketoglutarate is synthesized via reductive carboxylation of succinyl CoA. The finding that succinate was not incorporated into alanine and aspartate excludes that oxaloacetate and pyruvate are synthesized from α-ketoglutarate via isocitrate or citrate. This is taken as evidence that a complete reductive carboxylic acid cycle is not involved here in autotrophic CO2 fixation.

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.

Acetate thiokinase and the assimilation of acetate in Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum growing on H2 plus CO2 as sole carbon and energy source was found to contain acetate thiokinase (Acetyl CoA synthetase; EC 6.2.1.1): Acetate+ATP+CoA → Acetyl CoA+AMP+PPi. The apparent Km value for acetate was 40 μM. Acetate kinase (EC 2.7.2.1) and phosphotransacetylase (EC 2.3.1.8) could not be detected. The specific activity of acetate thiokinase was high in cells grown with limited H2 and CO2 supply (approximately 100nmol/min · mg protein), it was low in exponentially grown cells (2 nmol/min·mg protein). This corresponded with the finding that cells growing linearly in the presence of acetate assimilated the monocarboxylic acid in high amounts (>10% of the cell carbon was derived from acetate), whereas exponentially growing cells did not (<1% of cell carbon was derived from acetate). These latter observations indicated that acetate thiokinase and free acetate are not involved in autotrophic CO2 fixation in M. thermoautotrophicum. The presence and some kinetic properties of succinate thiokinase (EC 6.2.1.5), adenylate kinase (EC 2.7.4.3), and inorganic pyrophosphatase (EC 3.6.1.1.) are also described.

Autotrophic CO2 fixation in Chlorobium limicola. Evidence against the operation of the Calvin cycle in growing cells

Chlorobium limicola has been proposed to assimilate CO2 autotrophically via a reductive tricarboxylic acid cycle rather than via the Calvin cycle. This proposal has been a matter of considerable controversy. In order to determine which pathway is operative, the bacterium was grown on a mineral salts medium with CO2 as the main carbon source supplemented with specifically labeled 14C-pyruvate, and the incorporation of 14C into alanine (≙intracellular pyruvate), aspartate (≙oxaloacetate), glutamate (≙α-ketoglutarate), and glucose (≙hexosephosphate) was measured in exponentially growing cells in long term labeling experiments. During growth in presence of pyruvate, 20% of the cell carbon were derived from pyruvate in the medium, 80% from CO2. Since pyruvate was not oxidized to CO2, only those compounds should become labeled which were synthesized from CO2 via pyruvate.The three amino acids and glucose were found to be labeled. Alanine had one fifth the specific radioactivity of the extracellular pyruvate, indicating that 20% of the intracellular pyruvate pool were derived from pyruvate in the medium, 80% were synthesized from CO2. Glucose had twice the specific radioactivity of alanine, showing that hexosephosphate synthesis from CO2 proceeded via the pyruvate pool. The latter finding is not consistent with the operation of the Calvin cycle, in which pyruvate is not an intermediate. The specific radioactivities of aspartate (≙oxaloacetate) and of glutamate (≙α-ketoglutarate) were practically identical but considerably lower than that of alanine (≙ intracellular pyruvate). These findings are compatible with the operation of a reductive tricarboxylic acid cycle as mechanism of autotrophic CO2 fixation. Degradation studies of the cell components support this interpretation.

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.

Products of CO2 fixation and 14C labelling pattern of alanine in Methanobacterium thermoautotrophicum pulse-labelled with 14CO2

The incorporation of 14CO2 by an exponentially growing culture of the autotrophic bacterium Methanobacterium thermoautotrophicum has been studied. The distribution of radioactivity during 2s–120s incubation periods has been analyzed by chromatography and radioautography. After a 2 s incubation most of the radioactivity of the ethanolsoluble fraction was present in the amino acids alanine, glutamate, glutamine and aspartate, whereas phosphorylated compounds were only weakly labelled. The percentage of the total radioactivity fixed, which was contained in the principal early labelled amino acid alanine, increased in the first 20 s and only then decreased, indicating that alanine is derived from primary products of CO2 fixation.The labelling patterns of alanine produced during various incubation times have been determined by degradation. After a 2 s 14CO2 pulse, 61% of the radioactivity was located in C-1, 23% in C-2, and 16% in C-3. The results are consistent with the operation of a previously proposed autotrophic CO2 assimilation pathway which involves the formation of acetyl CoA from 2 CO2 via one-carbon unit intermediates, followed by the reductive carboxylation of acetyl CoA to pyruvate.