Erhard Stupperich

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.

Biosynthetic pathways of Vibrio succinogenes growing with fumarate as terminal electron acceptor and sole carbon source

Abstract1.With fumarate as the terminal electron acceptor and either H2 or formate as donor, Vibrio succinogenes could grow anaerobically in a mineral medium using fumarate as the sole carbon source. Both the growth rate and the cell yield were increased when glutamate was also present in the medium.2.Glutamate was incorporated only into the amino acids of the glutamate family (glutamate, glutamine, proline and arginine) of the protein. The residual cell constituents were synthesized from fumarate.3.Pyruvate and phosphoenolpyruvate, as the central intermediates of most of the cell constituents, were formed through the action of malic enzyme and phosphoenolpyruvate synthetase. Fructose-1,6-bisphosphate aldolase was present in the bacterium suggesting that this enzyme is involved in carbohydrate synthesis.4.In the absence of added glutamate the amino acids of the glutamate family were synthesized from fumarate via citrate. The enzymes involved in glutamate synthesis were present.5.During growth in the presence of glutamate, net reducing equivalents were needed for cell synthesis. Glutamate and not H2 or formate was used as the source of these reducing equivalents. For this purpose part of the glutamate was oxidized to yield succinate and CO2.6.The α-ketoglutarate dehydrogenase involved in this reaction was found to use ferredoxin as the electron acceptor. The ferredoxin of the bacterium was reoxidized by means of a NADP-ferredoxin oxidoreductase. Enzymes catalyzing the reduction of NAD, NADP or ferredoxin by H2 or formate were not detected in the bacterium.

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.

Carbon monoxide fixation into the carboxyl group of acetyl coenzyme A during autotrophic growth of Methanobacterium

Methanobacterium thermoautotrophicum is a methane-forming archaebacterium which grows on HZ. and CO2 as sole energy and carbon sources [ 1,2]. This autotrophic organism does not assimilate CO2 via the Calvin cycle (for literature see [3,4]). Acetyl CoA rather than 3-phosphoglycerate appears as the earliest detectable CO2 fixation product [3,5]. The operation of a reductive tricarboxylic acid cycle like that of the green sulfur bacteria [6,7] has also been excluded as the mechanism of acetyl CoA synthesis [8]. All available data have suggested instead a total synthesis of acetyl CoA via one-carbon intermediates [3-51. It has been recently suggested [9,10] that acetyl CoA synthesis in autotrophic methanogens could be mechanistically related to acetate formation from 2 CO2 in acetogenic bacteria. In the latter group of eubacteria experiments with 14C0 have shown that the carboxyl group of acetate is derived from CO, which itself is probably formed from CO2 via direct reduction [9,1 I]. To test for this possibility we grew Iki. thermoautotrophicum on 80% H2/20% CO2 in the presence of 5% 14C0 and then investigated the incorporation of 14C into cellular compounds. We found that 14C0 was specifically incorporated into carbon positions which are biosynthetically derived from the carboxy1 group of acetyl CoA. Under the above conditions, about 15% of the acetyl CoA was synthesized from 1 CO2 (methyl group) and 1 CO (carboxy1 group). Also, an exchange of unlabeled CO with r4C02 was observed. Growth: M. thermoautotrophicum (Marburg strain), was grown at 65°C on 40 ml mineral medium in a pressurized closed vessel with 1.1 1 gas phase (80% H2/20% CO2/0.1% H2S) at 1 bar overpressure [12]. Just before inoculation with 5% preculture, 50 ml 14C0 were added with a syringe. 14C0 was prepared from r4C-formate as in [ 131, and unlabeled CO was added as carrier to give a specific radioactivity of 49000 dpm/pmol CO. Samples (1 ml) of the culture were periodically withdrawn for determination of cell density and radiocarbon incorporation. The gas phase was simultaneously sampled for gaschromatographic analysis of CO, COZ, and CH4 (0.4 ml) and for determination of radioactivity in CO2 (1 ml). At an optical density of A ,457~ = 1.4, the culture was harvested by centrifugation. The cells were then washed and fractionated as in [7], and the radioactivity in each fraction was determined. From the protein and cell wall fraction L-alanine was isolated; its specific radioactivity was measured and the label distribution in each individual carbon atom was determined by complete chemical degradation. The fixation of 14C02 into CO was studied under identical conditions, except that CO2, rather than CO was labeled with 14C (final specific radioactivity 112000 dpm/pmol total COz). At the beginning and at the end of the experiment, 40 ml gas samples were transferred into an evacuated 60-ml stoppered vial containing 5 ml of 5 M KOH, and 14C02 was quantitatively absorbed by 3 h shaking. The remaining gas phase, which contain-

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.

Carbohydrate synthesis from acetyl CoA in the autotroph Methanobacterium thermoautotrophicum

AbstractMethanobacterium thermoautotrophicum assimilates CO2 via a novel pathway which involves the synthesis of acetyl CoA from 2 CO2. The pathway of carbohydrate synthesis in this autotroph starting from acetyl CoA and CO2 was studied using, (I) 14CO2 pulse-labeling, (II) [14C]pyruvate long term labelling, and (III) enzyme studies.(I)The distribution of radioactivity incorporated from 14CO2 by an exponentially growing culture during 2 s–120 s incubation periods has been analysed with respect to 3-phosphoglyceric acid and sugar phosphates. Radioactivity first appeared in 3-phosphoglyceric acid and only later in sugar phosphates. Fructose and glucose phosphates were among the earliest labeled carbohydrates followed by pentose phosphates and other sugar phosphates.(II)When the organism was grown in the presence of [2-14C]pyruvate during several generations, radioactivity was incorporated into alanine and glucosamine in a ratio of 1:2. Alanine contained the label at C-2, whilst glucosamine was equally labeled at C-2 and at C-5.(III)The following enzymatic activities were detected in cell extracts with specific activities being sufficiently high to account for the in vivo rate of carbohydrate synthesis: pyruvate synthase, phosphoenolpyruvate synthetase, phosphoenolpyruvate carboxylase, enolase, phosphoglycerate mutase, phosphoglycerate kinase, glyceraldehydephosphate dehydrogenase.nThe data indicate that glucogenesis from acetyl CoA and CO2 in Methanobacterium involves the pathway depicted in Fig 1.

Autotrophic acetyl coenzyme A synthesis in vitro from two CO2 in Methanobacterium

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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.