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 -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 -ketoglutarate.     

Different effects of 5-fluorouracil on Methanosarcina barkeri and on Methanobacterium thermoautotrophicum

Abstract Growth of Methanosarcina barkeri (strain Fusaro) was found to be inhibited by 5-fluorouracil (FU) only at relatively high concentrations (>50 μg / ml ). Inhibition could not be relieved by uracil. Therefore, FU probably did not exert its effect via inhibition of DNA synthesis as is the case in other organisms. Control experiments with Methanobacterium thermoautotrophicum (strain Marburg) on the other hand revealed that the effect of FU on this archaebacterium is probably exerted at the level of nucleic acid synthesis. The M. thermoautotrophicum cultures rapidly acquired resistance towards the pyramidine analog.     

Formyl-methanofuran synthesis in Methanobacterium thermoautotrophicum

Abstract The initial step of methanogenesis from CO₂ is the formation of formyl-methanofuran (formyl-MFR) from methanofuran (MFR), CO₂ and H₂. The enzymology of this novel type of CO₂ fixation reaction has been difficult to study because formyl-MFR synthesis is subject to a complex activation. Recently, however, a number of advances have made questions regarding formyl-MFR synthesis more approachable.     

Ammonia assimilation and glutamate incorporation in coenzyme F420 derivatives ofMethanosarcina barkeri

Methanosarcina barkeri was able to grow on L-alanine and L-glutamate as sole nitrogen sources. Cell yields were 0.5 g/l and 0.7 g/l (wet wt), respectively. The mechanism of ammonia assimilation inMethanosarcina barkeri strain MS was studied by analysis of enzyme activities. Activity levels of nitrogen-assimilating enzymes in extracts of cells grown on different nitrogen sources (ammonia, 0.05–100 mM; L-alanine, 10 mM; L-glutamate, 10 mM) were compared. Activities of glutamate dehydrogenase, glutamate synthase, glutamine synthetase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase could be measured in cells grown on these three nitrogen sources. Alanine dehydrogenase was not detected under the growth conditions used. None of the measured enzyme activities varied significantly in response to the NH₄ ⁺ concentration. The length of the poly--glutamyl side chain of F₄₂₀ derivatives turned out to be independent of the concentration of ammonia in the culture medium.Abbreviations ADH alanine dehydrogenase - FO 7,8-didemethyl-8-hydroxy-5-deazariboflavin - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase - GS glutamine synthetase - H₄MPT tetrahydromethanopterin     

Acetate assimilation pathway of Methanosarcina barkeri.

The pathway of acetate assimilation in Methanosarcina barkeri was determined from analysis of the position of label in alanine, aspartate, and glutamate formed in cells grown in the presence of [14C]acetate and by measurement of enzyme activities in cell extracts. The specific radioactivity of glutamate from cells grown on [1-14C]- or [2-14C]acetate was approximately twice that of aspartate. The methyl and carboxyl carbons of acetate were incorporated into aspartate and glutamate to similar extents. Degradation studies revealed that acetate was not significantly incorporated into the C1 of alanine, C1 or C4 of aspartate, or C1 of glutamate. The C5 of glutamate, however, was partially derived from the carboxyl carbon of acetate. Cell extracts were found to contain the following enzyme activities, in nanomoles per minute per milligram of protein at 37 degrees C: F420-linked pyruvate synthase, 170; citrate synthase, 0.7; aconitase, 55; oxidized nicotinamide adenine dinucleotide phosphate-linked isocitrate dehydrogenase, 75; and oxidized nicotinamide adenine dinucleotide-linked malate dehydrogenase, 76. The results indicate that M. barkeri assimilates acetate into alanine and aspartate via pyruvate and oxaloacetate and into glutamate via citrate, isocitrate, and alpha-ketoglutarate. The data reveal differences in the metabolism of M. barkeri and Methanobacterium thermoautotrophicum and similarities in the assimilation of acetate between M. barkeri and other anaerobic bacteria, such as Clostridium kluyveri.     

Formyl-methanofuran synthesis in Methanobacterium thermoautotrophicum

The initial step of methanogenesis from CO2 is the formation of formyl-methanofuran (formyl-MFR) from methanofuran (MFR), CO2 and H2. The enzymology of this novel type of CO2 fixation reaction has been difficult to study because formyl-MFR synthesis is subject to a complex activation. Recently, however, a number of advances have made questions regarding formyl-MFR synthesis more approachable.     

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 K _m value for acetate was 40 M. 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.     

Activation of formylmethanofuran synthesis in cell extracts of Methanobacterium thermoautotrophicum.

In cell extracts of Methanobacterium thermoautotrophicum, formylmethanofuran (formyl-MFR) synthesis (an essential CO2 fixation reaction that is an early step in CO2 reduction to methane) is subject to a complex activation that involves a heterodisulfide of coenzyme M and N-(7-mercaptoheptanoyl)threonine O3-phosphate (CoM-S-S-HTP). In this paper we report that titanium(III) citrate, a low-potential reducing agent, stimulated CO2 reduction to methane and activated formyl-MFR synthesis in cell extracts. Titanium(III) citrate functioned as the sole source of electrons for formyl-MFR synthesis and enabled this reaction to occur independently of CoM-S-S-HTP. In addition, CoM-S-S-HTP was found to activate an unknown electron carrier that reduced metronidazole. The activation of formyl-MFR synthesis by CoM-S-S-HTP may involve the activation of a low-potential electron carrier.     

15N-NMR studies of Methanobacterium thermoautotrophicum: comparison of assimilation of different nitrogen sources

Methanobacterium thermoautotrophicum can utilize glutamine and urea as well as ammonia as the sole nitrogen source during growth on H2 and CO2. High-field 15N-NMR has been used to compare the assimilation of these different nitrogen sources by this organism. The 15N-NMR spectra of extracts of cells grown in media containing [delta-15N]glutamine as the nitrogen source show that the glutamine amide nitrogen is rapidly converted to glutamate. The 15N-NMR spectra of cell extracts from cells grown on [15N]urea show a marked increase in the labeling of the alpha-NH2 of glutamate concurrent with a decrease in the urea resonance. These two nitrogen sources do not show the metabolic shift to alanine as the major resonance in stationary phase as is seen with 15NH4Cl. This behavior is discussed in terms of the enzymes of nitrogen metabolism.     

Methanogenesis and ATP synthesis in a protoplast system of Methanobacterium thermoautotrophicum.

When Methanobacterium thermoautotrophicum cells were incubated in 50 mM potassium phosphate buffer (pH 7.0) containing 1 M sucrose and autolysate from Methanobacterium wolfei, they were transformed into protoplasts. The protoplasts, which possessed no cell wall, lysed in buffer without sucrose. Unlike whole cells, the protoplasts did not show convoluted internal membrane structures. The protoplasts produced methane from H2-CO2 (approximately 1 mumol min-1 mg of protein-1) at about 50% the rate obtained for whole cells, and methanogenesis was coupled with ATP synthesis. Addition of the protonophore 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF-6847) to protoplast suspensions resulted in a dissipation of the membrane potential (delta psi), and this was accompanied by a parallel decrease in the rates of ATP synthesis and methanogenesis. In this respect protoplasts differed from whole cells in which ATP synthesis and methanogenesis were virtually unaffected by the addition of the protonophore. It is concluded that the insensitivity of whole cells to protonophores could be due to internal membrane structures. Membrane preparations produced from lysis of protoplasts or by sonication of whole cells gave comparatively low rates of methanogenesis (methylcoenzyme M methylreductase activity, less than or equal to 100 nmol of CH4 min-1 mg of protein-1), and no coupling with ATP synthesis could be demonstrated.     

Relationship of Intracellular Coenzyme F420 Content to Growth and Metabolic Activity of Methanobacterium bryantii and Methanosarcina barkeri

The use of F₄₂₀ as a parameter for growth or metabolic activity of methanogenic bacteria was investigated. Two representative species of methanogens were grown in batch culture: Methanobacterium bryantii (strain M.o.H.G.) on H₂ and CO₂, and Methanosarcina barkeri (strain Fusaro) on methanol or acetate. The total intracellular content of coenzyme F₄₂₀ was followed by high-resolution fluorescence spectroscopy. F₄₂₀ concentration in M. bryantii ranged from 1.84 to 3.65 μmol · g of protein⁻¹; and in M. barkeri grown with methanol it ranged from 0.84 to 1.54 μmol · g⁻¹ depending on growth conditions. The content of F₄₂₀ in M. barkeri was influenced by a factor of 2 depending on the composition of the medium (minimal or complex) and by a factor of 3 to 4 depending on whether methanol or acetate was used as the carbon source. A comparison of F₄₂₀ content with protein, cell dry weight, optical density, and specific methane production rate showed that the intracellular content of F₄₂₀ approximately followed the increase in biomass in both strains. In contrast, no correlation was found between specific methane production rate and intracellular F₄₂₀ content. However, qCH₄(F₄₂₀), calculated by dividing the methane production rate by the coenzyme F₄₂₀ concentration, almost paralleled qCH₄(protein). These results suggest that F₄₂₀ may be used as a specific parameter for estimating the biomass, but not the metabolic activity, of methanogens; hence qCH₄(F₄₂₀) determined in mixed populations with complex carbon substrates must be considered as measure of the actual methanogenic activity and not as a measure of potential activity.     

Tungstate does not support synthesis of active formylmethanofuran dehydrogenase in Methanosarcina barkeri

Cell extracts of Methanosarcina barkeri grown on methanol in media supplemented with molybdate exhibited a specific activity of formylmethanofuran dehydrogenase of approximately 1 U (1 mol/min)/mg protein. When the growth medium was supplemented with tungstate rather than with molybdate, the specific activity was only 0.04 U/mg. Despite this reduction in specific activity growth on methanol was not inhibited. An inhibition of both growth and synthesis of active formylmethanofuran dehydrogenase was observed, however, when H₂ and CO₂ were the energy substrates. The results indicate that, in contrast to Methanobacterium wolfei and Methanobacterium thermoautotrophicum, M. barkeri possesses only a molybdenum containing formylmethanofuran dehydrogenase and not in addition a tungsten isoenzyme.     

Ammonia assimilation and glutamate formation in Caulobacter crescentus.

In the dimorphic bacterium Caulobacter crescentus, ammonia assimilation occurs only via the combined action of the enzymes glutamine synthetase and glutamate synthase. Mutants auxotrophic for glutamate lacked glutamate synthase activity, and the mutations leading to the glutamate auxotrophy appeared to lie at two distinct genetic loci. Both glutamate synthase and glutamine synthetase activities were subject to regulation by repression. Glutamate synthase activity was highest in cultures grown in minimal medium with ammonia as sole nitrogen source and was about fivefold lower in rich broth. Glutamine synthetase activity was highest in cells grown with growth-rate-limiting amounts of ammonia as nitrogen source and was about fourfold lower in rich broth. In addition, glutamine synthetase activity appeared to be regulated by an adenylylation system like that described for Escherichia coli.     

Biosynthesis of nucleotides, flavins, and deazaflavins in Methanobacterium thermoautotrophicum

The biosynthesis of deazaflavins, flavins, ribonucleotides, and selected amino acids was studied in Methanobacterium thermoautotrophicum by incorporation of 13C-labeled acetate and pyruvate. 13C enrichments were monitored by 13C and 1H NMR spectroscopy. The biosynthesis of ribonucleotides follows the standard pathway. The xylene ring of riboflavin is formed from two pentose moieties in agreement with studies in yeasts and eubacteria. The pyrimidine ring and the ribityl side chain of the deazaflavin chromophore of coenzyme F420 are derived from the purine nucleotide pool. The phenolic ring and C-5 of the deazaflavin system are supplied by the shikimate pathway. A hypothetical mechanism for the assembly of the deazaflavin chromophore from 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione and 4-hydroxyphenyl-pyruvate is proposed.     

Biosynthesis of methanofuran in Methanobacterium thermoautotrophicum.

The 13C NMR signals of methanofuran were assigned by two-dimensional 1H and 13C NMR experiments. On this basis, the incorporation of 13C-labeled acetate and pyruvate into methanofuran by growing cells of Methanobacterium thermoautotrophicum was analyzed by one- and two-dimensional 13C NMR experiments. The data were analyzed by a retrobiosynthetic approach based on a comparison of labeling patterns in a variety of metabolites. The data show that the furan ring is formed by condensation of two molecules from the pyruvate/triose pool. The tetracarbocylic acid moiety is assembled from ketoglutarate, two molecules of acetyl CoA, and one molecule of carbon dioxide.