Autotrophic synthesis of activated acetic acid from two CO2 inMethanobacterium thermoautotrophicum

Stupperich, Erhard; Fuchs, Georg

doi:10.1007/bf00692704

Cited by 45 publications

(29 citation statements)

References 32 publications

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“…The pathway for the reduction of CO2 to the methyl level in methanogenesis is similar, in some ways, to this acetogenic path, in that THMP is used as the carbon carrier at three oxidation levels (8,28). CO2 is reduced to the levels of CH3-THMP, and serves as a donor of the acetate methyl group, and another CO2 is reduced by a different path to become the carboxyl (25). Thus, it is interesting that the methanogens differ from the acetogens in not being able to directly incorporate formate into the methyl group of acetate.…”

Section: Resultsmentioning

confidence: 99%

Source of carbon and hydrogen in methane produced from formate by Methanococcus thermolithotrophicus

Sparling¹,

Daniels²

1986

J Bacteriol

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Methanococcus thermolithotrophicus is able to produce methane either from H2-CO2 or from formate. The route of formate entry into the methanogenic pathway was investigated by using 2H20 or [I3C] With few exceptions, all methanogens can use H2-CO2 as their sole source of carbon and energy (6). The pathway of reduction of CO2 to methane is for the most part understood in Methanobacterium thermoautotrophicum (8) and is expected to be similar for all other C02-reducing methanogens.

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Section: Resultsmentioning

confidence: 99%

Source of carbon and hydrogen in methane produced from formate by Methanococcus thermolithotrophicus

Sparling¹,

Daniels²

1986

J Bacteriol

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confidence: 99%

“…The primary C02-fixing pathway in M. thermoautotrophicum, the pathway for the biosynthesis of acetyl coenzyme A, is well documented (8,11,25,26). Formate does not appear to be a free intermediate for incorporation into either carbon of the acetyl group (11,25,26). Determination of a biosynthetic role for formate in M. thermoautotrophicum by substitution with other compounds may be difficult, since this microorganism probably lacks appropriate uptake mechanisms (7,12), growth can be inhibited by relatively high (millimolar) concentrations of organic compounds in media (12,13), and more than one compound may be required.…”

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confidence: 99%

Formate auxotroph of Methanobacterium thermoautotrophicum Marburg

1989

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A formate-requiring auxotroph of Methanobacterium thermoautotrophicum Marburg was isolated after hydroxylamine mutagenesis and bacitracin selection. The requirement for formate is unique and specific; combined pools of other volatile fatty acids, amino acids, vitamins, and nitrogen bases did not substitute for formate. Compared with those of the wild type, cell extracts of the formate auxotroph were deficient in formate dehydrogenase activity, but cells of all of the strains examined catalyzed a formate-carbon dioxide exchange activity. All of the strains examined took up a small amount (200 to 260 ,umol/liter) of formate (3 mM) added to medium. The results of the study of this novel auxotroph indicate a role for formate in biosynthetic reactions in this methanogen. Moreover, because methanogenesis from H2-C02 is not impaired in the mutant, free formate is not an intermediate in the reduction of CO2 to CH4.The autotrophic, thermophilic archaebacterium Methanobacterium thermoautotrophicum has several attractive features for the development of a genetic system in a methanogen, including well-characterized mutants (19,21), the presence of a cryptic plasmid (17) which may be suitable for the development of a plasmid vector (18), and a primitive natural transformation system for genetic exchange (30). While much of the research into developing a genetic exchange system in M. thermoautotrophicum in our laboratory has focused on mutants resistant to nitrogen base analogs (19, 28, 30; V. E. Worrell and D. P. Nagle, Jr., Abstr. Annu. Meet. Am. Soc. Microbiol. 1989, I22, p. 221; D. P. Nagle, Jr., Dev. Ind. Microbiol., in press), mutants with auxotrophic markers would also be useful for these investigations into the genetics of M. thermoautotrophicum. In this report we describe the isolation and partial characterization of a unique auxotroph, a formate-requiring strain of M. thermoautotrophicum.(A portion of this work has been presented elsewhere [R. S. Tanner and D. P. Nagle, Jr., Abstr. Annu. Meet. Am. Soc. Microbiol. 1988, I10, p. 182].) MATERIALS AND METHODS Strains and media. Strains of M. thermoautotrophicum used in this study were AH (ATCC 29096), obtained from R. S. Wolfe; Marburg (DSM 2133), obtained from H. Hippe; Kr, a kanamycin-resistant derivative of Marburg; and RT-103, a formate-requiring derivative of Kr. M. thermoautotrophicum Kr was isolated after prolonged incubation of a culture in medium containing 500 ,ug of kanamycin per ml. The MICs of kanamycin sulfate in basal medium for strains Marburg and Kr were 175 and 1,000 ,ug/ml, respectively.The basal medium contained the following, in grams per liter: NaCl, 0.8; NH4Cl, 1.0; KCI, 0.1; KH2PO4, 0.1; MgSO4. 7H20, 0.2; CaCl2 -2H20, 0.02; NaHCO3, 5.0; resazurin, 0.0005; cysteine hydrochloride, 0.4; and Na2S. 9H20, 0.4, as well as 10 ml of trace metal solution. The trace metal solution was adapted from that of Wolin et al. (29) and contained the following, in grams per liter: nitrilotriacetic acid, 2.0 (pH adjusted to 6 with KOH); MnSO4-H20, 1.0; Fe(NH4)2(SO4...

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“…Methanogens which are unable to grow autotrophically lack CO dehydrogenase and show a dependency on acetate as a carbon source (3,23). The biosynthesis of acetyl-CoA and CO2 reduction to methane share a common C1 pool (21). The methyl group of the acetate is derived from methyltetrahydromethanopterin, an intermediate in methanogenesis (7,14,19,30).…”

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Pseudoauxotrophy of Methanococcus voltae for acetate, leucine, and isoleucine

1988

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Methanococcus voltae is a methanogenic bacterium which requires leucine, isoleucine, and acetate for growth. However, it also can synthesize these amino acids, and it is capable of low levels of autotrophic acetyl coenzyme A (acetyl-CoA) biosynthesis. When cells were grown in the presence of '4CO2, as well as in the presence of compounds required for growth, the alanine found in the cellular protein was radiolabeled. The percentages of radiolabel in the C-1, C-2, and C-3 positions of alanine were 64, 24, and 16%, respectively. The incorporation of radiolabel into the C-2 and C-3 positions of alanine demonstrated the autotrophic acetyl-CoA biosynthetic pathway in this bacterium. Additional evidence was obtained in cell extracts in which autotrophically synthesized acetyl-CoA was trapped into lactate. In these extracts, both CO and CH20 stimulated acetyl-CoA synthesis. 14CH20 was specifically incorporated into the C-3 of lactate. Cell extracts of M. voltae also contained low levels of CO dehydrogenase, 13 nmol min-' mg of protein-'. These results further confirmed the presence of the autotrophic acetyl-CoA biosynthetic pathway in M. voltae. Likewise, '4Co2 and [U-_4C]acetate were also incorporated into leucine and isoleucine during growth. During growth with[U_14C]leucine or [U-_4C]isoleucine, the specific radioactivity of these amino acids in the culture medium declined, and the specffic radioactivities of these amino acids recovered from the cellular protein were 32 to 40% lower than the initial specific radioactivities in the medium. Cell extracts of M. voltae also contained levels of isopropyl malate synthase, an enzyme that is specific to the leucine biosynthetic pathway, of 0.8 nmol min' mg of protein-1. Thus, M. voltae is capable of autotrophic CO2 fixation and leucine and isoleucine biosynthesis.

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Autotrophic synthesis of activated acetic acid from two CO2 inMethanobacterium thermoautotrophicum

Cited by 45 publications

References 32 publications

Source of carbon and hydrogen in methane produced from formate by Methanococcus thermolithotrophicus

Source of carbon and hydrogen in methane produced from formate by Methanococcus thermolithotrophicus

Formate auxotroph of Methanobacterium thermoautotrophicum Marburg

Pseudoauxotrophy of Methanococcus voltae for acetate, leucine, and isoleucine

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