Sodium dependence of growth and methane formation in Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum was found to require sodium for growth and for CO₂ reduction to methane. The dependence of the rate of growth and methane formation on the sodium concentration was hyperbolic with an apparent K_s for sodium of approximately 1 mM. The findings indicate that sodium has a specific function in the energy metabolism of this bacterium.     

Carbon monoxide production by Methanobacterium thermoautotrophicum

Abstract Methanobacterium thermoautotrophicum was grown in a fermenter gassed with an 80% H₂/20% CO₂ mixture. The effluent gas was found to contain between 30 ppm and 90 ppm carbon monoxide. Approx. 5 nmol CO were produced per min and mg cells (dry weight) by the culture. This is to our knowledge the first report on biological carbon monoxide formation under strictly anaerobic conditions.     

Origin of hydrogen in methane produced by Methanobacterium thermoautotrophicum.

The production of deuterated methane by Methanobacterium thermoautotrophicum in H2O-D2O mixtures was examined by high-resolution mass spectrometry. The hydrogen in the methane arose solely from water and not from hydrogen gas. Hydrogen gas served only as an electron source in methanogenesis. A whole-cell product isotope discrimination of 1.5 favoring hydrogen over deuterium was observed in methane production in 81 atom% deuterated water. The distribution of deuterated methane species is described by a simple model of the overall reaction.     

Tungstate can substitute for molybdate in sustaining growth of Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum (strain Marburg) was found to grow on media supplemented with tungstate rather than with molybdate. The Archaeon then synthesized a tungsten iron-sulfur isoenzyme of formylmethanofuran dehydrogenase. The isoenzyme was purified to apparent homogeneity and shown to be composed of four different subunits of apparent molecular masses 65 kDa, 53 kDa, 31 kDa, and 15 kDa and to contain per mol 0.4 mol tungsten, <0.05 mol molybdenum, 8 mol non-heme iron, 8 mol acid-labile sulfur and molybdopterin guanine dinucleotide. Its molecular and catalytic properties were significantly different from those of the molybdenum isoenzyme characterized previously. The two isoenzymes also differed in their metal specificity: the active molybdenum isoenzyme was only synthesized when molybdenum was available during growth whereas the active tungsten isoenzyme was also generated during growth of the cells on molybdate medium. Under the latter conditions the tungsten isoenzyme was synthesized containing molybdenum rather than tungsten.Abbreviations MFR methanofuran - CHO-MFR N-formylmethanofuran - MGD molybdopterin guanine dinucleotide - MAD molybdopterin adenine dinucleotide - MHD molybdopterin hypoxanthine dinucleotide - FPLC fast protein liquid chromatography - SDS/PAGE sodium dodecylsulfate/polyacrylamide gel electrophoresis - ICP-MS inductively coupled plasma mass spectrometry     

Photoreactivation in Methanobacterium thermoautotrophicum

The sensitivity of three methanogenic bacteria towards ultraviolet irradiation was similar to the UV-sensitivity of Escherichia coli. The lethal effects of UV-irradiation in Methanobacterium thermoautotrophicum Marburg and in Methanobacterium thermoautotrophicum H but not in Methanococcus vannielii were reversed by exposure to visible light. In cell suspensions of Methanobacterium thermoautotrophicum that had been irradiated to 0.1% survival, 90% of the UV-caused damage was photorepairable. The in vivo action spectrum for photoreactivation suggests that in this organism a deazaflavin, probably F₄₂₀, functions as the chromophore of the photoreactivating enzyme.     

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.     

Methane production by the membranous fraction of Methanobacterium thermoautotrophicum.

Intact membrane vesicles are required to synthesize methane from CO2 and H2 by disrupted preparations of Methanobacterium thermoautotrophicum cells. When membrane vesicles were removed by high-speed centrifugation at 226 600 g, the remaining supernatant fraction no longer synthesized methane. Alternatively, if vesicle structure was disrupted by passage through a Ribi cell fractionator at very high pressures (345 MPa), the bacterial cell extract, with all the particulate fraction in it, did not synthesize methane. Methyl-coenzyme M, a new coenzyme first described by McBride & Wolfe [(1971) Biochemistry 10, 2317--2324], was shown to stimulate methane production from CO2 and H2, as previously reported, but the methyl group of the coenzyme did not appear to be a precursor of methane in this reaction. No methyl-coenzyme M reductase activity was detected in the cytoplasmic fraction of M. thermoautotrophicum cells.     

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.     

Purification and use of Methanobacterium wolfei pseudomurein endopeptidase for lysis of Methanobacterium thermoautotrophicum.

The pseudomurein-degrading enzyme from autolysates of Methanobacterium wolfei was purified approximately 500-fold to electrophoretic homogeneity by ion-exchange chromatography under anaerobic conditions. Analysis of the soluble cell wall fragments produced by the pure enzyme from a cell wall preparation of M. thermoautotrophicum indicated that it is a peptidase hydrolyzing the epsilon-Ala-Lys bond of pseudomurein. A partially purified preparation of pseudomurein endopeptidase was free of nuclease activity and thus proved useful for the preparation in high yields of undegraded chromosomal and plasmid DNA from M. thermoautotrophicum. The partially purified enzyme was also used for the preparation of protoplasts, which were stabilized by 0.8 M sucrose. Under growth conditions the protoplasts produced methane and increased up to 100-fold in size, but failed to regenerate a cell wall.     

Tetrahydromethanopterin-dependent serine transhydroxymethylase from Methanobacterium thermoautotrophicum

Serine transhydroxymethylase of Methanobacterium thermoautotrophicum has been purified to within 95% of homogeneity. Activity was strictly dependent on tetrahydromethanopterin, tetrahydrofolate being unable to serve as the acceptor C₁ units from l-serine. The native protein has a molecular weight of about 102,000 daltons. The enzyme shows maximal activity at 60°C, has a pH optimum of 8.1, and required pyridoxal-5-phosphate and Mg²⁺ for optimal activity.     

5-Fluorouracil-resistant strain of Methanobacterium thermoautotrophicum.

Growth of Methanobacterium thermoautotrophicum Marburg is inhibited by the pyrimidine, 5-fluorouracil (FU). It was shown previously that methanogenesis is not inhibited to the same extent as growth. A spontaneously occurring FU-resistant strain (RTAE-1) was isolated from a culture of strain Marburg. The growth of both strains was inhibited by 5-fluorodeoxyuridine but not 5-fluorocytosine, and the wild type was more susceptible to inhibition by 5-azauracil and 6-azauracil than was strain RTAE-1. The cellular targets for the pyrimidine analogs are not known. When the accumulation of 14C-labeled uracil or FU by the two strains was compared, the wild type took up 15-fold more radiolabel per cell than did the FU-resistant strain. In the wild type, radiolabel from uracil was incorporated into the soluble pool, RNA, and DNA. The metabolism of uracil appeared to involve a uracil phosphoribosyltransferase activity. Strain Marburg extracts contained this enzyme, whereas FU-resistant strain RTAE-1 extracts had less than 1/10 as much activity. Although it is possible that a change in permeability to the compounds plays a role in the stable resistance of strain RTAE-1, the fact that it lacks the ability to metabolize pyrimidines to nucleotides is sufficient to account for its phenotype.     

Purification and properties of Methanobacterium thermoautotrophicum DNA photolyase

We have purified DNA photolyase from the autotrophic anaerobic archaebacterium Methanobacterium thermoautotrophicum to near homogeneity by a two-column affinity chromatography. The purified enzyme has an Mr = 60,000 and shows near UV absorption peak at 440 nm and a fluorescence emission maximum at 462 nm indicating that it contains 8-hydroxy-5-deazaflavin (coenzyme F420) as an intrinsic chromophore. The photolyase binds with high specificity to thymine dimer in DNA with an equilibrium binding constant, KA = 1.4 x 10(9) M-1, and a dissociation rate constant, koff = 1.4 x 10(-4) s-1 (t1/2 = 43 min). Despite 6-fold higher affinity compared to the folate-containing Escherichia coli photolyase the two enzymes apparently contact the same phosphates around the thymine dimer: the phosphate immediately 5' and the three phosphates immediately 3' to the dimer on the damaged strand and the phosphate across from the dimer in the minor groove on the complementary strand. The absolute action spectrum of the Methanobacterium photolyase in the 400-500-nm region closely matches the absorption of the enzyme-bound F420. The quantum yield (phi) over this region is constant and is approximately 0.2. The value is measurably smaller than the quantum yields reported for other DNA photolyases.     

Formate auxotroph of Methanobacterium thermoautotrophicum Marburg.

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 mumol/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-CO2 is not impaired in the mutant, free formate is not an intermediate in the reduction of CO2 to CH4.     

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.     

A plasmid in the archaebacterium Methanobacterium thermoautotrophicum

The archaebacterium Methanobacterium thermoautotrophicum Marburg (DSM 2133) was found to contain a plasmid (pME2001) in covalently closed circular form. It was isolated by CsCl gradient centrifugation of total DNA in the presence of ethidium bromide. Multimers up to the hexamer were observed upon agarose gel electrophoresis and electron microscopy of a purified plasmid preparation. A restriction map was constructed. The length of plasmid pME2001 was determined to be approximately 4,500 bp. Southern hybridization of plasmid DNA to DNA extracted from Methanobacterium thermoautotrophicum delta H (DSM1053) revealed the presence of a plasmid with homologous sequences in the delta H strain.     

DNA relatedness among some thermophilic members of the genus Methanobacterium: emendation of the species Methanobacterium thermoautotrophicum and rejection of Methanobacterium thermoformicicum as a synonym of Methanobacterium thermoautotrophicum.

DNA reassociation was used to determine levels of relatedness among four thermophilic Methanobacterium strains that are able to use formate and between these organisms and two representative strains of Methanobacterium thermoautotrophicum, strain delta HT (= DSM 1053T = ATCC 29096T) (T = type strain) and strain Marburg (= DSM 2133). Three homology groups were delineated, and these groups coincided with the clusters identified by antigenic fingerprinting. The first group, which had levels of cross hybridization that ranged from 73 to 99%, included M. thermoautotrophicum delta HT, Methanobacterium thermoformicicum Z-245, Methanobacterium sp. strain THF, and Methanobacterium sp. strain FTF. The second and third groups were each represented by only one strain, Methanobacterium sp. strain CB-12 and M. thermoautotrophicum Marburg, respectively (cross-hybridization levels, 13 to 30 and 29 to 33%, respectively). Our results indicate that the name M. thermoformicicum should be rejected as it is a synonym of M. thermoautotrophicum. The taxonomic positions of strains Marburg and CB-12 need further investigation.