Transduction in the archaebacterium Methanobacterium thermoautotrophicum Marburg.

The virulent bacteriophage psi M1 of Methanobacterium thermoautotrophicum Marburg mediated transduction of a resistance marker and of three biosynthesis markers. Transductants were observed at frequencies of 6 x 10(-4) to 5 x 10(-6)/PFU.     

Genetic and physiological characterization of the purine salvage pathway in the archaebacterium Methanobacterium thermoautotrophicum Marburg.

The enzymes involved in the purine interconversion pathway of wild-type and purine analog-resistant strains of Methanobacterium thermoautotrophicum Marburg were assayed by radiometric and spectrophotometric methods. Wild-type cells incorporated labeled adenine, guanine, and hypoxanthine, whereas mutant strains varied in their ability to incorporate these bases. Adenine, guanine, hypoxanthine, and xanthine were activated by phosphoribosyltransferase activities present in wild-type cell extracts. Some mutant strains simultaneously lost the ability to convert both guanine and hypoxanthine to the respective nucleotide, suggesting that the same enzyme activates both bases. Adenosine, guanosine, and inosine phosphorylase activities were detected for the conversion of base to nucleoside. Adenine deaminase activity was detected at low levels. Guanine deaminase activity was not detected. Nucleoside kinase activities for the conversion of adenosine, guanosine, and inosine to the respective nucleotides were detected by a new assay. The nucleotide-interconverting enzymes AMP deaminase, succinyl-AMP synthetase, succinyl-AMP lyase, IMP dehydrogenase, and GMP synthetase were present in extracts; GMP reductase was not detected. The results indicate that this autotrophic methanogen has a complex system for the utilization of exogenous purines.     

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.     

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.     

Physical map of the Methanobacterium thermoautotrophicum Marburg chromosome.

A physical map of the Methanobacterium thermoautotrophicum Marburg chromosome was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by NotI, PmeI, and NheI. The order of the fragments was deduced from Southern blot hybridization of NotI fragment probes to various restriction digests and from partial digests. The derived map is circular, and the genome size was estimated to be 1,623 kb. Several cloned genes were hybridized to restriction fragments to locate their positions on the map. Genes coding for proteins involved in the methanogenic pathway were located on the same segment of the circular chromosome. In addition, the genomes of a variety of thermophilic Methanobacterium strains were treated with restriction enzymes and analyzed by pulsed-field gel electrophoresis. The sums of the fragment sizes varied from 1,600 to 1,728 kb among the strains, and widely different macrorestriction patterns were observed.     

Halotolerance of Methanobacterium thermoautotrophicum delta H and Marburg.

Methanobacterium thermoautotrophicum delta H and Marburg were adapted to grow in medium containing up to 0.65 M NaCl. From 0.01 to 0.5 M NaCl, there was a lag before cell growth which increased with increasing external NaCl. The effect of NaCl on methane production was not significant once the cells began to grow. Intracellular solutes were monitored by nuclear magnetic resonance (NMR) spectroscopy as a function of osmotic stress. In the delta H strain, the major intracellular small organic solutes, cyclic-2,3-diphosphoglycerate and glutamate, increased at most twofold between 0.01 and 0.4 M NaCl and decreased when the external NaCl was 0.5 M. M. thermoautotrophicum Marburg similarly showed a decrease in solute (cyclic-2,3-diphosphoglycerate, 1,3,4,6-tetracarboxyhexane, and L-alpha-glutamate) concentrations for cells grown in medium containing > 0.5 M NaCl. At 0.65 M NaCl, a new organic solute, which was visible in only trace amounts at the lower NaCl concentrations, became the dominant solute. Intracellular potassium in the delta H strain, detected by atomic absorption and 39K NMR, was roughly constant between 0.01 and 0.4 M and then decreased as the external NaCl increased further. The high intracellular K+ was balanced by the negative charges of the organic osmolytes. At the higher external salt concentrations, it is suggested that Na+ and possibly Cl- ions are internalized to provide osmotic balance. A striking difference of strain Marburg from strain delta H was that yeast extract facilitated growth in high-NaCl-containing medium. The yeast extract supplied only trace NMR-detectable solutes (e.g., betaine) but had a large effect on endogenous glutamate levels, which were significantly decreased. Exogenous choline and glycine, instead of yeast extract, also aided growth in NaCl-containing media. Both solutes were internalized with the choline converted to betaine; the contribution to osmotic balance of these species was 20 to 25% of the total small-molecule pool. These results indicate that M. thermoautotrophicum shows little changes in its internal solutes over a wide range of external NaCl. Furthermore, they illustrate the considerable differences in physiology in the delta H and Marburg strains of this organism.     

Characterization of a superoxide dismutase gene from the archaebacterium Methanobacterium thermoautotrophicum

A gene encoding superoxide dismutase (SOD) was cloned from the archaebacterium Methanobacterium thermoautotrophicum, the first example from an anaerobic bacterium. The deduced amino acid sequence showed overall similarity to sequences of known Mn- and Fe-SODs from aerobic organisms. Judging from a detailed sequence comparison, the cloned SOD gene is classified as Mn-SOD. By comparison of Mn-SOD sequences among various species it was suggested that archaebacterial superoxide dismutase is a direct descendant of a primordial enzyme. Between a putative promoter and the start codon there is an inverted repeat sequence which is also found in the counterpart of Halobacterium halobium.     

Unique characteristics of superoxide dismutase of a strictly anaerobic archaebacterium Methanobacterium thermoautotrophicum

The superoxide dismutase (SOD) gene of Methanobacterium thermoautotrophicum (Takao, M., Oikawa, A., and Yasui, A. (1990) Arch. Biochem. Biophys. 283, 210-216), a strictly anaerobic archaebacterium, was expressed in Escherichia coli. The gene product accounted for more than 30% of the host's soluble protein. The purified protein was an active iron-containing tetrameric SOD with specific activity similar to known manganese-containing SODs (MnSODs) of aerobic archaebacteria. Although M. thermoautotrophicum SOD is an iron-containing SOD (FeSOD), it resembles MnSODs in amino acid sequence as judged by criteria distinguishing FeSODs from MnSODs. Moreover, M. thermoautotrophicum SOD is resistant to azide and hydrogen peroxide as MnSODs are, suggesting that its evolution is distinct from known eubacterial FeSODs.     

Coenzyme F390 synthetase from Methanobacterium thermoautotrophicum Marburg belongs to the superfamily of adenylate-forming enzymes.

Depending on the reduction-oxidation state of the cell, some methanogenic bacteria synthesize or hydrolyze 8-hydroxyadenylylated coenzyme F420 (coenzyme F390). These two reactions are catalyzed by coenzyme F390 synthetase and hydrolase, respectively. To gain more insight into the mechanism of the former reaction, coenzyme F390 synthetase from Methanobacterium thermoautotrophicum Marburg was purified 89-fold from cell extract to a specific activity of 0.75 mumol.min-1.mg of protein-1. The monomeric enzyme consisted of a polypeptide with an apparent molecular mass of 41 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ftsA, the gene encoding coenzyme F390 synthetase, was cloned and sequenced. It encoded a protein of 377 amino acids with a predicted M(r) of 43,280. FtsA was found to be similar to domains found in the superfamily of peptide synthetases and adenylate-forming enzymes. FtsA was most similar to gramicidin S synthetase II (67% similarity in a 227-amino-acid region) and sigma-(L-alpha-aminoadipyl)-L-cysteine-D-valine synthetase (57% similarity in a 193-amino-acid region). Coenzyme F390 synthetase, however, holds an exceptional position in the superfamily of adenylate-forming enzymes in that it does not activate a carboxyl group of an amino or hydroxy acid but an aromatic hydroxyl group of coenzyme F420.     

Characterization of the Superoxide dismutase gene and its upstream region from Methanobacterium thermoautotrophicum Marburg

Abstract A gene ( sod ) encoding Superoxide dismutase (SOD) was isolated from the strictly anaerobic archaeon Methanobacterium thermoautotrophicum Marburg. Its identity was confirmed by functional complementation of an Escherichia coli mutant strain lacking SOD activity and by DNA sequence analysis of a cloned fragment. Upstream of sod , separated by a 5-bp intergenic region, lies the open reading frame orfk which potentially codes for a protein of 209 amino acid residues. The amino acid sequence for this presumptive product had a similarity coefficient of 55.5% to a subunit of the alkyl hydroperoxide reductase (encoded by the ahpC gene) from Salmonella typhimurium .     

Purification and structural characterization of a flavoprotein induced by iron limitation in Methanobacterium thermoautotrophicum Marburg.

Cells of Methanobacterium thermoautotrophicum (strain Marburg) grown under iron-limiting conditions were found to synthesize a soluble polypeptide as one of the major cell proteins. This polypeptide purified as a homotetramer (170 kDa [subunit molecular mass, 43 kDa]) had a UV-visible spectrum typical of flavoproteins and contained 0.7 mol of flavin mononucleotide per mol of monomer. Quantitative analysis by immunoblotting with polyclonal antibodies indicated that the flavoprotein, which amounts to about 0.6% of soluble cell protein under iron-sufficient conditions (> or = 50 microM Fe2+), was induced fivefold by iron limitation (< 12 microM Fe2+). The flavoprotein-encoding gene, fprA, was cloned and sequenced. Sequence analysis revealed a well-conserved archaebacterial consensus promoter upstream of fprA, a flavodoxin signature within fprA, and 28% amino acid identity with a putative flavin mononucleotide-containing protein of Rhodobacter capsulatus which is found within an operon involved in nitrogen fixation. A possible physiological function for the flavoprotein is discussed.     

Two genetically distinct methyl-coenzyme M reductases in Methanobacterium thermoautotrophicum strain Marburg and delta H

Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step in methanogenic archaebacteria. The reductase has been characterized in detail from Methanobacterium thermoautotrophicum strain Marburg and delta H, which grow on H2 and CO2 as energy source. During purification of the enzyme we have now discovered a second methyl-coenzyme M reductase (MCR II) in the two strains, which elutes at lower salt concentration from anion-exchange columns than the enzyme (MCR I) previously characterized. MCR II is similar to MCR I in that it is also composed of three different subunits alpha, beta, and gamma but distinct from MCR I in that the gamma subunit is 5 kDa smaller, as revealed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of the alpha, beta, and gamma subunits of MCR II and MCR I were found to be different in several amino acid positions. The respective sequences showed, however, strong similarities indicating that MCR II was not derived from MCR I by limited proteolysis. The relative amounts of MCR I and MCR II present in the cells were affected by the growth conditions. When the cultures were supplied with sufficient H2 and and CO2 and the cells grew exponentially, essentially only MCR II was found. When growth was limited by the gas supply, MCR I predominated.     

Purification and properties of heterodisulfide reductase from Methanobacterium thermoautotrophicum (strain Marburg)

The reduction of the heterodisulfide of coenzyme M (H-S-CoM) and 7-mercaptoheptanoyl-L-threonine phosphate (H-S-HTP) is a key reaction in the metabolism of methanogenic bacteria. The heterodisulfide reductase catalyzing this step was purified 80-fold to apparent homogeneity from Methanobacterium thermoautotrophicum. The native enzyme showed an apparent molecular mass of 550 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of three different subunits of apparent molecular masses 80 kDa, 36 kDa, and 21 kDa. The enzyme, which was brownish yellow, contained per mg protein 7 +/- 1 nmol FAD, 130 +/- 10 nmol non-heme iron and 130 +/- 10 nmol acid-labile sulfur, corresponding to 4 mol FAD and 72 mol FeS/mol native enzyme. The purified heterodisulfide reductase catalyzed the reduction of CoM-S-S-HTP (app. Km = 0.1 mM) with reduced benzylviologen at a specific rate of 30 mumol.min-1.mg protein-1 (kcat = 68 s-1) and the reduction of methylene blue with H-S-CoM (app. Km = 0.2 mM) plus H-S-HTP (app. Km less than 0.05 mM) at a specific rate of 15 mumol.min-1.mg-1. The enzyme was highly specific for CoM-S-S-HTP and H-S-CoM plus H-S-HTP. The physiological electron donor/acceptor remains to be identified.     

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.     

Physical and genetic map of the Methanobacterium wolfei genome and its comparison with the updated genomic map of Methanobacterium thermoautotrophicum Marburg

A physical and genetic map of the chromosome of Methanobacterium wolfei was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by digestion with NotI and NheI. The chromosome was found to be circular and 1,729 kb in size. Twenty-eight genes were mapped to specific restriction enzyme fragments by performing hybridization experiments with gene probes from various Methanobacterium strains. The genomic map obtained was compared with the updated genomic map of Methanobacterium thermoautotrophicum Marburg. In spite of major restriction pattern dissimilarities, the overall genetic organization seemed to be conserved between the genomes of the two strains. In addition, the two rRNA operons of strain Marburg were precisely mapped on the chromosome, and it was shown that they are transcribed in the same direction.