Biochemical analysis of neomycin-resistance in the methanoarchaeon Methanothermobacter thermautotrophicus and some implications for energetic processes in this strain

Methanogenesis-driven ATP synthesis in a neomycin-resistant mutant of Methanothermobacter thermautotrophicus (formerly Methanobacterium thermoautotrophicum strain DeltaH) was strongly inhibited at both pH 6.8 and pH 8.5 by the uncoupler 3,3',4',5 -tetrachlorosalicylanilide (TCS) in the presence of either 1 or 10 mM NaCl. The generation of a membrane potential in the mutant cells at pH 6.8 was also strongly inhibited by TCS in the presence of 1 or 10 mM NaCl. On the other hand, at pH 8.5 in the presence of 10mM NaCl, a protonophore-resistant membrane potential of approximately 150 mV was found. These results indicate that in the mutant cells the process of energy transduction between methanogenesis and membrane potential generation is not impaired. In contrast to the wild-type strain, ATP synthesis in the mutant cells was driven by an electrochemical gradient of H(+) under alkaline conditions. Unlike wild-type cells, the mutant lacks the capacity to transduce an uncoupler-resistant membrane potential energy at pH 8.5 into ATP synthesis. Na(+)/H(+) exchange was comparable in the wild type and the mutant cells. Western blots of sub-cellular fractions with polyclonal antiserum reactive to the B-subunit of the halobacterial A-type H(+)-translocating ATPase confirmed the presence of A-type ATP synthase in the mutant cells. Furthermore, in the mutant cells a protein band of molecular mass about 45 kDa is absent but there was an abundant protein band at about 67 kDa. Based on the observed bioenergetic features of the mutant cells, neither the A(1)A(o) ATP synthase alone nor together with the Na(+)/H(+) antiporter seems to be responsible for ATP synthesis driven by sodium motive force. Rather, some other links between neomycin-resistance and failure of sodium motive force-dependent ATP synthesis in the neomycin resistant mutant are discussed.     

Structural polymorphism of Methanothermobacter thermautotrophicus MCM

The minichromosome maintenance (MCM) proteins are essential for replication initiation and elongation in eukarya and archaea. There are six MCM proteins in eukaryotes, and MCM complexes are believed to unwind DNA during chromosomal DNA replication. However, the mechanism and structure of the MCM complexes are not known. Only one MCM is found in the archaeon Methanothermobacter thermautotrophicus (mtMCM), and this provides a simpler system for study. The crystal structure of a mtMCM N-terminal fragment has been solved, but surprisingly only subtle structural changes were seen between the wild-type protein and one having a mutation corresponding to the yeast MCM5 bob1 mutation. The bob1 mutation bypasses the phosphorylation required for activation of MCM in yeast. We have used electron microscopy and three-dimensional reconstruction to examine a number of different fragments of mtMCM, and can visualize a large conformational change within the N-terminal fragment. This offers new insight into the conformational dynamics of MCM and the phosphorylation-bypass phenotype in yeast.     

DNA content and nucleoid distribution in Methanothermobacter thermautotrophicus

Flow cytometry and epifluorescence microscopy results for the euryarchaeon Methanothermobacter thermautotrophicus were consistent with filaments containing multiple cells. Filaments of one to four cells contained two to eight nucleoids. Single chromosome-containing cells were not observed. Filaments containing multiple genome copies displayed synchronous DNA replication initiation. Chromosome segregation occurred during replication or rapidly after replication termination.     

Structural basis of the Methanothermobacter thermautotrophicus MCM helicase activity

The MCM complex from the archaeon Methanother-mobacter thermautotrophicus is a model for the eukaryotic MCM2-7 helicase. We present electron-microscopy single-particle reconstructions of a DNA treated M.thermautotrophicus MCM sample and a ADP·AlF_x treated sample, respectively assembling as double hexamers and double heptamers. The electron-density maps display an unexpected asymmetry between the two rings, suggesting that large conformational changes can occur within the complex. The structure of the MCM N-terminal domain, as well as the AAA+ and the C-terminal HTH dom-ains of ZraR can be fitted into the reconstructions. Distinct configurations can be modelled for the AAA+ and the HTH domains, suggesting the nature of the conformational change within the complex. The pre-sensor 1 and the helix 2 insertions, important for the activity, can be located pointing towards the centre of the channel in the presence of DNA. We propose a mechanistic model for the helicase activity, based on a ligand-controlled rotation of the AAA+ subunits.     

A recombinant exonuclease III homologue of the thermophilic archaeon Methanothermobacter thermautotrophicus

AP endonucleases catalyse an important step in the base excision repair (BER) pathway by incising the phosphodiester backbone of damaged DNA immediately 5' to an abasic site. Here, we report the cloning and expression of the 774 bp Mth0212 gene from the thermophilic archaeon Methanothermobacter thermautotrophicus, which codes for a putative AP endonuclease. The 30.3 kDa protein shares 30% sequence identity with exonuclease III (ExoIII) of Escherichia coli and 40% sequence identity with the human AP endonuclease Ape1. The gene was amplified from a culture sample and cloned into an expression vector. Using an E. coli host, the thermophilic protein could be produced and purified. Characterization of the enzymatic activity revealed strong binding and Mg2+-dependent nicking activity on undamaged double-stranded (ds) DNA at low ionic strength, even at temperatures below the optimum growth temperature of M. thermautotrophicus (65 degrees C). Additionally, a much faster nicking activity on AP site containing DNA was demonstrated. Unspecific incision of undamaged ds DNA was nearly inhibited at KCl concentration of approximately 0.5 M, whereas incision at AP sites was still complete at such salt concentrations. Nicked DNA was further degraded at temperatures above 50 degrees C, probably by an exonucleolytic activity of the enzyme, which was also found on recessed 3' ends of linearized ds DNA. The enzyme was active at temperatures up to 70 degrees C and, using circular dichroism spectroscopy, shown to denature at temperatures approaching 80 degrees C. Considering the high intracellular potassium ion concentration in M. thermautotrophicus, our results suggest that the characterized thermophilic enzyme acts as an AP endonuclease in vivo with similar activities as Ape1.     

Isolation and characterization of an uncoupler-resistant mutant of Methanothermobacter thermautotrophicus

A spontaneous mutant of Methanothermobacter thermautotrophicus resistant to the protonophorous uncoupler TCS was isolated. The mutant strain exhibited increased CH(4) formation and elevated level of ATPase activity under non-growing conditions. ATP synthesis driven by methanogenic electron transport as well as by potassium diffusion potential in the presence of either H(+) or Na(+) ions was markedly diminished in the mutant strain. An abundant membrane-associated protein complex with molecular mass approximately 670 kDa was detected in the mutant strain after native PAGE. The results indicate that TCS resistance in this mutant has arisen as a consequence of mutation(s) that affects a specific locus coding for an uncoupler binding protein(s) and/or modulate the activity of unidentified ATPase.     

Bioenergetics of the formyl-methanofuran dehydrogenase and heterodisulfide reductase reactions in Methanothermobacter thermautotrophicus.

The synthesis of formyl-methanofuran and the reduction of the heterodisulfide (CoM-S-S-CoB) of coenzyme M (HS-CoM) and coenzyme B (HS-CoB) are two crucial, H2-dependent reactions in the energy metabolism of methanogenic archaea. The bioenergetics of the reactions in vivo were studied in chemostat cultures and in cell suspensions of Methanothermobacter thermautotrophicus metabolizing at defined dissolved hydrogen partial pressures ( pH2). Formyl-methanofuran synthesis is an endergonic reaction (DeltaG degrees ' = +16 kJ.mol-1). By analyzing the concentration ratios between formyl-methanofuran and methanofuran in the cells, free energy changes under experimental conditions (DeltaG') were found to range between +10 and +35 kJ.mol-1 depending on the pH2 applied. The comparison with the sodium motive force indicated that the reaction should be driven by the import of a variable number of two to four sodium ions. Heterodisulfide reduction (DeltaG degrees ' = -40 kJ.mol-1) was associated with free energy changes as high as -55 to -80 kJ.mol-1. The values were determined by analyzing the concentrations of CoM-S-S-CoB, HS-CoM and HS-CoB in methane-forming cells operating under a variety of hydrogen partial pressures. Free energy changes were in equilibrium with the proton motive force to the extent that three to four protons could be translocated out of the cells per reaction. Remarkably, an apparent proton translocation stoichiometry of three held for cells that had been grown at pH2<0.12 bar, whilst the number was four for cells grown above that concentration. The shift occurred within a narrow pH2 span around 0.12 bar. The findings suggest that the methanogens regulate the bioenergetic machinery involved in CoM-S-S-CoB reduction and proton pumping in response to the environmental hydrogen concentrations.     

Solubilized glycosyltransferases and biosynthesis in vitro of glycolipids

The assembly of most of the ceramide-linked glycolipids (GSLs) in eukaryotic cells occurs in Golgi bodies. At least 18 different glycolipid:glycosyltransferases (GSL:GLTs) have been characterized, 10 of which have been solubilized. These GLTs can be classified into 2 distinct groups: 1) GLTs dedicated to either Dol-P-P-sugar(s) or ceramide-linked sugar(s); and 2) GLTs with dual loyalties (i.e., they compete with glycolipid- and glycoprotein-bound oligosaccharides). Studies with solubilized and purified GalNAcT-1 and GalNAcT-2 from embryonic chicken brains prove that GalNAcT-1 (UDP-GalNAc:GM3 beta 1-4GalNAcT) is specific for GSL, whereas GalNAcT-2 (UDP-GalNAc:Gb3 beta 1-3GalNAcT) can transfer to an oligosaccharide containing the alpha-linked terminal galactose. Similarly, GalT-3 (UDP-Gal:GM2 beta 1-3GalT) is more specific for ganglio-oligosaccharide and GalT-4 (UDP-Gal:Lc3 beta 1-4GalT) can transfer galactose to N-acetylglucosamine linked to p-nitrophenol, glycolipid or glycoprotein. Both GalT-3 and GalT-4 have been separated and purified from embryonic chicken brains. Studies with solubilized SAT-4 and SAT-3, from bovine spleen and embryonic chicken brains, respectively, suggest the existence of 2 different gene-expressed alpha 2-3SATs. The newly discovered FucT-3 (GDP-Fuc:NeuGc-iLc6-alpha 1-3FucT) from human colon carcinoma (Colo-205) has also been solubilized and separated from other GSL:GLTs. Using a new activity gel-Western blot combined technique, the molecular mass of this FucT-3 was determined to be 105 kDa.     

Identification of amino acids important for the biochemical activity of Methanothermobacter thermautotrophicus MCM

Methanothermobacter thermautotrophicus minichromosomal maintenance protein (mtMCM) is a 75 kDa protein that self-assembles into a double hexamer structure. The double hexamer formed by the N-terminal region of mtMCM has a highly charged (overwhelmingly net positive) inner channel. Here we investigate the effects of point mutations of some of these charged residues on the biological activities of mtMCM. Although all of the mutants were similar to the wild type in protein folding and complex assembly, we found that mutations impaired helicase activity. The study of the DNA binding and ATPase activities of these mutants revealed that the impairment of the helicase activity was highly correlated with a decrease in DNA binding, providing evidence consistent with the role of these charged residues of the inner channel in interactions with DNA.     

Methanogenesis is Ca2+ dependent in Methanothermobacter thermautotrophicus strain DeltaH

The effect of Ca2+ ions on methanogenesis and growth of Methanothermobacter thermautotrophicus was investigated. The calcium chelator ethylene glycol bis(2-aminoethylether)-N,N,N',N'-tetra-acetic acid, calcium ionophore A23187 and ruthenium red all inhibited growth of this strain. Methane formation was strongly dependent on the external Ca2+ concentration in a resting cell suspension. In addition, methanogenesis of Ca2+ preloaded cells was stimulated by 400%. Inhibitor studies revealed that Co2+ and Ni2+, inorganic antagonists of Ca2+ transport, strongly inhibited methanogenesis in these cells. Interestingly, our findings imply that one of the enzymes of methanogenesis might catalyse a Ca2+ -dependent step and allow a direct activation of methanogenesis by Ca2+ ions.     

Methanothermobacter thermautotrophicus tRNA Gln confines the amidotransferase GatCAB to asparaginyl-tRNA Asn formation

Many prokaryotes form the amide aminoacyl-tRNAs glutaminyl-tRNA and asparaginyl-tRNA by tRNA-dependent amidation of the mischarged tRNA species, glutamyl-tRNA^Gln or aspartyl-tRNA^Asn. Archaea employ two such amidotransferases, GatCAB and GatDE, while bacteria possess only one, GatCAB. The Methanothermobacter thermautotrophicus GatDE is slightly more efficient using Asn as an amide donor than Gln (k_cat/K_M of 5.4 s⁻¹/mM and 1.2 s⁻¹/mM, respectively). Unlike the bacterial GatCAB enzymes studied to date, the M. thermautotrophicus GatCAB uses Asn almost as well as Gln as an amide donor (k_cat/K_M of 5.7 s⁻¹/mM and 16.7 s⁻¹/mM, respectively). In contrast to the initial characterization of the M. thermautotrophicus GatCAB as being able to form Asn-tRNA^Asn and Gln-tRNA^Gln, our data demonstrate that while the enzyme is able to transamidate Asp-tRNA^Asn (k_cat/K_M of 125 s⁻¹/mM) it is unable to transamidate M. thermautotrophicus Glu-tRNA^Gln. However, M. thermautotrophicus GatCAB is capable of transamidating Glu-tRNA^Gln from H. pylori or B. subtilis, and M. thermautotrophicus Glu-tRNA^Asn. Thus, M. thermautotrophicus encodes two amidotransferases, each with its own activity, GatDE for Gln-tRNA and GatCAB for Asn-tRNA synthesis.     

Electrochemical control of redox potential affects methanogenesis of the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus

To investigate the precise effect of the redox potential on the methanogenesis of the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus by using an electrochemical redox controlling system without adding oxidizing or reducing agents. A bioelectrochemical system was applied to control the redox conditions in culture and to measure the methane‐producing activity of M. thermautotrophicus at a constant potential from +0·2 to ?0·8 V (vs Ag/AgCl). Methane production and growth of M. thermautotrophicus were 1·6 and 3·5 times increased at ?0·8 V, compared with control experiments without electrolysis, respectively, while methanogenesis was suppressed between +0·2 and ?0·2 V. A clear relationship between an electrochemically regulated redox potential and methanogenesis was revealed.

Significance and Impact of the Study

A novel bioelectrochemical method can activate the methanogenesis of M. thermautotrophicus by controlling the redox potential in culture conditions at ?0·8 V, which is a difficult potential to achieve by conventional methods (e.g. by adding reducing agents). This study provides useful insights for the application of a bioelectrochemical system in industrial processes involving methanogens, such as in anaerobic digesters.     

Biochemical characterization of the Methanothermobacter thermautotrophicus minichromosome maintenance (MCM) helicase N-terminal domains

Minichromosome maintenance helicases are ring-shaped complexes that play an essential role in archaeal and eukaryal DNA replication by separating the two strands of chromosomal DNA to provide the single-stranded substrate for the replicative polymerases. For the archaeal protein it was shown that the N-terminal portion of the protein, which is composed of domains A, B, and C, is involved in multimer formation and single-stranded DNA binding and may also play a role in regulating the helicase activity. Here, a detailed biochemical characterization of the N-terminal region of the Methanothermobacter thermautotrophicus minichromosome maintenance helicase is described. Using biochemical and biophysical analyses it is shown that domain C of the N-terminal portion, located adjacent to the helicase catalytic domains, is required for protein multimerization and that domain B is the main contact region with single-stranded DNA. It is also shown that although oligomerization is not essential for single-stranded DNA binding and ATPase activity, the presence of domain C is essential for helicase activity.     

CDP-2,3-Di-O-geranylgeranyl-sn-glycerol:L-serine O-archaetidyltransferase (archaetidylserine synthase) in the methanogenic archaeon Methanothermobacter thermautotrophicus

CDP-2,3-di-O-geranylgeranyl-sn-glycerol:L-serine O-archaetidyltransferase (archaetidylserine synthase) activity in cell extracts of Methanothermobacter thermautotrophicus cells was characterized. The enzyme catalyzed the formation of unsaturated archaetidylserine from CDP-unsaturated archaeol and L-serine. The identity of the reaction products was confirmed by thin-layer chromatography, fast atom bombardment-mass spectrum analysis, and chemical degradation. The enzyme showed maximal activity in the presence of 10 mM Mn2+ and 1% Triton X-100. Among various synthetic substrate analogs, both enantiomers of CDP-unsaturated archaeols with ether-linked geranylgeranyl chains and CDP-saturated archaeol with ether-linked phytanyl chains were similarly active toward the archaetidylserine synthase. The activity on the ester analog of the substrate was two to three times higher than that on the corresponding ether-type substrate. The activity of D-serine with the enzyme was 30% of that observed for L-serine. A trace amount of an acid-labile, unsaturated archaetidylserine intermediate was detected in the cells by a pulse-labeling experiment. A gene (MT1027) in M. thermautotrophicus genome annotated as the gene encoding phosphatidylserine synthase was found to be homologous to Bacillus subtilis pssA but not to Escherichia coli pssA. The substrate specificity of phosphatidylserine synthase from B. subtilis was quite similar to that observed for the M. thermautotrophicus archaetidylserine synthase, while the E. coli enzyme had a strong preference for CDP-1,2-diacyl-sn-glycerol. It was concluded that M. thermautotrophicus archaetidylserine synthase belongs to subclass II phosphatidylserine synthase (B. subtilis type) on the basis of not only homology but also substrate specificity and some enzymatic properties. The possibility that a gene encoding the subclass II phosphatidylserine synthase might be transferred from a bacterium to an ancestor of methanogens is discussed.     

Protein complexes in the archaeon Methanothermobacter thermautotrophicus analyzed by blue native/SDS-PAGE and mass spectrometry

Methanothermobacter thermautotrophicus is a thermophilic archaeon that produces methane as the end product of its primary metabolism. The biochemistry of methane formation has been extensively studied and is catalyzed by individual enzymes and proteins that are organized in protein complexes. Although much is known of the protein complexes involved in methanogenesis, only limited information is available on the associations of proteins involved in other cell processes of M. thermautotrophicus. To visualize and identify interacting and individual proteins of M. thermautotrophicus on a proteome-wide scale, protein preparations were separated using blue native electrophoresis followed by SDS-PAGE. A total of 361 proteins, corresponding to almost 20% of the predicted proteome, was identified using peptide mass fingerprinting after MALDI-TOF MS. All previously characterized complexes involved in energy generation could be visualized. Furthermore the expression and association of the heterodisulfide reductase and methylviologen-reducing hydrogenase complexes depended on culture conditions. Also homomeric supercomplexes of the ATP synthase stalk subcomplex and the N5-methyl-5,6,7,8-tetrahydromethanopterin:coenzyme M methyltransferase complex were separated. Chemical cross-linking experiments confirmed that the multimerization of both complexes was not experimentally induced. A considerable number of previously uncharacterized protein complexes were reproducibly visualized. These included an exosome-like complex consisting of four exosome core subunits, which associated with a tRNA-intron endonuclease, thereby expanding the constituency of archaeal exosomes. The results presented show the presence of novel complexes and demonstrate the added value of including blue native gel electrophoresis followed by SDS-PAGE in discovering protein complexes that are involved in catabolic, anabolic, and general cell processes.     

DNA binding by the Methanothermobacter thermautotrophicus Cdc6 protein is inhibited by the minichromosome maintenance helicase

The Cdc6 proteins from the archaeon Methanothermobacter thermautotrophicus were previously shown to bind double-stranded DNA. It is shown here that the proteins also bind single-stranded DNA. Using minichromosome maintenance (MCM) helicase mutant proteins unable to bind DNA, it was found that the interaction of MCM with Cdc6 inhibits the DNA binding activity of Cdc6.