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Uwe Deppenmeier - One of the best experts on this subject based on the ideXlab platform.
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A Novel Inducible Protein Production System and Neomycin Resistance as Selection Marker for Methanosarcina mazei
Hindawi Limited, 2012Co-Authors: Sebastian Mondorf, Uwe Deppenmeier, Cornelia WelteAbstract:Methanosarcina mazei is one of the model organisms for the methanogenic order Methanosarcinales whose metabolism has been studied in detail. However, the genetic toolbox is still limited. This study was aimed at widening the scope of utilizable methods in this group of organisms. (i) Proteins specific to methanogens are oftentimes difficult to produce in E. coli. However, a protein production system is not available for methanogens. Here we present an inducible system to produce Strep-tagged proteins in Ms. mazei. The promoter p1687, which directs the transcription of methyl transferases that demethylate methylamines, was cloned into plasmid pWM321 and its activity was determined by monitoring β-glucuronidase production. The promoter was inactive during growth on methanol but was rapidly activated when trimethylamine was added to the medium. The gene encoding the β-glucuronidase from E. coli was fused to a Strep-tag and was cloned downstream of the p1687 promoter. The protein was overproduced in Ms. mazei and was purified in an active form by affinity chromatography. (ii) Puromycin is currently the only antibiotic used as a selectable marker in Ms. mazei and its relatives. We established neomycin resistance as a second selectable marker by designing a plasmid that confers neomycin resistance in Ms. mazei
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methanoferrodoxin represents a new class of superoxide reductase containing an iron sulfur cluster
FEBS Journal, 2011Co-Authors: Christian Krätzer, Cornelia U. Welte, Katerina Dörner, Thorsten Friedrich, Uwe DeppenmeierAbstract:),which are generated by the partial reduction of oxygen[1]. Bacteria deal with oxidative stress with a set ofdetoxifying enzymes. Superoxide dismutases (SODs)were the first enzymes known to eliminate superoxideby disproportionation to hydrogen peroxide and diox-ygen [1]. Superoxide reductases (SORs) are a new fam-ily of enzymes that were discovered in sulfate-reducingbacteria of the Desulfovibrio genus [2,3], and catalyzethe reduction of superoxide to peroxide. SORs are pre-dominantly found in anaerobic or microaerophilicbacteria such as Desulfovibrio desulfuricans [2] andClostridium acetobutylicum [4], or anaerobic archaeonssuch as Archaeoglobus fulgidus [5] and Pyrococcusfuriosus [6]. In the past decade, SORs from theseorganisms and others have been studied in detail, anda considerable amount of biochemical, crystallographicand spectroscopic information has been reported [7].Methanosarcina mazei is one of the methanogenicarchaeons, which are characterized by the ability togenerate methane as the major end product of energymetabolism [8]. Many Methanosarcina strains are ableto utilize H
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involvement of ech hydrogenase in energy conservation of Methanosarcina mazei
FEBS Journal, 2010Co-Authors: Cornelia U. Welte, Christian Krätzer, Uwe DeppenmeierAbstract:Methanosarcina mazei belongs to the group of aceticlastic methanogens and converts acetate into the potent greenhouse gases CO2 and CH4. The aceticlastic respiratory chain involved in methane formation comprises the three transmembrane proteins Ech hydrogenase, F420 nonreducing hydrogenase and heterodisulfide reductase. It has been shown that the latter two contribute to the proton motive force. The data presented here clearly demonstrate that Ech hydrogenase is also involved in energy conservation. ATP synthesis was observed in a cytoplasm-free vesicular system of Ms. mazei that was dependent on the oxidation of reduced ferredoxin and the formation of molecular hydrogen (as catalysed by Ech hydrogenase). Such an ATP formation was not observed in a Δech mutant strain. The protonophore 3,5-di-tert-butyl-4-hydroxybenzylidene-malononitrile (SF6847) led to complete inhibition of ATP formation in the Ms. mazei wild-type without inhibiting hydrogen production by Ech hydrogenase, whereas the sodium ion ionophore ETH157 did not affect ATP formation in this system. Thus, we conclude that Ech hydrogenase acts as primary proton pump in a ferredoxin-dependent electron transport system.
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function of ech hydrogenase in ferredoxin dependent membrane bound electron transport in Methanosarcina mazei
Journal of Bacteriology, 2010Co-Authors: Verena Kallnik, Marcel Grapp, Gunes Bender, Steve Ragsdale, Uwe DeppenmeierAbstract:Reduced ferredoxin is an intermediate in the methylotrophic and aceticlastic pathway of methanogenesis and donates electrons to membrane-integral proteins, which transfer electrons to the heterodisulfide reductase. A ferredoxin interaction has been observed previously for the Ech hydrogenase. Here we present a detailed analysis of a Methanosarcina mazei ech mutant which shows decreased ferredoxin-dependent membranebound electron transport activity, a lower growth rate, and faster substrate consumption. Evidence is presented that a second protein whose identity is unknown oxidizes reduced ferredoxin, indicating an involvement in methanogenesis from methylated C1 compounds. The aceticlastic pathway of methanogenesis creates approximately 70% (10) of the biologically produced methane and is of great ecological importance, as methane is a potent greenhouse gas. Organisms using this pathway to convert acetate to methane belong exclusively to the genera Methanosarcina and Methanosaeta. The two carbon atoms of acetate have different fates in the pathway. The methyl moiety is converted to methane, whereas the carbonyl moiety is further oxidized to CO2 and the electrons derived from this oxidation step are used to reduce ferredoxin (Fd) (6). During methanogenesis from methylated C1 compounds (methanol and methylamines), onequarter of the methyl groups are oxidized to obtain electrons for the reduction of heterodisulfide (27). A key enzyme in the oxidative part of methylotrophic methanogenesis is the formylmethanofuran dehydrogenase, which oxidizes the intermediate formylmethanofuran to CO2 (7). The electrons are transferred to Fd. It has been suggested that reduced ferredoxin (Fdred) donates electrons to the respiratory chain with the heterodisulfide (coenzyme M [CoM]-S-S-CoB) as the terminal electron acceptor and that the reaction is catalyzed by the Fdred:CoMS-S-CoB oxidoreductase system (7, 24). The direct membranebound electron acceptor for Fdred is still a matter of debate; for the Ech hydrogenase, a reduced ferredoxin-accepting, H2evolving activity has been observed for Methanosarcina barkeri (20), which implies that the H2:CoM-S-S-CoB oxidoreductase system is involved in electron transport (13). Direct electron flow from the Ech hydrogenase to the heterodisulfide reductase has not been shown to date (20, 21). In contrast to M. barkeri, Methanosarcina acetivorans lacks the Ech hydrogenase (11). It can nevertheless grow on acetate, which is why another complex present in this organism, the Rnf complex, is thought
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identification of genes involved in salt adaptation in the archaeon Methanosarcina mazei go1 using genome wide gene expression profiling
Fems Microbiology Letters, 2007Co-Authors: Katharina Pfluger, Robert P. Gunsalus, Uwe Deppenmeier, Gerhard Gottschalk, Armin Ehrenreich, Kirsty Salmon, Volker MullerAbstract:Methanosarcina mazei is a nonhalophilic methanogen that can adapt to 800 mM NaCl. Microarray studies have been used to examine the effect of elevated salinities on the regulation of gene expression in M. mazei. Eighty-four genes of different functional categories, such as solute transport and biosynthesis, Na+ export, stress response, ion, protein and phosphate transport, metabolic enzymes, regulatory proteins, DNA-modification systems, and cell-surface modulators, were found to be stronger expressed at high salinities. Moreover, 10 genes encoding different metabolic functions including potassium uptake and ATP synthesis were reduced in expression under high salt. The overall expression profiles suggest that M. mazei is able to adapt to high salinities by multiple upregulation of many different cellular functions including protective pathways such as solute transport and biosynthesis, import of phosphate, export of Na+, and upregulation of pathways for modification of DNA and cell surface architecture.
Volker Muller - One of the best experts on this subject based on the ideXlab platform.
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Genetic analysis of the role of the ABC transporter Ota and Otb in glycine betaine transport in Methanosarcina mazei Gö1
Archives of Microbiology, 2008Co-Authors: Regina Saum, Ana Mingote, Helena Santos, Volker MullerAbstract:The methanogenic archaeon Methanosarcina mazei Gö1 accumulates glycine betaine in response to hypersalinity but differs from most other methanoarchaea in having two gene clusters both encoding a potential glycine betaine transporter, Ota and Otb. We have created mutants with either ota or otb deleted to address their role in salt adaptation. The mutants were not impaired in growth at low or high salt, neither at 37°C nor at lower temperatures. However, the ∆ota mutant was completely defective in glycine betaine transport demonstrating that Ota is the only glycine betaine transporter in M. mazei . The mutation in otb led to increased transcription of ota and thus increased transport and accumulation of glycine betaine suggesting a cross talk between the two transporters.
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differential regulation of ota and otb two primary glycine betaine transporters in the methanogenic archaeon Methanosarcina mazei go1
Journal of Molecular Microbiology and Biotechnology, 2008Co-Authors: Regina Spanheimer, K Pfluger, Stephanie Kogl, M Hoffmann, Stefan Schmidt, Volker MullerAbstract:Methanogenic archaea accumulate glycine betaine in response to hypersalinity, but the regulation of proteins involved, their mechanism of activation and regulation of the corresponding genes are largely unknown. Methanosarcina mazei differs from most other methanoarchaea in having two gene clusters both encoding a potential glycine betaine transporter, Ota and Otb. Western blot as well as quantitative real-time PCR revealed that Otb is not regulated by osmolarity. On the other hand, cellular levels of Ota increased with increasing salt concentrations. A maximum was reached at 300–500 mM NaCl. Ota concentrations reached a maximum 4 h after an osmotic upshock. Hyperosmolarity also caused an increase in cellular Ota concentrations. In addition to osmolarity Ota expression was regulated by the growth phase. Expression of Ota as well as transport of betaine was downregulated in the presence of glycine betaine.
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the salt induced abc transporter ota of the methanogenic archaeon Methanosarcina mazei go1 is a glycine betaine transporter
Fems Microbiology Letters, 2007Co-Authors: Silke Schmidt, Volker Muller, K Pfluger, Stephanie Kogl, Regina SpanheimerAbstract:The genes encoding the three subunits of the primary ABC transporter Ota of the methanogenic archaeon Methanosarcina mazei Go1 were cloned in an expression vector (pBAD24) and transformed into the glycine betaine transport-negative mutant Escherichia coli MKH13. Ota was produced as demonstrated by Western blotting. Uptake studies revealed that Ota catalyzed the transport of glycine betaine in E. coli MKH13(pBAD-Ota) with a Km of 10±5 μM and a maximal velocity of 1.5±0.5 nmol min−1 mg protein−1. Transport was ATP dependent. Ota was activated by salinity gradients, but only marginally by sugar gradients across the membrane. Glycine betaine transport was inhibited to a small extent by an excess of dimethylglycin or proline betaine, but not by sarcosine or glycine.
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identification of genes involved in salt adaptation in the archaeon Methanosarcina mazei go1 using genome wide gene expression profiling
Fems Microbiology Letters, 2007Co-Authors: Katharina Pfluger, Robert P. Gunsalus, Uwe Deppenmeier, Gerhard Gottschalk, Armin Ehrenreich, Kirsty Salmon, Volker MullerAbstract:Methanosarcina mazei is a nonhalophilic methanogen that can adapt to 800 mM NaCl. Microarray studies have been used to examine the effect of elevated salinities on the regulation of gene expression in M. mazei. Eighty-four genes of different functional categories, such as solute transport and biosynthesis, Na+ export, stress response, ion, protein and phosphate transport, metabolic enzymes, regulatory proteins, DNA-modification systems, and cell-surface modulators, were found to be stronger expressed at high salinities. Moreover, 10 genes encoding different metabolic functions including potassium uptake and ATP synthesis were reduced in expression under high salt. The overall expression profiles suggest that M. mazei is able to adapt to high salinities by multiple upregulation of many different cellular functions including protective pathways such as solute transport and biosynthesis, import of phosphate, export of Na+, and upregulation of pathways for modification of DNA and cell surface architecture.
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coexistence of group i and group ii chaperonins in the archaeon Methanosarcina mazei
Journal of Biological Chemistry, 2003Co-Authors: Daniel Klunker, Uwe Deppenmeier, Angela Hirtreiter, Luis Figueiredo, Dean J Naylor, Gunter Pfeifer, Volker Muller, Bernd Haas, Gerhard Gottschalk, Ulrich F HartlAbstract:Abstract Two distantly related classes of cylindrical chaperonin complexes assist in the folding of newly synthesized and stress-denatured proteins in an ATP-dependent manner. Group I chaperonins are thought to be restricted to the cytosol of bacteria and to mitochondria and chloroplasts, whereas the group II chaperonins are found in the archaeal and eukaryotic cytosol. Here we show that members of the archaeal genus Methanosarcina co-express both the complete group I (GroEL/GroES) and group II (thermosome/prefoldin) chaperonin systems in their cytosol. These mesophilic archaea have acquired between 20 and 35% of their genes by lateral gene transfer from bacteria. In Methanosarcina mazei Go1, both chaperonins are similarly abundant and are moderately induced under heat stress. The M. mazei GroEL/GroES proteins have the structural features of their bacterial counterparts. The thermosome contains three paralogous subunits, α, β, and γ, which assemble preferentially at a molar ratio of 2:1:1. As shown in vitro, the assembly reaction is dependent on ATP/Mg2+ or ADP/Mg2+ and the regulatory role of the β subunit. The co-existence of both chaperonin systems in the same cellular compartment suggests the Methanosarcina species as useful model systems in studying the differential substrate specificity of the group I and II chaperonins and in elucidating how newly synthesized proteins are sorted from the ribosome to the proper chaperonin for folding.
Jason W Chin - One of the best experts on this subject based on the ideXlab platform.
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an evolved methanomethylophilus alvus pyrrolysyl trna synthetase trna pair is highly active and orthogonal in mammalian cells
Biochemistry, 2019Co-Authors: Vaclav Beranek, Julian C W Willis, Jason W ChinAbstract:We recently characterized a new class of pyrrolysyl-tRNA synthetase (PylRS)/PyltRNA pairs from Methanomassiliicocales that are active and orthogonal in Escherichia coli. The aminoacyl-tRNA synthetases (aaRSs) of these pairs lack the N-terminal domain that is essential for tRNA recognition and in vivo activity in the Methanosarcina mazei (Mm) PylRS but share a homologous active site with MmPylRS; this facilitates the transplantation of mutations discovered with existing PylRS systems into the new PylRS systems to reprogram their substrate specificity for the incorporation of noncanonical amino acids (ncAAs). Several of the new PylRS/PyltRNA pairs, or their evolved variants [including Methanomethylophilus alvus (Ma) PylRS/MaPyltRNA(6)CUA], are mutually orthogonal to the MmPylRS/MmPyltRNA pair, and the active sites of the Mm pair and Ma pair can be diverged to enable the incorporation of distinct ncAAs in response to distinct codons via orthogonal translation in E. coli. Here we demonstrate that MaPylRS/MaPy...
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genetic code expansion in stable cell lines enables encoded chromatin modification
Nature Methods, 2016Co-Authors: Simon J Elsasser, Russell J Ernst, Olivia S Walker, Jason W ChinAbstract:Genetically encoded unnatural amino acids provide powerful strategies for modulating the molecular functions of proteins in mammalian cells. However, this approach has not been coupled to genome-wide measurements, because efficient incorporation of unnatural amino acids is limited to transient expression settings that lead to very heterogeneous expression. We demonstrate that stable integration of the Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS)/tRNA(Pyl)CUA pair (and its derivatives) into the mammalian genome enables efficient, homogeneous incorporation of unnatural amino acids into target proteins in diverse mammalian cells, and we reveal the distinct transcriptional responses of embryonic stem cells and mouse embryonic fibroblasts to amber codon suppression. Genetically encoding N-ɛ-acetyl-lysine in place of six lysine residues in histone H3 enables deposition of pre-acetylated histones into cellular chromatin, via a pathway that is orthogonal to enzymatic modification. After synthetically encoding lysine-acetylation at natural modification sites, we determined the consequences of acetylation at specific amino acids in histones for gene expression.
Jaap S. Sinninghe Damsté - One of the best experts on this subject based on the ideXlab platform.
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A polyunsaturated irregular acyclic C25 isoprenoid in a methanogenic archaeon
Tetrahedron Letters, 1997Co-Authors: Jaap S. Sinninghe Damsté, Stefan Schouten, N. Herbert Van Vliet, Robert Huber, Jan A. J. GeenevasenAbstract:Abstract All trans 2,6,10,15,19-pentamethylicosa-2,6,14,18-tetraene 1 has been isolated from the methanogenic archaeon Methanosarcina mazei and identified by 1 H and 13 C NMR spectroscopy and mass spectrometry.
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2,6,10,15,19-pentamethylicosenes in Methanolobus bombayensis, a marine methanogenic archaeon
1997Co-Authors: Jaap S. Sinninghe Damsté, S. Schouten, M.j.e.c. Van Der Maarel, Robert HuberAbstract:2,6,10,15,19-Pentamethylicosenes (PMEs) containing three to five double bonds have been found in the methanogenic archaea Methanosarcina mazei (DSM 3338), a strain isolated from sewage sludge, and in Methanolobus bombayensis (OCM 438), a non-extremophilic archaeon isolated from a marine sediment. This finding gives additional support for the use of compounds with the PME carbon skeleton as markers for methanogenic activity in marine environments
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2,6,10,15,19-Pentamethylicosenes in Methanolobus bombayensis, a marine methanogenic archaeon, and in Methanosarcina mazei
Organic Geochemistry, 1997Co-Authors: Stefan Schouten, Robert Huber, M.j.e.c. Van Der Maarel, Jaap S. Sinninghe DamstéAbstract:2,6,10,15,19-Pentamethylicosenes (PMEs) containing three to five double bonds have been found in the methanogenic archaea Methanosarcina mazei (DSM 3338), a strain isolated from sewage sludge, and in Methanolobus bombayensis (OCM 438), a non-extremophilic archaeon isolated from a marine sediment. This finding gives additional support for the use of compounds with the PME carbon skeleton as markers for methanogenic activity in marine environments.
Gerhard Gottschalk - One of the best experts on this subject based on the ideXlab platform.
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identification of genes involved in salt adaptation in the archaeon Methanosarcina mazei go1 using genome wide gene expression profiling
Fems Microbiology Letters, 2007Co-Authors: Katharina Pfluger, Robert P. Gunsalus, Uwe Deppenmeier, Gerhard Gottschalk, Armin Ehrenreich, Kirsty Salmon, Volker MullerAbstract:Methanosarcina mazei is a nonhalophilic methanogen that can adapt to 800 mM NaCl. Microarray studies have been used to examine the effect of elevated salinities on the regulation of gene expression in M. mazei. Eighty-four genes of different functional categories, such as solute transport and biosynthesis, Na+ export, stress response, ion, protein and phosphate transport, metabolic enzymes, regulatory proteins, DNA-modification systems, and cell-surface modulators, were found to be stronger expressed at high salinities. Moreover, 10 genes encoding different metabolic functions including potassium uptake and ATP synthesis were reduced in expression under high salt. The overall expression profiles suggest that M. mazei is able to adapt to high salinities by multiple upregulation of many different cellular functions including protective pathways such as solute transport and biosynthesis, import of phosphate, export of Na+, and upregulation of pathways for modification of DNA and cell surface architecture.
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coexistence of group i and group ii chaperonins in the archaeon Methanosarcina mazei
Journal of Biological Chemistry, 2003Co-Authors: Daniel Klunker, Uwe Deppenmeier, Angela Hirtreiter, Luis Figueiredo, Dean J Naylor, Gunter Pfeifer, Volker Muller, Bernd Haas, Gerhard Gottschalk, Ulrich F HartlAbstract:Abstract Two distantly related classes of cylindrical chaperonin complexes assist in the folding of newly synthesized and stress-denatured proteins in an ATP-dependent manner. Group I chaperonins are thought to be restricted to the cytosol of bacteria and to mitochondria and chloroplasts, whereas the group II chaperonins are found in the archaeal and eukaryotic cytosol. Here we show that members of the archaeal genus Methanosarcina co-express both the complete group I (GroEL/GroES) and group II (thermosome/prefoldin) chaperonin systems in their cytosol. These mesophilic archaea have acquired between 20 and 35% of their genes by lateral gene transfer from bacteria. In Methanosarcina mazei Go1, both chaperonins are similarly abundant and are moderately induced under heat stress. The M. mazei GroEL/GroES proteins have the structural features of their bacterial counterparts. The thermosome contains three paralogous subunits, α, β, and γ, which assemble preferentially at a molar ratio of 2:1:1. As shown in vitro, the assembly reaction is dependent on ATP/Mg2+ or ADP/Mg2+ and the regulatory role of the β subunit. The co-existence of both chaperonin systems in the same cellular compartment suggests the Methanosarcina species as useful model systems in studying the differential substrate specificity of the group I and II chaperonins and in elucidating how newly synthesized proteins are sorted from the ribosome to the proper chaperonin for folding.
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coexistence of group i and group ii chaperonins in the archaeon Methanosarcina mazei
Journal of Biological Chemistry, 2003Co-Authors: Daniel Klunker, Uwe Deppenmeier, Angela Hirtreiter, Luis Figueiredo, Dean J Naylor, Gunter Pfeifer, Volker Muller, Bernd Haas, Gerhard Gottschalk, Ulrich F HartlAbstract:Two distantly related classes of cylindrical chaperonin complexes assist in the folding of newly synthesized and stress-denatured proteins in an ATP-dependent manner. Group I chaperonins are thought to be restricted to the cytosol of bacteria and to mitochondria and chloroplasts, whereas the group II chaperonins are found in the archaeal and eukaryotic cytosol. Here we show that members of the archaeal genus Methanosarcina co-express both the complete group I (GroEL/GroES) and group II (thermosome/prefoldin) chaperonin systems in their cytosol. These mesophilic archaea have acquired between 20 and 35% of their genes by lateral gene transfer from bacteria. In Methanosarcina mazei Go1, both chaperonins are similarly abundant and are moderately induced under heat stress. The M. mazei GroEL/GroES proteins have the structural features of their bacterial counterparts. The thermosome contains three paralogous subunits, alpha, beta, and gamma, which assemble preferentially at a molar ratio of 2:1:1. As shown in vitro, the assembly reaction is dependent on ATP/Mg2+ or ADP/Mg2+ and the regulatory role of the beta subunit. The co-existence of both chaperonin systems in the same cellular compartment suggests the Methanosarcina species as useful model systems in studying the differential substrate specificity of the group I and II chaperonins and in elucidating how newly synthesized proteins are sorted from the ribosome to the proper chaperonin for folding.
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identification of a salt induced primary transporter for glycine betaine in the methanogen Methanosarcina mazei go1
Applied and Environmental Microbiology, 2002Co-Authors: M Roesler, Gerhard Gottschalk, K Pfluger, H Flach, T Lienard, Volker MullerAbstract:The salt adaptation of the methanogenic archaeon Methanosarcina mazei Go1 was studied at the physiological and molecular levels. The freshwater organism M. mazei Go1 was able to adapt to salt concentrations up to 1 M, and the addition of the compatible solute glycine betaine to the growth medium facilitated adaptation to higher salt concentrations. Transport studies with cell suspensions revealed a salt-induced glycine betaine uptake activity in M. mazei Go1, and inhibitor studies argue for a primary transport device. Analysis of the genome of M. mazei Go1 identified a homolog of known primary glycine betaine transporters. This gene cluster was designated Ota (osmoprotectant transporter A). Its sequence and gene organization are very similar to those of the glycine betaine transporter OpuA of Bacillus subtilis. Northern blot analysis of otaC revealed a salt-dependent transcription of this gene. Ota is the first identified salt-induced transporter for compatible solutes in Archaea.
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identification and analysis of proton translocating pyrophosphatases in the methanogenic archaeon Methanosarcina mazei
Archaea, 2002Co-Authors: Sebastian Baumer, Gerhard Gottschalk, Sabine Lentes, Uwe DeppenmeierAbstract:Analysis of genome sequence data from the methanogenic archaeon Methanosarcina mazei Go1 revealed the existence of two open reading frames encoding proton-translocating pyrophosphatases (PPases). These open reading frames are linked by a 750-bp intergenic region containing TC-rich stretches and are transcribed in opposite directions. The corresponding polypeptides are referred to as Mvp1 and Mvp2 and consist of 671 and 676 amino acids, respectively. Both enzymes represent extremely hydrophobic, integral membrane proteins with 15 predicted transmembrane segments and an overall amino acid sequence similarity of 50.1%. Multiple sequence alignments revealed that Mvp1 is closely related to eukaryotic PPases, whereas Mvp2 shows highest homologies to bacterial PPases. Northern blot experiments with RNA from methanol-grown cells harvested in the mid-log growth phase indicated that only Mvp2 was produced under these conditions. Analysis of washed membranes showed that Mvp2 had a specific activity of 0.34 U mg (protein)–1. Proton translocation experiments with inverted membrane vesicles prepared from methanol-grown cells showed that hydrolysis of 1 mol of pyrophosphate was coupled to the translocation of about 1 mol of protons across the cytoplasmic membrane. Appropriate conditions for mvp1 expression could not be determined yet. The pyrophosphatases of M. mazei Go1 represent the first examples of this enzyme class in methanogenic archaea and may be part of their energy-conserving system. Abbreviations: DCCD, N,N′-dicyclohexylcarbodiimide; PPase, inorganic pyrophosphatase; PPi, inorganic pyrophosphate; Δp, proton motive force.