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PubMed1998

The vhuU gene encoding a small subunit of a selenium-containing [NiFe]-hydrogenase in Methanococcus voltae appears to be essential for the cell.

We developed a general method for the site-specific deletion of gene sequences to obtain new selectable markers in the archaeon Methanococcus voltae. Using a deletion in the hisA gene, a vector was integrated into the chromosome by homologous recombination, thereby reconstituting histidine prototrophy. The vector contained the beta-glucuronidase gene uidA of Escherichia coli as a reporter under the control of an M. voltae promoter that normally drives the expression of a selenium-free [NiFe]-hydrogenase after selenium deprivation. This construct has allowed us to check whether the selenium supply was sufficiently low to induce the transcription of the genes encoding the selenium-free hydrogenases. We tried to introduce a chromosomal deletion of the vhuU gene of the archaeon M. voltae by gene replacement and by keeping the cells under selenium deprivation. The gene vhuU encodes the very small, selenocysteine-containing subunit that is part of the primary reaction center of the Vhu hydrogenase. All transformants bearing the deletion also contained the vhuU wild-type gene. Therefore, the vhuU gene appears to be essential for the cell even under conditions that lead to the induction of the selenium-free homologue Vhc of the Vhu hydrogenase.

Aldose-Ketose Isomerases↗
PubMed2001

Identification of amino acids in the leader peptide of Methanococcus voltae preflagellin that are important in posttranslational processing.

Archaeal flagellins are made initially as preproteins with short, positively charged leader peptides. Analysis of all available archaeal preflagellin sequences indicates that the -1 position is always held by a glycine while the -2 and -3 positions are almost always held by charged amino acids. To evaluate the importance of these and other amino acids in the leader peptides of archaeal flagellins for processing by a peptidase, Methanococcus voltae mutant FlaB2 preflagellin genes were generated by PCR and the proteins tested in a methanogen preflagellin peptidase assay that detects the removal of the leader peptide from preflagellin. When the -1 position was changed from glycine to other amino acids tested, no cleavage was observed by the peptidase, with the exception of a change to alanine at which poor, partial processing was observed. Amino acid substitutions at the -2 lysine position resulted in a complete loss of processing by the peptidase, while changes at the -3 lysine resulted in partial processing. A mutant preflagellin with a leader peptide shortened from 12 amino acids to 6 amino acids was not processed. When the invariant glycine residue present at position +3 was changed to a valine, no processing of this mutant preflagellin was observed. The identification of critical amino acids in FlaB2 required for proper processing suggests that a specific preflagellin peptidase may cleave archaeal flagellins by recognition of a conserved sequence of amino acids.

Amino Acid Sequence↗
PubMed2001

Novel glucoamylase-type enzymes from Thermoactinomyces vulgaris and Methanococcus jannaschii whose genes are found in the flanking region of the alpha-amylase genes.

A region downstream of the gene for pullulan-hydrolyzing alpha-amylase, TVA II, of Thermoactinomyces vulgaris R-47 was sequenced, and an open reading frame encoding an enzyme homologous to glucoamylase was found. The nucleotide sequence of this enzyme, designated TGA, consists of 1,953 base pairs corresponding to a protein of 651 amino acid residues. The TGA gene was subcloned and expressed in Escherichia coli. Enzymatic analyses showed that, like other glucoamylases, TGA produced beta-D-glucose from its substrate. However, TGA hydrolyzed maltooligosaccharides such as maltotetraose and maltose more efficiently than starch, while fungal glucoamylases preferred starch to maltooligosaccharides. The primary structure of TGA resembled a putative glucoamylase from the hyperthermophilic archaeon Methanococcus jannaschii (MGA), while homologies between TGA and the fungal glucoamylases were low. The enzymatic properties of recombinant MGA produced in E. coli cells were similar to those of TGA. These findings indicate that TGA and MGA are novel glucoamy-lase-type enzymes with oligosaccaharide-metabolizing activity.

Amino Acid Sequence↗
PubMed2004

Mutational analysis of genes encoding chromatin proteins in the archaeon Methanococcus voltae indicates their involvement in the regulation of gene expression.

Several genes for chromatin proteins are known in Archaea. These include histones and histone-like proteins in Euryarchaeota, and a DNA binding protein, Alba, which was first detected in the crenarchaeote Sulfolobus solfataricus and is thought to be involved in transcriptional regulation. The methanogenic archaeon Methanococcus voltae harbors four genes coding for all these three types of chromatin proteins. Deletion mutants for the two histone genes ( hstAand hstB), the gene encoding the histone-like protein ( hmvA) and the gene for the Alba homologue ( albA) have now been constructed in this organism. Although all single mutants were viable, deletion of hstA resulted in slow growth. Two transcripts were detected for each of the two histone genes. These were expressed in different relative amounts, which were correlated with different growth phases. Cell extracts obtained from the different mutants exhibited altered protein patterns, as revealed by 2D gel electrophoresis, indicating that the chromatin proteins are involved in gene regulation in M. voltae.

Amino Acid Sequence↗
PubMed1998

Flagellin genes of Methanococcus vannielii: amplification by the polymerase chain reaction, demonstration of signal peptides and identification of major components of the flagellar filament.

The highly conserved nature of the 5'-termini of all archaeal flagellin genes was exploited by polymerase chain reaction (PCR) techniques to amplify the sequence of a portion of a flagellin gene family from the archaeon Methanococcus vannielii. Subsequent inverse PCR experiments generated fragments that permitted the sequencing of a total of three flagellin genes, which, by comparison with flagellin genes that have been sequenced, from other archaea appear to be equivalent to flaB1, flaB2, and flaB3 of M. voltae. Analysis of purified M. vannielii flagellar filaments by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed two major flagellins (Mr = 30,800 and 28,600), whose N-terminal sequences identified them as the products of the flaB1 and flaB2 genes, respectively. The gene product of flaB3 could not be detected in flagellar filaments by SDS-PAGE. The protein sequence data, coupled with the DNA sequences, demonstrated that both FlaB1 and FlaB2 flagellins are translated with a 12-amino acid signal peptide which is absent from the mature protein incorporated into the flagellar filament. These data suggest that archaeal flagellin export differs significantly from that of bacterial flagellins.

Amino Acid Sequence↗
PubMed2001

Insertional inactivation of the flaH gene in the archaeon Methanococcus voltae results in non-flagellated cells.

The marine methanogen Methanococcus voltae possesses two transcriptional units that encode a total of four flagellins. Immediately downstream of the flagellin genes are a number of ORFs, some of which are cotranscribed with the flagellin genes. These putative genes have been named flaCDEFGHIJ, although no biochemical data has implicated them in flagellar morphogenesis. None of the flaC-J genes has homology to any bacterial gene, with the exception of flaI, which shows homology to pilT, a gene that encodes a nucleotide binding protein of the type IV pilus family. In this study, insertional mutations in flaH of M. voltae were identified. The mutants were non-motile and non-flagellated as determined by electron microscopy. Southern hybridization experiments confirmed the insertion of a mutagenic vector into flaH and indicated that two, tandem, copies of the vector were present. It is believed that insertion of the vector into flaH should disrupt the transcription of flaIJ due to polar effects. The flaH mutant displayed the same pattern of multiple mRNA transcripts, all originating upstream of flaB1, as the wild-type cells. Northern hybridization experiments failed to detect a flaHIJ transcript in either wild-type or mutant cells. Immunoblotting experiments indicated, however, that the mutants produced similar amounts of flagellin, FlaD and FlaE to wild-type cells. Flagellin localization experiments suggest that the flaH mutant is deficient in flagellin secretion and/or assembly. The mutant also displayed similar preflagellin peptidase activity to the wild-type cells, indicating that none of the genes flaHIJ is likely to be the gene that encodes this enzyme, which is required for cleaving the leader peptide from the preflagellins prior to their incorporation into the flagellar filament. This is the first data indicating that the flaHIJ gene cluster is essential for flagellation in methanogens.

Archaeal Proteins↗
PubMed2001

Coordinate positive regulation of genes encoding [NiFe] hydrogenases in Methanococcus voltae.

Two transcription units encoding selenium-free [NiFe] hydrogenases in Methanococcus voltae are transcribed only upon selenium deprivation. Their products replace or complement selenocysteine-containing isoenzymes. The transcription units are linked by a 453-bp intergenic region, and are subject to both positive and negative transcriptional regulation. The mechanism of positive regulation was studied in detail. Mutations in identical 11-bp putative activator recognition sites close to each promoter showed that each site is involved in the activation of both promoters. Sequence-specific DNA-affinity chromatography yielded a 55-kDa protein which specifically recognized the 11-bp sequence. We consider this protein to be a transcriptional activator for both transcription units.

Base Sequence↗
PubMed2003

Isolation of a complete A1AO ATP synthase comprising nine subunits from the hyperthermophile Methanococcus jannaschii.

Archaeal A(1)A(O) ATP synthase/ATPase operons are highly conserved among species and comprise at least nine genes encoding structural proteins. However, all A(1)A(O) ATPase preparations reported to date contained only three to six subunits and, therefore, the study of this unique class of secondary energy converters is still in its infancy. To improve the quality of A(1)A(O) ATPase preparations, we chose the hyperthermophilic, methanogenic archaeon Methanococcus jannaschii as a model organism. Individual subunits of the A(1)A(O) ATPase from M. jannaschii were produced in E. coli, purified, and antibodies were raised. The antibodies enabled the development of a protocol ensuring purification of the entire nine-subunit A(1)A(O) ATPase. The ATPase was solubilized from membranes of M. jannaschii by Triton X-100 and purified to apparent homogeneity by sucrose density gradient centrifugation, ion exchange chromatography, and gel filtration. Electron micrographs revealed the A(1) and A(O) domains and the central stalk, but also additional masses which could represent a second stalk. Inhibitor studies were used to demonstrate that the A(1) and A(O) domains are functionally coupled. This is the first description of an A(1)A(O) ATPase preparation in which the two domains (A(1) and A(O)) are fully conserved and functionally coupled.

Adenosine Triphosphatases↗
PubMed2003

Effect of temperature and pressure on the proteolytic specificity of the recombinant 20S proteasome from Methanococcus jannaschii.

The hydrolytic specificity of the recombinant 20S proteasome from the deep-sea thermophile Methanococcus jannaschii was evaluated toward oxidized insulin B-chain across a range of temperatures (35 degrees, 55 degrees, 75 degrees, and 90 degrees C) and hydrostatic pressures (1, 250, 500, and 1,000 atm). Of the four temperatures considered, the same maximum overall hydrolysis rate was observed at both 55 degrees and 75 degrees C, which are much lower than the T(opt) of 116 degrees C previously observed for a small amide substrate (Michels and Clark 1997). At 35 degrees C the rates of cleavage were highest at the carboxyl side of glutamine and leucine, whereas at the three higher temperatures, the most rapid cleavages occurred after leucine and glutamic acid residues. The distribution of proteolytic fragments and the cleavage sequence also varied between the lowest and higher temperatures. Application of hydrostatic pressure did not increase proteasome activity, as observed previously for the amide substrate (Michels and Clark 1997), but instead significantly reduced the overall conversion of the polypeptide substrate. Overall cleavage patterns observed for the recombinant M. jannaschii proteasome were similar to those reported previously for Thermoplasma acidophilum (Akopian et al. 1997) and human proteasomes (Dick et al. 1991), indicating that proteasome specificity has been conserved despite significant environmental diversity.

Amino Acid Sequence↗
PubMed2003

Production of C35 isoprenoids depends on H2 availability during cultivation of the hyperthermophile Methanococcus jannaschii.

A series of five progressively saturated C35 isoprenoids has been identified in cell-free extracts of the deep-sea methanogen Methanococcus jannaschii. Production and relative abundance of the isoprenoids were dependent on culture conditions; significant production occurred in a 16-l fermentor (12-l working volume) and a 2.5-l fermentor (2-l working volume) but could not be duplicated in serum bottles. Several factors were investigated and shown not to account for the different production levels, including medium composition, pH, and temperature. However, the interphase mass transfer rate was shown to significantly affect the production of C35 isoprenoids in a fermentor. The structures of the novel isoprenoids were confirmed by hydrogenation reactions and mass spectra of the isoprenoids. Indirect evidence based on genomics and mass spectrometry data implicates head-to-head condensation of farnesyl pyrophosphate (C15) with geranylgeranyl pyrophosphate (C20) as the mechanism for C35 synthesis.

Culture Media↗
PubMed2000

MJ1647, an open reading frame in the genome of the hyperthermophile Methanococcus jannaschii, encodes a very thermostable archaeal histone with a C-terminal extension.

All archaeal histones studied to date have similar lengths, 66 to 69 amino acid residues that form three alpha-helices separated by two beta-strand loop regions which together constitute a histone fold. In contrast, the eukaryal nucleosome core histones are larger, 102 to 135 residues in length, with N-terminal and C-terminal extensions flanking the histone fold that participate in gene regulation and higher-order chromatin assembly. In the Methanococcus jannaschii genome, MJ1647 was annotated as an open reading frame predicted to encode an archaeal histone with an approximately 27-amino-acid C-terminal extension, and we here document the DNA binding and assembly properties and thermodynamic stability parameters of the recombinant product of MJ1647 synthesized in Escherichia coli with (rMJ1647) and without (rMJ1647delta) the C-terminal extension. The presence of the C-terminal extension did not prevent homodimer formation or inhibit DNA binding, but the complexes formed by rMJ1647, presumably archaeal nucleosomes containing a (rMJ1647)4 tetramer, were apparently less stable than those formed by (rMJ1647delta)4. The presence of the C-terminal extension increased the thermostability of rMJ1647 when compared with rMJ1647delta in 0.2 M KCl at pH 4 but not in the absence of KCl at pH 1. Based on thermal unfolding transitions, rMJ1647 and rHAfB generated by expression of AF0337 cloned from the genome of the related hyperthermophile Archaeoglobus fulgidus in E. coli were found to have higher thermodynamic stabilities than all previously studied archaeal histones.

Amino Acid Sequence↗
PubMed1993

Influence of illumination on the electronic interaction between 77Se and nickel in active F420-non-reducing hydrogenase from Methanococcus voltae.

The selenium-containing F420-non-reducing hydrogenase from Methanococcus voltae was anaerobically purified. The enzyme as isolated showed an EPR spectrum with gx,y,z = 2.21, 2.15 and 2.01. Upon illumination this spectrum disappeared and a new signal with the lowest g value at 2.05 arose. EPR studies were carried out either with the enzyme containing natural selenium or enriched in the nuclear isotope 77Se. The hyperfine splitting caused by 77Se in the 'dark' signal is shown to be highly anisotropic. In contrast the splitting is nearly isotropic after illumination. A new model for the nickel site is proposed to explain these observations.

Anaerobiosis↗
PubMed1993

Association of the mcrD gene product with methyl coenzyme M reductase in Methanococcus vannielii.

The mcrD gene, subcloned from the methyl coenzyme M reductase (MR) encoding mcrBDCGA operon in Methanococcus vannielii, has been expressed at a high level in Escherichia coli. Rabbit antibodies, raised against the product of this gene (rgpmcrD, recombinant gene product of mcrD) purified from E. coli, have been used to quantitate gpmcrD in M. vannielii and to follow its fate during MR purification. The molar ratio of gpmcrD to MR was found to be approx. 1:15 in cells of M. vannielii taken from batch cultures at all stages of growth. Sedimentation of lysates of M. vannielii cells through sucrose gradients and analyses of the fractions obtained by Western blotting and immunoprecipitation have demonstrated the presence of a macromolecular complex containing both gpmcrD and MR. Addition of mcrD antibodies or removal of gpmcrD from lysates of M. vannielii cells by immunoprecipitation decreased the rates of methanogenesis in vitro by approx. 20%. Addition of purified rgpmcrD to these lysates did not stimulate methanogenesis.

Blotting, Western↗
PubMed1994

In vivo transcripts of the S-layer-encoding structural gene of the archaeon Methanococcus voltae.

The 5' region of the S-layer-encoding structural gene (sla) of Methanococcus voltae was sequenced. The sequence information was then used to identify the in vivo transcription products of the gene. We observed three transcripts, and upstream from each transcription start point was a region with similarity to the Box A consensus sequence observed in archaeal promoters. In two of the three cases, two Box A sequences were present in tandem. This arrangement may play a role in the high level of gene expression expected for the sla gene. Presumptive archaeal Box B signatures were also identified.

Amino Acid Sequence↗
PubMed2003

The biosynthesis of cysteine and homocysteine in Methanococcus jannaschii.

The pathway for the biosynthesis of cysteine and homocysteine in Methanococcus jannaschii has been examined using a gas chromatography-mass spectrometry (GC-MS) stable isotope dilution method to identify and quantitate the intermediates in the pathways. The first step in the pathway, and the one responsible for incorporation of sulfur into both cysteine and methionine, is the reaction between O-phosphohomoserine and a presently unidentified sulfur source present in cell extracts, to produce L-homocysteine. This sulfur source was shown not to be sulfide. The resulting L-homocysteine then reacts with O-phosphoserine to form L-cystathionine, which is cleaved to L-cysteine. The pathway has elements of both the plant and mammalian pathways in that the sulfur is first incorporated into homocysteine using O-phosphohomoserine as the acceptor and the resulting homocysteine, via transsulfuration, supplies the sulfur for cysteine formation. The pathway leading to these two amino acids represents an example of metabolic thrift where the preexisting cellular metabolites O-phosphohomoserine and O-phosphoserine are used as the ultimate source of the carbon framework for the biosynthesis of these amino acids. These findings explain the absence of identifiable genes in the genome of this organism for the biosynthesis of cysteine and homocysteine.

Cystathionine↗
PubMed2005

Compatible solute effects on thermostability of glutamine synthetase and aspartate transcarbamoylase from Methanococcus jannaschii.

Methanococcus jannaschii accumulates alpha- and beta-glutamate as osmolytes. The effect of these and other solutes on the thermostability of two multisubunit metabolic enzymes from M. jannaschii, aspartate transcarbamoylase catalytic trimer (ATCase C3) and glutamine synthetase (GS), has been measured and compared to solute effects on bacterial mesophilic counterparts in order to explore if osmolytes accumulated by each organism can preferentially stabilize the proteins to thermal unfolding. For both ATCase enzymes and for the B. subtilis GS, the solutes normally accumulated by the organism were very effective in protecting the enzyme from losing activity at high temperatures, although solute effects on loss of secondary structure did not necessarily correlate with this thermoprotection of activity. The recombinant M. jannaschii GS exhibited quite different behavior. The pure enzyme had a thermal unfolding transition with a midpoint temperature (Tm) less than 60 degrees C, well under the growth temperature of the organism (85 degrees C). None of the small molecule solutes tested (including the K+-glutamate isomers accumulated by M. jannaschii) significantly stabilized the protein to incubation at 85 degrees C. Instead, protein-protein interactions, as illustrated by E. coli GroEL or ribosomal protein L2 stabilization of GS, appeared to be the dominant factor in stabilizing this archaeal enzyme at the growth temperature.

Aspartate Carbamoyltransferase↗
PubMed2004

Functional identification of the SecB homologue in Methanococcus jannaschii and direct interaction of SecB with trigger factor.

In this study Mj0357 protein, a hypothetical protein from Methanococcus jannaschii which shows an 18% sequence identity with SecB from E. coli, has been identified as a functional homologue of SecB in M. jannaschii through a number of biochemical and biophysical examinations. It is composed mostly of beta-strands and exists as a homotetramer in solution. Mj0357 protein exhibits in vitro chaperone-like activity, suppressing thermal aggregation of citrate synthase and binding to partially folded maltose-binding protein. Upon binding to a peptide ligand, the protein undergoes a conformational change to expose a hydrophobic patch on the protein surface. All these physicochemical properties are highly similar to those of E. coli SecB. In addition, E. coli trigger factor (TF) has been shown here for the first time to bind E. coli SecB and Mj0357 protein with low micromolar affinities, indicating that the TF could interact directly along the SecB-dependent translocation pathway. These results indicate that the translocation pathway is conserved and functionally homologous in at least one of the archaeal organisms.

Animals↗
PubMed2004

Continuous culture of Methanococcus maripaludis under defined nutrient conditions.

To study global regulation in the methanogenic archaeon Methanococcus maripaludis, we devised a system for steady-state growth in chemostats. New Brunswick Bioflo 110 bioreactors were equipped with controlled delivery of hydrogen, nitrogen, carbon dioxide, hydrogen sulfide, and anaerobic medium. We determined conditions and media compositions for growth with three different limiting nutrients, hydrogen, phosphate, and leucine. To investigate leucine limitation we constructed and characterized a mutant in the leuA gene for 2-isopropylmalate synthase, demonstrating for the first time the function of this gene in the Archaea. Steady state specific growth rates in these studies ranged from 0.042 to 0.24 h(-1). Plots of culture density vs. growth rate for each condition showed the behavior predicted by growth modeling. The results show that growth behavior is normal and reproducible and validate the use of the chemostat system for metabolic and global regulation studies in M. maripaludis.

2-Isopropylmalate Synthase↗