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PubMed2003

Function and regulation of the formate dehydrogenase genes of the methanogenic archaeon Methanococcus maripaludis.

Methanococcus maripaludis is a mesophilic species of Archaea capable of producing methane from two substrates: hydrogen plus carbon dioxide and formate. To study the latter, we identified the formate dehydrogenase genes of M. maripaludis and found that the genome contains two gene clusters important for formate utilization. Phylogenetic analysis suggested that the two formate dehydrogenase gene sets arose from duplication events within the methanococcal lineage. The first gene cluster encodes homologs of formate dehydrogenase alpha (FdhA) and beta (FdhB) subunits and a putative formate transporter (FdhC) as well as a carbonic anhydrase analog. The second gene cluster encodes only FdhA and FdhB homologs. Mutants lacking either fdhA gene exhibited a partial growth defect on formate, whereas a double mutant was completely unable to grow on formate as a sole methanogenic substrate. Investigation of fdh gene expression revealed that transcription of both gene clusters is controlled by the presence of H(2) and not by the presence of formate.

Formate Dehydrogenases↗
PubMed2004

Dimethylselenide demethylation is an adaptive response to selenium deprivation in the archaeon Methanococcus voltae.

The archaeon Methanococcus voltae needs selenium for optimal growth. A gene group most likely involved in the demethylation of dimethylselenide was discovered, the expression of which is induced upon selenium deprivation. The operon comprises open reading frames for a corrinoid protein and two putative methyltransferases. It is shown that the addition of dimethylselenide to selenium-depleted growth medium relieves the lack of selenium, as indicated by the repression of a promoter of a transcription unit encoding selenium-free hydrogenases which is normally active only upon selenium deprivation. Knockout mutants of the corrinoid protein or one of the two methyltransferase genes did not show repression of the hydrogenase promoter in the presence of dimethylselenide. The mutation of the other methyltransferase gene had no effect. Growth rates of the two effective mutants were reduced compared to wild-type cells in selenium-limited medium in the presence of dimethylselenide.

Adaptation, Physiological↗
PubMed2004

Two biosynthetic pathways for aromatic amino acids in the archaeon Methanococcus maripaludis.

Methanococcus maripaludis is a strictly anaerobic, methane-producing archaeon. Aromatic amino acids (AroAAs) are biosynthesized in this autotroph either by the de novo pathway, with chorismate as an intermediate, or by the incorporation of exogenous aryl acids via indolepyruvate oxidoreductase (IOR). In order to evaluate the roles of these pathways, the gene that encodes the third step in the de novo pathway, 3-dehydroquinate dehydratase (DHQ), was deleted. This mutant required all three AroAAs for growth, and no DHQ activity was detectible in cell extracts, compared to 6.0 +/- 0.2 mU mg(-1) in the wild-type extract. The growth requirement for the AroAAs could be fulfilled by the corresponding aryl acids phenylacetate, indoleacetate, and p-hydroxyphenylacetate. The specific incorporation of phenylacetate into phenylalanine by the IOR pathway was demonstrated in vivo by labeling with [1-(13)C]phenylacetate. M. maripaludis has two IOR homologs. A deletion mutant for one of these homologs contained 76, 74, and 42% lower activity for phenylpyruvate, p-hydoxyphenylpyruvate, and indolepyruvate oxidation, respectively, than the wild type. Growth of this mutant in minimal medium was inhibited by the aryl acids, but the AroAAs partially restored growth. Genetic complementation of the IOR mutant also restored much of the wild-type phenotype. Thus, aryl acids appear to regulate the expression or activity of the de novo pathway. The aryl acids did not significantly inhibit the activity of the biosynthetic enzymes chorismate mutase, prephenate dehydratase, and prephenate dehydrogenase in cell extracts, so the inhibition of growth was probably not due to an effect on these enzymes.

Amino Acids, Aromatic↗
PubMed2004

Complete genome sequence of the genetically tractable hydrogenotrophic methanogen Methanococcus maripaludis.

The genome sequence of the genetically tractable, mesophilic, hydrogenotrophic methanogen Methanococcus maripaludis contains 1,722 protein-coding genes in a single circular chromosome of 1,661,137 bp. Of the protein-coding genes (open reading frames [ORFs]), 44% were assigned a function, 48% were conserved but had unknown or uncertain functions, and 7.5% (129 ORFs) were unique to M. maripaludis. Of the unique ORFs, 27 were confirmed to encode proteins by the mass spectrometric identification of unique peptides. Genes for most known functions and pathways were identified. For example, a full complement of hydrogenases and methanogenesis enzymes was identified, including eight selenocysteine-containing proteins, with each being paralogous to a cysteine-containing counterpart. At least 59 proteins were predicted to contain iron-sulfur centers, including ferredoxins, polyferredoxins, and subunits of enzymes with various redox functions. Unusual features included the absence of a Cdc6 homolog, implying a variation in replication initiation, and the presence of a bacterial-like RNase HI as well as an RNase HII typical of the Archaea. The presence of alanine dehydrogenase and alanine racemase, which are uniquely present among the Archaea, explained the ability of the organism to use L- and D-alanine as nitrogen sources. Features that contrasted with the related organism Methanocaldococcus jannaschii included the absence of inteins, even though close homologs of most intein-containing proteins were encoded. Although two-thirds of the ORFs had their highest Blastp hits in Methanocaldococcus jannaschii, lateral gene transfer or gene loss has apparently resulted in genes, which are often clustered, with top Blastp hits in more distantly related groups.

Archaeal Proteins↗
PubMed2005

Site-directed mutagenesis analysis of amino acids critical for activity of the type I signal peptidase of the archaeon Methanococcus voltae.

Site-directed mutagenesis studies of the signal peptidase of the methanogenic archaeon Methanococcus voltae identified three conserved residues (Ser52, His122, and Asp148) critical for activity. The requirement for one conserved aspartic acid residue distinguishes the archaeal enzyme from both the Escherichia coli and yeast Sec11 enzymes.

Amino Acid Sequence↗
PubMed2005

Markerless mutagenesis in Methanococcus maripaludis demonstrates roles for alanine dehydrogenase, alanine racemase, and alanine permease.

Among the archaea, Methanococcus maripaludis has the unusual ability to use L- or D-alanine as a nitrogen source. To understand how this occurs, we tested the roles of three adjacent genes encoding homologs of alanine dehydrogenase, alanine racemase, and alanine permease. To produce mutations in these genes, we devised a method for markerless mutagenesis that builds on previously established genetic tools for M. maripaludis. The technique uses a negative selection strategy that takes advantage of the ability of the M. maripaludis hpt gene encoding hypoxanthine phosphoribosyltransferase to confer sensitivity to the base analog 8-azahypoxanthine. In addition, we developed a negative selection method to stably incorporate constructs into the genome at the site of the upt gene encoding uracil phosphoribosyltransferase. Mutants with in-frame deletion mutations in the genes for alanine dehydrogenase and alanine permease lost the ability to grow on either isomer of alanine, while a mutant with an in-frame deletion mutation in the gene for alanine racemase lost only the ability to grow on D-alanine. The wild-type gene for alanine dehydrogenase, incorporated into the upt site, complemented the alanine dehydrogenase mutation. Hence, the permease is required for the transport of either isomer, the dehydrogenase is specific for the L isomer, and the racemase converts the D isomer to the L isomer. Phylogenetic analysis indicated that all three genes had been acquired by lateral gene transfer from the low-moles-percent G+C gram-positive bacteria.

Alanine↗
PubMed2006

Disruption of the operon encoding Ehb hydrogenase limits anabolic CO2 assimilation in the archaeon Methanococcus maripaludis.

Methanococcus maripaludis is a mesophilic archaeon that reduces CO2 to methane with H2 or formate as an energy source. It contains two membrane-bound energy-conserving hydrogenases, Eha and Ehb. To determine the role of Ehb, a deletion in the ehb operon was constructed to yield the mutant, strain S40. Growth of S40 was severely impaired in minimal medium. Both acetate and yeast extract were necessary to restore growth to nearly wild-type levels, suggesting that Ehb was involved in multiple steps in carbon assimilation. However, no differences in the total hydrogenase specific activities were found between the wild type and mutant in either cell extracts or membrane-purified fractions. Methanogenesis by resting cells with pyruvate as the electron donor was also reduced by 30% in S40, suggesting a defect in pyruvate oxidation. CO dehydrogenase/acetyl coenzyme A (CoA) synthase and pyruvate oxidoreductase had higher specific activities in the mutant, and genes encoding these enzymes, as well as AMP-forming acetyl-CoA synthetase, were expressed at increased levels. These observations support a role for Ehb in anabolic CO2 assimilation in methanococci.

Acetate-CoA Ligase↗
PubMed1994

Biotransformation of 2,4,6-trinitrotoluene (TNT) by a Methanococcus sp. (strain B) isolated from a lake sediment.

A mesophilic, irregular coccoid methanogen, which shows close resemblance to Methanococcus sp., was isolated from a sediment sample of St. Joseph Lake located in the University of Notre Dame campus. Formate or hydrogen plus carbon dioxide served as substrate for methanogenesis in a mineral salt medium. This organism was studied for its ability to metabolize 2,4,6-trinitrotoluene (TNT). The result showed that this isolate could transform 100 ppm of TNT within 40-60 days of incubation at 30 degrees C. The main intermediate produced was 2,4-diamino-6-nitrotoluene. The TNT transformation rates were higher in cells grown in hydrogen plus carbon dioxide than in cells grown in formate. The isolate did not use acetate and methanol as sole source of carbon and energy. The organism had an optimal pH range of 6.8-7.2. The optimal growth conditions for this isolate are described.

Biotransformation↗
PubMed2004

Automated metabolic reconstruction for Methanococcus jannaschii.

We present the computational prediction and synthesis of the metabolic pathways in Methanococcus jannaschii from its genomic sequence using the PathoLogic software. Metabolic reconstruction is based on a reference knowledge base of metabolic pathways and is performed with minimal manual intervention. We predict the existence of 609 metabolic reactions that are assembled in 113 metabolic pathways and an additional 17 super-pathways consisting of one or more component pathways. These assignments represent significantly improved enzyme and pathway predictions compared with previous metabolic reconstructions, and some key metabolic reactions, previously missing, have been identified. Our results, in the form of enzymatic assignments and metabolic pathway predictions, form a database (MJCyc) that is accessible over the World Wide Web for further dissemination among members of the scientific community.

Computational Biology↗
PubMed2005

Role of the precorrin 6-X reductase gene in cobamide biosynthesis in Methanococcus maripaludis.

In Methanococcus maripaludis strain JJ, deletion of the homolog to cbiJ, which encodes the corrin biosynthetic enzyme precorrin 6-X reductase, yielded an auxotroph that required either cobamide or acetate for good growth. This phenotype closely resembled that of JJ117, a mutant in which tandem repeats were introduced into the region immediately downstream of the homolog of cbiJ. Mutant JJ117 also produced low quantities of cobamides, about 15 nmol g(-1) protein or 1-2% of the amount found in wild-type cells. These results confirm the role of the cbiJ homolog in cobamide biosynthesis in the Archaea and suggest the presence of low amounts of a bypass activity in these organisms.

Base Sequence↗
PubMed2001

Characterization of RNase P holoenzymes from Methanococcus jannaschii and Methanothermobacter thermoautotrophicus.

The partial purification and basic biochemical characterization of the RNase P holoenzymes of two species of methanogenic Archaea, Methanothermobacter thermoautotrophicus (previously Methanobacterium thermoautotrophicum strain deltaH) and Methanococcus jannaschii, are described. The properties of these enzymes, particularly buoyant density in Cs2SO4 and recent information about the subunit composition of the archaeal enzymes, suggest that RNase P enzymes in Archaea are much more alike than earlier studies in Sulfolobus acidocaldarius and Haloferax volcanii suggested.

Centrifugation, Density Gradient↗
PubMed2000

A tRNA pseudogene in the archaeon Methanococcus jannaschii.

While searching the first completely sequenced genome of the archaeon Methanococcus jannaschii for a small RNA gene, we discovered a 5' truncated gene of a transfer RNA (tRNA(Ser-UCR)) at position 334,431-334,486; including the CCA-end that exactly matched the 3' terminal domain of the annotated M. jannaschii tRNA(Ser-UCR) gene located at position 303,992-304,081. This truncated tRNA gene covering 56 nucleotides (about 2/3) of the genuine tRNA represents, to the best of our knowledge, the first described tRNA pseudogene in the archaeal domain.

Base Sequence↗
PubMedDate not supplied

[Immunoelectron-microscopic determination of the localization of tryptophanyl-tRNA synthetase in eubacterial cells of Escherichia coli and Methanococcus halophilus archaebacteria].

Localization of tryptophanyl-tRNA-synthetase (TRS) was studied in halophilic archaebacterium Methanococcus halophilus and eubacterium E. coli. Ultrathin sections of the cells, fixed with glutaraldehyde and embedded in "Lowicryl K4M" at -35 degrees C, were treated with colloidal gold complexes containing monoclonal antibodies Aml against TRS. The latter bind specifically to TRS isolated both from eucaryotes, archae- and eubacteria. According to the label distribution three zones in M. halophilus and E. coli can be distinguished: (i) about 75% of the whole amount of gold particles are localized in the cytoplasm, the distribution of label being more or less homogeneous; (ii) cytoplasmic regions, adjacent to nucleoid, are intensively labelled (about 20% of the whole amount of label); (iii) very few gold particles (not more than 10% of the whole amount) are present in the nucleoid. The data obtained show, that the distribution of TRS in the nucleoid and cytoplasm of archaebacterium M. halophilus is close to the distribution of TRS, found in E. coli. It supports our previous conclusion that the structural organization of transcription-translation apparatus in methanogen and halophilic archaebacteria is similar to that in eubacteria.

Antibodies, Monoclonal↗
PubMed1993

Purification and characterization of a general transcription factor, aTFB, from the archaeon Methanococcus thermolithotrophicus.

We have recently shown that cell-free transcription of homologous templates from the archaeon Methanococcus thermolithotrophicus requires an archaeal transcription factor (aTFA) that separated from the RNA polymerase during phosphocellulose chromatography. We report here the identification and extensive purification of a second activity, aTFB, required for in vitro transcription. This activity copurified with RNA polymerase during initial chromatographic steps but was positively identified as a distinct transcription factor after Superdex 200 sizing chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the intensity of a M(r) = 28,000 polypeptide in silver-stained gels is correlated with transcription factor activity. The same polypeptide, when eluted from a denaturing polyacrylamide gel and subsequently renatured, showed the functional properties of the transcription factor. In conjunction with gel filtration and sedimentation studies, which indicated a molecular mass of 54,000 Da for the native protein, these results suggested that aTFB is a dimer with polypeptide chains of identical molecular mass. Functional studies with highly purified aTFB demonstrated that it is a general factor required for transcription of genes encoding tRNA and proteins.

Cell-Free System↗
PubMedDate not supplied

An all sulfur analogue of the smallest subunit of F420-non-reducing hydrogenase from Methanococcus voltae--metal binding and structure.

The 25 amino acid long subunit VhuU of the F420-non-reducing hydrogenase from Methanococcus voltae contains selenocysteine within the consensus sequence of known [NiFe] hydrogenases DP(C or U)CxxCxxH (U = selenocysteine). The sulfur-analogue VhuUc was chemically synthesized, purified and its metal binding capability, the catalytic properties, and structural features were investigated. The polypeptide was able to bind nickel, but did not catalyse the heterolytic activation of H2. 2D-NMR spectroscopy revealed an alpha-helical secondary structure for the 15 N-terminal amino acids in 50% TFE. Nickel only binds to the C-terminus, which contains the conserved amino acid motif. Structures derived from the NMR data are compatible with the participation of both sulfur atoms from the conserved cysteine residues in a metal ion binding. Structures obtained from the data sets for Ni.VhuUc as well as Zn.VhuUc showed no further ligands. The informational value for Ni.VhuUc was low due to paramagnetism.

Amino Acid Sequence↗
PubMed1996

Methanococcus jannaschii genome: revisited.

Analysis of genomic sequences is necessarily an ongoing process. Initial gene assignments tend (wisely) to be on the conservative side (Venter, 1996). The analysis of the genome then grows in an iterative fashion as additional data and more sophisticated algorithms are brought to bear on the data. The present report is an emendation of the original gene list of Methanococcus jannaschii (Bult et al., 1996). By using a somewhat more updated database and more relaxed (and operator-intensive) pattern matching methods, we were able to add significantly to, and in a few cases amend, the gene identification table originally published by Bult et al. (1996).

Databases as Topic↗
PubMed1985

Non-repetitive AT-rich sequences are found in intergenic regions of Methanococcus voltae DNA.

AT-rich DNA, which constitutes a distinct fraction of the cellular DNA of the archaebacterium Methanococcus voltae, was shown to consist of non-repetitive sequences dispersed on the chromosome and to lack continuous open reading frames in five out of six randomly analyzed cases. Upon subsequent analysis of intergenic regions, AT-rich sequences were again detected. Transcription start points were mapped in front of three open reading frames. The 5' ends of the transcripts were found in variable positions relative to the AT-rich sequences in the different cases. Shine-Dalgarno type sequences complementary to the 3' end of 16S rRNA were discovered at suitable distances from the 5' ends of the genes.

Base Sequence↗
PubMed1990

Construction of an integration vector for use in the archaebacterium Methanococcus voltae and expression of a eubacterial resistance gene.

An integration vector for use in Methanococcus voltae was constructed, based on the Escherichia coli vector pUC18. It carries the structural gene for puromycin transacetylase from Streptomyces alboniger, which is flanked by expression signals of M. voltae structural genes and hisA gene sequences of this bacterium. Transformed M. voltae cells are puromycin resistant. Several types of integration of the vector into the chromosome were found. Only one case was due to nonhomologous recombination. The integrated sequences were stable under selective pressure but were slowly lost in some cases in the absence of the selective drug. The vector could be excised from M. voltae chromosomal DNA, recircularized and transformed back into E. coli.

Blotting, Southern↗