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PubMedDate not supplied

Molecular evolution of H+-ATPases. I. Methanococcus and Sulfolobus are monophyletic with respect to eukaryotes and Eubacteria.

The classification of methanogenic bacteria as archaebacteria based on 16 s rRNA sequence analysis is currently in dispute. To provide an alternative molecular marker, the polymerase chain reaction technique was used to amplify a 930 bp fragment of Methanococcus thermolithotrophicus genomic DNA corresponding to the catalytic domain of the membrane H+-ATPase. The deduced amino acid sequence was 54-58% identical to the approximately 70 kDa subunits of Sulfolobus acidocaldarius and the eukaryotic vacuolar-type H+-ATPase, and only 29% identical to the beta subunit of the eubacterial-type F0F1-ATPases. Interestingly, a highly conserved aspartate residue in the phosphorylation domain of E1E2-ATPases (P-type) is conserved in the Methanococcus sequence, but is absent from all other known vacuolar and F0F1-ATPases. This suggests that the H+-ATPase of M. thermolithotrophicus, like that of M. voltae, may have a phosphorylated intermediate, despite belonging to the vacuolar-type class of proton pumps. Phylogenetic analysis using Felsenstein's maximum likelihood method and Lake's evolutionary parsimony method confirmed that the H+-ATPases of the two archaebacteria, Methanococcus and Sulfolobus, when compared to eukaryotic vacuolar-type ATPases and eubacterial F0F1-ATPases, form a monophyletic group.

Amino Acid Sequence↗

PubMed1997

Selenoprotein synthesis in archaea: identification of an mRNA element of Methanococcus jannaschii probably directing selenocysteine insertion.

Selenocysteine is encoded by a UGA codon in all organisms that synthesise selenoproteins. This codon is specified as a selenocysteine codon by an mRNA secondary structure, which is located immediately 3' of the UGA in the reading frame of selenoprotein genes in Gram-negative bacteria, whereas it is located in the 3' untranslated region of eukaryal selenoprotein genes. The location and the structure of a similar mRNA signal in archaea has so far not been determined. Seven selenoproteins were identified for the archaeon Methanococcus jannaschii by labelling with 75Se and by SDS/polyacrylamide electrophoresis. Their size could be correlated with open reading frames possessing internal UGA codons from the total genomic sequence. One of the open reading frames, that of the VhuD subunit of a hydrogenase, possesses two UGA codons and appears to code for a selenoprotein with two selenocysteine residues. A strongly conserved mRNA element was identified that is exclusively linked to selenoprotein genes. It is located in the 3' untranslated region in six of the mRNAs and in the 5' untranslated region of the fdhA mRNA. This element, which is present in the 3' non-translated region of two selenoprotein mRNAs from Methanococcus voltae, is proposed to act in decoding of the UGA with selenocysteine.

Amino Acid Sequence↗

PubMed1997

Unidentified open reading frames in the genome of Methanococcus jannaschii are similar in sequence to an archaebacterial gene for tRNA nucleotidyltransferase.

The protein sequence of ATP/CTP:tRNA nucleotidyltransferase (cca) from Sulfolobus shibatae was used to search open reading frames in the genome of Methanococcus jannaschii. Translations of two unidentified open reading frames showed significant sequence similarity to portions of the Sulfolobus cca protein. When the two open reading frames were joined together, the expanded open reading frame was similar in sequence to the entire Sulfolobus cca protein and displayed features of the active site signature sequence proposed for members of class I enzymes within the superfamily of nucleotidyltransferases (Yue et al., 1996, RNA 2, 895-908). A possible UUG start codon was identified based on significant sequence similarity of the resulting amino-terminal region to that of Sulfolobus, and on a six-base complementarity between an adjacent upstream sequence and Methanococcus 16S rRNA.

Amino Acid Sequence↗

PubMed1991

Control regions of an archaeal gene. A TATA box and an initiator element promote cell-free transcription of the tRNA(Val) gene of Methanococcus vannielii.

To identify the DNA sequences required for initiation of transcription in archaea, the 5'-flanking region of the tRNA(Val) gene of Methanococcus vannielii was modified by deletions, restructuring and site-directed mutagenesis, and the tRNA encoding sequence was replaced by a fortuitous Escherichia coli sequence. The effects of these mutations on promoter function were tested in an homologous cell-free transcription system. The DNA region from position -35 to +9 relative to the transcription start site was sufficient for maximal initiation of cell-free transcription. Removal of the DNA region between -35 and -30 reduced initiation by a factor of 2. Deletions extending to position -24 almost completely abolished specific transcription. Analysis of 16 site-specific mutations in the region from -33 to +2 provided evidence that a conserved A + T-rich sequence (TATA box), centered at -25, is essential for initiation of transcription. Single point mutations in six positions of the TATA box reduced initiation of transcription from 0.2 to 0.01 of wild-type levels. A second conserved motif at the transcription start site (consensus ATGC) could be replaced by some sequences containing a pyrimidine-purine dinucleotide but appeared necessary for a maximal rate of gene transcription. Mutations altering the spacing between the two conserved elements demonstrated that initiation occurs at a strictly defined distance of 22 to 27 base-pairs downstream from the TATA box. Our results support the conclusion that the TATA box is the major DNA region mediating promoter recognition, influencing the efficiency of transcription and specifying the site of transcription initiation. This Methanococcus promoter element closely resembles in structure and function the TATA box of promoters of eukaryotic protein-encoding genes transcribed by RNA polymerase II.

Base Sequence↗

PubMed1997

Cloning and phylogenetic analysis of the genes encoding acetohydroxyacid synthase from the archaeon Methanococcus aeolicus.

The gene for acetohydroxyacid synthase (AHAS) was cloned from the archaeon Methanococcus aeolicus. Contrary to biochemical studies [Xing, R. and Whitman, W.B. (1994) J. Bacteriol. 176, 1207-1213] the enzyme was encoded by two open reading frames (ORFs). Based on sequence homology, these ORFs were designated ilvB and ilvN for the large and small subunits of AHAS, respectively. A putative methanogen promoter preceded ilvB-ilvN, and a potential internal promoter was found upstream of ilvN. ilvB encoded a 65-kDa protein, which agreed well with the measured value for the purified enzyme. ilvN encoded a 19-kDa protein, which fell within the range of M(r) of small subunits from other sources. Phylogenetic analysis of the deduced amino acid sequence of ilvB showed a close relationship between the AHAS of Bacteria and Archaea, to the exclusion of other enzymes in this family, including pyruvate oxidase, glyoxylate carboligase, pyruvate decarboxylase, and the acetolactate synthase found in fermentative Bacteria. Thus, this family of enzymes probably arose prior to the divergence of the Bacteria and Archaea. Moreover, the higher plant AHAS and the red algal AHAS were related to the AHAS II of Escherichia coli and the cyanobacterial AHAS, respectively. For this reason, these genes appear to have been acquired by the Eucarya during the endosymbiosis that gave rise to the mitochondrion and chloroplast, respectively. One of the ORFs in the Methanococcus jannaschii genome possesses high similarity to the M. aeolicus ilvB, indicating that it is an authentic AHAS.

Acetolactate Synthase↗

PubMed1999

Genetics of Methanococcus: possibilities for functional genomics in Archaea.

Although the genomic sequences of a number of Archaea have been completed in the last three years, genetic systems in the sequenced organisms are absent. In contrast, genetic studies of the mesophiles in the archaeal genus Methanococcus have become commonplace following the recent developments of antibiotic resistance markers, DNA transformation methods, reporter genes, shuttle vectors and expression vectors. These developments have led to investigations of the transcription of the genes for hydrogen metabolism, nitrogen fixation and flagellin assembly. These genetic systems can potentially be used to analyse the genomic sequence of the hyperthermophile Methanococcus jannaschii, addressing questions of its physiology and the function of its many uncharacterized open reading frames. Thus, the sequence of M. jannaschii can serve as a starting point for gene isolation, while in vivo genetics in the mesophilic methanococci can provide the experimental systems to test the predictions from genomics.

Anaerobiosis↗

PubMed1999

Thermal adaptation analyzed by comparison of protein sequences from mesophilic and extremely thermophilic Methanococcus species.

The genome sequence of the extremely thermophilic archaeon Methanococcus jannaschii provides a wealth of data on proteins from a thermophile. In this paper, sequences of 115 proteins from M. jannaschii are compared with their homologs from mesophilic Methanococcus species. Although the growth temperatures of the mesophiles are about 50 degrees C below that of M. jannaschii, their genomic G+C contents are nearly identical. The properties most correlated with the proteins of the thermophile include higher residue volume, higher residue hydrophobicity, more charged amino acids (especially Glu, Arg, and Lys), and fewer uncharged polar residues (Ser, Thr, Asn, and Gln). These are recurring themes, with all trends applying to 83-92% of the proteins for which complete sequences were available. Nearly all of the amino acid replacements most significantly correlated with the temperature change are the same relatively conservative changes observed in all proteins, but in the case of the mesophile/thermophile comparison there is a directional bias. We identify 26 specific pairs of amino acids with a statistically significant (P < 0.01) preferred direction of replacement.

Acclimatization↗

PubMed1995

The translation product of the presumptive Thermococcus celer TATA-binding protein sequence is a transcription factor related in structure and function to Methanococcus transcription factor B.

A gene for a putative homolog of TATA-binding protein (TBP) from Thermococcus celer has been expressed in Escherichia coli, and the function of the purified recombinant protein was studied in a Methanococcus-derived cell-free transcription system. Thermococcus TBP can replace archaeal transcription factor B (aTFB) in cell-free transcription reactions. This transcriptional activation is TATA box-dependent and occurs both on tRNA(Val) and protein-encoding genes as templates indicating that Thermococcus TBP is a general transcription factor. Antibodies raised against Thermococcus TBP bind to Methanococcus aTFB and inhibit a TFB activity. These findings demonstrate that Thermococcus TBP (like eucaryal TBPs) can direct specific transcription from TATA boxes.

Archaea↗

PubMed2000

Crystal structure of a fibrillarin homologue from Methanococcus jannaschii, a hyperthermophile, at 1.6 A resolution.

Fibrillarin is a phylogenetically conserved protein essential for efficient processing of pre-rRNA through its association with a class of small nucleolar RNAs during ribosomal biogenesis. The protein is the antigen for the autoimmune disease scleroderma. Here we report the crystal structure of the fibrillarin homologue from Methanococcus jannaschii, a hyperthermophile, at 1.6 A resolution. The structure consists of two domains, with a novel fold in the N-terminal region and a methyltransferase-like domain in the C-terminal region. Mapping temperature-sensitive mutations found in yeast fibrillarin Nop1 to the Methanococcus homologue structure reveals that many of the mutations cluster in the core of the methyltransferase-like domain.

Amino Acid Sequence↗

PubMed1999

Genetics of nitrogen regulation in Methanococcus maripaludis.

We have used genetic methods in Methanococcus maripaludis to study nitrogen metabolism and its regulation. We present evidence for a "nitrogen regulon" in Methanococcus and Methanobacterium species containing genes of nitrogen metabolism that are regulated coordinately at the transcriptional level via a common repressor binding site sequence, or operator. The implied mechanism for regulation resembles the general bacterial paradigm for repression, but contrasts with well-known mechanisms of nitrogen regulation in bacteria, which occur by activation. Genes in the nitrogen regulons include those for nitrogen fixation, glutamine synthetase, (methyl)ammonia transport, the regulatory protein GlnB, and ammonia-dependent NAD synthetase, as well as a gene of unknown function. We also studied the function of two novel GlnB homologues that are encoded within the nif gene cluster of diazotrophic methanogens. The phenotype resulting from a glnB null mutation in M. maripaludis provides direct evidence that glnB-like genes are involved in "ammonia switch-off," the post-transcriptional inhibition of nitrogen fixation upon addition of ammonia. Finally, we show that the gene nifX is not required for nitrogen fixation, in agreement with findings in several bacteria. These studies illustrate the utility of genetic methods in M. maripaludis and show the enhanced perspective that studies in the Archaea can bring to known biological systems.

Amino Acid Sequence↗

PubMed1999

Expression vectors for Methanococcus maripaludis: overexpression of acetohydroxyacid synthase and beta-galactosidase.

A series of integrative and shuttle expression vectors was developed for use in Methanococcus maripaludis. The integrative expression vectors contained the Methanococcus voltae histone promoter and multiple cloning sites designed for efficient cloning of DNA. Upon transformation, they can be used to overexpress specific homologous genes in M. maripaludis. When tested with ilvBN, which encodes the large and small subunits of acetohydroxyacid synthase, transformants possessed specific activity 13-fold higher than that of the wild type. An expression shuttle vector, based on the cryptic plasmid pURB500 and the components of the integrative vector, was also developed for the expression of heterologous genes in M. maripaludis. The beta-galactosidase gene from Escherichia coli was expressed to approximately 1% of the total cellular protein using this vector. During this work, the genes for the acetohydroxyacid synthase (ilvBN) and phosphoenolpyruvate synthase (ppsA) were sequenced from a M. maripaludis genomic library.

Acetolactate Synthase↗

PubMed1998

Methanococcus infernus sp. nov., a novel hyperthermophilic lithotrophic methanogen isolated from a deep-sea hydrothermal vent.

An autotrophic, extremely thermophilic methanogen (ME(T)) was isolated from a deep-sea hydrothermal chimney sample collected on the Mid-Atlantic Ridge at a depth of 3000 m. The heavily flagellated cells are motile and coccoid shaped. The new strain growths between 55 and 91 degrees C, with an optimum growth temperature at 85 degree C. The optimum pH for growth is 6.5, and the optimum sea salt concentration for growth is around 25 g l-1. The organism uses H2 and CO2 as the only substrate for growth and methane production. Tungsten, selenium and yeast extract stimulate growth significantly. In the presence of CO2 and H2, the organism reduces elemental sulphur to hydrogen sulphide. The G+C content of the genomic DNA is 33 mol%. As determined by 16S gene sequence analysis, this organism is closely related to Methanococcus jannaschii strain JAL-1T. However, no significant homology was observed between them with DNA-DNA hybridization. It is proposed that this organism should be placed in a new species, Methanococcus infernus. The type strain is ME(T) (= DSM 11812T).

Anti-Bacterial Agents↗

PubMed1996

Neomycin resistance as a selectable marker in Methanococcus maripaludis.

We cloned the aminoglycoside phosphotransferase genes APH3'I and APH3'II between the Methanococcus voltae methyl reductase promoter and terminator in a plasmid containing a fragment of Methanococcus maripaludis chromosomal DNA. The resulting plasmids encoding neomycin resistance transformed M. maripaludis at frequencies similar to those observed for pKAS102 encoding puromycin resistance. The antibiotic geneticin was not inhibitory to M. maripaludis.

Anti-Bacterial Agents↗

PubMed1997

Characterization of pURB500 from the archaeon Methanococcus maripaludis and construction of a shuttle vector.

The complete sequence of the 8,285-bp plasmid pURB500 from Methanococcus maripaludis C5 was determined. Sequence analysis identified 18 open reading frames as well as two regions of potential iterons and complex secondary structures. The shuttle vector, pDLT44, for M. maripaludis JJ was constructed from the entire pURB500 plasmid and pMEB.2, an Escherichia coli vector containing a methanococcal puromycin-resistance marker (P. Gernhardt, O. Possot, M. Foglino, L. Sibold, and A. Klein, Mol. Gen. Genet. 221:273-279, 1990). By using polyethylene glycol transformation, M. maripaludis JJ was transformed at a frequency of 3.3 x 10(7) transformants per microg of pDLT44. The shuttle vector was stable in E. coli under ampicillin selection but was maintained at a lower copy number than pMEB.2. Based on the inability of various restriction fragments of pURB500 to support maintenance in M. maripaludis JJ, multiple regions of pURB500 were required. pDLT44 did not replicate in Methanococcus voltae.

Amino Acid Sequence↗

PubMed1984

Apparent operon for a 5S ribosomal RNA gene and for tRNA genes in the archaebacterium Methanococcus vannielii.

The nucleotide sequence of the chromosomal segment from Methanococcus vannielii previously shown to encode a gene for 5S ribosomal RNA (rRNA) unlinked to any other rRNA genes (Jarsch et al. 1983) was determined. It was found that the 5S rRNA gene is flanked by seven genes for tRNA (tRNAPro, tRNAThr, tRNATyr, tRNALys and tRNAAsp). Two of the tRNA genes (tRNAAsp and tRNALys) are repeated in the cluster. Only the tRNAPro cistron encodes the 3'-CCA tRNA sequence. The 5S rRNA/tRNA gene cluster probably represents one transcriptional unit. The 5'- and 3'-flanking sequences of the tRNA/5S rRNA gene cluster bear some similarity with initiation and termination signals in eubacteria. There is indication that the different 5S rRNA genes from Methanococcus exhibit sequence polymorphism.

Base Sequence↗

PubMed1989

The H+ ATPase regulatory subunit of Methanococcus thermolithotrophicus: amplification of an 800 bp fragment by polymerase chain reaction.

An 800 bp fragment of Methanococcus thermolithotrophicus genomic DNA was amplified by the polymerase chain reaction method using primers designed from conserved regions of the V-type H+ ATPase regulatory subunits from the archaebacterium Sulfolobus, and several eukaryotes. Although more than one product was obtained, only one of them had the expected size and was exclusively amplified in the presence of the left and right primers. The DNA and the deduced protein sequences of the putative Methanococcus H+ ATPase subunit revealed homology to the corresponding sequences in Sulfolobus and eukaryotes (about 60% identical residues) and a less evident homology to the eubacterial F1-ATPase alpha-subunit (22% identical residues with E. coli).

Amino Acid Sequence↗

PubMed1985

Transcription in methanogens. Evidence for specific in vitro transcription of the purified DNA-dependent RNA polymerase of Methanococcus thermolithotrophicus.

The purification of the DNA-dependent RNA polymerase of Methanococcus thermolithotrophicus is described. As the first step of purification the endogenous template was removed from the enzyme by hydrophobic interaction chromatography. The purified enzyme consists of seven components with different molecular masses. Transcription studies on T7 DNA and the recombinant plasmid pMV15, containing rRNA genes of Methanococcus vannielii, revealed that only the methanogen DNA is transcribed specifically, indicating a principal structural difference between archaebacterial and eubacterial promoters. This could be shown both by analysis of ternary transcription complexes and Southern hybridization. The site of initiation was found within a restriction fragment harbouring the first 390 nucleotides of the sequence coding for mature 16S rRNA and 1100 base pairs of upstream sequences. The specific initiation on this fragment strongly suggests that the enzyme can start in vitro transcription from the promoter(s) of rRNA synthesis.

Adenosine Monophosphate↗

PubMed1985

Amino acid biosynthesis and sodium-dependent transport in Methanococcus voltae, as revealed by 13C NMR.

Of several methanogenic bacteria examined only Methanococcus voltae readily incorporated exogenous amino acids into cell protein. This was easily shown, since growth in the presence of exogenous amino acids resulted in a loss of signal intensities from those carbon atoms normally labelled by [13C]acetate during biosynthesis. From 80% to 95% of the Ser, Lys, Pro or Val incorporated into protein could be supplied directly from the growth medium. In contrast, Asp and Glu, if supplied to the medium, accounted for only a small percentage of the total acidic amino acid used in protein synthesis. Constitutive transport systems took up a wide range of amino acids at rates of 0.1-4.1 nmol min-1 mg-1. The transport systems required Na+, with the possible exception of the basic amino acid lysine, and were inhibited by N-ethylmaleimide or 3,3',4',5-tetrachlorosalicylanilide. No interconversion of Ile to other amino acids was detected when cells were given [13C]Ile during growth, whereas the expected labelling of the Asp and Glu families of amino acids resulted when [13C]Asp was provided to the culture. Mc. voltae synthesized its amino acids from acetate via routes fully consistent with those found in Methanospirillum hungatei [Ekiel, I., Smith, I.C.P. & Sprott, G.D. (1983) J. Bacteriol. 156, 316-326]. Propionate could substitute for an auxotrophic requirement for Ile, resulting in the synthesis of Ile with the beta-carbon originating from the carboxyl of acetate and the alpha-carbon from the carboxyl of propionate. No labelling of Ile from [13C]acetate could occur without the fatty acid. These results provide strong evidence for the carboxylation of propionate to form 2-oxobutyrate as intermediate in Ile biosynthesis, and show that the metabolic defect in Ile biosynthesis occurs prior to 2-oxobutyrate synthesis. The presence of constitutive amino acid transport systems and multiple routes for ile biosynthesis make Methanococcus voltae an attractive methanogen for genetic studies.

Amino Acids↗