Hyperthermophilic Methanothermus Fervidus Research Articles

Histones and chromatin structure in hyperthermophilicArchaea

HMf is a histone from the hyperthermophile Methanothermus fervidus. It is the archetype and most studied member of a family of archaeal histones that have primary sequences and three-dimensional structures in common with the eukaryal nucleosome core histones and that bind and compact DNA molecules into nucleosome-like structures (NLS). HMf preparations are mixtures of two similar, small (approximately 7.5 kDa) polypeptides designated HMfA and HMfB that in vivo form both homodimers and heterodimers. HMfA synthesis predominates during exponential growth but the relative amount of HMfB increases as M. fervidus cells enter the stationary growth phase. Analyses of homogeneous preparations of recombinant (r) (HMfA)2 and (rHMfB)2 have demonstrated that these proteins have different DNA-binding and compaction properties in vitro, consistent with different roles in vivo for the (HMfA)2, (HMfB)2 and HMfA. HmfB dimers, and for the NLS that they form, in regulating gene expression and in genome compaction and stability.

Cloning, sequencing and expression of the gene encoding 2-phosphoglycerate kinase from Methanothermus fervidus

The gene encoding 2-phosphoglycerate kinase (2PGK), which catalyses the first step in the biosynthesis of cyclic 2,3-diphosphoglycerate in methanogens, was cloned and sequenced from the hyperthermophilic Methanothermus fervidus. The 2pgk gene codes for 304 amino acids, corresponding to a relative molecular mass of 35040. The 2pgk mRNA was estimated to be 1600 nucleotides in size. Putative transcription signals and the ribosome-binding site of 2pgk are discussed. Production of 2PGK from M. fervidus in Es-cherichia coli reveals the same apparent molecular weights for the native enzyme and its denatured subunit as those shown by the 2PGK purified from M. fervidus. Also the kinetic parameters of 2PKG produced in E. coli correspond well with those from the enzyme isolated from the natural host M. fervidus.

Cloning, sequencing and transcript analysis of the gene encoding formylmethanofuran: tetrahydromethanopterin formyltransferase from the hyperthermophilic Methanothermus fervidus.

The formylmethanofuran:tetrahydromethanopterin formyltransferase (FTR) from Methanothermus fervidus was partially purified and its N-terminal amino acid sequence determined. Using as probe a mixture of oligonucleotides derived from the FTR N-terminus, the corresponding gene (ftr) was cloned and sequenced. The ftr gene codes for 297 amino acids, corresponding to a molecular mass of 31,836 daltons, in contrast to the 41,000 daltons estimated for the protein by sodium dodecylsulphate-polyacrylamide gel electrophoresis. The deduced amino acid sequence of the hyperthermophilic FTR from M. fervidus is 76% identical to the thermophilic FTR from Methanobacterium thermoautotrophicum and has a larger number of lysine residues. A putative ATP-binding site of the FTR is reported. The size of the ftr mRNA was estimated as 1000 nucleotides indicating monocistronic transcription of the 891 bp gene. The ftr mRNA starts 27 bp downstream of the centre of a putative archaeal box A motif and terminates at an oligo-dT stretch. In vitro transcription of the ftr gene, utilizing a transcription system developed for the distantly related Sulfolobus shibatae, is discussed with respect to the functional conservation of the basal transcription apparatus of Archaea.

Primary structure of the heterosaccharide of the surface glycoprotein of Methanothermus fervidus.

The outer surface of the cells of the hyperthermophile Methanothermus fervidus is covered by crystalline glycoprotein subunits (S-layer). From the purified S-layer glycoprotein, a heterosaccharide was isolated. The heterosaccharide consists of D-3-O-methylmannose, D-mannose, and D-N-acetylgalactosamine in a molar ratio of 2:3:1 corresponding to a relative molecular mass of 1061.83 Da. 3-O-methylmannose could be partly replaced by 3-O-methylglucose. The primary structure of the glycan was revealed by methylation analysis, by plasma desorption mass spectrometry, and by high field NMR spectroscopy. The purified heterosaccharide is linked via N-acetylgalactosamine to an asparagine residue of the peptide moiety. The following structure is proposed for the heterosaccharide: alpha-D-3-O-MetManp-(1-->6)-alpha-D-3-O-MetManp-((1-->2)-alp ha-D-Manp)3-(1-->4) - D-GalNAc.

HMt, a histone-related protein from Methanobacterium thermoautotrophicum delta H.

HMt, a histone-related protein, has been isolated and characterized from Methanobacterium thermoautotrophicum delta H. HMt preparations contain two polypeptides designated HMt1 and HMt2, encoded by the hmtA and hmtB genes, respectively, that have been cloned, sequenced, and expressed in Escherichia coli. HMt1 and HMt2 are predicted to contain 68 and 67 amino acid residues, respectively, and have calculated molecular masses of 7,275 and 7,141 Da, respectively. Aligning the amino acid sequences of HMt1 and HMt2 with the sequences of HMf1 and HMf2, the subunit polypeptides of HMf, a histone-related protein from the hyperthermophile Methanothermus fervidus, revealed that 40 amino acid residues (approximately 60%) are conserved in all four polypeptides. In pairwise comparisons, these four polypeptides are 66 to 84% identical. The sequences and locations of the TATA box promoter elements and ribosome binding sites are very similar upstream of the hmtA and hmtB genes in M. thermoautotrophicum and upstream of the hmfA and hmfB genes in M. fervidus. HMt binding compacted linear pUC19 DNA molecules in vitro and therefore increased their electrophoretic mobilities through agarose gels. At protein/DNA mass ratios of < 0.2:1, HMt binding caused an increase in the overall negative superhelicity of relaxed, circular DNA molecules, but at HMt/DNA mass ratios of > 0.2:1, positive supercoils were introduced into these molecules. HMt and HMf are indistinguishable in terms of their abilities to compact and constrain DNA molecules in positive toroidal supercoils in vitro. Histone-related proteins with these properties are therefore not limited to reverse gyrase-containing hyperthermophilic species.

Transcription in vitro and in vivo of the 7S RNA gene associated with the ribosomal RNA operon in the hyperthermophilic archaeonMethanothermus fervidus

The gene encoding the archaeal 7S RNA in the hyperthermophile Methanothermus fervidus is linked to a tRNA(Ser) and rRNA operon in the arrangement 5'-7S RNA-14nt-tRNA(Ser)-196nt-16S rRNA and the promoter directing transcription of this 7S RNA gene has now been identified. Initiation of transcription of the 7S RNA gene has been shown to occur both in vivo in M. fervidus and in vitro, using a Methanococcus thermolithotrophicus derived cell-free transcription system, at the first G residue within the initiator sequence ATGG, located 6 bp upstream of the 5' end of the 7S RNA coding region. Cotranscription of the 7S RNA and tRNA(Ser) has been demonstrated in vitro.

Transcription in vitro and in vivo of the 7S RNA gene associated with the ribosomal RNA operon in the hyperthermophilic archaeon Methanothermus fervidus.

The gene encoding the archaeal 7S RNA in the hyperthermophile Methanothermus fervidus is linked to a tRNA(Ser) and rRNA operon in the arrangement 5'-7S RNA-14nt-tRNA(Ser)-196nt-16S rRNA and the promoter directing transcription of this 7S RNA gene has now been identified. Initiation of transcription of the 7S RNA gene has been shown to occur both in vivo in M. fervidus and in vitro, using a Methanococcus thermolithotrophicus derived cell-free transcription system, at the first G residue within the initiator sequence ATGG, located 6 bp upstream of the 5' end of the 7S RNA coding region. Cotranscription of the 7S RNA and tRNA(Ser) has been demonstrated in vitro.