Polyadenylated RNA isolated from the archaebacterium Halobacterium halobium.

Polyadenylated [poly(A)+] RNA has been isolated from the halophilic archaebacterium Halobacterium halobium by binding, at 4 degrees C, to oligo(dT)-cellulose. H. halobium contains approximately 12 times more poly(A) per unit of RNA than does the methanogenic archaebacterium Methanococcus vannielii. The 3' poly(A) tracts in poly(A)+ RNA molecules are approximately twice as long (average length of 20 nucleotides) in H. halobium as in M. vannielii. In both archaebacterial species, poly(A)+ RNAs are unstable.     

MvnI: A restriction enzyme in the archaebacterium Methanococcus vannielii

Abstract The methanogenic archaebacerium Methanococcus vannielii contains a type II restriction endonuclease. The enzyme was purified by a simple three-step procedure resulting in enzyme preparations free of contaminating unspecific nucleases. The restriction enzyme recognizes and cleaves the sequence 5'-CG ↓ CG-3' ( Fnu DII and Tha I isoschizomer) and generates DNA fragments with blunt ends. Due to its purity and activity at moderate temperatures, Mvn I might be a useful alternative to Fnu DII and Tha I active at 60°C.     

Aminoglycoside sensitivity of ribosomes from the archaebacterium Methanococcus vannielii: Structure-activity relationship

Abstract The effect of about 20 aminoglycoside antibiotics comprising compounds with specific 70S or with 70S plus 80S activity on polypeptide synthesis and translational misreading by ribosomes from the archaebacterium Methanococcus vannielii was investigated. A clear structure-activity relationship was found: sensitivity was observed only to the class of 4,5-disubstituted deoxystreptamine compounds, with neomycin and paromomycin as the most active ones. The streptomycin class aminoglycosides were completely inactive whereas the gentamicin group compounds solely affected misreading and only at high concentrations. Viomycin, a specific inhibitor of the translocation reaction at the eubacterial ribosome which competes with binding of 2-deoxystreptamine aminoglycosides was inactive as well.     

Antibiotic resistance caused by permeability changes of the archaebacterium Methanococcus vannielii

Abstract Spontaneous mutants of the methane-producing archaebacterium Methanococcus vannielii have been isolated which are resistant to bromoethane-sulphonate, A2315-A (a virginiamycin), neomycin and thiostrepton. Ultraviolet (UV) irradiation followed by growth in bacitracin led to the isolation of bacitracin-resistant mutants. In vitro translation reactions using ribosomes prepared from neomycin or A2315-A-resistant mutants were sensitive to neomycin or A2315-A indicating that the in vivo resistance of the mutants must reflect changes in the permeability of the M. vannielii proteinaceous cell envelope.     

Sequence of the gene for ribosomal protein L23 from the archaebacterium Methanococcus vannielii

The N-terminal sequence of HPLC-purified protein L23 from the Methanococcus vannielii ribosome has been determined by automated liquid-phase Edman degradation. Using the N-terminal amino acid sequence, an oligonucleotide probe complementary to the 5'-end of the gene was synthesized. The 26-mer oligonucleotide, containing two inosines, was used for hybridization with digested M. vannielii chromosomal DNA. The hybridizing band from HpaII-digested genomic DNA was ligated into pUC18 to yield plasmid pMvaZ1 containing the entire gene of protein L23. The nucleotide sequence complemented the partial amino acid sequence, and the gene codes for a protein of 9824 Da. The amino acid sequence of protein L23 form M. vannielii was compared to that of ribosomal proteins from other archaebacteria as well as from eubacteria and eukaryotes. The number of identical amino acids is highest when the M. vannielii protein is compared to the homologous protein from yeast and lowest vs that from tobacco chloroplasts. Interestingly, the secondary structures of the proteins as predicted by computer programs are more conserved than the primary structures.     

Autogenous translational regulation of the ribosomal MvaL1 operon in the archaebacterium Methanococcus vannielii.

The mechanisms for regulation of ribosomal gene expression have been characterized in eukaryotes and eubacteria, but not yet in archaebacteria. We have studied the regulation of the synthesis of ribosomal proteins MvaL1, MvaL10, and MvaL12, encoded by the MvaL1 operon of Methanococcus vannielii, a methanogenic archaebacterium. MvaL1, the homolog of the regulatory protein L1 encoded by the L11 operon of Escherichia coli, was shown to be an autoregulator of the MvaL1 operon. As in E. coli, regulation takes place at the level of translation. The target site for repression by MvaL1 was localized by site-directed mutagenesis to a region within the coding sequence of the MvaL1 gene commencing about 30 bases downstream of the ATG initiation codon. The MvaL1 binding site on the mRNA exhibits similarity in both primary sequence and secondary structure to the L1 regulatory target site of E. coli and to the putative binding site for MvaL1 on the 23S rRNA. In contrast to other regulatory systems, the putative MvaL1 binding site is located in a sequence of the mRNA which is not in direct contact with the ribosome as part of the initiation complex. Furthermore, the untranslated leader sequence is not involved in the regulation. Therefore, we suggest that a novel mechanism of translational feedback regulation exists in M. vannielii.     

Composition and Characterization of tRNA from Methanococcus vannielii.

Purified bulk tRNA from Methanococcus vanielii (carbon source, formate) showed variation in the modified nucleoside pattern reported for Escherichia coli as analyzed by both ion-exchange and thin-layer chromatography. Ribothymidine and 7-methylguanosine were absent; 1-methyladenosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, thiolated nucleosides, pseudouridine, dihydrouridine, and O2'-methylcytidine were quantitated. In vitro methylation by M. Vannielii extracts with S-adenosylmethionine and undermethylated E. coli tRNA revealed active tRNA methyltransferases for formation of methylated residues found in native M. vannielii tRNA, but none for the formation of 7-methylguanosine or ribothymidine. The native M. vannielii tRNA became methylated in the 7-methylguanosine position by E. Coli extracts, but ribothymidine was not formed. Both M. vannielii and E. coli tRNA methyltransferases produced unidentified methylated residues in tRNA's lacking or deficient in ribothymidine.     

Purine metabolism in Methanococcus vannielii.

Methanococcus vannielii is capable of degrading purines to the extent that each of these purines may serve as the sole nitrogen source for growth. Results presented here demonstrate that purine degradation by M. vannielii is accomplished by a route similar to that described for clostridia. Various characteristics of the purine-degrading pathway of M. vannielii are described. Additionally, it is shown that M. vannielii does not extensively degrade exogenously supplied guanine if that compound is present at levels near or lower than those required to supply the cellular guanine requirement. Under those conditions, M. vannielii incorporates the intact guanine molecule into its guanine nucleotide pool. The benefits of a purine-degrading pathway to methanogens are discussed.     

Chemotaxis in the archaebacterium Methanococcus voltae.

The archaebacterium Methanococcus voltae, was shown to be chemotactic. Acetate, isoleucine, and leucine were identified as attractants; whereas histidine was not an attractant. A motile, generally nonchemotactic mutant was isolated.     

Purification and properties of carbon monoxide dehydrogenase from Methanococcus vannielii.

Carbon monoxide dehydrogenase was purified to homogeneity from Methanococcus vannielii grown with formate as the sole carbon source. The enzyme is composed of subunits with molecular weights of 89,000 and 21,000 in an alpha 2 beta 2 oligomeric structure. The native molecular weight of carbon monoxide dehydrogenase, determined by gel electrophoresis, is 220,000. The enzyme from M. vannielii contains 2 g-atoms of nickel per mol of enzyme. Except for its relatively high pH optimum of 10.5 and its slightly greater net positive charge, the enzyme from M. vannielii closely resembles carbon monoxide dehydrogenase isolated previously from acetate-grown Methanosarcina barkeri. Carbon monoxide dehydrogenase from M. vannielii constitutes 0.2% of the soluble protein of the cell. By comparison the enzyme comprises 5% of the soluble protein in acetate-grown cells of M. barkeri and approximately 1% in methanol-grown cells.     

Gene for the diphtheria toxin-susceptible elongation factor 2 from Methanococcus vannielii.

Protein synthesis elongation factor 2 (EF-2) from all archaebacteria so far analysed, is susceptible to inactivation by diphtheria toxin, a property which it shares with EF-2 from the eukaryotic 8OS translation system. To resolve the structural basis of diphtheria toxin susceptibility, the structural gene for the EF-2 from an archaebacterium, Methanococcus vannielii, was cloned and its nucleotide sequence determined. It was found that (i) this gene is closely linked to that coding for elongation factor 1 alpha-(EF-1 alpha), (ii) the size of the gene product, as derived from the nucleotide sequence, lies between those for EF-2 from eukaryotes and eubacteria, (iii) it displays a higher sequence similarity to eukaryotic EF-2 than to eubacterial homologues, and (iv) the histidine residue which is modified to diphthamide and then ADP-ribosylated by diphtheria toxin is present in a sequence context similar to that of eukaryotic EF-2 but it is not conserved in eubacterial EF-G. The EF-2 gene from Methanococcus is expressed in transformed Saccharomyces cerevisiae but is not ADP-ribosylated by diphtheria toxin. This indicates that the Saccharomyces enzyme system is unable to post-translationally convert the respective histidine residue from the Methanococcus EF-2 into diphthamide.     

Polyadenylated RNA from Acetabularia

Polyadenylated RNA was isolated from whole cells and from anucleate cytoplasm of Acetabularia mediterranea. It is synthesised in nucleate but not in anucleate cells. This RNA has a high molecular weight ranging from 0.5 to 3.0 × 10⁶ daltons. In contrast to the RNA of 80 S ribosomes the synthesis of polyadenylated RNA is only slightly enhanced in nucleated cell fragments during the initial phase of regeneration. Newly synthesised polyadenylated RNA migrates from the nucleus through the stalk at about 5 mm a day. The data suggest that polyadenylated RNA migrates independently of 80 S ribosomes.     

Conservation of primary structure in the hisI gene of the archaebacterium, Methanococcus vannielii, the eubacterium Escherichia coli, and the eucaryote Saccharomyces cerevisiae

Summary A 2.7 kilobase pair (Kb) fragment of DNA, which complements mutations in the hisI locus of Escherichia coli, has been cloned and sequenced from the genome of the methanogenic archaebacterium Methanococcus vannielii. The cloned DNA directs the synthesis of three polypeptides, with molecular weights of 71,000, 29,000 and 15,600 in minicells of E. coli. Subcloning and mutagenesis demonstrates that hisI complementation results from the activity of the 15,600 molecular weight polypeptide. The primary structure of this archaebacterial gene and its gene product have been compared with the functionally equivalent gene and protein from the eubacterium E. coli (hisI) (Chiariotti et al. 1986) and from the eucaryote Saccharomyces cerevisiae (his4A) (Donahue et al. 1982). The DNA sequences of the archaebacterial and eubacterial genes are 40% homologous, the archaebacterial and eucaryotic DNA sequences are 47% homologous and, as previously reported (Bruni et al. 1986) the eubacterial and eucaryotic DNA sequences are 45% homologous. In E. coli the hisI locus is part of a bifunctional gene (hisI/E) within the single his operon. In S. cerevisiae the his4A locus is part of a multifunctional gene (his4) which encodes a protein with at least four enzymatic activities. The his genes of S. cerevisiae do not form an operon and are not physically linked. The M. vannielii hisI gene does not appear to be part of a multifunctional DNA sequence and, although it does appear to be within an operon, the open reading frames (ORFs) 5 and 3 to the M. vannielii hisI gene are not related to any published his sequences. The hisI and hisA genes (Cue et al. 1985) of M. vannielii are not closely linked in its genome.     

Solution NMR structure of selenium-binding protein from Methanococcus vannielii

Selenium is an important nutrient. The lack of selenium will suppress expression of various enzymes that will lead to cell abnormality and diseases. However, high concentrations of free selenium are toxic to the cell because it adversely affects numerous cell metabolic pathways. In Methanococcus vannielii, selenium transport in the cell is established by the selenium-binding protein, SeBP. SeBP sequesters selenium during transport, thus regulating the level of free selenium in the cell, and delivers it specifically to the selenophosphate synthase enzyme. In solution, SeBP is an oligomer of 8.8-kDa subunits. It is a symmetric pentamer. The solution structure of SeBP was determined by NMR spectroscopy. Each subunit of SeBP is composed of an alpha-helix on top of a 4-stranded twisted beta-sheet. The stability of the five subunits stems mainly from hydrophobic interactions and supplemented by hydrogen bond interactions. The loop containing Cys(59) has been shown to be important for selenium binding, is flexible, and adopts multiple conformations. However, the cysteine accessibility is restricted in the structure, reducing the possibility of the binding of free selenium readily. Therefore, a different selenium precursor or other factors might be needed to facilitate opening of this loop to expose Cys(59) for selenium binding.     

Heat shock response of the archaebacterium Methanococcus voltae.

The general properties of the heat shock response of the archaebacterium Methanococcus voltae were characterized. The induction of 11 heat shock proteins, with apparent molecular weights ranging from 18,000 to 90,000, occurred optimally at 40 to 50 degrees C. Some of the heat shock proteins were preferentially enriched in either the soluble (cytoplasm) or particulate (membrane) fraction. Alternative stresses (ethanol, hydrogen peroxide, NaCl) stimulated the synthesis of subsets of the heat shock proteins as well as unique proteins. Western blot (immunoblot) analysis, in which antisera to Escherichia coli heat shock proteins (DnaK and GroEL) were used, did not detect any immunologically cross-reactive proteins. In addition, Southern blot analysis did not reveal any homology between M. voltae and four highly conserved heat shock genes, mopB and dnaK from E. coli and hsp70 genes from Drosophila species and Saccharomyces cerevisiae.