Single crystals of large ribosomal particles from Halobacterium marismortui diffract to 6 A

Large, well-ordered three-dimensional crystals of 50 S ribosomal subunits from Halobacterium marismortui have been obtained by seeding. The crystals have been characterized with synchrotron X-ray radiation as monoclinic, space group P2(1), with unit cell dimensions of a = 182(+/- 5) A, b = 584(+/- 10) A, c = 186(+/- 5) A, beta = 109 degrees. At 4 degrees C, the crystals (0.6 mm X 0.6 mm X 0.1 mm) diffract to 6 A resolution and are stable in the synchrotron beam for several hours. Compact packing is reflected from the crystallographic unit cell parameters and from electron micrographs of positively stained thin sections of embedded crystals.     

Polypeptide elongation factor Tu from Halobacterium marismortui

A GDP-binding protein of 60 kDa from Halobacterium marismortui has been purified to homogeneity. The purification has been carried out in high-salt buffers or in 50% glycerol buffers to protect the halophilic protein from denaturation. Evidence that this protein is the halophilic elongation factor Tu (hEF-Tu) is provided by the high homology of its N terminus with the corresponding sequences of other EF-Tus, and by immunological studies. Like some other EF-Tus the native protein can be cleaved with trypsin without concomitant loss of GDP-binding ability. The molecular mass of this hEF-Tu is higher than that for the corresponding factors from other sources including the halobacterium Halobacterium cutirubrum. The protein possesses typical halophilic characteristics, in that it is stable and active in 3 M KCl or 2 M (NH4)2SO4. Some other properties, like autofragmentation under sample treatment before SDS-PAGE, are described.     

Primary structures of ribosomal proteins from the archaebacterium Halobacterium marismortui and the eubacterium Bacillus stearothermophilus.

Approximately 40 ribosomal proteins from each Halobacterium marismortui and Bacillus stearothermophilus have been sequenced either by direct protein sequence analysis or by DNA sequence analysis of the appropriate genes. The comparison of the amino acid sequences from the archaebacterium H marismortui with the available ribosomal proteins from the eubacterial and eukaryotic kingdoms revealed four different groups of proteins: 24 proteins are related to both eubacterial as well as eukaryotic proteins. Eleven proteins are exclusively related to eukaryotic counterparts. For three proteins only eubacterial relatives-and for another three proteins no counterpart-could be found. The similarities of the halobacterial ribosomal proteins are in general somewhat higher to their eukaryotic than to their eubacterial counterparts. The comparison of B stearothermophilus proteins with their E coli homologues showed that the proteins evolved at different rates. Some proteins are highly conserved with 64-76% identity, others are poorly conserved with only 25-34% identical amino acid residues.     

Crystallization of halophilic malate dehydrogenase from Halobacterium marismortui

Malate dehydrogenase from the extreme halophile Halobacterium marismortui crystallizes in highly concentrated phosphate solution in space group 12 with cell dimensions a = 113.8 A, b = 122.8 A, c = 126.7 A, beta = 98.1 degrees. The halophilic enzyme was found to be unstable at lower concentrations of phosphate. It associates with unusually large amounts of water and salt, and the combined particle volume shows a tight fit in the unit cell.     

Primary structures of five ribosomal proteins from the archaebacterium Halobacterium marismortui and their structural relationships to eubacterial and eukaryotic ribosomal proteins

The complete amino acid sequences of ribosomal proteins L9, L20, L21/22, L24 and L32 from the archaebacterium Halobacterium marismortui were determined. The comparison of the sequences of these proteins with those from other organisms revealed that proteins L21/22 and L24 are homologous to ribosomal protein Yrp29 from yeast and L19 from rat, respectively, and that H. marismortui L20 is homologous to L30 from eubacteria. H. marismortui ribosomal protein L9 showed sequence homology to both L29 from yeast and L15 from eubacteria. No homologous protein was found for H. marismortui L32. These results are discussed with respect to the phylogenetic relationship between eubacteria, archaebacteria and eukaryotes.     

Purification and characterization of ribosomal proteins from the 30 S subunit of the extreme halophile Halobacterium marismortui

Ribosomal proteins were extracted from 30 S subunits of Halobacterium marismortui under native conditions.Their separation was based on gel filtration and hydrophobic chromatography, performed at a concentration of 3.2 M KC1 to avoid denaturation. A total of nine proteins were isolated, purified and identified by partial amino-terminal sequences and two-dimension a gel electrophoresis. There is a high degree of sequence homology with 30 S proteins from H. cutirubrum, and also some with 30 (S) proteins of eubacteria.Proton NMR data indicate unfolding of the proteins in low salt. One of the proteins, however, retains its secondary structure at a salt concentration as low as 0.1 M NaCl, and even in 8 M urea. One reason for this outstanding stability could be the high proportion (50%) of β-structure in this protein as determined from circular dichroism measurements. In general, there is a higher β-sheet content than for 30 S proteins from Escherichia coli. Measurements of Stokes radii indicate several of the proteins to have a rather elongated shape. One of these is a complex consisting of L3/L4 and L20, similar to the LI-complex from E. co&.The presence of this 50 S complex in the preparation of the small subunit suggests a location on the interface between the subunits.     

Lactate dehydrogenase from the extreme halophilic archaebacterium Halobacterium marismortui

D-Lactate dehydrogenase from the extreme halophilic archaebacterium Halobacterium marismortui has been partially purified by ammonium-sulfate fractionation, hydrophobic and ion exchange chromatography. Catalytic activity of the enzyme requires salt concentrations beyond 1M NaCl: optimum conditions are 4M NaCl or KCl, pH 6-8, 50 degrees C. Michaelis constants for NADH and pyruvate under optimum conditions of enzymatic activity are 0.070 and 4.5mM, respectively. As for other bacterial D-specific lactate dehydrogenases, fructose 1,6-bisphosphate and divalent cations (Mg2+, Mn2+) do not affect the catalytic activity of the enzyme. As shown by gel-filtration and ultracentrifugal analysis, the enzyme under the conditions of the enzyme assay is a dimer with a subunit molecular mass close to 36 kDa. At low salt concentrations (less than 1M), as well as high concentrations of chaotropic solvent components and low pH, the enzyme undergoes reversible deactivation, dissociation and denaturation. The temperature dependence of the enzymatic activity shows non-linear Arrhenius behavior with activation energies of the order of 90 and 25 kJ/mol at temperatures below and beyond ca. 30 degrees C. In the presence of high salt, the enzyme exhibits exceptional thermal stability; denaturation only occurs at temperatures beyond 55 degrees C. The half-time of deactivation at 70 and 75 degrees C is 300 and 15 min, respectively. Maximum stability is observed at pH 7.5-9.0.     

The primary structures of ribosomal proteins L16, L23 and L33 from the archaebacterium Halobacterium marismortui

The complete amino acid sequences of ribosomal proteins L16, L23 and L33 from the archaebacterium Halobacterium marismortui were determined. The sequences were established by manual sequencing of peptides produced with several proteases as well as by cleavage with dilute HCl. Proteins L16, L23 and L33 consist of 119, 154 and 69 amino acid residues, and their molecular masses are 13538, 16812 and 7620 Da, respectively. The comparison of their sequences with those of ribosomal proteins from other organisms revealed that L23 and L33 are related to eubacterial ribosomal proteins from Escherichia coli and Bacillus stearothermophilus, while protein L16 was found to be homologous to a eukaryotic ribosomal protein from yeast. These results provide information about the special phylogenetic position of archaebacteria.     

Molecular cloning and nucleotide sequence of the gene for the ribosomal protein S11 from the archaebacterium Halobacterium marismortui

The gene encoding ribosomal protein S11 (Escherichia coli S15 homologue) from Halobacterium marismortui was cloned employing two synthetic oligonucleotide mixtures, 23 and 32 bases in length, as hybridization probes. The nucleotide sequence of the gene and the adjacent 5'- and 3'-flanking regions (1300 base pairs) were then determined by the dideoxy chain termination method. Comparison of the nucleotide sequence of the H. marismortui S11 gene with that of the E. coli S15 gene (rpsO) showed that the 3'-end of the S11 gene can be aligned with the entire E. coli S15 gene, sharing 44% identical nucleotides. It has been found that the S11 gene has a higher G + C content (G + C = 65%) than that of the E. coli S15 gene (G + C = 53%). This increase in G + C content specifically shows up as a preference for G + C in the 3rd position of the codon. Upstream of the S11 gene, an archaebacterial promoter sequence (GGACTTTCA) and a putative ribosomal binding site (GCGGT) have been found, 88 and 15 (or 24) base pairs from the initiation codon of the gene. In addition, an open reading frame could be identified immediately after the stop codon for the S11 gene. Northern blotting analysis using the S11 coding region as probe has shown that the S11 gene is located on a 2.4-kilobase mRNA, suggesting that it is cotranscribed with other downstream gene(s).     

The N-terminal sequence of ribosomal protein L10 from the archaebacterium Halobacterium marismortui and its relationship to eubacterial protein L6 and other ribosomal proteins

The amino-terminal sequence of ribosomal protein L10 from Halobacterium marismortui has been determined up to residue 54, using both a liquid- and a gas-phase sequenator. The two sequences are in good agreement. The protein is clearly homologous to protein HcuL10 from the related strain Halobacterium cutirubrum. Furthermore, a weaker but distinct homology to ribosomal protein L6 from Escherichia coli and Bacillus stearothermophilus can be detected. In addition to 7 identical amino acids in the first 36 residues in all four sequences a number of conservative replacements occurs, of mainly hydrophobic amino acids. In this common region the pattern of conserved amino acids suggests the presence of a beta-alpha fold as it occurs in ribosomal proteins L12 and L30. Furthermore, several potential cases of homology to other ribosomal components of the three ur-kingdoms have been found.     

Distribution of non-electrolytes in Halobacterium cells. I. Halobacterium marismortui.

Halobacterium marismortui is an obligatorily halophilic species isolated from the Dead Sea. When inulin, fructose or glycerol are added to suspensions of bacteria, the amounts of these substances recovered from centrifuges pellets are more than could have been present in the extracellular space. Thus a certain amount becomes associated with the bacteria, though not enough to equilibrate with all the cell water. The inulin or fructose concentration found after uptake of these substances was correlated with the cell sodium concentration. It is argued that inulin, fructose or glycerol is unlikely to be adsorbed on the outside of the bacteria and more probably crosses the plasma membrane. A possible scheme for explaining the data is presented.     

Complete amino acid sequences of the ribosomal proteins L25, L29 and L31 from the archaebacterium Halobacterium marismortui

Ribosomal proteins were extracted from 50S ribosomal subunits of the archaebacterium Halobacterium marismortui by decreasing the concentration of Mg2+ and K+, and the proteins were separated and purified by ion-exchange column chromatography on DEAE-cellulose. Ten proteins were purified to homogeneity and three of these proteins were subjected to sequence analysis. The complete amino acid sequences of the ribosomal proteins L25, L29 and L31 were established by analyses of the peptides obtained by enzymatic digestion with trypsin, Staphylococcus aureus protease, chymotrypsin and lysylendopeptidase. Proteins L25, L29 and L31 consist of 84, 115 and 95 amino acid residues with the molecular masses of 9472 Da, 12293 Da and 10418 Da respectively. A comparison of their sequences with those of other large-ribosomal-subunit proteins from other organisms revealed that protein L25 from H. marismortui is homologous to protein L23 from Escherichia coli (34.6%), Bacillus stearothermophilus (41.8%), and tobacco chloroplasts (16.3%) as well as to protein L25 from yeast (38.0%). Proteins L29 and L31 do not appear to be homologous to any other ribosomal proteins whose structures are so far known.     

Organization and nucleotide sequence of a gene cluster coding for eight ribosomal proteins in the archaebacterium Halobacterium marismortui

A DNA fragment containing the genes for the eight ribosomal proteins HmaL3, HL6, HmaL23, HmaL2, HmaS19, HmaL22, HmaS3, and HmaL29 from Halobacterium marismortui has been cloned and sequenced. The organization of this gene cluster in general corresponds to the S10 operon of Escherichia coli although there exists some differences between them. The sequence analysis of the 5'- and 3'-region of the gene cluster revealed three open reading frames (orf1, orf2, and orf3) which do not code for any ribosomal protein whose structure is known. A putative promoter is located upstream of orf1. Out of the eight ribosomal proteins five have counterparts in eubacteria only, two in both eubacteria and eukaryotes, and one is exclusively related to an eukaryotic ribosomal protein.     

Nucleotide sequence of four genes encoding ribosomal proteins from the 'S10 and spectinomycin' operon equivalent region in the archaebacterium Halobacterium marismortui

Four genes encoding ribosomal proteins HmaS17, HmaL14, HmaL24 and HS3, have been identified in the lambda EMBL3 clone PP*7 from a genomic library of the archaebacterium Halobacterium marismortui. The clone contains genes from the 'S10 and spectinomycin' operon equivalent region. Three of the deduced proteins are homologous to the corresponding Escherichia coli and Methancoccus vannielii S17, L14 and L24 proteins, as well as to eukaryotic proteins from rat or yeast. HS3 was identified as an extra protein corresponding to the gene product for orfc in M. vannielii and the eukaryotic ribosomal protein RS4 from rat. The equivalence of HmaL24 (HL16) and E. coli L24, which share only 28% identical amino acid residues, could now be shown by localizing the HmaL24 gene at the same position in the cluster.     

Molecular basis for the special properties of proteins and enzymes from Halobacterium marismortui

Abstract Three proteins from Halobacterium marismortui , malate dehydrogenase (hMDH), glutamate dehydrogenase (hGDH) and ferredoxin (hFD) were purified and characterized with respect to their molecular masses, amino acid composition and, for hFD only, primary structure. Striking features of halophilic proteins are: the high excess of acidic over basic residues; acidic clusters in the sequence. Low-salt concentration causes inactivation and changes in structural parameters of hMDH and hGDH. Reactivation of hMDH involves long-lived stable intermediates. The salt concentration optimum of enzymic activity is independent of salt nature. The high capacity of halophilic proteins to retain water and salt is due to unique molecular properties, studied by physico-chemical techniques.     

The taxonomic status of "Halobacterium marismortui" from the Dead Sea: a comparison with Halobacterium vallismortis

A Halobacterium strain, isolated by Ginzburg et al. from the Dead Sea in the late 1960's, often referred to as "Halobacterium marismortui" or "Halobacterium of the Dead Sea" (deposited in the American Type Culture Collection as ATCC 43049) was compared with Halobacterium (Haloarcula) vallismortis ATCC 29715. The strains appeared to be very closely related, as shown by the near identity of their 5S and 16S ribosomal RNA's, and a large number of other common properties. Distinct differences exist, however, in cell morphology, and in their potency to utilize different sugars and other compounds.