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PubMed1986

The cell surface glycoprotein of Halobacterium halobium. Physico-chemical characterization in the absence and presence of salt.

The cell surface glycoprotein of Halobacterium halobium is soluble in dilute buffer at neutral pH. At low counterion concentrations, the protein is monomeric (Ms,D = 209 kDa) and exhibits the characteristics of a highly charged polyelectrolyte. Evidence obtained from intrinsic fluorescence and far-UV circular dichroism shows that the monomer at low salt loses both its native conformation and its inherent tendency to form high molecular mass assemblies. In 4M NaCl, 25 mM KCl, and in the presence of divalent ions (greater than or equal to 50mM Mg2+ or Ca2+), association to well-defined assemblies of up to approximately 4 X 10(6) Da occurs. At low Mg2+ concentration and in the presence of Ba2+, a wide size-distribution of aggregates is observed. The assembly pattern of the protein may be correlated with salt-dependent alterations in the morphology of the bacterium.

Buffers↗
PubMed1990

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.

Archaea↗
PubMed1994

Genes for DNA cytosine methyltransferases and structural proteins, expressed during lytic growth by the phage phi H of the archaebacterium Halobacterium salinarium.

Lytic genes and transcription from the Halobacterium salinarium phage phi H were studied. Genes for three structural proteins were located to the left arm of the linear phage genome. The right arm was shown to encode three DNA cytosine methyltransferases, the first such sequences reported from an archaebacterium. One cytosine methyltransferase is of the N(4)-methyltransferase type. The other two open reading frames (ORFs) seem to be parts of the same gene, which has been split by a recombination event. This gene product is of the C5-methyltransferase type. The methyltransferase genes are the first phi H genes detected showing high homology to eubacterial proteins. Five of the six described gene products have a higher proportion acidic over basic amino acid residues, a common characteristic of halobacterial proteins. Lytic phi H transcription was shown to produce three RNA species, two shorter species encoding the methyltransferase genes and one large species transcribed from both the right and the left phage arm and subsequently being processed upstream of the region encoding the structural proteins.

Amino Acid Sequence↗
PubMed2003

Color-specific conditioning effects due to both orange and blue stimuli are observed in a Halobacterium salinarum strain devoid of putative methylatable sites on HtrI.

Behavioral responses of Halobacterium salinarum appear as changes in the frequency of motion reversals. Turning on orange light decreases the reversal frequency, whereas blue light induces reversals. Light pulses normally induce the same response as step-up stimuli. However, anomalous behavioral reactions, including inverse responses, are seen when stimuli are applied in sequence. The occurrence of a prior stimulus is conditioning for successive stimulation on a time scale of the same order of adaptational processes. These prolonged conditioning effects are color-specific. The only adaptation process identified so far is methylation of the transducers, and this could be somehow color-specific. Therefore we tested for the behavioral anomalies in a mutant in which all methylation sites on the transducer have been eliminated. The results show that behavioral anomalies are unaffected by the absence of methylation processes on the transducer.

Adaptation, Physiological↗
PubMed1985

Active uptake of glutamate in vesicles of Halobacterium salinarium.

Uptake of glutamate into vesicles of Halobacterium salinarium has been studied during respiration and in the nonrespiring state. Uptake requires respiration or a minimum gradient in NaCl, which is consistent with an Na+ symport mechanism for uptake, as proposed for H. halobium. By replacing KCl or NaCl by choline chloride, it has been possible to distinguish between the effects of gradients and/or absolute concentration effects of NaCl and KCl. Uptake depends on the concentration of KCl on the inside, but not on a gradient in KCl. This points to a role for K+ as a regulator of uptake rate, but not of total uptake. The uptake of glutamate is not inhibited by a number of acids with similar chemical groups. Inhibition is, however, caused by D-glutamate. This indicates a specific transport site for glutamate. Parallel results are obtained for binding of glutamate to a Triton extract of the vesicle membrane. The variation in binding and uptake properties with the salt concentration is discussed with reference to transport kinetics.

Bacterial Proteins↗
PubMed2001

Molecular cloning, sequencing analysis and expression of the catalase-peroxidase gene from Halobacterium salinarum.

The gene encoding catalase-peroxidase was cloned from chromosomal DNA from the Archaea, Halobacterium salinarum. The nucleotide sequence of a 3.5 kb fragment, containing the catalase-peroxidase gene and its flanking regions was determined. A 2.16 kb open reading frame was obtained, encoding the enzyme which was comprised of 720 amino acid residues with a calculated molecular weight of 80 kDa. The deduced amino acid sequence of the H. salinarum catalase-peroxidase showed a high degree of identity to other bifunctional catalase-peroxidases. A transcriptional start site was identified 183 bp upstream of the translational start codon. Southern blot analysis indicated that catalase-peroxidase was a single copy gene. The Archaeal catalase-peroxidase gene was expressed in Escherichia coli, and the expressed fusion protein exhibited both catalase and peroxidase activities.

Amino Acid Sequence↗
PubMed1991

Halobacterium halobium Mn-SOD gene: archaebacterial and eubacterial features.

A 1.8 kb PstI fragment from Halobacterium halobium DNA was found to hybridize to synthetic oligonucleotide probes constructed by using the sequence of the N-terminus of a Mn-containing superoxide dismutase purified from H. halobium. The entire insert containing a 600-bp coding sequence for Mn-SOD and its 5' and 3' flanking regions was sequenced. The derived amino acid sequence of the structural gene showed a similarity to other manganic and iron-containing superoxide dismutases in normally conserved regions. Primer extension analysis of the H. halobium Mn-SOD mRNA showed that gene transcription begins 14 bases upstream of the translational start. A Shine-Dalgarno sequence and archaebacterial consensus promoter sequences were observed. Several other promoter and terminator nucleotide sequences homologous to prokaryotic and eukaryotic organisms were found.

Amino Acid Sequence↗
PubMed2006

A non-discriminating aspartyl-tRNA synthetase from Halobacterium salinarum.

The tRNA-dependent transamidation pathway is the essential route for Asn-tRNA(Asn) formation in organisms that lack an asparaginyl-tRNA synthetase. This pathway relies on a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS encoded by aspS), an enzyme with relaxed tRNA specificity, to form Asp-tRNA(Asn). The misacylated tRNA is then converted to Asn-tRNA(Asn) by the action of an Asp-tRNA(Asn) amidotransferase. Here we show that Asn-tRNA(Asn) formation in the extreme halophile Halobacterium salinarum also occurs by this transamidation mechanism, and we explore the property of the haloarchaeal AspRS to aspartylate tRNA(Asn) in vivo and in vitro. Transformation of the E. coli trpA34 strain with the H. salinarum aspS and tRNA(Asn) genes led to restoration of tryptophan prototrophy by missense suppression of the trpA34 mutant with heterologously in vivo formed Asp-tRNA(Asn). The haloarchaeal AspRS works well at low and high (0.1-3 M) salt concentrations but it is unable to use Escherichia coli tRNA as substrate. We show that mutations of two amino acids (H26 and P84) located in the AspRS anticodon binding domain limit the specificity of this nondiscriminating enzyme towards tRNA(Asn). Thus, as was observed in an archaeal discriminating AspRS and a bacterial ND-AspRS, amino acids in these positions influence the enzyme's tRNA selection.

Aspartate-tRNA Ligase↗
PubMed1976

Formation of lipid-linked sugar compounds in Halobacterium salinarium. Presumed intermediates in glycoprotein synthesis.

The ability of bacitracin to inhibit the growth of Halobacterium salinarium suggested that glycosylation of the major envelope component, a high molecular weight glycoprotein, might occur via a pathway involving lipid intermediates. This report demonstrates that the cells have enzymatic activities for formation of lipid-linked sugar compounds having the expected properties of such intermediates. Whole cell homogenate catalyzed the transfer of sugar from UDP-glucose, GDP-mannose, and UDP-N-acetyglucosamine to endogenous lipid acceptors. Two lipid products were formed from UDP-glucose, two from GDP-mannose, and one from UDP-N-acetylglucosamine. Characterization of the partially purified lipids by ion exchange chromatography, thin layer chromatography, and mild acid and base hydrolysis showed the major product in each case to have the properties expected for polyisoprenyl phosphoglucose, polyisoprenyl phosphomannose, and polyisoprenyl pyrophospho-N-acetylglucosamine. Estimates of chain length by thin layer chromatography indicate that the lipid has 11 to 12 isoprene identity as a C55-60-polyisoprenyl pyrophospho-N-acetylglucosamine. The N-acetylglucosamine transferase, present in cell envelope preparations, was partially characterized. The enzyme was found to be extremely halophilic, specifically requiring a high concentration of KCl. Optimum activity was obtained at 4 m KCl and partial substitution of K+ by Na+ resulted in a decrease in activity.

Cell Membrane↗
PubMed1999

Effect of Halococcus morrhuae and Halobacterium saccharovorum on the activation of human peripheral blood lymphocytes.

The immunomodulator properties of two species of halophilic Archaebacteria, Halobacterium saccharovorum and Halococcus rnorrhuae, were analysed by the study of lymphocyte activation. Two methods were used to detect activation in lymphocytes, namely incorporation of the radioactive nucleotide [3H]-thymidine, and CD25 expression. H. morrhuae had a stimulatory effect on human lymphocytes, but this action was observed only with the [3H]-thymidine uptake method, whereas H. saccharovorum produced no immunomodulator effect.

Adult↗
PubMed1999

Kinetic mechanism of glucose dehydrogenase from Halobacterium salinarum.

The kinetic mechanism of glucose dehydrogenase (EC 1.1.1.47) from Halobacterium salinarum was studied by initial velocity and product inhibition methods. The results suggest that both, in the forward and reverse direction, the reaction mechanism is of Bi Bi sequential ordered type involving formation of ternary complexes. NADP+ adds first and NADPH formed dissociates from the enzyme last. For the reverse direction, NADPH adds first and NADP+ leaves last. Product inhibition experiments indicate that (a), the coenzymes compete for the same site and form of the enzyme and (b), ternary abortive complexes of enzyme-NADP(+)-glucono-delta-lactone and enzyme-NADPH-glucose are formed. All the other inhibitions are noncompetitive.

Glucose 1-Dehydrogenase↗
PubMedDate not supplied

[Magnesium orthophosphate, a new form of reserve phosphates in the halophilic archaeon Halobacterium salinarium].

The accumulation and utilization of reserve phosphates in the extremely halophilic archaeon Halobacterium salinarium were studied. The growth of H. salinarium was found to depend on the initial concentration of inorganic phosphate (Pi) in the culture medium and its content in the inoculum. Growing cells consumed 85-95% of Pi from the medium. Unlike the reserve phosphates of many other microorganisms, which are mainly polyphosphates, the reserve phosphates of H. salinarium cells contain no more than 15% polyphosphates, the rest being magnesium orthophosphate. The excessive consumption of Pi from the medium changed cell morphology and caused the death of part of the cell population. The cells that remained viable could grow in a Pi-deficient medium, utilizing about 70% of reserve magnesium phosphate as the phosphorus source.

Culture Media↗
PubMed2001

[Separation of DNA fragments from halobacterium halobium by capillary electrophoresis with non-gel sieving media].

The optimum electrophoresis separation conditions of Lambda DNA/EcoR I + Hind III fragments were investigated using the capillary column DB-1 coated with polysiloxane. When hydroxyethylcellulose (HEC, 1.0%, mass percentage) was used as the only non-gel sieving medium solution, some Lambda DNA fragments could not be separated. After polyvinylpyrrolidone (PVP, 2%, mass percentage) being added into the medium solution, PVP and HEC formed a network colloidal solution in buffer, and changed the network hole size of sieving media. In addition, the results showed that PVP could restrain the adsorption of capillary to DNA, reduced electroosmotic flow, and improved the selectivity of separation. For the first time, all fragments of Lambda DNA marker was separated completely under the same condition of mixed sieving medium solution. The method was applied to separate two groups of DNA fragments of halobacterium halobium, and the base pair number was conjectured.

Cellulose↗
PubMed1999

[Different protein forms of purple membrane produced by Halobacterium halobium under various nutritional condition].

The extremely halophilic bacterium, purple membrane producing Halobacterium halobium strain R1 were cultured in four liquid media which consist of different constituents. After the purple membrane is harvested by ultracentrification and purified by sucrose density gradient ultracentrification, and run on SDS-PAGE, the result of Coomassie blue staining showed different bR protein forms: three bands with molecule weight ranging from 26.0 to 27.5 kD in peptone medium, while only one band with MW 26.0 kD in other three media-complex, synthetic and artificial seawater media, which corresponded to the mature form in peptone medium. The result of Western blotting not only confirmed the different protein forms of Coomassie blue staining in the four media, but also gave additional bands that Coomassie blue can not detect due to its lower sensitivity: 1) in the complex, synthetic and artificial seawater media, a faint 28.0 kD band existed, while in the peptone medium it did not exist; and 2) in the four media, it appeared that a faint 23.0-24.0 kD band exited. The different bR protein forms of purple membrane which result from the difference of nutritional constituents in these four media might be caused by different precursor processing enzymes or different activities of precursor processing enzyme(s) under these different nutritional conditions.

Culture Media↗
PubMedDate not supplied

[Luminescence of bacteriorhodopsin in purple membranes from Halobacterium haolbium cells].

Red luminescence of purple membranes from Halobacterium halobium cells was found out, and its emission, excitation and polarization spectra were investigated. Simultaneous parallel measurements of absorption and luminescence changes in one sample brought about by the action of light were also carried out. The bands in the spectra can be attributed to a number of bacteriorhodpsin (BR) forms: BR(595,520), BR(650,575),BR(600-620), BR(700,625), BR(730,660) BR(780,695), where the number above is the position of the luminescence maxima, below--that of absorption. Proceding from the quantum yield of the luminescence (10(-3)) and of photoreaction (10(-1)) of BR, the photoisomerization rate constant of the latter was estimated (10(11) sec(-1). The temperature dependence of the luminescence quantum yield points to the existence of two or three quenching processes with different activation energies. BR phosphorescence was not observed in the region 500-1100 nm. High degree (36%) os luminescence polarization shows that there is no homogeneous energy transfer between BR molecules, or there is regularity in orientation of their dipoles. Energy migration from the bulk of carotenoids to BR was not found. However limited heterogeneous transfer between the different BR forms cannot be ruled out. The absence (or limitation) of migration indicated that there is a spatial separation of the chromophores. Data on possible participation of triplet states in the BR photoconversions are discussed.

Bacteriorhodopsins↗
PubMed1976

Purification and characterization of a prokaryotic glycoprotein from the cell envelope of Halobacterium salinarium.

The glycoprotein which accounts for approximately 50% of the protein and all of the nonlipid carbohydrate of the cell envelope of Halobacterium salinarium (Mescher, M. F., Strominger, J. L., and Watson S. W. (1974) J. Bacteriol. 120, 945-954) has been purified and partially characterized. The glycoprotein has an apparent molecular weight of 200,000, is extremely acidic, and has a carbohydrate content of approximately 10 to 12%. The carbohydrate included neutral hexoses, amino sugar, and uronic acid. Information regarding the number, composition, and mode of attachment of the carbohydrate chains was obtained by isolation and examination of the glycopeptides derived from degradation of cell envelope protein with trypsin and pronase. Trypsin digestion resulted in two glycopeptides. One of these was large (approximately 55,000 daltons) and had most of the neutral hexose linked to it. The carbohydrate moieties consisted of di- and trisaccharides of glucosylgalactose and (uronic acid, glucose)-galactose attached via O-glycosidic linkages between galactose and threonine. The other tryptic glycopeptide had a relatively large heterosaccharide attached to it via an alkaline-stable linkage. The heterosaccharide contained 1 glucose, 8 to 9 galactose, 1 mannose, and 10 to 11 glucosamine residues, and approximately 6 residues of an unidentified amino augar. The alkaline stability of the linkage and the amino acid composition of glycopeptides resulting from Pronase digestion of the tryptic glycopeptide showed that the heterosaccharide was attached to an asparagine residue, presumably via an N-glycosylamine bond to the amide group. The intact glycoprotein has a single N-linked heterosaccharide, 22 to 24 O-linked disaccharides, and 12 to 14 O-linked trisaccharides per molecule. N- and O-glycosidic linkages are the most common carbohydrate-protein linkages in mammalian glycoproteins but, to our knowledge, this is the first report of either type of linkage in a prokaryotic cell envelope protein.

Amino Acids↗
PubMed1992

Involvement of thiol groups in the reaction mechanism of Mn(2+)-activated alkaline p-nitrophenylphosphate phosphatase of the extreme halophilic archaebacterium Halobacterium halobium.

Halobacterium halobium contains a cytosolic alkaline p-nitrophenylphosphate phosphatase which is selectively activated by Mn2+ ions (Bonet et al., 1991: Int. J. Biochem. 12, 1445-1451). We investigated the reaction mechanism of the enzyme in the presence and the absence of Mn2+ by means of pH studies and the application of some group-specific reagents. A pKes 8.4 of a group in the Mn(2+)-enzyme-substrate complex involved in catalysis could not be detected when catalysis in the absence of the cation was analysed. Only the enzyme with bound Mn2+ was inhibited by p-hydroxymercuribenzoate. Reducing agents stimulated the Mn(2+)-dependent activity but had no effect on the native enzyme activity. Our results indicate that the reaction mechanism is different whether the activating cation is present or not, and that the reaction mechanism in the presence of Mn2+ involves thiol groups.

4-Nitrophenylphosphatase↗
PubMed1992

The binding of a second divalent metal ion is necessary for the activation of ATP hydrolysis and its inhibition by tightly bound ADP in the ATPase from Halobacterium saccharovorum.

A binding site for divalent metal ions on the ATPase from Halobacterium saccharovorum is proposed. This site is different from the catalytic site which binds ATP and a complexed divalent metal ion. Occupation of the second site greatly stimulates the rate of ATP hydrolysis and the affinity of the catalytic site for the metal ion-ATP complex. The time-dependent inhibition of the ATPase, which occurs during catalysis and which is known to be caused by the retention of ADP, is also dependent on the occupation of this metal ion binding site. The binding of the metal ion apparently induces extremely tight binding of ADP after the departure of Pi. Mg2+, Mn2+, Zn2+, Co2+, and Ca2+ were tested and showed both the activating and the inhibitory effects, although their binding constants for ATP and the second metal ion binding site were quite different. The characteristic shapes of the nonlinear ATP hydrolysis curves obtained with different metal ions, and different ratios of metal ion and ATP, could be explained with the established dissociation constants. On this basis, a model for the ATPase was developed with two catalytic cycles: one in which the second metal ion binding site is occupied, and another in which it is empty. These pathways are connected by metal ion-dependent equilibria.

Adenosine Diphosphate↗