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PubMed2004

[Site-directed mutagenesis and promoter functional analysis of RM07 DNA fragment from Halobacterium halobium in Escherichia coli].

The RM07 DNA fragment from Halobacterium halobium was inserted upstream of chloramphenicol acetyltransferase (cat) reporter gene in pKK232-8 in two different orientations (positively or negatively), generating the RM07-cat fusion plasmids pRM07-1 (+) and pRM07-1 (-). These two plasmids and pKK232-8 were transformed into Escherichia coli HB101 respectively, then the antibiotic resistance level and the chloramphenicol acetyltransferase protein concentration of different transformants were detected. The research results revealed that HB101/pRM07-1 (+) was resistant to chloramphenicol and could grow on the plate containing chloramphenicol, but HB101/pRM07-1 (-) and HB101/pKK232-8 were sensitive to it. Therefore, the results suggested that the positive RM07 fragment had promoter activity in Escherichia coli while the negative RM07 fragment did not. Site-directed mutagenesis of RM07 was performed by PCR mutagenesis method. The effect of specific nucleotide mutations on the chloramphenicol resistance level of different transformants was detected. The different transformants containing different nucleotide mutations were inoculated on the plates containing various concentrations of chloramphenicol and then incubated at the same time. The change of chloramphenicol resistance level reflected the change of promoter activity. By using this method, the important nucleotide responsible for the promoter function of RM07 in Escherichia coli was determined. The promoter activity of RM07 in Escherichia coli was also improved greatly by modifying the nucleotide component.

Chloramphenicol O-Acetyltransferase↗
PubMed1977

Light-dependent cation gradients and electrical potential in Halobacterium halobium cell envelope vesicles.

Vesicles can be prepared from Halobacterium halobium cell envelopes, which contain properly oriented bacteriorhodopsin and which extrude H+ during illumination. The pH difference that is generated across the membranes is accompanied by an electrical potential of 90-100 mV (interior negative) and the movements of other cations. Among these is the efflux of Na+, which proceeds against its electrochemical potential. The relationship between the size and direction of the light-induced pH gradient and the rate of depletion of Na+ from the vesicles, as well as other evidence, suggest that the active Na+-extrusion is facilitated by a membrane component that exchanges H+ for Na+ with a stoichiometry greater than 1. The gradients of H+ and Na+ are thus coupled to one another. The Na+-gradient (Na+out greater than Na+in), which arises during illumination, plays a major role in energizing the active transport of amino acids.

Bacteriorhodopsins↗
PubMed1977

Light-activated amino acid transport in Halobacterium halobium envelope vesicles.

Vesicles prepared from Halobacterium halobium cell envelopes accumulate amino acids in response to light-induced electrical and chemical gradients. Nineteen of 20 commonly occurring amino acids have been shown to be actively accumulated by these vesicles in response to illumination or in response to an artificially created Na-gradient. Sodium-activated amino acid transport for 18 of these amino acids has been shown to occur in direct response to the protonmotive force generated. Glutamate is transported only in response to a sodium gradient. Michaelis constants for the uptake of these amino acids are close or identical whether the amino acids are accumulated in response to a sodium gradient or a protonmotive force (i.e., electrical gradient). On the basis of shared common carriers the transport systems can be divided into eight classes, each responsible for the transport of one or several amino acids, i.e., arginine, lysine, histidine; asparagine, glutamine; alanine, glycine, threonine, serine; leucine, valine, isoleucine, methionine; phenylalanine, tyrosine, tryptophan; aspartate; glutamate; proline. Available evidence suggests that these carriers are symmetrical in that amino acids can be transported equally well in both directions across the vesicle membranes. A tentative working model to account for these observations is presented.

Amino Acids↗
PubMed1977

Light energy conservation processes in Halobacterium halobium cells.

In Halobacterium halobium, proton pumping driven by light or by respiration generates an electrochemical potential difference across the membrane. Energy storage in this form is only transient. Cellular energy transducers competing with proton leaks stabilize this free energy as high energy phosphate bonds, electrochemical potential of other ions, and chemical potential of amino acids and possibly other chemical species. The pH changes induced by light or by respiration in cell suspensions are complicated by proton flows associated with the functioning of the cellular energy transducers. Dominant is the proton inflow coupled to the synthesis of ATP, which has been kinetically resolved. A proton-per-ATP ratio of about 3 is calculated from simultaneous measurements of photophosphorylation and the proton inflow. This value is compatible with the chemiosmotic coupling hypothesis. The time course of the light-induced changes in membrane potential indicates that light-driven pumping increases a dark preexisting potential of about 130 mV only by a small amount (20-30 mV). The complex kinetic features of the membrane potential changes do not closely follow those of the pH changes, indicating that flows of ions other than protons are involved. A qualitative model consistent with the available data is presented. A salient feature of this model is a sudden relaxation of the protonmotive force by a proton inflow through the ATPase when the preexisting protonmotive force is increased by light or respiration and reaches a critical value. The trigger could be either the proton-motive force, the pH gradient, or possibly the internal pH.

Aerobiosis↗
PubMed1990

Recognition of exon-intron boundaries by the Halobacterium volcanii tRNA intron endonuclease.

The intron-containing tRNA(Trp) precursor from Halobacterium volcanii, like many intron-containing archaebacterial precursor tRNAs, can assume a structure in which the two intron endonuclease cleavage sites are localized in two three-nucleotide loops separated by four base pairs. To investigate the role of this structure in cleavage by the halophilic endonuclease, a series of mutant tRNA(Trp) RNAs were prepared and evaluated as substrates. We find that alterations in this structure result in the loss of cleavage at both 5' and 3' sites. Cleavage of a 35-nucleotide model RNA substrate, containing only these features, demonstrates that sequences and structures present at the exon-intron boundaries are sufficient for recognition and cleavage. We have also examined the mechanism used by the halophilic endonuclease to identify the cleavage sites. Addition of a single base, or a base pair in the anticodon stem above the cleavage sites, does not affect the cleavage site selection. The addition of nucleotides between the two cleavage sites significantly decreases cleavage efficiency and has an effect on the cleavage site selection. These results demonstrate that the halophilic endonuclease requires a defined structure at the exon-intron boundaries and does not identify its cleavage sites by a measurement mechanism like that employed by eukaryotic tRNA intron endonucleases.

Bacterial Proteins↗
PubMed1991

F1-like properties of an ATPase from the archaebacterium Halobacterium saccharovorum.

For F1-ATPases from mitochondria and chloroplasts, tight binding of Mg2+ and ADP without Pi at a catalytic site had been reported as a cause of enzyme inhibition. The time dependence of this inhibition and the effect of various agents on this process have been described (Du, Z., and Boyer, P. D. (1990) Biochemistry 29, 402-407, and references therein). Similar results are now reported for the ATPase from Halobacterium saccharovorum. The nonlinear hydrolysis kinetics were modulated by nitrate, azide, sulfite, GTP, ADP in the absence of ATP, or Pi in characteristic ways, in good analogy with the effects of these agents on F1 enzymes. The similarity to the F1 systems suggests that it is tight ADP binding that is affected. Although these reactions of the H. saccharovorum ATPase occurred on different time and concentration scales than those of F1-ATPases, the two systems do not appear to be fundamentally different. The hydrolytic mechanism of the H. saccharovorum ATPase thus identifies this enzyme as a member of the F0F1-ATPase family.

Adenosine Diphosphate↗
PubMed1991

Halobacterial S9 operon. Three ribosomal protein genes are cotranscribed with genes encoding a tRNA(Leu), the enolase, and a putative membrane protein in the archaebacterium Haloarcula (Halobacterium) marismortui.

In the eubacterium Escherichia coli the genes for the ribosomal proteins L13 and S9 form an operon consisting of two genes. The corresponding operon of the archaebacterium Haloarcula marismortui (Halobacterium marismortui was recently reassigned to the genus Haloarcula [Oren, A., Ginzburg, M., Ginzburg, B. Z., Hochstein, L. I., and Volcani, B. E. (1990) Int. J. Syst. Bacteriol. 40, 209-210] and is now called Haloarcula marismortui) which is presented here, is much larger encoding three ribosomal proteins (HL29, HmaL13, HmaS9), a tRNA(Leu), the glycolytic enzyme enolase (HmaEno), a putative membrane protein (OrfMSG), and two not yet identified open reading frames (OrfMMV, OrfMNA). The nucleotide sequence of 3931 base pairs has been established. Northern analysis revealed the existence of a polycistronic mRNA (3.7 kilobases) demonstrating that the transcription of a gene of the glycolytic pathway is coupled to the transcription of ribosomal protein genes. Upstream of the first gene of the operon (tRNA(Leu)) a promoter structure typical for the extreme halophilic archaebacteria was detected and downstream of the last gene (OrfMSG) a terminator structure was present. As shown by S1-nuclease mapping, a tRNA-mRNA transcript as well as the mRNA alone was present in vivo. The transcriptional start point and the tRNA-mRNA cleavage point are shown to be almost identical to the 5' and 3' ends, respectively, of the putative mature tRNA. The C-terminal part of the OrfMSG protein shows a significant similarity to the vertebrate laminin receptor protein.

Bacterial Proteins↗
PubMedDate not supplied

[Energy characteristics of the stages of photo-dependent transport of 14C-alanine in Halobacterium halobium R1 cells].

The energy of light is utilized by Halobacterium halobium R1 only at the very beginning of illumination at the same time as the transport of protons into the cells, as follows from comparing the data of photophosphorylation and light-dependent transport. The release of protons by the cells on further illumination suggests that the photoprocess and the accumulation of energy by the cells are being uncoupled. The primary transport of 15C-alanine in the course of illumination is caused directly by electrochemical gradient (i) whereas the secondary transport in the dark involves the energy liberated on hydrolysis of ATP which has been formed in the process of photophosphorylation (II). An indirect technique of assaying ATP from the transport of 14C-alanine into the cells makes it possible to characterize the distribution of utilized light energy between photophosphorylation (II) and transport processes (I). The bulk of energy is accumulated as ATP. The incorporation of 14C-alanine into the cells is stimulated twofold by illumination for a long time under physiological conditions in the presence of respiration. Therefore, light plays an important role in the energy balance of H. halobium R1.

Adenosine Triphosphate↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1989

Dihydrofolate reductase of the extremely halophilic archaebacterium Halobacterium volcanii. The enzyme and its coding gene.

Halobacterium volcanii mutants that are resistant to the dihydrofolate reductase inhibitor trimethoprim contain DNA sequence amplifications. This paper describes the cloning and nucleic acid sequencing of the amplified DNA sequence of the H. volcanii mutant WR215. This sequence contains an open reading frame that codes for an amino acid sequence that is homologous to the amino acid sequences of dihydrofolate reductases from different sources. As a result of the gene amplification, the trimethoprim-resistant mutant overproduces dihydrofolate reductase. This enzyme was purified to homogeneity using ammonium sulfate-mediated chromatographies. It is shown that the enzyme comprises 5% of the cell protein. The amino acid sequence of the first 15 amino acids of the enzyme fits the coding sequence of the gene. Preliminary biochemical characterization shows that the enzyme is unstable at salt concentrations lower than 2 M and that its activity increases with increase in the KCl or NaCl concentrations.

Amino Acid Sequence↗
PubMed1989

Hysteretic behavior of an ATPase from the archaebacterium, Halobacterium saccharovorum.

The membrane-bound detergent-activated ATPase from Halobacterium saccharovorum was purified at a physiological salt concentration (4 M NaCl) in the presence of nonionic detergents. The preparation contains putative subunits of 110, 71, 31, 22, and 14 kDa. The enzyme activity required high salt concentration but was not dependent on any one specific monovalent cation or any anion. The hydrolysis of ATP was nonlinear with time; the data are consistent with a kinetic model where the enzyme is irreversibly converted from an initial into a final stable form during the first few minutes of the reaction. The model thus contains a rate constant (k) for the transition and hydrolytic rates, v1 and v2, for the two forms of the enzyme. We found that this hysteretic behavior was influenced differently by various conditions and inhibitors. The constant k was smaller with Mn2+ than with Mg2+ as the divalent cation, showed negative temperature dependence, and a distinct pH optimum between 7.5 and 8.5. Thiols decreased k, but nitrate, a specific inhibitor of archaebacterial ATPases, increased it. ADP showed competitive inhibition against both the initial and the final form of the enzyme. Nitrate reversibly inhibited only the latter and in a manner dependent on whether Mn2+ or Mg2+ was used. The kinetic data suggest that all agents tested, with effects on the hydrolytic activity, seem to act at or near the catalytic site of the enzyme.

Adenosine Diphosphate↗
PubMed1989

Evolution and regulation of the gene encoding superoxide dismutase from the archaebacterium Halobacterium cutirubrum.

The gene encoding the manganese-containing superoxide dismutase (SOD) of Halobacterium cutirubrum was isolated and characterized. The gene and 5'- and 3'-untranslated regions were located on a genomic DNA fragment of 1127 nucleotides. The deduced amino acid sequence is 200 residues long and has 39-42% identity with manganese-containing SODs of eubacteria and mitochondria. This homology may be due to either lateral transfer of the gene between eubacteria and archaebacteria or to high amino acid sequence conservation in the enzyme during the separate evolution of eubacteria and archaebacteria. Transcription of the gene initiates only about three nucleotides upstream of the translation initiation codon. The 5' end of the transcript does not contain a purine-rich Shine-Dalgarno sequence, and the promoter region does not contain consensus sequences found in other archaebacterial promoters. Termination of transcription occurs at 5 consecutive thymine residues that are preceded by a GC-rich region. The gene is basally expressed in anaerobically grown cells but is also inducible by paraquat, a generator of oxygen radicals. The same transcription initiation site is used in both types of expression, suggesting that one promoter is responsible for both basal and regulated expression. In addition to the single copy of the authentic SOD gene, the genome of H. cutirubrum contains a sequence that is very closely related to but does not code for the previously purified SOD of this organism.

Amino Acid Sequence↗
PubMed1989

The function of gamma-glutamylcysteine and bis-gamma-glutamylcystine reductase in Halobacterium halobium.

gamma-Glutamylcysteine and bis-gamma-glutamylcystine reductase appear to function in the halobacteria in a fashion analogous to GSH and glutathione reductase in other cells. Bis-gamma-glutamylcystine reductase (GCR), a NADPH-dependent dimer of Mr 122,000 recently purified to homogeneity from Halobacterium halobium (Sundquist, A.R., and Fahey, R.C. (1988) J. Bacteriol., 170, 3459-3467), was found to be highly specific for bis-gamma-glutamylcystine and to be present in cell extract at a level sufficient to maintain gamma-glutamylcysteine predominantly in its thiol form [( thiol]/[disulfide] approximately 50). Bis-gamma-glutamylcystine reductase is similar to glutathione reductase in many respects; GCR demonstrated a FAD:subunit stoichiometry of 1, inhibition by heavy metal ions, and a pH optimum near neutrality. However, GCR exhibited no activity with GSSG and was most active at salt levels exceeding 2 M. A turnover number of 1,700 mumol min-1 mumol-1 FAD and apparent Km values of 0.8 mM for bis-gamma-glutamylcystine and 0.29 mM for NADPH were determined for GCR. The effect of salt on the autoxidation rates of gamma-glutamylcysteine, GSH, and Cys was also studied. In the absence of added salt, Cys oxidized more rapidly than gamma-glutamylcysteine, which in turn oxidized more rapidly than GSH. The presence of 4.3 M chloride (K+ and Na+) significantly slowed the autoxidation of all three thiols. The rate of autoxidation of gamma-glutamylcysteine in 4.3 M chloride proved slower than that of GSH in the absence of added chloride. Thus, gamma-glutamylcysteine is at least as stable under halophilic conditions as GSH is under nonhalophilic conditions, explaining why halobacteria utilize gamma-glutamylcysteine rather than GSH.

Dipeptides↗
PubMed1988

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).

Amino Acid Sequence↗
PubMed1988

A tRNA(Trp) intron endonuclease from Halobacterium volcanii. Unique substrate recognition properties.

We report on the properties of a partially purified tRNA intron endonuclease from the archaebacterium Halobacterium volcanii. This enzyme is capable of precise excision of the 104-nucleotide intron from halo-bacterial pre-tRNA(Trp) substrates generated in vitro by T7 RNA polymerase transcription. The reaction requires divalent cations (Mg2+ or Ca2+) or spermidine, is inhibited by monovalent cations, and produces 5'-hydroxyl and 2',3'-cyclic phosphate termini. Unlike the universal substrate recognition properties characteristic of the eukaryotic tRNA intron endonucleases, this enzyme is specific for halophilic tRNA(Trp) substrates. The partially purified enzyme is not capable of removing the intron from a yeast pre-tRNA(Phe) substrate. Analysis of the enzyme's ability to cleave tRNA(Trp) substrates lacking exon sequences demonstrated that the mature tRNA-like structure is not required in the substrate. A substrate retaining the intact intron and only the anticodon stem and loop exon regions was efficiently cleaved. Deletions within the intron indicated that the intron was not a primary site for recognition by the endonuclease; however, its presence affects the efficiency of the cleavage reaction. The possible relationship of this enzyme to other RNA endonucleases is discussed.

Base Sequence↗
PubMed1987

The primary structures of ribosomal proteins S14 and S16 from the archaebacterium Halobacterium marismortui. Comparison with eubacterial and eukaryotic ribosomal proteins.

The amino acid sequences of two ribosomal proteins, S14 and S16, from the archaebacterium Halobacterium marismortui have been determined. Sequence data were obtained by the manual and solid-phase sequencing of peptides derived from enzymatic digestions with trypsin, chymotrypsin, pepsin, and Staphylococcus aureus protease as well as by chemical cleavage with cyanogen bromide. Proteins S14 and S16 contain 109 and 126 amino acid residues and have Mr values of 11,964 and 13,515, respectively. Comparison of the sequences with those of ribosomal proteins from other organisms demonstrates that S14 has a significant homology with the rat liver ribosomal protein S11 (36% identity) as well as with the Escherichia coli ribosomal protein S17 (37%), and that S16 is related to the yeast ribosomal protein YS22 (40%) and proteins S8 from E. coli (28%) and Bacillus stearothermophilus (30%). A comparison of the amino acid residues in the homologous regions of halophilic and nonhalophilic ribosomal proteins reveals that halophilic proteins have more glutamic acids, asparatic acids, prolines, and alanines, and less lysines, arginines, and isoleucines than their nonhalophilic counterparts. These amino acid substitutions probably contribute to the structural stability of halophilic ribosomal proteins.

Amino Acid Sequence↗
PubMed1986

Isoprenoid synthesis in Halobacterium halobium. Modulation of 3-hydroxy-3-methylglutaryl coenzyme a concentration in response to mevalonate availability.

Halobacterium halobium was evaluated as a potentially simpler biological model to study the regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity (content) in response to mevalonate availability. H. halobium's HMG-CoA reductase was soluble and required NADPH as its reduced coenzyme. Maximum HMG-CoA reductase activity (4-10 nmol/min/mg of soluble protein) was obtained in buffers which contained 3.5 M KCl. Mevinolin (a) blocked growth of H. halobium, (b) was a competitive inhibitor of HMG-CoA reductase (Ki = 20 nM), (c) did not cause the paradoxical increase in assayable reductase activity, as reported for eukaryotic cells, and (d) caused a rapid (within 30 min) 8-12-fold accumulation of intracellular HMG-CoA. Mevalonate blocked and reversed mevinolin-mediated HMG-CoA accumulation. Although mevinolin-treated cell's growth was restored by mevalonate, HMG-CoA reductase's activity was not. Thus, H. halobium is a unique biological model which allows one to study the regulation of intracellular HMG-CoA concentration and not HMG-CoA reductase activity (content) in response to mevalonate availability.

Acyl Coenzyme A↗