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At least 19 recordsLinked to original sources
PubMed2006

Halophile aldehyde dehydrogenase from Halobacterium salinarum.

Halobacterium salinarum is a member of the halophilic archaea. In the present study, H. salinarum was cultured at various NaCl concentrations (3.5, 4.3, and 6.0 M NaCl), and its proteome was determined and identificated via proteomics technique. We detected 14 proteins which were significantly down-regulated in 3.5 M and/or 6 M NaCl. Among the identified protein spots, aldehyde dehydrogenase (ALDH) was selected for evaluation with regard to its potential applications in industry. The most effective metabolism function exhibited by ALDH is the oxidation of aldehydes to carboxylic acids. The ALDH gene from H. salinarum (1.5 kb fragment) was amplified by PCR and cloned into the E. coli strain, BL21 (DE3), with the pGEX-KG vector. We subsequently analyzed the enzyme activity of the recombinant ALDH (54 kDa) at a variety of salt concentrations. The purified recombinant ALDH from H. salinarum exhibited the most pronounced activity at 1 M NaCl. Therefore, the ALDH from H.salinarum is a halophilic enzyme, and may prove useful for applications in hypersaline environments.

Aldehyde Dehydrogenase↗
PubMed2005

A microprobe analysis of inorganic elements in Halobacterium salinarum.

Halobacterium salinarum were grown on peptone agar containing 4.28 M NaCl, 0.036 M K and other salts. Stationary phase organisms were lifted onto carbon planchets, freeze-dried, carbon coated and examined in a scanning electron microscope equipped with an X-ray spectrometer. Intracellular element concentrations (mol/kg H(2)O) were determined using a bulk analysis program with appropriate standards. The cell K concentration was 110 times that of the medium. For Na this value was 0.3 and for Cl, 1.1. When Rb was present in the medium, its intracellular concentration was 77 times higher than the external value. The cation minus anion value suggests a high fixed negative charge, 0.72 equivalents. Intracellular apparent dielectric constants were calculated using cellular EMFs derived from the literature, and sodium concentration. The determined values ranged from 22-28 (vs 80 for normal water) suggesting phases of structured cell water. Ionic distributions in these extremophiles are treated according to the classical principles elucidated by Willard Gibbs and represents a heterogeneous system in thermodynamic equilibrium with the hypersaline environment. Factors to be considered are: (1) composition of Halobacterium and its immobile negative charge; (2) the physicochemical properties of the individual ions (charge, ionic radius, hydration energy, standard chemical potential); (3) the dielectric constant of the dispersion medium (water); and (4) the binding of ions, particularly potassium.

Chlorides↗
PubMed2005

Ca(2+)-dependent cell aggregation of halophilic archaeon, Halobacterium salinarum.

Halobacterium salinarum CCM 2090 exhibits a Ca(2+)-dependent cellular aggregation system. This process is caused by the interaction between Ca(2+) and aggregation factor(s). Although the aggregates formed are encased in matrix-like substances, such as bacterial biofilms, they are not attached to the abiotic substratum. Therefore, these substances may be distinguished from typical biofilms.

Biofilms↗
PubMed2003

Repair of UV damage in Halobacterium salinarum.

Halobacterium is one of the few known Archaea that tolerates high levels of sunlight in its natural environment. Photoreactivation is probably its most important strategy for surviving UV irradiation and we have shown that both of the major UV photoproducts, cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts, can be very efficiently repaired by photoreactivation in this organism. There are two putative photolyase gene homologues in the published genome sequence of Halobacterium sp. NRC-1. We have made a mutant deleted in one of these, phr2, and confirmed that this gene codes for a CPD photolyase. (6-4) photoproducts are still photoreactivated in the mutant so we are currently establishing whether the other homologue, phr1, codes for a (6-4) photolyase. We have also demonstrated an excision repair capacity that operates in the absence of visible light but the nature of this pathway is not yet known. There is probably a bacteria-type excision-repair mechanism, since homologues of uvrA, uvrB, uvrC and uvrD have been identified in the Halobacterium genome. However, there are also homologues of eukaryotic nucleotide-excision-repair genes ( Saccharomyces cerevisiae RAD3, RAD25 and RAD2 ) so there may be multiple repair mechanisms for UV damage in Halobacterium.

Animals↗
PubMed2005

MpcT is the transducer for membrane potential changes in Halobacterium salinarum.

In Halobacterium salinarum mutants containing either of the light-driven ion pumps bacteriorhodopsin (H(+)) or halorhodopsin (Cl(-)) as their only retinal protein, a decrease of irradiance in the absence of respiration causes a phototactic response. The conversion of the causal event, a decrease of proton motive force across the cell membrane, into a reversal of flagellar motor rotational direction was expected to involve a transducer. Via deletion analysis of all 18 known and putative halobacterial transducer (htr) genes, we found that Htr14, a methylatable membrane-bound transducer lacking an extracellular domain, mediates the biological response, which includes adaptive methylation. Based on a minimal stimulus length of 200 ms and the determined cytoplasmic buffering capacity, we conclude that the change in the membrane potential (DeltaPsi), and not that of the internal pH, is the signal-generating event. Htr14 was therefore renamed to Membrane potential change Transducer, or MpcT. It is the first transducer for which the causative stimulus could be narrowed to a change in DeltaPsi, as opposed to a change in pH or cellular redox state.

Adaptation, Physiological↗
PubMed2004

Identification and characterization of inosine monophosphate dehydrogenase from Halobacterium salinarum.

Extremely halophilic Archaea, Halobacterium salinarum live in hypersaline habitats and maintain an osmotic balance of their cytoplasm by accumulating high concentrations of salt (mainly KCl). Therefore, their enzymes adapted to high NaCl concentrations offer a multitude of acutal or potential applications such as biocatalysts in the presence of high salt concentrations. In this study, the protein expression profile of H. salinarum cultured under different NaCl concentrations (3.5 M, 4.3 M, and 6.0 M) was investigated using two-dimensional gel electrophoresis (2-DE). As a result of 2-DE, the protein spots concentrated in acidic range at pH 3-10 were separated effectively using pH 3.5-4.5 ultrazoom IPG DryStrips. The proteins which proved to be upregulated or downregulated in 2-DE gel were digested with trypsin and identified with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization quadrupole (ESI-Q) TOF-mass spectrometry. Most proteins were identified as known annotated proteins based on sequence homology and few as unknown hypothetical proteins. Among proteins identified, an enzyme named inosine monophosphate dehydrogenase (IMPDH) was selected based on the possibility of its industrial application. IMPDH gene (1.6 kb fragment) expected to exist in H. salinarum was amplified by polymerase chain reaction (PCR) and expressed in Escherichia coli strain, BL21 (DE3) using a pGEX-KG vector. Recombinant IMPDH purified from H. salinarum has a higher activity in the presence of salt than in the absence of salt.

Base Sequence↗
PubMed2002

Cell cycle-dependent expression of an essential SMC-like protein and dynamic chromosome localization in the archaeon Halobacterium salinarum.

The genome of Halobacterium salinarum encodes four proteins of the structural maintenance of chromosomes (SMC) protein superfamily. Two proteins form a novel subfamily and are named 'SMC-like proteins of H. salinarum' (Sph1 and Sph2). Northern blot analyses revealed that sph1 and hp24, the adjacent gene, are solely transcribed in exponentially growing, but not in stationary phase, cells. A synchronization procedure was developed, which makes use of the DNA polymerase inhibitor aphidicolin and leads to highly synchronous cultures. It allowed us for the first time to study cell cycle-dependent transcription in an archaeon. The sph1 transcript was found to be highly cell cycle regulated, with its maximal accumulation around the time of septum formation. The Sph1 protein level was also elevated at that time, but a basal protein level was found throughout the cell cycle. The hp24 transcript was sharply upregulated about 1 h before sph1 and had already declined at the time of sph1 induction. These and additional transcript patterns revealed that precisely controlled transcriptional regulation is involved in haloarchaeal cell cycle progression. A DNA staining protocol was developed, which opened the possibility of following the dynamic intracellular localization of haloarchaeal nucleoids using synchronized cultures. After an initial dispersed localization, the nucleoid is condensed at mid-cell. Subsequently, DNA is rapidly transported to the 1/4 and 3/4 positions. All staining patterns were also observed in untreated exponentially growing cells, excluding synchronization artifacts. The Sph1 concentration is elevated when segregation of the new chromosomes is nearly complete; therefore, it is proposed to play a role in a late step of replication, e.g. DNA repair, similar to eukaryotic Rad18 proteins.

Aphidicolin↗
PubMed2005

An efficient proteomics based strategy for the functional characterization of a novel halophilic enzyme from Halobacterium salinarum.

The extremely halophilic archaeon, Halobacterium salinarum grows in environments containing over 25% NaCl. The enzymes of this organism have thus been adapted to be active and stable in hypersaline conditions, which makes them strong candidates as robust industrial enzymes. In this study, the proteomics approach was applied to screen novel halophilic enzymes. We focused initially on proteins that are differentially expressed under different salt concentrations in culture media. After two-dimensional gel electrophoresis over a pH 3.5-4.5 range, 29 differentially expressed protein spots were identified by tandem mass spectrometry and six of these had no similarity to preexisting genes of known function. To predict the function of them, we used various bioinformatic methods. Among other proteins, we selected Vng0487h, which showed a high similarity to acetyltransferases. As a step toward assaying the enzymatic activity of this protein, we cloned the Vng0487h gene of H. salinarum and expressed and purified the recombinant protein with a glutathione-S-transferase (GST) tag in Escherichia coli. Using a GST-pulldown assay, a protein fragment derived from E. coli could interact with recombinant Vng0487h, and was identified to be the ribosomal protein L3. This protein showed high sequence homology with ribosomal protein L7/12 from E. coli and ribosomal protein L13p from H. salinarum. This suggests that Vng0487h acetylates a subunit of ribosomal protein, possibly L13p, in H. salinarum. During the present study, an efficient procedure was established to screen novel halophilic enzymes, and to predict and assess their functions.

Acetyltransferases↗
PubMed1998

Photoresponses of Halobacterium salinarum to repetitive pulse stimuli.

Halobacterium salinarum cells from 3-day-old cultures have been stimulated with different patterns of repetitive pulse stimuli. A short train of 0.6-s orange light pulses with a 4-s period resulted in reversal peaks of increasing intensity. The reverse occurred when blue light pulses were delivered as a finite train: with a 3-s period, the response declined in sequence from the first to the last pulse. To evaluate the response of the system under steady-state conditions of stimulation, continuous trains of pulses were also applied; whereas blue light always produced a sharply peaked response immediately after each pulse, orange pulses resulted in a declining peak of reversals that lasted until the subsequent pulse. An attempt to account for these results in terms of current excitation/adaptation models shows that additional mechanisms appear to be at work in this transduction chain.

Biophysical Phenomena↗
PubMed2002

A bZIP protein from halophilic archaea: structural features and dimer formation of cGvpE from Halobacterium salinarum.

The cGvpE protein of Halobacterium salinarum PHH4 has been identified as transcriptional activator for the promoter of the c-gvpA gene encoding the major gas vesicle structural protein cGvpA. Molecular modelling of the carboxy-terminal region of cGvpE suggests that this protein resembles a basic leucine-zipper protein, and mutations in the putative DNA binding domain DNAB completely abolish the activator function in Haloferax volcanii transformants. Mutations in the key residues of the putative leucine-zipper region AH6 of cGvpE confirmed that the three residues V159, L166 and L173 were essential for the activator function of cGvpE at the c-gvpA promoter, whereas the cysteine residue C180 could be altered to a leucine or an aspartate residue without the loss of this function. Mutations in basic residues of helix AH4 demonstrated the importance of the lysine K104 for the activator function of cGvpE. A cGvpE protein containing a his-tag at the C-terminus was still able to activate the expression of c-gvpA in vivo. The cGvpE his-purified from Hf. volcanii formed a dimer in Blue-native polyacrylamide gels that could be resolved into monomers by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Dimers of cGvpE were already seen using SDS-PAGE, but not with cGvpE mutant proteins containing the alterations L166E or L173E/C180L in the leucine zipper. These results imply that the hydrophobic surface of helix AH6 is indeed required for the establishment of cGvpE dimers.

Amino Acid Sequence↗
PubMed2004

Identification of a novel protein D3UPCA from Halobacterium salinarum and prediction of its function.

Halobacterium salinarum is an extremely halophilic archaea, which is able to live in highly saline environments. In a recent study, several halophilic archaea were found to have the ability to biodegrade organic hydrocarbon pollutants, but protein information regarding hydrocarbon degradation and tolerance in halophilic archaea has been relatively rare. In this study, the protein expression profile of H. salinarum cultured under different diesel concentrations (0, 2 and 4%) was investigated by two-dimensional gel electrophoresis. Proteins which increased their expression levels in diesel media were identified using matrix-assisted laser desorption/ionization-time of flight and electrospray ionization-tandem mass spectrometry analysis. Among these, a protein spot (named D3UPCA) which was up-regulated about nine-fold and found to have COG3388, an uncharacterized protein conserved in archaea, was selected in order to further characterize its functions. The D3UPCA coding gene (named d3upca) was cloned and expressed in Escherichia coli, and purified by the glutathione-S-transferase-fusion method. The function of the protein was estimated using various bioinformatics tools and was predicted to be related to the regulation of transcription and/or translation of genes needed to tolerate stresses associated within the presence of diesel oil.

Amino Acid Sequence↗
PubMed2003

Osmotic shock induces the presence of glycocardiolipin in the purple membrane of Halobacterium salinarum.

In the purple membrane (PM) of Halobacterium salinarum is present a phospholipid dimer consisting of sulfo-triglycosyl-diether (S-TGD-1) esterified to the phosphate group of phosphatidic acid (PA), i.e., S-TGD-1-PA, called glycocardiolipin (GlyC) (Corcelli, A., M. Colella, G. Mascolo, F. P. Fanizzi, and M. Kates. A novel glycolipid and phospholipid in the purple membrane. 2000. Biochemistry. 39: 3318-3326). The GlyC content of whole cells, PM, and other cell fractions of H. salinarum have been analyzed. GlyC is a nonabundant phospholipid in H. salinarum cells, and it represents one of the major phospholipids of isolated PM. In this report, we show that a) GlyC is formed during the isolation of PM, b) GlyC increase in H. salinarum cells is specifically induced by osmotic shock, and c) in correspondence with GlyC increase, a decrease of S-TGD-1 levels occurs. The changes in membrane lipid composition observed during the isolation of PM are due to de novo synthesis of GlyC from S-TGD-1.

Cardiolipins↗
PubMed2000

Salt dependent stability and unfolding of [Fe2-S2] ferredoxin of Halobacterium salinarum: spectroscopic investigations.

Ferredoxin from the haloarchaeon Halobacterium salinarum is a 14. 6-kDa protein with a [Fe2-S2] center and is involved in the oxidative decarboxylation of 2-oxoacids. It possesses a high molar excess of acidic amino acid residues and is stable at high salt concentration. We have purified the protein from this extreme haloarchaeon and investigated its salt-dependent stability by circular dichroism, fluorescence, and absorption techniques. The predominantly beta-sheeted protein is stable in salt concentrations of >/=1.5 M NaCl. At lower concentrations a time-dependent increase in fluorescence intensity ratio (I(360):I(330)), a decrease in the absorption at 420 nm, and a decrease in ellipticity values are observed. The rate of fluorescence intensity change at any low salt concentration is the highest, followed by absorption and ellipticity. This suggests that at low salt the unfolding of ferredoxin starts with the loss of tertiary structure, which leads to the disruption of the [Fe2-S2] center, resulting in the loss of secondary structural elements.

Binding Sites↗
PubMed2002

Cl(-) concentration dependence of photovoltage generation by halorhodopsin from Halobacterium salinarum.

The photovoltage generation by halorhodopsin from Halobacterium salinarum (shR) was examined by adsorbing shR-containing membranes onto a thin polymer film. The photovoltage consisted of two major components: one with a sub-millisecond range time constant and the other with a millisecond range time constant with different amplitudes, as previously reported. These components exhibited different Cl(-) concentration dependencies (0.1-9 M). We found that the time constant for the fast component was relatively independent of the Cl(-) concentration, whereas the time constant for the slow component increased sigmoidally at higher Cl(-) concentrations. The fast and the slow processes were attributed to charge (Cl(-)) movements within the protein and related to Cl(-) ejection, respectively. The laser photolysis studies of shR-membrane suspensions revealed that they corresponded to the formation and the decay of the N intermediate. The photovoltage amplitude of the slow component exhibited a distorted bell-shaped Cl(-) concentration dependence, and the Cl(-) concentration dependence of its time constant suggested a weak and highly cooperative Cl(-)-binding site(s) on the cytoplasmic side (apparent K(D) of approximately 5 M and Hill coefficient > or =5). The Cl(-) concentration dependence of the photovoltage amplitude and the time constant for the slow process suggested a competition between spontaneous relaxation and ion translocation. The time constant for the relaxation was estimated to be >100 ms.

Biophysical Phenomena↗
PubMed2001

Structural stabilization of [2Fe-2S] ferredoxin from Halobacterium salinarum.

The ferredoxin of the extreme haloarchaeon Halobacterium salinarum requires high (>2 M) concentration of salt for its stability. We have used a variety of spectroscopic probes for identifying the structural elements which necessitate the presence of high salt for its stability. Titration of either the fluorescence intensity of the tryptophan residues or the circular dichroism (CD) at 217 nm with salt has identified a structural form at low (<0.1 M) concentration of salt. This structural form (L) exhibits increased solvent exposure of W side chain(s) and decreased level of secondary structure compared to the native (N) protein at high concentrations of salt. The L-form, however, contains significantly higher levels of both secondary and tertiary structures compared to the form (U) found in highly denaturing conditions such as 8 M urea. The structural integrity of the L-form was highly pH dependent while that of N- or U-form was not. The pH dependence of either fluorescence intensity or CD of the L-form showed the presence of two apparent pK values: approximately 5 and approximately 10. The structural integrity of the L-form at low (<5) pH was very similar to that of the N-form. However, titration with denaturants showed that the low pH L-form is significantly less stable than the N-form. The increased destabilization of the L-form with the increase in pH was interpreted to be due to mutual Coulombic repulsion of carboxylate side chains (pK approximately 6) and due to the disruption of salt bridge(s) between ionized carboxylates and protonated amino groups (pK approximately 10). Estimation of solvent accessibility of W residues by fluorescence quenching, and measurement of decay kinetics of fluorescence intensity and anisotropy strongly support the above model. Polylysine interacted stoichiometrically with the L-form of ferredoxin resulting in nativelike structure. In conclusion, our studies show that high concentration of salt stabilizes the haloarchaeal ferredoxin in two ways: (i) neutralization of Coulombic repulsion among carboxyl groups of the acidic residues, and (ii) salting out of hydrophobic residues leading to their burial and stronger interaction.

Circular Dichroism↗
PubMed2000

The effects of ultraviolet radiation on the moderate halophile Halomonas elongata and the extreme halophile Halobacterium salinarum.

Both the moderately halophilic bacterium, Halomonas elongata, and the extremely halophilic archaea, Halobacterium salinarum, can be found in hypersaline environments (e.g., salterns). On complex media, H. elongata grows over a salt range of 0.05-5.2 M, whereas, H. salinarum multiplies over a salt range of 2.5-5.2 M. The purpose of this study was to illustrate the effect that solar (UV-A and UV-B) and germicidal radiation (UV-C) had on the growth patterns of these bacteria at varied salt concentrations. Halomonas elongata grown on a complex medium at 0.05, 1.37, and 4.3 M NaCl was found to be more sensitive to UV-A and UV-B radiation, as the salt concentration of the medium increased. Halobacterium salinarum grown on a complex medium at 3.0 and 4.3 M NaCl did not show a significant drop in viability after 39.3 kJ.m-2 of UV-A and UV-B exposure. When exposed to UV-C, H. elongata exhibited substantially more sensitivity than H. salinarum. In H. elongata, differential sensitivity to UV-C was observed. At 0.05 M NaCl, H. elongata was less sensitive to UV-C than at 1.37 and 4.3 M NaCl. Both bacteria showed some photoreactivation when incubated under visible light following both UV-A, UV-B, and UV-C exposure. Mutagenesis following UV-C exposure was demonstrated by both organisms.

Anti-Bacterial Agents↗
PubMed2000

Archaeal promoter-directed expression of the Halobacterium salinarum catalase-peroxidase gene.

The Halobacterium salinarum catalase-peroxidase gene was subcloned into shuttle vectors pWL102 and pWL202 and expressed under the control of different archaeal promoters. When Hbt. salinarum was transformed with the catalase-peroxidase gene under the control of its own promoter, catalase-peroxidase activity increased twofold. Catalase-peroxidase activity increased threefold when Hbt. salinarum was transformed with the catalase-peroxidase gene under the control of a tRNA promoter. This bifunctional enzyme in Hbt. salinarum was not induced by environmental stresses such as H2O2, intense light, darkness, high temperature, low temperature, redox inhibitors, heavy metals, or ions.

Base Sequence↗
PubMed2000

Differential transport properties of D-leucine and L-leucine in the archaeon, Halobacterium salinarum.

The transport of D-leucine was compared with that of L-leucine in Halobacterium salinarum. When a high-outside/low-inside Na+ gradient was imposed, D-leucine as well as L-leucine accumulated in envelope vesicles, supporting the hypothesis that D-leucine is transported via a symport system along with Na+. Kinetic analyses, including inhibition experiments, indicated that both enantiomers are transported via a common carrier. However, a Hill plot indicated a single binding site for Na+ during L-leucine transport, but dual binding sites for Na+ during D-leucine transport. Furthermore, D-leucine transport was dependent on electrical membrane potential, suggesting that a transporter bound with D-leucine is positively charged. L-leucine transport was slightly, if at all, dependent on membrane potential, suggesting that a transporter bound with L-leucine is electrically neutral. These results indicate that the leucine carrier in Halobacterium salinarum translocates two moles of Na+ per mole of D-leucine, and one mole of Na+ per mole of L-leucine.

Biological Transport↗