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Enhanced production and growth of Halobacterium salinarum are achieved by direct supplement of essential amino acids in the modified nutrient culture medium. As arginine (R) and aspartate (D) are the main amino acid sources for producing bacteriorhodopsin efficiently from Halobacterium salinarum, both individual and combined effects of these two amino acids (in different compositions) in the basal medium were studied. The BR production is enhanced by 83% on the eighth day (saturated) for all individual and combined amino acid supplements. Maximum production of 201 mg/l is observed for combined amino acid (R3D2)-supplemented culture which is 4.46-fold higher than the conventional culture growth from the basal medium. The obtained results suggest the efficient method to enhance BR production at low cost and thus, open up the possibility to utilize this potential biomolecule for various photonics applications which were earlier restricted due to the high cost of protein molecules. [ABSTRACT FROM AUTHOR]

Spray drying is appropriate for the preservation of halophilic microorganisms due to the nature of these microorganisms, as they survive in adverse environmental conditions by being encapsulated in salt crystals. Artificial neural networks were in this study used to optimize practically significant spray-drying regimes of the C50-carotenoids producer Halobacterium salinarum. Immediately after drying, the samples contained up to 54% halobacterial biomass and less than 5% moisture, and the level of preservation of carotenoids was 95-97%. The storage of biomass at 4 °C resulted in the gradual degradation of the carotenoids, which reached 58-64% in the best samples after 1 year. A comprehensive study of changes in halobacteria biomass after spray drying and the nature of the damage provided new data on the survival and preservation of cells and biologically active substances in the various spray-drying regimes and at different storage times. [ABSTRACT FROM AUTHOR]

Bacteriorhodopsin (bR) is a trans-membrane proton pump found in the purple membrane of Halobacterium salinarum. This protein has high photochemical and photoelectric conversion efficiency and thermal stability, allowing it to withstand high temperatures, high salinity, and nutritionally-limited environments. The ability of this protein to convert light energy into chemical energy has applications that are mainly therapeutic/diagnostic and research-oriented. There is increasing demand for bacteriorhodopsin production in different fields. The present study maximized bacteriorhodopsin production using H. salinarum. The physical parameters of illumination, agitation speed, temperature, and nitrogen source were studied using a fractional factorial design to determine the optimal levels of each. The most suitable nitrogen source was determined to be peptone from meat. The optimal temperature was 39 °C, agitation speed was 150 rpm, and light intensity was 6300 lux for bR production. Under these conditions, the maximum bR yield was 196 mg/l, which is about 4.23 fold greater than those obtained with basal medium. The proposed strategies could be used for bR production using this archaeobacterium; the results are the highest reported thus far from a batch culture of H. salinarum. [ABSTRACT FROM AUTHOR]

Many members of the Halobacteriaceae were found to produce halocins, molecules that inhibit the growth of other halophilic archaea. Halocin H4 that is produced by Haloferax mediterranei and inhibits the growth of Halobacterium salinarum is one of the best studied halocins to date. The gene encoding this halocin had been previously identified as halH4, located on one of Hfx. mediterranei megaplasmids. We generated a mutant of the halH4 gene and examined the killing ability of the Haloferax mediterranei halH4 mutant with respect to both Halobacterium salinarum and Haloferax volcanii. We showed that both wild-type Hfx. mediterranei and the halH4 mutant strain efficiently inhibited the growth of both species, indicating halocin redundancy. Surprisingly, the halH4 deletion mutant exhibited faster growth in standard medium than the wild type, and is likely to have a better response to several nucleotides, which could explain this phenotype. [ABSTRACT FROM AUTHOR]

Nucleoside diphosphate kinase (HsNDK) from an extremely halophilic archaea, Halobacterium salinarum, is composed of a homo hexamer, assembled as a trimer of basic dimeric units. It requires >2 M NaCl for refolding, although it does not require NaCl for stability or enzymatic activity below 30 °C. A HisN111L mutant with an N-terminal extension sequence containing hexa-His tag, in which Asn111 was replaced with Leu, was designed to be less stable between basic dimeric units. This mutant can lose between 6 and 12 hydrogen bonds between basic dimeric units in the hexamer structure. The HisN111L mutant had enhanced salt requirements for enzymatic activity and refolding even though the secondary structure of the HisN111L mutant was confirmed to be similar to the control, HisNDK, in low and high salt solutions using circular dichroism. We reported previously that G114R and D148C mutants, which had enhanced interactions between basic dimeric units, showed facilitated refolding and stabilization in low salt solution. The results of this study help to elucidate the process for engineering industrial enzymes by controlling subunit-subunit interactions through mutations. [ABSTRACT FROM AUTHOR]

Three pigmented strains of halophilic archaea (RS75, RS77, RS79) were isolated from the monoliths of mottled sylvinite from the Verkhnekamsk salt deposit (Solikamsk, Russia). The cells were nonmotile, gram-negative, pleomorphic, disk-shaped or ovoid, 0.8-1.0 × 1.5-2.5 μm. The organism was a chemoorganotrophic obligate aerobe producing catalase and oxidase. A number of carbohydrates and carboxylic acids were used as growth substrates. Growth occurred in the presence of 7-27 % NaCl (with the optimum at 15-18 %), 0.02-20 % KCl (0.2-1 %), 0.2-16 % MgCl (2-3 %), in the temperature range from 23 to 51 °C (40-45 °C), and pH 5.5-8.0 (6.8-7.0). The membranes contained carotenoids of the bacterioruberin series. Phosphatidylglyceromethylphosphate (PGP-Me), phosphatidylglycerol (PG), sulfated diglycosyl diether (S-DGD-1) predominated among the polar lipids. The DNA G + C content was 64.0-65.0 mol %. Phylogenetic analysis of the 16S rRNA gene sequences showed high similarity of the new strains to Haloferax species: H. denitrificans (99.2 %) and H. volcanii (99.1 %), H. larsenii (96.9 %) and H. elongans (96.6 %). DNA-DNA hybridization revealed 93-95 % similarity between strain RS75 and strains RS77 and RS79; the similarity levels between strain RS75 and the type strains of Haloferax denitrificans VKM B-1754 and Halobacterium salinarum VKM B-1769 were 50 and 10 %, respectively. According to its phenotypic and genotypic characteristics, the organism was classified as a member of the genus Haloferax, forming a new species with the proposed name Haloferax chudinovii sp. nov. type strain is RS75 (=VKPM B-11279). [ABSTRACT FROM AUTHOR]

Ionizing radiation (IR) is of particular interest in biology because its exposure results in severe oxidative stress to the cell's macromolecules. Our recent work with extremophiles supports the idea that IR resistance is most likely achieved by a metabolic route, effected by manganese (Mn) antioxidants. Biochemical analysis of 'super-IR resistant' mutants of H. salinarum, evolved over multiple cycles of exposure to high doses of IR, confirmed the key role for Mn antioxidants in the IR resistance of this organism. Analysis of the proteome of H. salinarum 'super-IR resistant' mutants revealed increased expression for proteins involved in energy metabolism, replenishing the cell with reducing equivalents depleted by the oxidative stress inflicted by IR. Maintenance of redox homeostasis was also activated by the over-expression of coenzyme biosynthesis pathways involved in redox reactions. We propose that in H. salinarum, increased tolerance to IR is a combination of metabolic regulatory adjustments and the accumulation of Mn-antioxidant complexes. [ABSTRACT FROM AUTHOR]

In this study, we examined the high-pressure survival of a range of prokaryotes not found in high-pressure environments to determine the effects of adaptations to osmotic and oxidative stresses on piezo-resistance. The pressure survivals of Halobacterium salinarum NRC-1, Deinococcus radiodurans R1, and Chromohalobacter salexigens were compared to that of Escherichia coli MG1655. C. salexigens, which uses the compatible solute ectoine as an osmolyte, was as piezo-sensitive as E. coli MG1655, suggesting that ectoine is not a piezolyte. D. radiodurans R1 and H. salinarum NRC-1, both resistant to oxidative stress, were found to be highly piezo-resistant. H. salinarum NRC-1 showed nearly full survival after pressurization up to 400 MPa; a survival 3.5 log units higher than E. coli MG1655. This piezo-resistance was maintained in H. salinarum NRC-1 for pressurizations up to 1 h. We hypothesize that the high-pressure resistance of H. salinarum NRC-1 is due to a combination of factors including cell envelope structure and the presence of intracellular salts. [ABSTRACT FROM AUTHOR]

The genome of the halophilic archaeon Halobacterium salinarum encodes the high-affinity ATP-dependent K uptake system Kdp. Previous studies have shown that the genes coding for the KdpFABC complex are arranged in a kdpFABCQ gene cluster together with an additional gene kdpQ. In bacteria, expression of the kdpFABC genes is generally regulated by the dedicated sensor kinase/response regulator pair KdpD/KdpE, which are absent in H. salinarum. Surprisingly, H. salinarum expresses the kdp genes in a manner which is strikingly similar to Escherichia coli. In this study, we show that the halobacterial kdpFABCQ genes constitute an operon and that kdpFABCQ expression is subject to a complex regulatory mechanism involving a negative transcriptional regulator and is further modulated via a so far unknown mechanism. We describe how the regulation of kdp gene expression is facilitated in H. salinarum in contrast to its bacterial counterparts. Whereas the Kdp system fulfils the same core function as an ATP-driven K uptake system in both archaea and bacteria, the different mechanisms involved in the regulation of gene expression appear to have evolved separately, possibly reflecting a different physiological role of ATP-driven K uptake in halophilic archaea. [ABSTRACT FROM AUTHOR]

Halobacterium species balance high external osmolality by the accumulation of almost equimolar amounts of KCl. Thus, steady K+ supply is a vital prerequisite for life of these extreme halophiles. So far, K+ is reported to enter the halobacterial cell only passively by use of potential-driven uniporters. However, the genome of both the extreme halophilic archaeon Halobacterium sp. NRC-1 and H. salinarum R1 comprises one single gene cluster containing the genes kdpFABC coding for homologs of the bacterial ATP-driven K+ uptake system KdpFABC, together with an additional ORF so far annotated as cat3 in Halobacterium sp. NRC-1 and as UspA protein in H. salinarum R1 (the ORF is only referred to as cat3 in the following). Deletion of the kdpFABCcat3 genes led to a reduced ability to grow under limiting K+ concentrations, whereas real-time RT-PCR measurements revealed cat3-dependent high expression rates of the Kdp system in case of external K+ depletion. Synthesis of the KdpFABC complex enables H. salinarum R1 to grow under extreme potassium-limiting conditions of >20 μM K+. These results provide the first experimental evidence of an ATP-driven K+ uptake system in Halobacterium. Moreover, H. salinarum R1 was shown to further adapt to K+ limitation by a significant decrease of the intracellular K+ level, which suggests a rather complex mechanism of K+ homeostasis, in which the adaptation of cellular K+ concentrations and the concomitant transcriptional regulation of genes coding for a high-affinity ATP-driven K+ uptake system ensure the essential potassium supply under limiting conditions. [ABSTRACT FROM AUTHOR]

Halobacterium species balance high external osmolality by the accumulation of almost equimolar amounts of KCl. Thus, steady K+ supply is a vital prerequisite for life of these extreme halophiles. So far, K+ is reported to enter the halobacterial cell only passively by use of potential-driven uniporters. However, the genome of both the extreme halophilic archaeon Halobacterium sp. NRC-1 and H. salinarum R1 comprises one single gene cluster containing the genes kdpFABC coding for homologs of the bacterial ATP-driven K+ uptake system KdpFABC, together with an additional ORF so far annotated as cat3 in Halobacterium sp. NRC-1 and as UspA protein in H. salinarum R1 (the ORF is only referred to as cat3 in the following). Deletion of the kdpFABCcat3 genes led to a reduced ability to grow under limiting K+ concentrations, whereas real-time RT-PCR measurements revealed cat3-dependent high expression rates of the Kdp system in case of external K+ depletion. Synthesis of the KdpFABC complex enables H. salinarum R1 to grow under extreme potassium-limiting conditions of >20 μM K+. These results provide the first experimental evidence of an ATP-driven K+ uptake system in Halobacterium. Moreover, H. salinarum R1 was shown to further adapt to K+ limitation by a significant decrease of the intracellular K+ level, which suggests a rather complex mechanism of K+ homeostasis, in which the adaptation of cellular K+ concentrations and the concomitant transcriptional regulation of genes coding for a high-affinity ATP-driven K+ uptake system ensure the essential potassium supply under limiting conditions. [ABSTRACT FROM AUTHOR]

The [2Fe–2S] ferredoxin from the extreme haloarchaeon Halobacterium salinarum is stable in high (>1.5 M) salt concentration. At low salt concentration the protein exhibits partial unfolding. The kinetics of unfolding was studied in low salt and in presence of urea in order to investigate the role of salt ions on the stability of the protein. The urea dependent unfolding, monitored by fluorescence of the tryptophan residues and circular dichroism, suggests that the native protein is stable at neutral pH, is destabilized in both acidic and alkaline environment, and involves the formation of kinetic intermediate(s). In contrast, the unfolding kinetics in low salt exhibits enhanced rate of unfolding with increase in pH value and is a two state process without the formation of intermediate. The unfolding at neutral pH is salt concentration dependent and occurs in two stages. The first stage, involves an initial fast phase (indicative of the formation of a hydrophobic collapsed state) followed by a relatively slow phase, and is dependent on the type of cation and anion. The second stage is considerably slower, proceeds with an increase in fluorescence intensity and is largely independent of the nature of salt. Our results thus show that the native form of the haloarchaeal ferredoxin (in high salt concentration) unfolds in low salt concentration through an apparently hydrophobic collapsed form, which leads to a kinetic intermediate. This intermediate then unfolds further to the low salt form of the protein. [ABSTRACT FROM AUTHOR]

Two moderately halophilic low G + C Gram-positive bacteria were isolated from a sample of salted skate (Class Chondrychthyes, Genus Raja). Phylogenetic analysis of the 16S rRNA gene sequence of strains RH1T and RH4 showed that these organisms represented a novel species of the genus Salinicoccus. The new isolates formed pink–red colonies and flocculated in liquid media, with optimum growth in media containing 4% NaCl and pH of about 8.0. These organisms are aerobic but reduce nitrate to nitrite under anaerobic conditions. Acid is produced from several carbohydrates. Oxidase and catalase were detected. Menaquinone 6 was the major respiratory quinone. The major fatty acids of strains RH1T and RH4 were 15:0 anteiso and 15:0 iso. The G + C contents of DNA were 46.2 and 46.0 mol%, respectively. The peptidoglycan was of A3alpha L-Lys-Gly5–6 type. On the basis of the phylogenetic analyses, physiological and biochemical characteristics, we suggest that strain RH1T (=LMG 22840 = CIP 108576) represents a new species of the genus Salinicoccus, for which we propose the name Salinicoccus salsiraiae. [ABSTRACT FROM AUTHOR]

The stability of malate dehydrogenase (hMDH) from Halobacterium salinarum in aqueous medium at low salt concentrations (1 and 0.5 M NaCl) was studied at 4° and 25°C. The results showed that hMDH was more stable at the higher salt concentration and the low temperature. hMDH was introduced into reverse micelles of hexadecyltrimethylammonium bromide in cyclohexane with 1-butanol as co-surfactant. The hMDH stability in this system was studied at two ω0 ([H2O]/[surfactant]) values and the effects of salt concentration, presence of substrate and dilution before or after its introduction into reverse micelles were examined. The results showed that the half-life of hMDH dissolved in buffer with 1 M NaCl was 12–50 days in reverse micelles (depending on the various conditions), in contrast to only about 1 day in aqueous medium at 25°C. These observations indicate that reverse micelles provide a microenvironment that allows a much greater stability of this enzyme compared with an aqueous medium. [ABSTRACT FROM AUTHOR]

We have recently discovered heme-containing signal transducers from the archaeon Halobacterium salinarum (HemAT-Hs) and the gram-positive bacterium Bacillus subtilis (HemAT-Bs). These proteins bind diatomic oxygen and trigger aerotactic responses. We identified that HemAT oxygen-sensing domains contain a globin-coupled sensor (GCS) motif, which exists as a two-domain transducer, having no similarity to the PAS domain (Period circadian protein, Ah receptor nuclear translocator protein, Single-minded protein) superfamily transducers. Using the GCS motif, we predicted that a 439-amino-acid protein annotated as a methyl-accepting chemotaxis protein (MCP) in the facultatively alkaliphilic bacterium Bacillus halodurans is a globin-coupled oxygen sensor. We cloned, expressed, and purified GCSBh and performed its spectral analysis. GCSBh binds heme and shows myoglobin-like spectra. This suggests that GCSBh acts as an oxygen sensor and transmits a conformational signal through a linked signaling domain to trigger an aerotactic response in B. halodurans. [ABSTRACT FROM AUTHOR]