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Halophilic proteases are extremely useful in harsh environments during industrial manufacturing processes. Production of the enzymes by halophiles has been hampered with their slow growth and complicated purification processes. Here, we developed a rapid method for extremozyme production by recombinant Bacillus subtilis . HProPRW1, a halophilic protease from an extreme haloarchaeon Halobacterium salinarum PRW1, was expressed from a salt-inducible expression system in B. subtilis . Three different signal peptides were evaluated for HProPRW1 secretion, revealing an efficient function of the HProPRW1 native signal peptide in B. subtilis . At optimal conditions, HProPRW1 could be produced within 22 h by recombinant B. subtilis instead of 96 h by wild type. The recombinant enzyme functions most optimally at 60 °C, pH 9% and 12% NaCl. The crude enzyme was stable for at least 1 month at 4 °C. This new protocol can be applied for production of other high-value halophilic enzymes. [ABSTRACT FROM AUTHOR]

Gas vesicles (GVs) are a new and unique class of biologically derived ultrasound contrast agents with sub-micron size whose acoustic properties have not been fully elucidated. In this study, we investigated the acoustic collapse pressure and behavior of Halobacterium salinarum gas vesicles at transmit center frequencies ranging from 12.5 to 27.5 MHz. The acoustic collapse pressure was found to be above 550 kPa at all frequencies, nine-fold higher than the critical pressure observed under hydrostatic conditions. We illustrate that gas vesicles behave non-linearly when exposed to ultrasound at incident pressure ranging from 160 kPa to the collapse pressure and generate second harmonic amplitudes of −2 to −6 dB below the fundamental in media with viscosities ranging from 0.89 to 8 mPa·s. Simulations performed using a Rayleigh–Plesset-type model accounting for buckling and a dynamic finite-element analysis suggest that buckling is the mechanism behind the generation of harmonics. We found good agreement between the level of second harmonic relative to the fundamental measured at 20 MHz and the Rayleigh–Plesset model predictions. Finite-element simulations extended these findings to a non-spherical geometry, confirmed that the acoustic buckling pressure corresponds to the critical pressure under hydrostatic conditions and support the hypothesis of limited gas flow across the GV shell during the compression phase in the frequency range investigated. From simulations, estimates of GV bandwidth-limited scattering indicate that a single GV has a scattering cross section comparable to that of a red blood cell. These findings will inform the development of GV-based contrast agents and pulse sequences to optimize their detection with ultrasound. [ABSTRACT FROM AUTHOR]

Recent advances in lipidomic analysis in combination with various physiological experiments set the stage for deciphering the structure-function of haloarchaeal membrane lipids. Here we focused primarily on changes in lipid composition of Haloferax volcanii , but also performed a comparative analysis with four other haloarchaeal species ( Halobacterium salinarum , Halorubrum lacusprofundi, Halorubrum sodomense and Haloplanus natans ) all representing distinctive cell morphologies and behaviors (i.e., rod shape vs. pleomorphic behavior). Common to all five haloarchaea, our data reveal an extraordinary high level of menaquinone, reaching up to 72% of the total lipids. This ubiquity suggests that menaquinones may function beyond their ordinary role as electron and proton transporter, acting simultaneously as ion permeability barriers and as powerful shield against oxidative stress. In addition, we aimed at understanding the role of cations interacting with the characteristic negatively charged surface of haloarchaeal membranes. We propose for instance that by bridging the negative charges of adjacent anionic phospholipids, Mg 2+ acts as surrogate for cardiolipin, a molecule that is known to control curvature stress of membranes. This study further provides a bioenergetic perspective as to how haloarchaea evolved following oxygenation of Earth's atmosphere. The success of the aerobic lifestyle of haloarchaea includes multiple membrane-based strategies that successfully balance the need for a robust bilayer structure with the need for high rates of electron transport – collectively representing the molecular basis to inhabit hypersaline water bodies around the planet. [ABSTRACT FROM AUTHOR]

Submerged growth of Halobacterium salinarum and therefore synthesis of bacteriorhodopsin (BR) and carotenoids depend greatly on products of both chemical and/or photochemical oxidation of medium components and cellular metabolism which act as inhibitors. Some cultivation variants which allowed eliminating an adverse effect of inhibitors on biomass accumulation and BR synthesis are reviewed. The application of activated charcoal or ion exchange resin as adsorbents at preparing inoculums and the main cultivation stages was shown to allow controlling, namely lowering overstress of the halobacterial cells by metabolites. The halobacterial biomass containing BR up to 1,750 mg L −1 and the minimum amount of carotinoids that would BR greatly facilitate isolation was accumulated up to 45 g L −1 during eight-day cultivation with cell recycling through adsorbent suspension in a fed-batch mode. To control BR biosynthesis the express method of BR quantification based on colour shades of cell suspension was developed. [ABSTRACT FROM AUTHOR]

Herein we report for the first time on efficient and environmentally friendly bioenergy production from bacteriorhodopsin (bR) and green florescent protein (GFP) as co-sensitizers. bR as a transmembrane protein, acts like a light-driven proton pump in Halobacterium salinarum , converting light energy into a proton gradient. Employing GFP beside bR can enhance the photo-bioenergy production efficiency in two aspects: GFP can increase short circuit current by improvement in light absorption either by extending the sensitizingspectrumor making fluorescence in absorption region of bR. It can also enhance open circuit voltage more than 150 mV by improvement in photoelectrode converging and extending electron lifetime in photoelectrode. Maximum photovoltage of 680 mV and photocurrent of 1.2 mA cm −2 have been achieved upon co-sensitization with bR/GFP. With the power conversion efficiency of 0.45%, the highest efficiency of photovoltaic cell based on bR has been reported in this research. [ABSTRACT FROM AUTHOR]

Archaerhodopsin 4 (AR4), a new member of the microbial rhodopsin family, is isolated from Halobacterium species xz515 in a Tibetan salt lake. AR4 functions as a proton pump similar to bacteriorhodopsin (BR) but with an opposite temporal order of proton uptake and release at neutral pH. However, further studies to elucidate the mechanism of the proton pump and photocycle of AR4 have been inhibited due to the difficulty of establishing a suitable system in which to express recombinant AR4 mutants. In this paper, we report a reliable method for expressing recombinant AR4 in Halobacterium salinarum L33 with a high yield of up to 20 mg/l. Experimental results show that the recombinant AR4 retains the light-driven proton pump characteristics and photo-cycling kinetics, similar to that in the native membrane. The functional role of bacterioruberin in AR4 and the trimeric packing of AR4 in its native and recombinant forms are investigated through light-induced kinetic measurements, two-dimensional solid-state NMR experiments, dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FTIR). Such approaches provide new insights into structure–function relationships of AR4, and form a basis for other archaeal rhodopsins. [ABSTRACT FROM AUTHOR]

There are a lot of bacterial and eukaryotic DNA repair gene homologs among sequenced archaeal genomes but there is little information about DNA repair mechanisms and the interaction of involved repair proteins. In order to study DNA repair mechanisms in the third domain of life, we studied these processes in the model archaeon, Halobacterium salinarum . H . salinarum has homologs of eukaryotic nucleotide excision repair genes such as rad2 gene. A functional analysis of rad2 was performed by knocking down of this gene. We introduced an antisense RNA expression vector into the cells and the sensitivity of transformants against ultraviolet light exposure was measured to determine whether rad2 gene performs any role in the repair of the DNA lesions induced by UV light or not. Our data suggests that rad2 is functional in this pathway and knocked down strains were unable to completely repair the UV induced DNA damages. In this study, for the first time antisense RNA is used for functional analysis of a gene in H. salinarum and it is shown that antisense RNA could be used as a reliable genetic tool for understanding of the archaeal genetics. [ABSTRACT FROM AUTHOR]

Abstract: The model archaeon Halobacterium salinarum ssp. NRC-1 is an excellent system for the study of archaeal molecular biology. Unlike many other archaea, its only special growth requirement is high levels of sodium chloride and other salts; it requires neither high-temperature incubation nor anaerobic environments. Additionally, there are a number of well-developed post-genomic tools available, including whole-genome microarrays and a ura3-based gene deletion system. While some tools are available for protein expression, a system for measurement and purification of protein expressed from native promoters is lacking. We have adapted the established H. salinarum gene deletion system for this purpose, and have used this to place 8×-histidine tags on either the carboxyl or amino terminus of the protein encoded by the chromosomal rfa3 gene. To demonstrate the utility of this approach, we used Western blot analysis to determine levels of the Rfa3 protein under different conditions. This system provides another powerful molecular tool for studies of native protein expression and for simple protein purification in H. salinarum. [Copyright &y& Elsevier]

Abstract: Recently, there has been an interest in fabrication of X-ray sensors based on bacteriorhodopsin, a proton pump protein in cell membrane of Halobacterium salinarium. Therefore, a better understanding of interaction of X-ray photons with bacteriorhodopsin is required. We use WinXCom program to calculate the mass attenuation coefficient of bacteriorhodopsin and its comprising amino acids for photon energies from 1keV to 100GeV. These amino acids include alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, Asx1, Asx2, Glx1 and Glx2. We then use that data to calculate effective atomic number and electron densities for the same range of energy. We also emphasize on two ranges of energies (10–200keV and 1–20MeV) in which X-ray imaging and radiotherapy machines work. [Copyright &y& Elsevier]

Abstract: Bacteriorhodopsin is a proton-pump protein in the cell membrane of Halobacterium salinarium. Unlike many other biomaterials, bacteriorhodopsin keeps most of its physical, chemical and electrical properties in the dry thin film form. This makes bacteriorhodopsin an excellent candidate for fabrication of electro-mechanical devices in the scale of micrometer and possibly nanometer. In this paper, we report on employing a film of dry oriented bacteriorhodopsin for sensing kilovoltage (kV) energy X-ray beam. [Copyright &y& Elsevier]

Dodecins, a group of flavin-binding proteins with a dodecameric quaternary structure, are able to incorporate two flavins within each of their six identical binding pockets building an aromatic tetrade with two tryptophan residues. Dodecin from the archaeal Halobacterium salinarum is a riboflavin storage device. We demonstrate that unwanted side reactions induced by reactive riboflavin species and degradation of riboflavin are avoided by ultrafast depopulation of the reactive excited state of riboflavin. Intriguingly, in this process, the staggered riboflavin dimers do not interact in ground and photoexcited states. Rather, within the tetrade assembly, each riboflavin is kept under the control of the respective adjacent tryptophan, which suggests that the stacked arrangement is a matter of optimizing the flavin load. We further identify an electron transfer in combination with a proton transfer as a central element of the effective excited state depopulation mechanism. Structural and functional comparisons of the archaeal dodecin with bacterial homologs reveal diverging evolution. Bacterial dodecins bind the flavin FMN instead of riboflavin and exhibit a clearly different binding pocket design with inverse incorporations of flavin dimers. The different adoption of flavin changes photochemical properties, making bacterial dodecin a comparably less efficient quencher of flavins. This supports a functional role different for bacterial and archaeal dodecins. [ABSTRACT FROM AUTHOR]

Abstract: Sensory rhodopsin II from Halobacterium salinarum (HsSRII) is a retinal protein in which retinal binds to a specific lysine residue through a Schiff base. Here, we investigated the photobleaching of HsSRII in the presence of hydroxylamine. For identification of intermediate(s) attacked by hydroxylamine, we employed the flash-induced bleaching method. In order to change the concentration of intermediates, such as M- and O-intermediates, experiments were performed under varying flashlight intensities and concentrations of azide that accelerated only the M-decay. We found the proportional relationship between the bleaching rate and area under the concentration–time curve of M, indicating a preferential attack of hydroxylamine on M. Since hydroxylamine is a water-soluble reagent, we hypothesize that for M, hydrophilicity or water-accessibility increases specifically in the moiety of Schiff base. Thus, hydroxylamine bleaching rates may be an indication of conformational changes near the Schiff base. We also considered the possibility that azide may induce a small conformational change around the Schiff base. We compared the hydroxylamine susceptibility between HsSRII and NpSRII (SRII from Natronomonas pharaonis) and found that the M of HsSRII is about three times more susceptible than that of the stable NpSRII. In addition, long illumination to HsSRII easily produced M-like photoproduct, P370. We thus infer that the instability of HsSRII under illumination may be related to this increase of hydrophilicity at M and P370. [Copyright &y& Elsevier]

Abstract: Salinarum halorhodopsin (HsHR), a light-driven chloride ion pump of haloarchaeon Halobacterium salinarum, was heterologously expressed in Escherichia coli. The expressed HsHR had no color in the E. coli membrane, but turned purple after solubilization in the presence of all-trans retinal. This colored HsHR was purified by Ni-chelate chromatography in a yield of 3–4mg per liter culture. The purified HsHR showed a distinct chloride pumping activity by incorporation into the liposomes, and showed even in the detergent-solubilized state, its typical behaviors in both the unphotolyzed and photolyzed states. Upon solubilization, HsHR expressed in the E. coli membrane attains the proper folding and a trimeric assembly comparable to those in the native membranes. [Copyright &y& Elsevier]

Abstract: We have shown before that mutation of Gly114 to Arg enhances folding of hexameric nucleoside diphosphate kinase (HsNDK) from Halobacterium salinarum. In this study, we constructed three mutant forms, Gly114Lys (G114K), Gly114Ser (G114S) and Gly114Asp (G114D), to further clarify the role residue 114 plays in the stability and folding of HsNDK. While expression of G114D mutant resulted in inactive enzyme, other mutant HsNDKs were successfully expressed in active form. The G114K mutant, similar to Gly114Arg (G114R) mutant, refolded in 1M NaCl after heat-denaturation, under which the wild-type HsNDK and G114S proteins showed no refolding. [Copyright &y& Elsevier]

This paper is concerned with medium optimization for the production of bacteriorhodopsin (BR) by Halobacterium salinarium PTCC 1685 by one-factor-at-a-time and Taguchi's array methods. The one-factor-at-a-time method was used to study the effects of various carbon and nitrogen sources, temperature and initial pH on bacteriorhodopsin production. Among these variables, corn steep powder, universal peptone and yeast extract were the most suitable sources, respectively. The optimal temperature and initial pH for bacteriorhodopsin production were 38 °C and 7.5, respectively. Subsequently, the concentration of corn steep powder and other medium components were optimized using Taguchi's method. The results showed that corn steep powder, glycerol and meat extract had significant effect on bacteriorhodopsin production by H. salinarium PTCC 1685. The optimal concentrations of nutritional components for improved bacteriorhodopsin production were determined as 10 g l−1 meat extract, 3.75 g l−1 casamino acids, 10 g l−1 glycerol, 50 g l−1 corn steep powder, 1 g l−1 Na3-citrate, 6 g l−1 KCl, 0.2 g l−1 FeSO47H2O, 0.2 g l−1 MnSO47H2O and 10 g l−1 MgSO47H2O. The maximum bacteriorhodopsin production in shake flask culture was 191.7 mg l−1 about 2.85-fold higher than those obtained with the basal medium. Under optimal culture conditions, the maximum bacteriorhodopsin production in a 15-l stirred-tank bioreactor indicated 234.6 mg l−1 about 3.49-fold higher than the basal medium. [Copyright &y& Elsevier]

Abstract: As part of a programme to optimize the use of archaeal-lipid liposomes (archaeosomes) as vaccine adjuvants, we present the synthesis and immunological testing of an oligomeric series of mannose glycolipids (Manp 1–5). To generate the parent archaeol alcohol precursor, the polar lipids extracted from the archaeon Halobacterium salinarum were hydrolyzed to remove polar head groups, and the archaeol so generated partitioned into diethyl ether. This alcohol was then iteratively glycosylated with the donor 2-O-acetyl-3,4,6-tri-O-benzyl-α/β-d-mannopyranosyl trichloroacetimidate to yield α-Manp-(1→2) oligomers. A starch-derived trimer was also synthesized as a control. To promote hydration and form stable archaeosomes, an archaeal anionic lipid archaetidylglycerol (AG) was included in a 4:1 molar ratio. Archaeosomes prepared from Manp 1–2–AG were recovered at only 34–37%, whereas Manp 3−4–AG recoveries were 72–77%. Lipid recovery following hydration of Manp 5–AG archaeosomes declined to 34%, indicating an optimum of 3–4 Manp units for bilayer formation. The CD8+ T cell response in mice immunized with Manp 3–5 archaeosomes containing ovalbumin was highest for Manp 4 and declined for Manp 3 and Manp 5, revealing an optimum length of four unbranched units. The starch-derived trimer was more active than the Manp oligomers, suggesting the involvement of either a general binding lectin on antigen-presenting cells with highest affinity for triglucose or multiple lectin receptors. [Copyright &y& Elsevier]

The spectroscopic identification of sensory rhodopsin I by Bogomolni and Spudich in 1982 provided a molecular link between the light environment and phototaxis in Halobacterium salinarum, and thus laid the foundation for the study of signal transducing photosensors in prokaryotes. In recent years, a number of new prokaryotic photosensory receptors have been discovered across a broad range of taxa, including dozens in chemotrophic species. Among these photoreceptors are new classes of rhodopsins, BLUF-domain proteins, bacteriophytochromes, cryptochromes, and LOV-family photosensors. Genetic and biochemical analyses of these receptors have demonstrated that they can regulate processes ranging from photosynthetic pigment biosynthesis to virulence. [Copyright &y& Elsevier]

Abstract: Experimental results on generation of hydrogen from pure culture of Halobacterium halobium NCIM 2852 without any external support system investigated under different solar intensity, light intensity and wavelength are presented in this paper. The rate of hydrogen generation increases from to with the increase in solar intensity from 3 to , for an exposed surface area of of the bowl of the bioreactor. Further, with the same exposed surface area, the rate of generation of hydrogen increases to and at 1000 and 4000lx of light intensity, respectively. The rate of generation of hydrogen exhibits sharp maxima at 600nm light wavelength. [Copyright &y& Elsevier]

Ca2+ was found to be essential for initiating Halobacterium salinarum CCM 2090 cell aggregation. The floc formed from such aggregation could easily be dissociated without cellular lysis by sodium citrate. Cr2+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, and Zn2+ could replace Ca2+. However, Mg2+, Sr2+, Mo2+, Cd2+, Sn2+, Hg2+, and Pb2+ induced no flocculation of cells of this halophilic archaeon. Mg2+ acted antagonistically against Ca2+-induced aggregation. Such aggregation might be directly caused by the interaction of Ca2+ and aggregation factors from 55°C-treated cell extract. [Copyright &y& Elsevier]

Abstract: We reported in a previous study that proteomic approach, coupled with genomic techniques, could be used to screen and develop multiple candidates for halophilic enzymes from Halobacterium salinarum. In order to evaluate the biodegradation of isopropyl alcohol (IPA) by H. salinarum, the amounts of residual IPA and acetone generated in the growth media were determined using a gas chromatography-flame ionization detector (GC-FID). The protein expression profiles of cells which had been cultured with IPA were obtained with the two-dimensional gel electrophoresis. Proteins evidencing different expression levels in the presence of 0.5% IPA were identified by electrospray ionization-quadruple-time of flight (ESI-Q-TOF) mass spectrometry. We found 12 proteins which were down-regulated, and another 12 proteins which were up-regulated, in the presence of 0.5% IPA and we further identified 17 proteins among them using ESI-TOF MS/MS. Among these identified proteins, we selected glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for further characterization as a halophilic enzyme. We have demonstrated for the first time that H. salinarum possesses the ability to degrade IPA and GAPDH was both stable and active at high salt concentrations, with maximum activity occurring at 1 M NaCl, although the optimal salt concentration with regard to the growth of H. salinarum is 4.3 M. [Copyright &y& Elsevier]

Sensory rhodopsins are photoactive, membrane-embedded seven-transmembrane helix receptors that use retinal as a chromophore. They are widespread in the microbial world in each of the three domains of life: Archaea, Bacteria and Eukarya. A striking characteristic of these photoreceptors is their different modes of signaling in different organisms, including interaction with other membrane proteins, interaction with cytoplasmic transducers and light-controlled Ca2+ channel activity. More than two decades since the discovery of the first sensory rhodopsins in the archaeon Halobacterium salinarum, genome projects have revealed a widespread presence of homologous photosensors. New work on cyanobacteria, algae, fungi and marine proteobacteria is revealing how evolution has modified the common design of these proteins to produce a remarkably rich diversity in their signaling biochemistry. [Copyright &y& Elsevier]

Clinically significant bacterial resistance to drugs and cytotoxic compounds can be conferred by the energy-dependent efflux of toxicants, catalyzed by proteins embedded in the bacterial cell membrane. One such group of proteins, the small multidrug resistance family, are drug/proton antiporters that must oligomerize to function, a process that requires the assembly of at least two inactive monomers by intermolecular association of their four transmembrane helices. Here, we have used peptides that correspond to each of the four wild type transmembrane helices of the Halobacterium salinarum protein Hsmr and a corresponding library of mutant peptides to determine the interactive surfaces that likely contribute to protein oligomerization. Hsmr peptides were examined for strong (sodium dodecyl sulfate-resistant) and weaker (perfluorooctanoate-resistant) helix-helix interactions, in conjunction with circular dichroism, fluorescence energy transfer measurements, and molecular modeling. The results are compatible with a scheme in which two faces of helix four permit self-assembly via a higher affinity asymmetric pairing and a lower affinity symmetric interaction, resulting in a discrete tetramer. Our finding that two surfaces of helix four can contribute to the stability of small multidrug resistance protein assembly provides a molecular basis for the design of therapeutics that target this antibiotic resistance mechanism. [ABSTRACT FROM AUTHOR]

Halobacterium salinarum CCM 2090 exhibits a Ca2+-dependent cellular aggregation system. This process is caused by the interaction between Ca2+ 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. [Copyright &y& Elsevier]

Abstract: In earlier work, two glutamate dehydrogenase (GDH) proteins were purified from a strain of the halophilic archaeon Halobacterium salinarum (NRC-36014). One of these, an NAD+-specific enzyme, was matched to a cloned gene from H. salinarum (GenBank accession number:X63837 S75579) by sequencing peptide fragments. Analysis of enzymatic digests of the NADP+-GDH and database searching have now established that a gene encoding this protein exists in the full genomic sequence of Halobacterium sp. NRC-1 as gdhA1, together with two other distinct gdh genes, gdhA2 and gdhB. From N-terminal sequence, it is clear that the genomic listing incorrectly assigns the start codon for gdhA1 and the corresponding protein is 43 amino acids longer than previously indicated. The three genes could be amplified by PCR either from NRC-1, as expected, or from NRC-36014 (GenBank accession numbers: AY840085–AY840087 AY840085 AY840086 AY840087 ). A gene encoding the previously purified NAD+-GDH, is absent from the NRC-1 genome but can be successfully amplified from genomic DNA of NRC-36014 (GenBank accession number: AY840088). This establishes that NRC-36014 contains four gdh genes. [Copyright &y& Elsevier]

Transducer-free sensory rhodopsins carry out lightdriven proton transport in Halobacterium salinarum membranes. Transducer binding converts the proton pumps to signal-relay devices in which the transport is inhibited. In sensory rhodopsin I (SRI) binding of its cognate transducer HtrI inhibits transport by closing a cytoplasmic proton-conducting channel necessary for proton uptake during the SRI photochemical reaction cycle. To investigate the channel closure, a series of HtrI mutants truncated in the membrane-proximal cytoplasmic portion of an SRI-HtrI fusion were constructed and expressed in H. salinarum membranes. We found that binding of the membrane-embedded portion of HtrI is insufficient for channel closure, whereas cytoplasmic extension of the second HtrI transmembrane helix by 13 residues blocks proton conduction through the channel as well as full-length HtrI. Specifically the closure activity is localized in this 13-residue membrane-proximal cytoplasmic domain to the 5 final residues, each of which incrementally contributes to reduction of proton conductivity. Moreover, these same residues in the dark incrementally and proportionally increase the pKa of the Asp-76 counterion to the protonated Schiff base chromophore in the membrane-embedded photoactive site. We conclude that this critical region of HtrI alters the dark conformation of SRI as well as light-induced channel opening. The 5 residues in HtrI correspond in position to 5 residues demonstrated on the homologous NpHtrII to interact with the E-F loop of its cognate receptor NpSRII in the accompanying article (Yang, C.-S., Sineshchekov, O., Spudich, E. N., and Spudich, J. L. (2004) J. Biol. Chem. 279, 42970-42976). These results strongly suggest that the membrane-proximal region of Htr proteins interact with their cognate sensory rhodopsin cytoplasmic domains as part of the signal-relay coupling between the proteins. [ABSTRACT FROM AUTHOR]

Aim of this work is to study biological materials as innovative “smart” materials to implement new functions on Si. We characterized the structural and optical properties of a bacterial protein, the bacterio-rhodopsin (bR), located in the purple membrane (PM) of the Halobacterium salinarium, living in salty waters. When it is irradiated with a light at 570 nm a complex photochemical cycle starts. It consists of intermediate states with different absorption spectra and lifetimes. We prepared water solutions containing the protein and studied both the solution and the films formed after deposition on glass, Si and SiO2 surfaces. The films realized have thickness ranging from 5 to 45 μm and, more important, maintain bR optical properties. Finally, the PM confers a high stability to the bR with respect to thermal degradation. Our measurement show a denaturation temperature strongly dependent on the solution and film characteristics. In particular, bR can be stable up to 100 °C in solution and up to 190 °C in thick films, values much higher than the best literature data. [Copyright &y& Elsevier]

Pharaonis halorhodopsin (phR) is an inward light-driven chloride ion pump from Natronobacterium pharaonis. In order to clarify the role of Ser-130phR residue which corresponds to Ser-115shR for salinarum hR on the anion-binding affinity, the wild-type and Ser-130 mutants substituted with Thr, Cys and Ala were expressed in E. coli cells and solubilized with 0.1% n-dodecyl β-d-maltopyranoside The absorption maximum (λmax) of the S130T mutant indicated a blue shift from that of the wild type in the absence and presence of chloride. For S130A, a large red shift (12 nm) in the absence of chloride was observed. The wild-type and all mutants showed the blue-shift of λmax upon Cl− addition, from which the dissociation constants of Cl− were determined. The dissociation constants were 5, 89, 153 and 159 mM for the wild-type, S130A, S130T and S130C, respectively, at pH 7.0 and 25 °C. Circular dichroic spectra of the wild-type and the Ser-130 mutants exhibited an oligomerization. The present study revealed that the Ser-130 of N. pharaonis halorhodopsin is important for the chloride binding. [Copyright &y& Elsevier]

Reports on the characterization of an archael multidrug transporter with a unique amino acid composition. Cloning and characterization of an Smr protein from the archaeon Halobacterium solinarum; Amino acid composition of the protein, Hsmr; Sequence elements in small multidrug transporters; Salt dependence of Hsmr.

We have developed a method for time-lapse video photography and line scanning of optical densities for analysis of aerotactic responses of Halobacterium salinarum. This automated digital technique, along with line scan analysis of selected frames, gives a unique profile of the aerotactic migration of halobacterial cells. [Copyright &y& Elsevier]

A novel, 68 amino acid long flavoprotein called dodecin has been discovered in the proteome of Halobacterium salinarum by inverse structural genomics. The 1.7 A˚ crystal structure of this protein shows a dodecameric, hollow sphere-like arrangement of the protein subunits. Unlike other known flavoproteins, which bind only monomeric flavin cofactors, the structure of the dodecin oligomer comprises six riboflavin dimers. The dimerization of these riboflavins along the re-faces is mediated by aromatic, antiparallel π staggering of their isoalloxazine moieties. A unique aromatic tetrade is formed by further sandwiching of the riboflavin dimers between the indole groups of two symmetry-related Trp36s. So far, the dodecins represent the smallest known flavoproteins. Based on the structure and the wide spread occurrences in pathogenic and soil eubacteria, a function in flavin storage or protection against radical or oxygenic stress is suggested for the dodecins. [Copyright &y& Elsevier]

The lifetime of the M-state of Bacteriorhodopsin (BR) is increased by genetic and chemical modifications and by solubilizing purple membranes with detergent. Chemically modified D96E as well as D96N films, possess close to 100% bleaching efficiency, which makes them attractive for use in image storage media. The mutant S35C has spectral and Kinetic properties identical to the WT, both in aqueous suspensions and in film and on antimony–tin-oxide electrodes. This suggests that substitution provides an attachment site that does not interfere with the function of BR. The magnitude of photocurrent transients generated by WT and mutant BR proteins both with natural chromophores and analogs were used to measure the efficiency of the ground-to-M-state transitions. [Copyright &y& Elsevier]

An extracellular endopeptidase has been isolated and purified by a single step protocol using Bacitracin–NHS (N-hydroxysuccinimide) Sepharose affinity system. Halobacterium halobium S9 endopeptidase has very high salt requirements in the range of 4 M NaCl. Molecular weight as determined by SDS-PAGE and gel filtration analysis was approximately 71 and 45 kDa respectively. N-terminal amino acid sequence analysis shows LVPNDAR as the probable residues. The enzyme has optimal activity at pH 8.7 and at a temperature of 40 °C. H. halobium endopeptidase exhibited significant stability under wide variations of pH and temperature. The effect of divalent cations on the enzyme is positive and Ca2+ is the most significant of all the divalent salts tested. Inhibitor studies show inhibition by Ser, His and Asp specific inhibitors. Substrate specificity studies using peptides reveal high specificity for peptides containing Pro in the penultimate position and preference for hydrophobic amino acids in the carboxy terminal side of the peptides. The enzyme exhibits azocasein activity at low salt concentrations as against activities at higher salt concentrations for peptides. The action of endopeptidase on the complex peptide mixture of a tryptic digest of β-casein resulted in the degradation of hydrophobic peptides. Thus, these characteristics of the purified peptidase make it suitable for an array of industrial applications with special impetus in debittering of meso, cheese and protein hydrolysates in food processing industry. [Copyright &y& Elsevier]

An iron-rich protein, DpsAHsal, was isolated from the archaeon Halobacterium salinarum sharing a sequence identity of 35% with the starvation-induced DNA-binding protein, DpsA, of Synechecoccus sp. PCC7942. It consists of 20-kDa subunits forming a dodecameric structure. The protein exhibits a ferric iron loading of up to 100 Fe ions per mole of holoprotein. CD spectra and secondary structure calculations are consistent with an α-helical contribution of 60%. The UV/VIS spectrum provides no evidence for the presence of heme groups. This protein exhibits features of a non-heme type bacterial ferritin (Ftn) although it shares only little sequence homology with Ftn. Molecular modelling disclosed a high structural similarity to E. coli Dps. [Copyright &y& Elsevier]