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The individual and mutual effects of the key parameters for hydrogen production in a photo-bioreactor containing Halobacterium salinarium MTCC 1626 immobilized in calcium alginate and E. coli MTCC 40 were investigated through factorial and the Box-Behnken experimental design. A full factorial design was utilized to determine the optimal ratio of H. salinarium and E. coli for maximum hydrogen production. The Box-Behnken design was used to optimize temperature, pH, light intensity, and stirrer speed in a photo-bioreactor. The quadratic model adequately predicted the individual and interaction effects of the variables. The optimal rate of hydrogen production of 53.22 mL/L.h was obtained with 50% E. coli and 15% immobilized H. salinarium with calcium alginate. The maximum experimental rate of hydrogen production of 75.8 mL/L.h was obtained at an initial pH of 7.38, a temperature of 35.8°C, a light intensity of 12186 lux, and a stirring speed of 104 rpm. [ABSTRACT FROM AUTHOR]

Α new, testable, interpretation of the function of Aegean Early Bronze Age 'frying pan' ceramics that combines some previously suggested uses is presented experimentally. The proposed function involves a well‐documented natural phenomenon responsible for the rapid change in colour of a natural salt marsh or salt pan surface due to the explosive population growth of the extremophile Archaea Halobacterium salinarium when the salt concentration approaches saturation. The cyclical nature of the appearance of red pigmentation on the salt marsh surface every late summer or every few weeks in a domestic 'frying pan' presents an obvious analogy with the female menstruation cycle and provides an explanation for the fact that several ceramic vessels used for this purpose bear the characteristics of the female body. Furthermore, the resulting red pigment lends itself for use as a cosmetic colorant. [ABSTRACT FROM AUTHOR]

Halophilic proteins subjected to below about 15% salt in vitro denature through misfolding, aggregation and/or precipitation. Halobacteria, however, have been detected in environments of fluctuating salinity such as coastal salterns and even around fresh water springs in the depths of the Dead Sea. In order to identify the underlying mechanisms of low salt survival, we explored the reactivation capacity of Halobacterium (Hbt) salinarum sub-populations after incubation in low salt media and recovery in physiological salt. Respiratory oxygen consumption was assessed in stressed cells and cell viability was estimated by Live/Dead staining and flow cytometry. In vivo neutron scattering experiments showed that the recovery of Hbt salinarum sub-populations exposed to severe low salt conditions is related to a rapid retrieval of functional molecular dynamics in the proteome. In the hypothesis that the observations on Hbt salinarum have wider relevance, they could be of key ecological significance for the dispersion of extremophiles when environmental fluctuations become severe. [ABSTRACT FROM AUTHOR]

Proton translocation across membranes is vital to all kingdoms of life. Mechanistically, it relies on characteristic proton flows and modifications of hydrogen bonding patterns, termed protonation dynamics, which can be directly observed by fast magic angle spinning (MAS) NMR. Here, we demonstrate that reversible proton displacement in the active site of bacteriorhodopsin already takes place in its equilibrated dark-state, providing new information on the underlying hydrogen exchange processes. In particular, MAS NMR reveals proton exchange at D85 and the retinal Schiff base, suggesting a tautomeric equilibrium and thus partial ionization of D85. We provide evidence for a proton cage and detect a preformed proton path between D85 and the proton shuttle R82. The protons at D96 and D85 exchange with water, in line with ab initio molecular dynamics simulations. We propose that retinal isomerization makes the observed proton exchange processes irreversible and delivers a proton towards the extracellular release site. Daniel Friedrich et al. show that reversible proton translocation occurs in the dark–state of bacteriorhodopsin, involving the retinal Schiff base and D85 exchanging protons with H2O. They find evidence of an active site proton cage and possible proton transfer via R82. [ABSTRACT FROM AUTHOR]

Halophilic archaea are known as the novel producers of natural products and their supernatant metabolites could have cytotoxic effects on cancer cells. In the present study, we screened the anticancer potential of supernatant metabolites from eight native haloarchaeal strains obtained from a culture collection in Iran. Five human cancer cell lines including breast, lung, prostate and also human fibroblast cells as the normal control were used in the present study. Moreover, to evaluate the anti-tumor effect of the selected supernatant, inhibition of sphere formation and tumor development was assessed in-vitro and in-vivo, respectively. Among all strains, supernatant metabolites from Halobacterium salinarum IBRC M10715 had the most potent cytotoxic effect on prostate cancer cell lines (IC50 = 0.5 mg/mL) without any effects on normal cells. It significantly increased both early and late apoptosis (about 11% and 9%, respectively) in the androgen-dependent PC3 cell line, reduced sphere formation ability of DU145 and PC3 cells with down-regulation of SOX2 gene expression. Furthermore, our results revealed that tumors developed in nude mice significantly shrank post intratumor injection of metabolites of the haloarchaeal strain. In conclusion, we suggested here for the first time that supernatant metabolites from Halobacterium salinarum IBRC M10715 could be a novel component against prostate cancer in-vitro and in-vivo with remarkable reduction in stem-like properties of tumor. [ABSTRACT FROM AUTHOR]

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]

Graphical abstract Highlights • How protein-DNA interactions are maintained in hyper-saline environs is a mystery. • The DNA-binding domain of VNG0258/RosR shows adaptation to hyper-saline medium. • Ions in the first shell around the protein bind tightly to the DNA-binding site. • Ions are also buried in a cavity connected by a tunnel to the external medium. • The results pave the way to deciphering principles driving DNA-binding in high salt. Abstract Protein-DNA interactions are highly dependent on salt concentration. To gain insight into how such interactions are maintained in the highly saline cytoplasm of halophilic archaea, we determined the 3-D structure of VNG0258H/RosR, the first haloarchaeal DNA-binding protein from the extreme halophilic archaeon Halobactrium salinarum. It is a dimeric winged-helix-turn-helix (wHTH) protein with unique features due to adaptation to the halophilic environment. As ions are major players in DNA binding processes, particularly in halophilic environments, we investigated the solution structure of the ionic envelope and located anions in the first shell around the protein in the crystal using anomalous scattering. Anions that were found to be tightly bound to residues in the positively charged DNA-binding site would probably be released upon DNA binding and will thus make significant contribution to the driving force of the binding process. Unexpectedly, ions were also found in a buried internal cavity connected to the external medium by a tunnel. Our structure lays a solid groundwork for future structural, computational and biochemical studies on complexes of the protein with cognate DNA sequences, with implications to protein-DNA interactions in hyper-saline environments. [ABSTRACT FROM AUTHOR]

Cell suspensions of the haloarchaea Halorubrum sodomense and Halobacterium salinarum and the extremely halophilic bacterium Salinibacter ruber (Bacteroidetes) in saturated solutions of chlorides and sulfates (NaCl, KCl, MgSO4·7H2O, K2SO4, and (NH4)Al(SO4)2·12H2O) were left to evaporate to produce micrometric inclusions in laboratory-grown crystals. Raman spectra of these pinkish inclusions were obtained using a handheld Raman spectrometer with green excitation (532 nm). This portable instrument does not include any microscopic tool. Acceptable Raman spectra of carotenoids were obtained in the range of 200-4000 cm−1. This detection achievement was related to the mode of illumination and collection of scattered light as well as due to resonance Raman enhancement of carotenoid signals under green excitation. The position of diagnostic Raman carotenoid bands corresponds well to those specific carotenoids produced by a given halophile. To our best knowledge, this is the first study of carotenoids included in the laboratory in crystalline chlorides and sulfates, using a miniature portable Raman spectrometer.ᅟ [ABSTRACT FROM AUTHOR]

Abstract: Aspartic acid 103 (D103) of sensory rhodopsin II from Halobacterium salinarum (HsSRII, or also called phoborhodopsin) corresponds to D115 of bacteriorhodopsin (BR). This amino acid residue is functionally important in BR. This work reveals that a substitution of D103 with asparagine (D103N) or glutamic acid (D103E) can cause large changes in HsSRII photocycle. These changes include (1) shortened lifetime of the M intermediate in the following order: the wild‐type > D103N > D103E; (2) altered decay pathway of a 13‐cis O‐like species. The 13‐cis O‐like species, tentatively named Px, was detected in HsSRII photocycle. Px appeared to undergo branched reactions at 0°C, leading to a recovery of the unphotolyzed state and formation of a metastable intermediate, named P370, that slowly decayed to the unphotolyzed state at room temperature. In wild‐type HsSRII at 0°C, Px mainly decayed to the unphotolyzed state, and the decay reaction toward P370 was negligible. In mutant D103E at 0°C, Px decayed to P370, while the recovery of the unphotolyzed state became unobservable. In mutant D103N, the two reactions proceeded at comparable rates. Thus, D103 of HsSRII may play an important role in regulation of the photocycle of HsSRII. [ABSTRACT FROM AUTHOR]

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]

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]

Photoinduced biochemical reactions are ubiquitously governed by derivatives of flavin, which is a key player in a manifold of cellular redox reactions. The photoreactivity of flavins is also one of their greatest disadvantages as the molecules are sensitive to photodegradation. To prevent this unfavorable reaction, UV-light-exposed archaea bacteria, such as Halobacterium salinarum, manage the task of protecting flavin derivatives by dodecin, a protein which stores flavins and efficiently photoprotects them. In this study, we shed light on the photoprotection mechanism, i.e., the excited state quenching mechanism by dodecin using computational methodology. Molecular dynamics (MD) simulations unraveled the hydrogen bond network in the flavin binding pocket as a starting point for proton transfer upon preceding electron transfer. Using high-level ab initio quantum chemical methods, different proton transfer channels have been investigated and an energetically feasible Mg2+-promoted channel has been identified fully explaining previous experimental observations. This is the first extensive theoretical study of archaeal dodecin, furthering the understanding of its photocycle and manipulation. [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]

Three halophilic archaea, Halobacterium salinarum NRC-1, Halococcus hamelinensis, and Halococcus morrhuae, have been exposed to different regimes of simulated outer space ionizing radiation. Strains were exposed to high-energy heavy ion (HZE) particles, namely iron and argon ions, as well as to γ radiation (60Co) and X-rays, and the survival and the genetic integrity of the 16S rRNA gene were evaluated. Exposure to 1 kGy of argon or iron ions at the Heavy Ion Medical Accelerator in Chiba (HIMAC) facility at the National Institute for Radiological Sciences (NIRS) in Japan did not lead to a detectable loss in viability; only after exposure to 2 kGy of iron ions a decline in survival was observed. Furthermore, a delay in growth was manifested following exposure to 2 kGy iron ions. DNA integrity of the 16S rRNA was not compromised up to 1 kGy, with the exception of Hcc. hamelinensis following exposure to argon particles. All three strains showed a high resistance toward X-rays (exposed at the DLR in Cologne, Germany), where Hcc. hamelinensis and Hcc. morrhuae displayed better survival compared to Hbt. salinarum NRC-1. In all three organisms the DNA damage increased in a dose-dependent manner. To determine a biological endpoint for survival following exposure to γ radiation, strains were exposed to up to 112 kGy at the Beta-Gamma-Service GmbH (BGS) in Germany. Although all strains were incubated for up to 4 months, only Hcc. hamelinensis and Hcc. morrhuae recovered from 6 kGy of γ radiation. In comparison, Hbt. salinarum NRC-1 did not recover. The 16S rRNA gene integrity stayed remarkably well preserved up to 48 kGy for both halococci. This research presents novel data on the survival and genetic stability of three halophilic archaea following exposure to simulated outer space radiation. Key Words: Halophilic archaea-Radiation-Survival. Astrobiology 17, 110-117. [ABSTRACT FROM AUTHOR]

The interaction of semiconductor quantum dots and silver nanoparticles (AgNPs) with bacteriorhodopsin (BR), a membrane protein contained in the purple membrane (PM) of Halobacterium salinarum, is studied. It is shown that both types of nanoparticles are adsorbed efficiently on the surface of the purple membranes, modulating the parameters of the bacteriorhodopsin photocycle. Electrostatic interactions are found to be the main cause of the effect of nanoparticles on the bacteriorhodopsin photocycle. These results explain our earlier data on the 'fixation' of the bacteriorhodopsin photocycle for protein molecules trapped after incubation of the purple membranes with silver nanoparticles near the location of the 'hot spots' of the effect of surface-enhanced Raman scattering (SERS). It is demonstrated that exposure of silver nanoparticles with bacteriorhodopsin in SERS-active regions lowers the amount of bacteriorhodopsin molecules involved in phototransformations. [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]

Purple membranes (PM) of the bacteria Halobacterium salinarum are a unique natural membrane where bacteriorhodopsin (BR) can convert photon energy and pump protons. Elucidating the electronic properties of biomembranes is critical for revealing biological mechanisms and developing new devices. We report here the electric properties of PMs studied by using multi-functional electric force microscopy (EFM) at the nanoscale. The topography, surface potential, and dielectric capacity of PMs were imaged and quantitatively measured in parallel. Two orientations of PMs were identified by EFM because of its high resolution in differentiating electrical characteristics. The extracellular (EC) sides were more negative than the cytoplasmic (CP) side by 8 mV. The direction of potential difference may facilitate movement of protons across the membrane and thus play important roles in proton pumping. Unlike the side-dependent surface potentials observed in PM, the EFM capacitive response was independent of the side and was measured to be at a dC/dz value of ~5.25 nF/m. Furthermore, by modification of PM with de novo peptides based on peptide-protein interaction, directional oriented PM assembly on silicon substrate was obtained for technical devices. This work develops a new method for studying membrane nanoelectronics and exploring the bioelectric application at the nanoscale. [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]

Haloferax volcanii AglM and Halobacterium salinarum VNG1048G are UDP-glucose dehydrogenases involved in N-glycosylation in each species. Despite sharing >60% sequence identity and the ability of VNG1048G to functionally replace AglM in vivo, these proteins behaved differently as salinity changed. Whereas AglM was active in 2-4 M NaCl, VNG1048G lost much of its activity when salinity dropped below 3 M NaCl. To understand the molecular basis of this phenomenon, each protein was examined by size exclusion chromatrography in 2MNaCl. Whereas AglM appeared as a dodecamer, VNG1048G was essentially detected as a dodecamer and a dimer. The specific activity of the VNG1048G dodecamer was only a sixth of that of AglM, while the dimer was inactive. As such, not only was the oligomeric status of VNG1048G affected by lowered salinity, so was the behavior of the individual dodecamer subunits. Analyzing surface-exposed residues in homology models of the two UDP-glucose dehydrogenases revealed the more acidic and less basic VNG1048G surface, further explaining the greater salt-dependence of the Hbt. salinarum enzyme. [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]

Different modes of bacterial taxis play important roles in environmental adaptation, survival, colonization and dissemination of disease. One mode of taxis is flotation due to the production of gas vesicles. Gas vesicles are proteinaceous intracellular organelles, permeable only to gas, that enable flotation in aquatic niches. Gene clusters for gas vesicle biosynthesis are partially conserved in various archaea, cyanobacteria, and some proteobacteria, such as the enterobacterium, S erratia sp. ATCC 39006 (). Here we present the first systematic analysis of the genes required to produce gas vesicles in , identifying how this differs from the archaeon H alobacterium salinarum. We define 11 proteins essential for gas vesicle production. Mutation of gvpN or gvpV produced small bicone gas vesicles, suggesting that the cognate proteins are involved in the morphogenetic assembly pathway from bicones to mature cylindrical forms. Using volumetric compression, gas vesicles were shown to comprise 17% of cells, whereas in E scherichia coli heterologously expressing the gas vesicle cluster in a deregulated environment, gas vesicles can occupy around half of cellular volume. Gas vesicle production in and E . coli was exploited to calculate the instantaneous turgor pressure within cultured bacterial cells; the first time this has been performed in either strain. [ABSTRACT FROM AUTHOR]

The archaeon Halobacterium salinarum can produce energy using three different processes, namely photosynthesis, oxidative phosphorylation and fermentation of arginine, and is thus a model organism in bioenergetics. Compared to its bacteriorhodopsin-driven photosynthesis, less attention has been devoted to modeling its respiratory pathway. We created a system of ordinary differential equations that models its oxidative phosphorylation. The model consists of the electron transport chain, the ATP synthase, the potassium uniport and the sodium-proton antiport. By fitting the model parameters to experimental data, we show that the model can explain data on proton motive force generation, ATP production, and the charge balancing of ions between the sodium-proton antiporter and the potassium uniport. We performed sensitivity analysis of the model parameters to determine how the model will respond to perturbations in parameter values. The model and the parameters we derived provide a resource that can be used for analytical studies of the bioenergetics of H. salinarum. [ABSTRACT FROM AUTHOR]

An intein from Halobacterium salinarum can be isolated as an unspliced precursor protein with exogenous exteins after Escherichia coli overexpression. The intein promotes protein splicing and uncoupled N-terminal cleavage in vitro, conditional on incubation with NaCl or KCl at concentrations of >1.5 M. The protein splicing reaction also is conditional on reduction of a disulfide bond between two active site cysteines. Conditional protein splicing under these relatively mild conditions may lead to advances in intein-based biotechnology applications and hints at the possibility that this H. salinarum intein could serve as a switch to control extein activity under physiologically relevant conditions. [ABSTRACT FROM AUTHOR]

Solar radiation is among the most prominent stress factors organisms face during space travel and possibly on other planets. Our analysis of three different halophilic archaea, namely Halobacterium salinarum NRC-1, Halococcus morrhuae, and Halococcus hamelinensis, which were exposed to simulated solar radiation in either dried or liquid state, showed tremendous differences in tolerance and survivability. We found that Hcc. hamelinensis is not able to withstand high fluences of simulated solar radiation compared to the other tested organisms. These results can be correlated to significant differences in genomic integrity following exposure, as visualized by random amplified polymorphic DNA (RAPD)-PCR. In contrast to the other two tested strains, Hcc. hamelinensis accumulates compatible solutes such as trehalose for osmoprotection. The addition of 100 m M trehalose to the growth medium of Hcc. hamelinensis improved its survivability following exposure. Exposure of cells in liquid at different temperatures suggests that Hbt. salinarum NRC-1 is actively repairing cellular and DNA damage during exposure, whereas Hcc. morrhuae exhibits no difference in survival. For Hcc. morrhuae, the high resistance against simulated solar radiation may be explained with the formation of cell clusters. Our experiments showed that these clusters shield cells on the inside against simulated solar radiation, which results in better survival rates at higher fluences when compared to Hbt. salinarum NRC-1 and Hcc. hamelinensis. Overall, this study shows that some halophilic archaea are highly resistant to simulated solar radiation and that they are of high astrobiological significance. Key Words: Halophiles-Solar radiation-Stress resistance-Survival. Astrobiology 15, 987-997. [ABSTRACT FROM AUTHOR]

Permafrost-affected soils are characterized by a high abundance and diversity of methanogenic communities, which are considered suitable model organisms for potential life on Mars. Methanogens from Siberian permafrost have been proven to be highly resistant against divers stress conditions such as subzero temperatures, desiccation, and simulated thermophysical martian conditions. Here, we studied the radiation resistance of the currently described new species Methanosarcina soligelidi SMA-21, which was isolated from a Siberian permafrost-affected soil, in comparison to Methanosarcina barkeri, which is used as a reference organism from a nonpermafrost soil environment. Both strains were exposed to solar UV and ionizing radiation to assess their limits of survival. Methanosarcina soligelidi exhibit an increase in radiation resistance to UV (2.5- to 13.8-fold) and ionizing radiation (46.6-fold) compared to M. barkeri. The F10 (UVC) and D10 (X-rays) values of M. soligelidi are comparable to values for the well-known, highly radioresistant species Deinococcus radiodurans. In contrast, the radiation response of M. barkeri was highly sensitive to UV and ionizing radiation comparably to Escherichia coli and other radiosensitive microorganisms. This study showed that species of the same genus respond differently to UV and ionizing radiation, which might reflect the adaptation of Methanosarcina soligelidi SMA-21 to the harsh environmental conditions of the permafrost habitat. Key Words: Methanogenic archaea-Environmental UV-Ionizing radiation-Permafrost-Radiation resistance-Mars. Astrobiology 15, 951-960. [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]

Halorhodopsin from the halophilic archaeon Halobacterium salinarum is a membrane located light-driven chloride pump. Upon illumination Halorhodopsin undergoes a reversible photocycle initiated by the all- trans to 13- cis isomerization of the covalently bound retinal chromophore. The photocycle consists of several spectroscopically distinct intermediates. The structural basis of the chloride transport mechanism remains elusive, presumably because packing contacts have so far precluded protein conformational changes in the available crystals. With the intention to structurally characterize late photocycle intermediates by X-ray crystallography we crystallized Halorhodopsin in a new crystal form using the vesicle fusion method. In the new crystal form lateral contacts are mediated by helices A and G. Helices E and F that were suggested to perform large movements during the photocycle are almost unrestrained by packing contacts. This feature might permit the displacement of these helices without disrupting the crystal lattice. Therefore, this new crystal form might be an excellent system for the structural characterization of late Halorhodopsin photocycle intermediates by trapping or by time resolved experiments, especially at XFELs. [ABSTRACT FROM AUTHOR]

Fed batch bioelectrochemical systems (BESs) based on electrical stimulation were used to treat p-fluoronitrobenzene (p-FNB) wastewater at high salinities. At a NaCl concentration of 40 g/liter, p-FNB was removed 100% in 96 h in the BES, whereas in the biotic control (BC) (absence of current), p-FNB removal was only 10%. By increasing NaCl concentrations from 0 g/liter to 40 g/liter, defluorination efficiency decreased around 40% in the BES, and in the BC it was completely ceased. p-FNB was mineralized by 30% in the BES and hardly in the BC. Microorganisms were able to store 3.8 and 0.7 times more K+ and Na+ intracellularly in the BES than in the BC. Following the same trend, the ratio of protein to soluble polysaccharide increased from 3.1 to 7.8 as the NaCl increased from 0 to 40 g/liter. Both trends raise speculation that an electrical stimulation drives microbial preference toward K+ and protein accumulation to tolerate salinity. These findings are in accordance with an enrichment of halophilic organisms in the BES. Halobacterium dominated in the BES by 56.8% at a NaCl concentration of 40 g/liter, while its abundance was found as low as 17.5% in the BC. These findings propose a new method of electrical stimulation to improve microbial salinity resistance. [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]

Dye-sensitized solar cells ( DSSCs), which are proposed as a substitute for silicon crystalline solar cells, have received considerable attention in the recent decade. They could be produced from inexpensive materials through low-cost processes. In the current work, a bio-sensitized solar cell is designed using abundant, cheap, and nontoxic materials. Bacteriorhodopsin and bacterioruberin are two natural biomolecules found in the cytoplasmic membrane of Halobacterium salinarum. These two pigments were immobilized on nanoporous titanium dioxide films successfully and employed as molecular sensitizers in DSSC with efficient photocurrent generation. The photovoltaic performance of DSSCs based on bacteriorhodopsin and bacterioruberin sensitizers was investigated. Under AM1.5 irradiation a short-circuit current of 0.45 mA cm−2, open circuit voltages of 0.57 V, fill factor of 0.62, and an overall energy conversion efficiency of 0.16% are achieved by employing a mixture of biomolecules as a sensitizer. [ABSTRACT FROM AUTHOR]

It was recently shown that halo archaeal strains of different genera are able to adhere to surfaces and form surface-attached bio films. However, the surface structures mediating the adhesion were still unknown. We have identified a novel surface structure with Halobacterium salinarum strain R1, crucial for surface adhesion. Electron microscopic studies of surface-attached cells frequently showed pili-like surface structures of two different diameters that were irregularly distributed on the surface. The thinner filaments, 7-8nm in diameter, represented a so far unobserved novel pili-like structure. Examination of the Hbt. salinarum R1 genome identified two putative gene loci (pil-1 and pil-2) encoding type IV pilus biogenesis complexes besides the archaellum encoding fla gene locus. Both pil-1 and pil-2 were expressed as transcriptional units, and the transcriptional start of pil-1 was identified. In silico analyses revealed that the pil-1 locus is present with other euryarchaeal genomes whereas the pil-2 is restricted to halo archaea. Comparative real time qRT-PCR studies indicated that the general transcriptional activity was reduced in adherent vs. planktonic cells. In contrast, the transcription of pilB1 and pilB2, encoding putative type IV pilus assembly ATPases, was induced in comparison to the archaella assembly/motor ATPase (flaI) and the ferredoxin gene. Mutant strains were constructed that incurred a flaI deletion or flaI/pilB1 gene deletions. The absence of flaI caused the loss of the archaella while the additional absence of pilB1 led to loss of the novel pili-like surface structures. The ΔflaI/ΔpilB1 double mutants showed a 10-fold reduction in surface adhesion compared to the parental strain. Since surface adhesion was not reduced with the non-archaella ted ΔflaI mutants, the pil-1 filaments have a distinct function in the adhesion process. [ABSTRACT FROM AUTHOR]

Introduction: Excessive use of tellurite nowadays, has suffered the environment from the toxic effects of the oxyanion. Hence, biological treatment of polluted areas is considered as an environmentally friendly and inexpensive method. Although the toxic effects of tellurite for most microorganisms have been reported, but several species of the bacteria including the halophilic bacteria used in this project can overcome the toxicity of the oxyanion by its reduction to the elemental form. The aim of this study was to identify the mechanism (s) involved in tellurite detoxification. Materials and methods: In order to enhance and maintain enzymatic activity during purification, the test conditions and enzyme production by the strain were optimized. The optimization was done by One Factor at A Time (OFAT) method. Several factors, including: time, various percentages of inoculum, range of pH, concentration of tellurite and various salts effects were optimized. For assurance tellurite removal was examined during the experiment. The enzyme was purified by various methods, including ammonium sulphate precipitation and hydrophobic interaction column chromatography in which the Concentration of 1.4 M saturated ammonium sulphate was applied. The purity of the enzyme was assessed by SDS-PAGE during each phase. Results: The optimum conditions obtained showed that at 30 hours, 3% inoculum, pH 7.5, without tellurite and with 5% NaCl the highest enzyme activity and tellurite removal are observed. Purification of the enzyme greatly reduced the concentration of unrelated proteins and caused a concentrated band which could be one subunit of the enzyme targeted. The partially purified enzyme's fraction was shown to have nitrate and selenite reductase activity other than tellurite reductase activity. Discussion and conclusion: This study is an approach to the identification of the halophilic microorganisms Physiology and enzymes involved in restoring tellurite. The production of the enzyme responsible for this phenomenon has been optimized and partially purified. [ABSTRACT FROM AUTHOR]

Photoactivation of attractant phototaxis receptor sensory rhodopsin I (SRI) in Halobacterium salinarum entails transfer of a proton from the retinylidene chromophore's Schiff base (SB) to an unidentified acceptor residue on the cytoplasmic half-channel, in sharp contrast to other microbial rhodopsins, including the closely related repellent phototaxis receptor SRII and the outward proton pump bacteriorhodopsin, in which the SB proton acceptor is an aspartate residue salt-bridged to the SB in the extracellular (EC) half-channel. Hisl66 on the cytoplasmic side of the SB in SRI has been implicated in the SB proton transfer reaction by mutation studies, and mutants of His 166 result in an inverted SB proton release to the EC as well as inversion of the protein's normally attractant phototaxis signal to repellent. Here we found by difference Fourier transform infrared spectroscopy the appearance of Fermi-resonant X-H stretch modes in light-minus-dark difference spectra; their assignment with 15N labeling and site-directed mutagenesis demonstrates that Hisl66 is the SB proton acceptor during the photochemical reaction cycle o f the wild-type SRI-Htrl complex. [ABSTRACT FROM AUTHOR]

Widespread microbial genome sequencing presents an opportunity to understand the gene regulatory networks of non-model organisms. This requires knowledge of the binding sites for transcription factors whose DNA-binding properties are unknown or difficult to infer. We adapted a protein structure-based method to predict the specificities and putative regulons of homologous transcription factors across diverse species. As a proof-of-concept we predicted the specificities and transcriptional target genes of divergent archaeal feast/famine regulatory proteins, several of which are encoded in the genome of Halobacterium salinarum. This was validated by comparison to experimentally determined specificities for transcription factors in distantly related extremophiles, chromatin immunoprecipitation experiments, and cis-regulatory sequence conservation across eighteen related species of halobacteria. Through this analysis we were able to infer that Halobacterium salinarum employs a divergent local trans-regulatory strategy to regulate genes (carA and carB) involved in arginine and pyrimidine metabolism, whereas Escherichia coli employs an operon. The prediction of gene regulatory binding sites using structure-based methods is useful for the inference of gene regulatory relationships in new species that are otherwise difficult to infer. [ABSTRACT FROM AUTHOR]

Genomic analysis of H. salinarum indicated that the de novo pathway for aromatic amino acid (AroAA) biosynthesis does not follow the classical pathway but begins from non-classical precursors, as is the case for M. jannaschii. The first two steps in the pathway were predicted to be carried out by genes OE1472F and OE1475F, while the 3rd step follows the canonical pathway involving gene OE1477R. The functions of these genes and their products were tested by biochemical and genetic methods. In this study, we provide evidence that supports the role of proteins OE1472F and OE1475F catalyzing consecutive enzymatic reactions leading to the production of 3-dehydroquinate (DHQ), after which AroAA production proceeds via the canonical pathway starting with the formation of DHS (dehydroshikimate), catalyzed by the product of ORF OE1477R. Nutritional requirements and AroAA uptake studies of the mutants gave results that were consistent with the proposed roles of these ORFs in AroAA biosynthesis. DNA microarray data indicated that the 13 genes of the canonical pathway appear to be utilised for AroAA biosynthesis in H. salinarum, as they are differentially expressed when cells are grown in medium lacking AroAA. [ABSTRACT FROM AUTHOR]

Microbes can tailor transcriptional responses to diverse environmental challenges despite having streamlined genomes and a limited number of regulators. Here, we present data-driven models that capture the dynamic interplay of the environment and genome-encoded regulatory programs of two types of prokaryotes: Escherichia coli (a bacterium) and Halobacterium salinarum (an archaeon). The models reveal how the genome-wide distributions of cis-acting gene regulatory elements and the conditional influences of transcription factors at each of those elements encode programs for eliciting a wide array of environment-specific responses. We demonstrate how these programs partition transcriptional regulation of genes within regulons and operons to re-organize gene-gene functional associations in each environment. The models capture fitness-relevant co-regulation by different transcriptional control mechanisms acting across the entire genome, to define a generalized, system-level organizing principle for prokaryotic gene regulatory networks that goes well beyond existing paradigms of gene regulation. An online resource () has been developed to facilitate multiscale exploration of conditional gene regulation in the two prokaryotes. [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]

The HschiA1 gene of the archaeon Halobacterium salinarum CECT 395 was cloned and overexpressed as an active protein of 66.5 kDa in Escherichia coli. The protein called HsChiA1p has a modular structure consisting of a glycosyl hydrolase family 18 catalytic region, as well as a N-terminal family 5 carbohydrate-binding module and a polycystic kidney domain. The purified recombinant chitinase displayed optimum catalytic activity at pH 7.3 and 40 °C and showed high stability over broad pH (6-8.5) and temperature (25-45 °C) ranges. Protein activity was stimulated by the metal ions Mg, K, and Ca and strongly inhibited by Mn. HsChiA1p is salt-dependent with its highest activity in the presence of 1.5 M of NaCl, but retains 20 % of its activity in the absence of salt. The recombinant enzyme hydrolysed p-NP-(GlcNAc), p-NP-(GlcNAc), crystalline chitin, and colloidal chitin. From its sequence features and biochemical properties, it can be identified as an exo-acting enzyme with potential interest regarding the biodegradation of chitin waste or its bioconversion into biologically active products. [ABSTRACT FROM AUTHOR]

Proteins from extremophiles have the ability to fold and remain stable in their extreme environment. Here, we investigate the presence of this effect in the cysteinyl-tRNA synthetase from Halobacterium salinarum ssp. NRC-1 (NRC-1), which was used as a model halophilic protein. The effects of salt on the structure and stability of NRC-1 and of E. coli CysRS were investigated through far-UV circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and thermal denaturation melts. The CD of NRC-1 CysRS was examined in different group I and group II chloride salts to examine the effects of the metal ions. Potassium was observed to have the strongest effect on NRC-1 CysRS structure, with the other group I salts having reduced strength. The group II salts had little effect on the protein. This suggests that the halophilic adaptations in this protein are mediated by potassium. CD and fluorescence spectra showed structural changes taking place in NRC-1 CysRS over the concentration range of 0–3 M KCl, while the structure of E. coli CysRS was relatively unaffected. Salt was also shown to increase the thermal stability of NRC-1 CysRS since the melt temperature of the CysRS from NRC-1 was increased in the presence of high salt, whereas the E. coli enzyme showed a decrease. By characterizing these interactions, this study not only explains the stability of halophilic proteins in extremes of salt, but also helps us to understand why and how group I salts stabilize proteins in general. [ABSTRACT FROM AUTHOR]