Halobacterium hubeiense sp. nov., a haloarchaeal species isolated from a bore core drilled in Hubei Province, PR China.

Eight colonies of live microbes were isolated from an extensively surface-sterilized halite sample which had been retrieved from a depth of 2000 m from a salt mine in the Qianjiang Depression, Hubei Province, PR China. The eight colonies, obtained after 4 weeks of incubation, were named JI20-1T-JI20-8 and JI20-1T was selected as the type strain. The strains have been previously described, including a genomic analysis based on the complete genome for strain JI20-1T and draft genomes for the other strains. In that study, the name Halobacterium hubeiense was suggested, based on the location of the drilling site. Previous phylogenomic analysis showed that strain JI20-1T is most closely related to the Permian isolate Halobacterium noricense from Alpine rock salt. The orthologous average nucleotide identity (orthoANI) and digital DNA-DNA hybridization (dDDH) percentages between the eight strains are 100-99.6 % and 99.8-96.4 %, respectively. The orthoANI and dDDH values of these strains with respect to the type strains of species of the genus Halobacterium are 89.9-78.2 % and 37.3-21.6 %, respectively, supporting their placement in a novel extremely halophilic archaeal species. The phylogenomic tree based on the comparison of sequences of 632 core-orthologous proteins confirmed the novel species status for these haloarchaea. The polar lipid profile includes phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, and sulfated galactosyl mannosyl galactosyl glucosyl diether, a profile compatible with that of Halobacterium noricense. Based on genomic, phenotypic, and chemotaxonomic characterization, we propose strain JI20-1T (=DSM 114402T = HAMBI 3616T) as the type strain of a novel species in the genus Halobacterium, with the name Halobacterium hubeiense sp. nov.

Halobacterium yunchengense sp. nov., Natronomonas amylolytica sp. nov., Halorientalis halophila sp. nov., Halobellus salinisoli sp. nov., halophilic archaea isolated from a saline lake and inland saline soil.

Four halophilic archaeal strains YCN1T, YCN58T, LT38T, and LT62T were isolated from Yuncheng Salt Lake (Shanxi, China) and Tarim Basin (Xinjiang, China), respectively. Phylogenetic and phylogenomic analyses showed that these four strains tightly cluster with related species of Halobacterium, Natronomonas, Halorientalis, and Halobellus, respectively. The AAI, ANI, and dDDH values between these four strains and their related species of respective genera were lower than the proposed threshold values for species delineation. Strains YCN1T, YCN58T, LT38T, and LT62T could be differentiated from the current species of Halobacterium, Natronomonas, Halorientalis, and Halobellus, respectively, based on the comparison of diverse phenotypic characteristics. The polar lipid profiles of these four strains were closely similar to those of respective relatives within the genera Halobacterium, Natronomonas, Halorientalis, and Halobellus, respectively. The phenotypic, phylogenetic, and genome-based analyses indicated that strains YCN1T, YCN58T, LT38T, and LT62T represent respective novel species within the genera Halobacterium, Natronomonas, Halorentalis, and Halobellus, for which the names Halobacterium yunchengense sp. nov., Natronomonas amylolytica sp. nov., Halorientalis halophila sp. nov., and Halobellus salinisoli sp. nov. are proposed, respectively.

Halobacterium wangiae sp. nov. and Halobacterium zhouii sp. nov., two extremely halophilic archaea isolated from sediment of a salt lake and saline soil of an inland saltern.

Two novel halophilic archaeal strains, Gai3-17T and XZYJT26T, were isolated from the sediment of Gaize salt lake and the saline soil of Mangkang ancient solar saltern in Tibet, PR China, respectively. Strains Gai3-17T and XZYJT26T were related to each other (96.5 and 89.7% similarity, respectively) and showed 97.5-95.4 and 91.5-87.7% similarities to the current members of Halobacterium based on 16S rRNA and rpoB' genes. The phylogenomic analysis indicated that strains Gai3-17T and XZYJT26T formed two distinct clades and clustered with the Halobacterium species. The two strains can be differentiated from the type strains of the six species with validly published names based on several phenotypic characteristics. The phospholipids of the two strains were phosphatidic acid, phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. One major glycolipid, sulphated galactosyl mannosyl glucosyl diether, was detected in strain Gai3-17T, while four glycolipids, mannosyl glucosyl diether, sulphated mannosyl glucosyl diether, disulphated mannosyl glucosyl diether and sulphated galactosyl mannosyl glucosyl diether were observed in strain XZYJT26T. The average nucleotide identity, digital DNA-DNA hybridization and amino acid identity values among the two strains and the members of Halobacterium were no more than 81, 25 and 77 %, respectively. These overall genome-related indices were below the threshold values for species boundary, indicating that strains Gai3-17T and XZYJT26T represent two novel species of Halobacterium. Thus, two novel species, Halobacterium wangiae sp. nov. and Halobacterium zhouii sp. nov., are proposed to accommodate strains Gai3-17T (=CGMCC 1.16101T=JCM 33551T) and XZYJT26T (=CGMCC 1.16682T=JCM 33556T), respectively.

Bioengineering of air-filled protein nanoparticles by genetic and chemical functionalization.

BACKGROUND: Various bacteria and archaea, including halophilic archaeon Halobacterium sp. NRC-1 produce gas vesicle nanoparticles (GVNPs), a unique class of stable, air-filled intracellular proteinaceous nanostructures. GVNPs are an attractive tool for biotechnological applications due to their readily production, purification, and unique physical properties. GVNPs are spindle- or cylinder-shaped, typically with a length of 100 nm to 1.5 µm and a width of 30-250 nm. Multiple monomeric subunits of GvpA and GvpC proteins form the GVNP shell, and several additional proteins are required as minor structural or assembly proteins. The haloarchaeal genetic system has been successfully used to produce and bioengineer GVNPs by fusing several foreign proteins with GvpC and has shown various applications, such as biocatalysis, diagnostics, bioimaging, drug delivery, and vaccine development. RESULTS: We demonstrated that native GvpC can be removed in a low salt buffer during the GVNP purification, leaving the GvpA-based GVNP's shell intact and stable under physiological conditions. Here, we report a genetic engineering and chemical modification approach for functionalizing the major GVNP protein, GvpA. This novel approach is based on combinatorial cysteine mutagenesis within GvpA and genetic expansion of the N-terminal and C-terminal regions. Consequently, we generated GvpA single, double, and triple cysteine variant libraries and investigated the impact of mutations on the structure and physical shape of the GVNPs formed. We used a thiol-maleimide chemistry strategy to introduce the biotechnological relevant activity by maleimide-activated streptavidin-biotin and maleimide-activated SpyTag003-SpyCatcher003 mediated functionalization of GVNPs. CONCLUSION: The merger of these genetic and chemical functionalization approaches significantly extends these novel protein nanomaterials' bioengineering and functionalization potential to assemble catalytically active proteins, biomaterials, and vaccines onto one nanoparticle in a modular fashion.

Divergent degeneration of creA antitoxin genes from minimal CRISPRs and the convergent strategy of tRNA-sequestering CreT toxins.

Aside from providing adaptive immunity, type I CRISPR-Cas was recently unearthed to employ a noncanonical RNA guide (CreA) to transcriptionally repress an RNA toxin (CreT). Here, we report that, for most archaeal and bacterial CreTA modules, the creA gene actually carries two flanking 'CRISPR repeats', which are, however, highly divergent and degenerated. By deep sequencing, we show that the two repeats give rise to an 8-nt 5' handle and a 22-nt 3' handle, respectively, i.e., the conserved elements of a canonical CRISPR RNA, indicating they both retained critical nucleotides for Cas6 processing during divergent degeneration. We also uncovered a minimal CreT toxin that sequesters the rare transfer RNA for isoleucine, tRNAIleCAU, with a six-codon open reading frame containing two consecutive AUA codons. To fully relieve its toxicity, both tRNAIleCAU overexpression and supply of extra agmatine (modifies the wobble base of tRNAIleCAU to decipher AUA codons) are required. By replacing AUA to AGA/AGG codons, we reprogrammed this toxin to sequester rare arginine tRNAs. These data provide essential information on CreTA origin and for future CreTA prediction, and enrich the knowledge of tRNA-sequestering small RNAs that are employed by CRISPR-Cas to get addictive to the host.

Resurrection of Ancestral Malate Dehydrogenases Reveals the Evolutionary History of Halobacterial Proteins: Deciphering Gene Trajectories and Changes in Biochemical Properties.

Extreme halophilic Archaea thrive in high salt, where, through proteomic adaptation, they cope with the strong osmolarity and extreme ionic conditions of their environment. In spite of wide fundamental interest, however, studies providing insights into this adaptation are scarce, because of practical difficulties inherent to the purification and characterization of halophilic enzymes. In this work, we describe the evolutionary history of malate dehydrogenases (MalDH) within Halobacteria (a class of the Euryarchaeota phylum). We resurrected nine ancestors along the inferred halobacterial MalDH phylogeny, including the Last Common Ancestral MalDH of Halobacteria (LCAHa) and compared their biochemical properties with those of five modern halobacterial MalDHs. We monitored the stability of these various MalDHs, their oligomeric states and enzymatic properties, as a function of concentration for different salts in the solvent. We found that a variety of evolutionary processes, such as amino acid replacement, gene duplication, loss of MalDH gene and replacement owing to horizontal transfer resulted in significant differences in solubility, stability and catalytic properties between these enzymes in the three Halobacteriales, Haloferacales, and Natrialbales orders since the LCAHa MalDH. We also showed how a stability trade-off might favor the emergence of new properties during adaptation to diverse environmental conditions. Altogether, our results suggest a new view of halophilic protein adaptation in Archaea.

Bioengineering of Halobacterium sp. NRC-1 gas vesicle nanoparticles with GvpC fusion protein produced in E. coli.

Gas vesicle nanoparticles (GVNPs) are hollow, buoyant prokaryotic organelles used for cell flotation. GVNPs are encoded by a large gas vesicle protein (gvp) gene cluster in the haloarchaeon, Halobacterium sp. NRC-1, including one gene, gvpC, specifying a protein bound to the surface of the nanoparticles. Genetically engineered GVNPs in the Halobacterium sp. have been produced by fusion of foreign sequences to gvpC. To improve the versatility of the GVNP platform, we developed a method for displaying exogenously produced GvpC fusion proteins on the haloarchaeal nanoparticles. The streptococcal IgG-binding protein domain was fused at or near the C-terminus of GvpC, expressed and purified from E. coli, and shown to bind to wild-type GVNPs. The two fusion proteins, GvpC3GB and GvpC4GB, without or with a highly acidic GvpC C-terminal region, were found to be able to bind nanoparticles equally well. The GVNP-bound GvpC-IgG-binding fusion protein was also capable of binding to an enzyme-linked IgG-HRP complex which retained enzyme activity, demonstrating the hybrid system capability for display and delivery of protein complexes. This is the first report demonstrating functional binding of exogenously produced GvpC fusion proteins to wild-type haloarchaeal GVNPs which significantly expands the capability of the platform to produce bioengineered nanoparticles for biomedical applications. KEY POINTS: • Haloarchaeal gas vesicle nanoparticles (GVNPs) constitute a versatile display system. • GvpC-streptococcal IgG-binding fusion proteins expressed in E. coli bind to GVNPs. • IgG-binding proteins displayed on floating GVNPs bind and display IgG-HRP complex.

Microbial gas vesicles as nanotechnology tools: exploiting intracellular organelles for translational utility in biotechnology, medicine and the environment.

A range of bacteria and archaea produce gas vesicles as a means to facilitate flotation. These gas vesicles have been purified from a number of species and their applications in biotechnology and medicine are reviewed here. Halobacterium sp. NRC-1 gas vesicles have been engineered to display antigens from eukaryotic, bacterial and viral pathogens. The ability of these recombinant nanoparticles to generate an immune response has been quantified both in vitro and in vivo. These gas vesicles, along with those purified from Anabaena flos-aquae and Bacillus megaterium, have been developed as an acoustic reporter system. This system utilizes the ability of gas vesicles to retain gas within a stable, rigid structure to produce contrast upon exposure to ultrasound. The susceptibility of gas vesicles to collapse when exposed to excess pressure has also been proposed as a biocontrol mechanism to disperse cyanobacterial blooms, providing an environmental function for these structures.

Halobacterium bonnevillei sp. nov., Halobaculum saliterrae sp. nov. and Halovenus carboxidivorans sp. nov., three novel carbon monoxide-oxidizing Halobacteria from saline crusts and soils.

Three novel carbon monoxide-oxidizing Halobacteria were isolated from Bonneville Salt Flats (Utah, USA) salt crusts and nearby saline soils. Phylogenetic analysis of 16S rRNA gene sequences revealed that strains PCN9T, WSA2T and WSH3T belong to the genera Halobacterium, Halobaculum and Halovenus, respectively. Strains PCN9T, WSA2T and WSH3T grew optimally at 40 °C (PCN9T) or 50 °C (WSA2T, WSH3T). NaCl optima were 3 M (PCN9T, WSA2T) or 4 M NaCl (WSH3T). Carbon monoxide was oxidized by all isolates, each of which contained a molybdenum-dependent CO dehydrogenase. G+C contents for the three respective isolates were 66.75, 67.62, and 63.97 mol% as derived from genome analyses. The closest phylogenetic relatives for PCN9T, WSA2T and WSH3T were Halobacterium noricense A1T, Halobaculum roseum D90T and Halovenus aranensis EB27T with 98.71, 98.19 and 95.95 % 16S rRNA gene sequence similarities, respectively. Genome comparisons of PCN9T with Halobacterium noricense A1T yielded an average nucleotide identity (ANI) of 82.0% and a digital DNA-DNA hybridization (dDDH) value of 25.7 %; comparisons of WSA2T with Halobaculum roseum D90T yielded ANI and dDDH values of 86.34 and 31.1 %, respectively. The ANI value for a comparison of WSH3T with Halovenus aranensis EB27T was 75.2 %. Physiological, biochemical, genetic and genomic characteristics of PCN9T, WSA2T and WSH3T differentiated them from their closest phylogenetic neighbours and indicated that they represent novel species for which the names Halobaculum bonnevillei, Halobaculum saliterrae and Halovenus carboxidivorans are proposed, respectively. The type strains are PCN9T (=JCM 32472=LMG 31022=ATCC TSD-126), WSA2T (=JCM 32473=ATCC TSD-127) and WSH3T (=JCM 32474=ATCC TSD-128).

Extremophilic models for astrobiology: haloarchaeal survival strategies and pigments for remote sensing.

Recent progress in extremophile biology, exploration of planetary bodies in the solar system, and the detection and characterization of extrasolar planets are leading to new insights in the field of astrobiology and possible distribution of life in the universe. Among the many extremophiles on Earth, the halophilic Archaea (Haloarchaea) are especially attractive models for astrobiology, being evolutionarily ancient and physiologically versatile, potentially surviving in a variety of planetary environments and with relevance for in situ life detection. Haloarchaea are polyextremophilic with tolerance of saturating salinity, anaerobic conditions, high levels of ultraviolet and ionizing radiation, subzero temperatures, desiccation, and toxic ions. Haloarchaea survive launches into Earth's stratosphere encountering conditions similar to those found on the surface of Mars. Studies of their unique proteins are revealing mechanisms permitting activity and function in high ionic strength, perchlorates, and subzero temperatures. Haloarchaea also produce spectacular blooms visible from space due to synthesis of red-orange isoprenoid carotenoids used for photoprotection and photorepair processes and purple retinal chromoproteins for phototrophy and phototaxis. Remote sensing using visible and infrared spectroscopy has shown that haloarchaeal pigments exhibit both a discernable peak of absorption and a reflective "green edge". Since the pigments produce remotely detectable features, they may influence the spectrum from an inhabited exoplanet imaged by a future large space-based telescope. In this review, we focus primarily on studies of two Haloarchaea, Halobacterium sp. NRC-1 and Halorubrum lacusprofundi.

Key amino acid residues conferring enhanced enzyme activity at cold temperatures in an Antarctic polyextremophilic ß-galactosidase.

The Antarctic microorganism Halorubrum lacusprofundi harbors a model polyextremophilic ß-galactosidase that functions in cold, hypersaline conditions. Six amino acid residues potentially important for cold activity were identified by comparative genomics and substituted with evolutionarily conserved residues (N251D, A263S, I299L, F387L, I476V, and V482L) in closely related homologs from mesophilic haloarchaea. Using a homology model, four residues (N251, A263, I299, and F387) were located in the TIM barrel around the active site in domain A, and two residues (I476 and V482) were within coiled or ß-sheet regions in domain B distant to the active site. Site-directed mutagenesis was performed by partial gene synthesis, and enzymes were overproduced from the cold-inducible cspD2 promoter in the genetically tractable Haloarchaeon, Halobacterium sp. NRC-1. Purified enzymes were characterized by steady-state kinetic analysis at temperatures from 0 to 25 °C using the chromogenic substrate o-nitrophenyl-ß-galactoside. All substitutions resulted in altered temperature activity profiles compared with wild type, with five of the six clearly exhibiting reduced catalytic efficiency (kcat/Km) at colder temperatures and/or higher efficiency at warmer temperatures. These results could be accounted for by temperature-dependent changes in both Km and kcat (three substitutions) or either Km or kcat (one substitution each). The effects were correlated with perturbation of charge, hydrogen bonding, or packing, likely affecting the temperature-dependent flexibility and function of the enzyme. Our interdisciplinary approach, incorporating comparative genomics, mutagenesis, enzyme kinetics, and modeling, has shown that divergence of a very small number of amino acid residues can account for the cold temperature function of a polyextremophilic enzyme.

Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent.

The 3D structure determination of biological macromolecules by X-ray crystallography suffers from a phase problem: to perform Fourier transformation to calculate real space density maps, both intensities and phases of structure factors are necessary; however, measured diffraction patterns give only intensities. Although serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has been steadily developed since 2009, experimental phasing still remains challenging. Here, using 7.0-keV (1.771 Å) X-ray pulses from the SPring-8 Angstrom Compact Free Electron Laser (SACLA), iodine single-wavelength anomalous diffraction (SAD), single isomorphous replacement (SIR), and single isomorphous replacement with anomalous scattering (SIRAS) phasing were performed in an SFX regime for a model membrane protein bacteriorhodopsin (bR). The crystals grown in bicelles were derivatized with an iodine-labeled detergent heavy-atom additive 13a (HAD13a), which contains the magic triangle, I3C head group with three iodine atoms. The alkyl tail was essential for binding of the detergent to the surface of bR. Strong anomalous and isomorphous difference signals from HAD13a enabled successful phasing using reflections up to 2.1-Å resolution from only 3,000 and 4,000 indexed images from native and derivative crystals, respectively. When more images were merged, structure solution was possible with data truncated at 3.3-Å resolution, which is the lowest resolution among the reported cases of SFX phasing. Moreover, preliminary SFX experiment showed that HAD13a successfully derivatized the G protein-coupled A2a adenosine receptor crystallized in lipidic cubic phases. These results pave the way for de novo structure determination of membrane proteins, which often diffract poorly, even with the brightest XFEL beams.

Phyletic Distribution and Lineage-Specific Domain Architectures of Archaeal Two-Component Signal Transduction Systems.

The two-component signal transduction (TCS) machinery is a key mechanism of sensing environmental changes in the prokaryotic world. TCS systems have been characterized thoroughly in bacteria but to a much lesser extent in archaea. Here, we provide an updated census of more than 2,000 histidine kinases and response regulators encoded in 218 complete archaeal genomes, as well as unfinished genomes available from metagenomic data. We describe the domain architectures of the archaeal TCS components, including several novel output domains, and discuss the evolution of the archaeal TCS machinery. The distribution of TCS systems in archaea is strongly biased, with high levels of abundance in haloarchaea and thaumarchaea but none detected in the sequenced genomes from the phyla Crenarchaeota, Nanoarchaeota, and Korarchaeota The archaeal sensor histidine kinases are generally similar to their well-studied bacterial counterparts but are often located in the cytoplasm and carry multiple PAS and/or GAF domains. In contrast, archaeal response regulators differ dramatically from the bacterial ones. Most archaeal genomes do not encode any of the major classes of bacterial response regulators, such as the DNA-binding transcriptional regulators of the OmpR/PhoB, NarL/FixJ, NtrC, AgrA/LytR, and ActR/PrrA families and the response regulators with GGDEF and/or EAL output domains. Instead, archaea encode multiple copies of response regulators containing either the stand-alone receiver (REC) domain or combinations of REC with PAS and/or GAF domains. Therefore, the prevailing mechanism of archaeal TCS signaling appears to be via a variety of protein-protein interactions, rather than direct transcriptional regulation.IMPORTANCE Although the Archaea represent a separate domain of life, their signaling systems have been assumed to be closely similar to the bacterial ones. A study of the domain architectures of the archaeal two-component signal transduction (TCS) machinery revealed an overall similarity of archaeal and bacterial sensory modules but substantial differences in the signal output modules. The prevailing mechanism of archaeal TCS signaling appears to involve various protein-protein interactions rather than direct transcription regulation. The complete list of histidine kinases and response regulators encoded in the analyzed archaeal genomes is available online at http://www.ncbi.nlm.nih.gov/Complete_Genomes/TCSarchaea.html.

Isolation and characterization from solar salterns of North Algeria of a haloarchaeon producing a new halocin.

Halophilic archaea, thriving in hypersaline environments, synthesize antimicrobial substances with an unknown role, called halocins. It has been suggested that halocin production gives transient competitive advantages to the producer strains and represents one of the environmental factors influencing the microbial community composition. Herein, we report on the antibacterial activity of a new haloarchaeon selected from solar salterns of the northern coast of Algeria. A total of 81 halophilic strains, isolated from the microbial consortia, were screened for the production of antimicrobial compounds by interspecies competition test and against a collection of commercial haloarchaea. On the basis of the partial 16S rRNA sequencing, the most efficient halocin producer was recognized as belonging to Haloferax (Hfx) sp., while the best indicator microorganism, showing high sensitivity toward halocin, was related to Haloarcula genus. The main morphological, physiological and biochemical properties of Hfx were investigated and a partial purification of the produced halocin was allowed to identify it as a surface membrane protein with a molecular mass between 30 and 40 kDa. Therefore, in this study, we isolated a new strain belonging to Haloferax genus and producing a promising antimicrobial compound useful for applications in health and food industries.

Spectroscopic characteristics of Rubricoccus marinus xenorhodopsin (RmXeR) and a putative model for its inward H+ transport mechanism.

A new group of microbial rhodopsins named xenorhodopsins (XeR), which are closely related to the cyanobacterial Anabaena sensory rhodopsin, show a light-driven "inward" proton transport activity, as reported for one representative of this group from Parvularcula oceani (PoXeR). In this study, we functionally and spectroscopically characterized a new member of the XeR clade from a marine bacterium Rubricoccus marinus SG-29T (RmXeR). Escherichia coli cells expressing recombinant RmXeR showed a light-induced alkalization of the cell suspension, which was strongly impaired by a protonophore, suggesting that RmXeR is a light-driven "inward" proton pump as is PoXeR. The spectroscopic properties of purified RmXeR were investigated and compared with those of PoXeR and a light-driven "outward" proton pump, bacteriorhodopsin (BR) from the archaeon Halobacterium salinarum. Action spectroscopy revealed that RmXeR with all-trans retinal is responsible for the light-driven inward proton transport activity, but not with 13-cis retinal. From pH titration experiments and mutational analysis, we estimated the pKa values for the protonated Schiff base of the retinal chromophore and its counterion as 11.1 ± 0.07 and 2.1 ± 0.07, respectively. Of note, the direction of both the retinal composition change upon light-dark adaptation and the acid-induced spectral shift was opposite that of BR, which is presumably related to the opposite directions of ion transport (from outside to inside for RmXeR and from inside to outside for BR). Flash photolysis experiments revealed the appearances of three intermediates (L, M and O) during the photocycle. The proton uptake and release were coincident with the formation and decay of the M intermediate, respectively. Together with associated findings from other microbial rhodopsins, we propose a putative model for the inward proton transport mechanism of RmXeR.

Mediation mechanism of tyrosine 185 on the retinal isomerization equilibrium and the proton release channel in the seven-transmembrane receptor bacteriorhodopsin.

Electrostatic coupling leading to conformational changes in proteins is challenging to demonstrate directly, it requires that both the local, discrete electronic details and dynamic information relevant to the functional descriptions are probed. Here, as a novel study to address this challenge, the roles of an aromatic residue in influencing the functional conformational changes of a membrane receptor in its natural membrane environment are reported. Previously intractable discrete electronic details have been obtained using 2D solid-state NMR of specifically labelled receptor, reinforced with molecular dynamic simulations, mutational analysis and functional assays, supported by and compared with rigid-atom crystal structural models. Hydrogen bonding and hydrophobic interactions are identified as the mechanistic origin for direct electromechanical coupling to the dynamics of conformational changes within the receptor.

Halobacterium litoreum sp. nov., isolated from a marine solar saltern.

Halophilic archaeal strain ZS-54-S2T was isolated from Zhoushan marine solar saltern, China. Cells were rod-shaped, Gram-stain-negative and formed red-pigmented colonies on an agar plate. Strain ZS-54-S2T was able to grow at 20-50 °C (optimum 35 °C), at 1.7-4.8 M NaCl (optimum 3.9 M), at 0.005-1.0 M MgCl2 (optimum 0.05 M) and at pH 5.0-9.5 (optimum pH 7.0). The cells lysed in distilled water and the minimal NaCl concentration to prevent cell lysis was found to be 5 % (w/v). The major polar lipids of the strain were phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, two glycolipids, which were chromatographically identical to sulfated galactosyl mannosyl galactofuranosyl glucosyl diether and galactosyl mannosyl glucosyl diether, and an unidentified glycolipid, which was chromatographically identical to one detected in Halobacterium salinarum ATCC 33171T. The 16S rRNA gene and rpoB' gene of strain ZS-54-S2T were phylogenetically related to the corresponding genes of Halobacterium noricense JCM 15102T (97.5 % and 90.6 % relatedness, respectively), Halobacterium jilantaiense CGMCC 1.5337T (96.9 and 91.2 %), Halobacterium rubrum CGMCC 1.12575T (96.8 and 90.3 %) and Halobacterium salinarum CGMCC 1.1958T (96.5 and 88.4 %). The DNA G+C content of strain ZS-54-S2T was 66.7 mol%. The phenotypic, chemotaxonomic and phylogenetic properties suggested that strain ZS-54-S2T (=CGMCC 1.12562T=JCM 30038T) represents a new species of Halobacterium, for which the name Halobacteriumlitoreum sp. nov. is proposed.

Cryo-electron microscopy of an extremely halophilic microbe: technical aspects.

Most halophilic Archaea of the class Halobacteriaceae depend on the presence of several molar sodium chloride for growth and cell integrity. This poses problems for structural studies, particularly for electron microscopy, where the high salt concentration results in diminished contrast. Since cryo-electron microscopy of intact cells provides new insights into the cellular and molecular organization under close-to-live conditions, we evaluated strategies and conditions to make halophilic microbes available for investigations in situ. Halobacterium salinarum, the test organism for this study, usually grows at 4.3 M NaCl. Adaptation to lower concentrations and subsequent NaCl reduction via dialysis led to still vital cells at 3 M salt. A comprehensive evaluation of vitrification parameters, thinning of frozen cells by focused-ion-beam micromachining, and cryo-electron microscopy revealed that structural studies under high salt conditions are possible in situ.

Radiation resistance in thermophiles: mechanisms and applications.

The study of prokaryotic life in high temperature environments viz., geothermal areas, hot, acidic geysers and undersea hydrothermal vents has revealed the existence of thermophiles (or hyperthermophiles). These microorganisms possess various stress adaptation mechanisms which enable them to bypass multiple physical and chemical barriers for survival. The discovery of radiation resistant thermophile Deinococcus geothermalis has given new insights into the field of radiation microbiology. The ability of radiation resistant thermophiles to deal with the lethal effects of ionizing radiations like DNA damage, oxidative bursts and protein damage has made them a model system for exobiology and interplanetary transmission of life. They might be an antiquity of historical transport process that brought microbial life on Earth. These radiation resistant thermophiles are resistant to desiccation as well and maintain their homeostasis by advance DNA repair mechanisms, reactive oxygen species (ROS) detoxification system and accumulation of compatible solutes. Moreover, engineered radioresistant thermophilic strains are the best candidate for bioremediation of radionuclide waste while the extremolytes produced by these organisms may have predicted therapeutic uses. So, the present article delineate a picture of radiation resistance thermophiles, their adaptive mechanisms to evade stress viz., radiation and desiccation, their present applications along with new horizons in near future.

Efflux by small multidrug resistance proteins is inhibited by membrane-interactive helix-stapled peptides.

Bacterial cell membranes contain several protein pumps that resist the toxic effects of drugs by efficiently extruding them. One family of these pumps, the small multidrug resistance proteins (SMRs), consists of proteins of about 110 residues that need to oligomerize to form a structural pathway for substrate extrusion. As such, SMR oligomerization sites should constitute viable targets for efflux inhibition, by disrupting protein-protein interactions between helical segments. To explore this proposition, we are using Hsmr, an SMR from Halobacter salinarum that dimerizes to extrude toxicants. Our previous work established that (i) Hsmr dimerization is mediated by a helix-helix interface in Hsmr transmembrane (TM) helix 4 (residues (90)GLALIVAGV(98)); and (ii) a peptide comprised of the full TM4(85-105) sequence inhibits Hsmr-mediated ethidium bromide efflux from bacterial cells. Here we define the minimal linear sequence for inhibitor activity (determined as TM4(88-100), and then "staple" this sequence via Grubbs metathesis to produce peptides typified by acetyl-A-(Sar)3-(88)VVGLXLIZXGVVV(100)-KKK-NH2 (X = 2-(4'-pentenyl)alanine at positions 92 and 96; Z = Val, Gly, or Asn at position 95)). The Asn(95) peptide displayed specific efflux inhibition and resensitization of Hsmr-expressing cells to ethidium bromide; and was non-hemolytic to human red blood cells. Stapling essentially prevented peptide degradation in blood plasma and liver homogenates versus an unstapled counterpart. The overall results confirm that the stapled analog of TM4(88-100) retains the structural complementarity required to disrupt the Hsmr TM4-TM4 locus in Hsmr, and portend the general validity of stapled peptides as therapeutics for the disruption of functional protein-protein interactions in membranes.