Synthesizing glycine betaine via choline oxidation pathway as an osmoprotectant strategy in Haloferacales.

The accumulation of organic compatible solutes, such as glycine betaine, is one of the osmoprotective strategies used by halophilic archaea to adapt to high salinity. The uptake of glycine betaine from the external environment using various transporters has been widely studied in different halophilic archaea. However, the de novo biosynthesis of glycine betaine and its distribution in halophilic archaea remain unclear. In this study, an extremely halophilic archaea strain, named Halorubrum sp. 2020YC2 and previously isolated from a salt-lake sample, was identified with complete choline oxidation pathway genes. Halorubrum sp. 2020YC2 could synthesize and accumulate 1.56-4.25 µmol per mg of protein of glycine betaine in a defined medium, with its content increasing along with increasing salinity. The intracellular content of glycine betaine remained relatively stable at different salinities when another exogenous solute such as trehalose was provided. The metabolic profile and transcriptional results strongly suggested that the intracellular glycine betaine was derived from serine, which came from the glycolytic intermediate 3-phosphoglycerate when glucose was used as the sole carbon source. Out of 205 available genomes of halophilic archaea, genes encoding the choline oxidation pathway were identified in 30 genomes, and more than half of the strains belonging to order Haloferacales contained the choline oxidation pathway. Phylogenetic analysis further indicated that this pathway evolved from halophilic Proteobacteria, and its absence in some genera indicated a possible gene loss event during evolution. The analysis of reported culture data of halophilic archaea strains eventually demonstrated that the presence of the choline oxidation pathway had no significant effects on the adaptation of Haloferacales to high salinity habitats. Therefore, the de novo biosynthesis of glycine betaine via the choline oxidation pathway could be an auxiliary osmoprotective strategy in halophilic archaea.

Biosynthesis of Silver Chloride Nanoparticles (AgCl-NPs) from Extreme Halophiles and Evaluation of Their Biological Applications.

The biosynthesis of nanoparticles (NPs) has gained an overwhelming interest due to their biological applications. However, NPs synthesis by pigmented extreme halophiles remains underexplored. The NPs synthesis using pigmented halophiles is inexpensive and less toxic than other processes. In this study, pigmented halophilic microorganisms (n = 77) were screened to synthesize silver chloride nanoparticles (AgCl-NPs) with silver nitrate as metal precursors, and their biological applications were assessed. The synthesis of AgCl-NPs was possible using the crude extract from cellular lysis (CECL) of six extreme halophiles. Two of the AgCl-NPs viz. AK2-NPs and MY6-NPs synthesized by the CECL of Haloferax alexandrinus RK_AK2 and Haloferax lucentense RK_MY6, respectively, exhibited antimicrobial, antioxidative, and anti-inflammatory activities. The surface plasmon resonance of the AgCl-NPs was determined with UV spectroscopy. XRD analysis of AK2-NPs and MY6-NPs confirmed the presence of silver in the form of chlorargyrite (silver chloride) having a cubic structure. The crystallite size of AK2-NPs and MY6-NPs, estimated with the Scherrer formula, was 115.81 nm and 137.50 nm. FTIR analysis verified the presence of diverse functional groups. Dynamic light-scattering analysis confirmed that the average size distribution of NPs was 71.02 nm and 117.36 nm for AK2-NPs and MY6-NPs, respectively, with monodisperse nature. The functional group in 1623-1641 cm-1 indicated the presence of protein ß-sheet structure and shifting of amino and hydroxyl groups from the pigmented CECL, which helps in capping and stabilizing nanoparticles. The study provides evidence that CECL of Haloferax species can rapidly synthesize NPs with unique characteristics and biological applications.

Ubiquitousness of Haloferax and Carotenoid Producing Genes in Arabian Sea Coastal Biosystems of India.

This study presents a comparative analysis of halophiles from the global open sea and coastal biosystems through shotgun metagenomes (n = 209) retrieved from public repositories. The open sea was significantly enriched with Prochlorococcus and Candidatus pelagibacter. Meanwhile, coastal biosystems were dominated by Marinobacter and Alcanivorax. Halophilic archaea Haloarcula and Haloquandratum, predominant in the coastal biosystem, were significantly (p < 0.05) enriched in coastal biosystems compared to the open sea. Analysis of whole genomes (n = 23,540), retrieved from EzBioCloud, detected crtI in 64.66% of genomes, while cruF was observed in 1.69% Bacteria and 40.75% Archaea. We further confirmed the viability and carotenoid pigment production by pure culture isolation (n = 1351) of extreme halophiles from sediments (n = 410 × 3) sampling at the Arabian coastline of India. All red-pigmented isolates were represented exclusively by Haloferax, resistant to saturated NaCl (6 M), and had >60% G + C content. Multidrug resistance to tetracycline, gentamicin, ampicillin, and chloramphenicol were also observed. Our study showed that coastal biosystems could be more suited for bioprospection of halophiles rather than the open sea.

Analysis of Gene Expression Changes in Plants Grown in Salty Soil in Response to Inoculation with Halophilic Bacteria.

Soil salinity is an increasing problem facing agriculture in many parts of the world. Climate change and irrigation practices have led to decreased yields of some farmland due to increased salt levels in the soil. Plants that have tolerance to salt are thus needed to feed the world's population. One approach addressing this problem is genetic engineering to introduce genes encoding salinity, but this approach has limitations. Another fairly new approach is the isolation and development of salt-tolerant (halophilic) plant-associated bacteria. These bacteria are used as inoculants to stimulate plant growth. Several reports are now available, demonstrating how the use of halophilic inoculants enhance plant growth in salty soil. However, the mechanisms for this growth stimulation are as yet not clear. Enhanced growth in response to bacterial inoculation is expected to be associated with changes in plant gene expression. In this review, we discuss the current literature and approaches for analyzing altered plant gene expression in response to inoculation with halophilic bacteria. Additionally, challenges and limitations to current approaches are analyzed. A further understanding of the molecular mechanisms involved in enhanced plant growth when inoculated with salt-tolerant bacteria will significantly improve agriculture in areas affected by saline soils.

Expansion segments in bacterial and archaeal 5S ribosomal RNAs.

The large ribosomal RNAs of eukaryotes frequently contain expansion sequences that add to the size of the rRNAs but do not affect their overall structural layout and are compatible with major ribosomal function as an mRNA translation machine. The expansion of prokaryotic ribosomal RNAs is much less explored. In order to obtain more insight into the structural variability of these conserved molecules, we herein report the results of a comprehensive search for the expansion sequences in prokaryotic 5S rRNAs. Overall, 89 expanded 5S rRNAs of 15 structural types were identified in 15 archaeal and 36 bacterial genomes. Expansion segments ranging in length from 13 to 109 residues were found to be distributed among 17 insertion sites. The strains harboring the expanded 5S rRNAs belong to the bacterial orders Clostridiales, Halanaerobiales, Thermoanaerobacterales, and Alteromonadales as well as the archael order Halobacterales When several copies of a 5S rRNA gene are present in a genome, the expanded versions may coexist with normal 5S rRNA genes. The insertion sequences are typically capable of forming extended helices, which do not seemingly interfere with folding of the conserved core. The expanded 5S rRNAs have largely been overlooked in 5S rRNA databases.

Physiological response to silver toxicity in the extremely halophilic archaeon Halomicrobium mukohataei.

Adaptive strategies responsible for heavy metal tolerance were explored in the extremely halophilic archaeon Halomicrobium mukohataei DSM 12286. The tested strain was seemingly able to overcome silver-induced oxidative stress (assessed by malondialdehyde quantification, catalase assay and total antioxidant capacity measurement) mainly through non-enzymatic antioxidants. Energy dispersive spectrometry analysis illustrated the presence of colloidal silver in Hmc. mukohataei cultures exposed to AgNO3. Bright-field and transmission electron microscopy images, as well as dynamic light scattering analysis, demonstrated the presence of intracellular nanoparticles, mostly spherical, within a size range of 20-100 nm. As determined by the zeta potential measurement, the biosynthesized nanoparticles were highly stable, with a negative surface charge. Our research is a first attempt in the systematic study of the oxidative stress and intracellular silver nanoparticle accumulation, generated by exposure to silver ions, in members of Halobacteria class, thus broadening our knowledge on mechanisms supporting heavy metal tolerance of microbial cells living under saline conditions.

Halophile, an essential platform for bioproduction.

Industrial biotechnology aims to compete as a stronger alternative ensuring environmental friendly microbial-based production that seeks to curb the predicament of pollution. However, the high cost of bioprocessing is a severe drawback, and therefore, new approaches must be developed to overcome this challenge. Halophiles have shown potentials of overcoming this challenge and are of much preference for unsterile and continuous contamination-free bioprocess due to their unique ability to grow under harsh environmental conditions. Recent advances in genetic manipulations have been established to better the performance of halophiles for industrial applications. Many researchers produced various products such as polyhydroxyalkanoates (PHA), ectoines, biosurfactants, and antioxidants using halophiles, and further efforts have been established to develop halophiles as the foundation for low-cost bioprocess. This paper provides a useful reference for researchers on the merits, drawbacks, achievements, and application of halophiles for bioproduction.

Species Widely Distributed in Halophilic Archaea Exhibit Opsin-Mediated Inhibition of Bacterioruberin Biosynthesis.

Halophilic Archaea are a distinctive pink color due to a carotenoid pigment called bacterioruberin. To sense or utilize light, many halophilic Archaea also produce rhodopsins, complexes of opsin proteins with a retinal prosthetic group. Both bacterioruberin and retinal are synthesized from isoprenoid precursors, with lycopene as the last shared intermediate. We previously described a regulatory mechanism by which Halobacterium salinarum bacterioopsin and Haloarcula vallismortis cruxopsin inhibit bacterioruberin synthesis catalyzed by lycopene elongase. In this work, we found that opsins in all three major Halobacteria clades inhibit bacterioruberin synthesis, suggesting that this regulatory mechanism existed in the common Halobacteria ancestor. Halophilic Archaea, which are generally heterotrophic and aerobic, likely evolved from an autotrophic, anaerobic methanogenic ancestor by acquiring many genes from Bacteria via lateral gene transfer. These bacterial "imports" include genes encoding opsins and lycopene elongases. To determine if opsins from Bacteria inhibit bacterioruberin synthesis, we tested bacterial opsins and found that an opsin from Curtobacterium, in the Actinobacteria phylum, inhibits bacterioruberin synthesis catalyzed by its own lycopene elongase, as well as that catalyzed by several archaeal enzymes. We also determined that the lycopene elongase from Halococcus salifodinae, a species from a family of Halobacteria lacking opsin homologs, retained the capacity to be inhibited by opsins. Together, our results indicate that opsin-mediated inhibition of bacterioruberin biosynthesis is a widely distributed mechanism found in both Archaea and Bacteria, possibly predating the divergence of the two domains. Further analysis may provide insight into the acquisition and evolution of the genes and their host species.IMPORTANCE All organisms use a variety of mechanisms to allocate limited resources to match their needs in their current environment. Here, we explore how halophilic microbes use a novel mechanism to allow efficient production of rhodopsin, a complex of an opsin protein and a retinal prosthetic group. We previously demonstrated that Halobacterium salinarum bacterioopsin directs available resources toward retinal by inhibiting synthesis of bacterioruberin, a molecule that shares precursors with retinal. In this work, we show that this mechanism can be carried out by proteins from halophilic Archaea that are not closely related to H. salinarum and those in at least one species of Bacteria Therefore, opsin-mediated inhibition of bacterioruberin synthesis may be a highly conserved, ancient regulatory mechanism.

Biochemical and thermodynamic analyses of energy conversion in extremophiles.

A variety of extreme environments, characterized by extreme values of various physicochemical parameters (temperature, pressure, salinity, pH, and so on), are found on Earth. Organisms that favorably live in such extreme environments are called extremophiles. All living organisms, including extremophiles, must acquire energy to maintain cellular homeostasis, including extremophiles. For energy conversion in harsh environments, thermodynamically useful reactions and stable biomolecules are essential. In this review, I briefly summarize recent studies of extreme environments and extremophiles living in these environments and describe energy conversion processes in various extremophiles based on my previous research. Furthermore, I discuss the correlation between the biological system of electrotrophy, a third biological energy acquisition system, and the mechanism underlying microbiologically influenced corrosion. These insights into energy conversion in extremophiles may improve our understanding of the "limits of life". Abbreviations: PPi: pyrophosphate; PPase: pyrophosphatase; ITC: isothermal titration microcalorimetry; SVNTase: Shewanella violacea 5'-nucleotidase; SANTase: Shewanella amazonensis 5'-nucleotidase.

A genome-scale metabolic network alignment method within a hypergraph-based framework using a rotational tensor-vector product.

Biological network alignment aims to discover important similarities and differences and thus find a mapping between topological and/or functional components of different biological molecular networks. Then, the mapped components can be considered to correspond to both their places in the network topology and their biological attributes. Development and evolution of biological network alignment methods has been accelerated by the rapidly increasing availability of such biological networks, yielding a repertoire of tens of methods based upon graph theory. However, most biological processes, especially the metabolic reactions, are more sophisticated than simple pairwise interactions and contain three or more participating components. Such multi-lateral relations are not captured by graphs, and computational methods to overcome this limitation are currently lacking. This paper introduces hypergraphs and association hypergraphs to describe metabolic networks and their potential alignments, respectively. Within this framework, metabolic networks are aligned by identifying the maximal Z-eigenvalue of a symmetric tensor. A shifted higher-order power method was utilized to identify a solution. A rotational strategy has been introduced to accelerate the tensor-vector product by 250-fold on average and reduce the storage cost by up to 1,000-fold. The algorithm was implemented on a spark-based distributed computation cluster to significantly increase the convergence rate further by 50- to 80-fold. The parameters have been explored to understand their impact on alignment accuracy and speed. In particular, the influence of initial value selection on the stationary point has been simulated to ensure an accurate approximation of the global optimum. This framework was demonstrated by alignments among the genome-wide metabolic networks of Escherichia coli MG-1655 and Halophilic archaeon DL31. To our knowledge, this is the first genome-wide metabolic network alignment at both the metabolite level and the enzyme level. These results demonstrate that it can supply quite a few valuable insights into metabolic networks. First, this method can access the driving force of organic reactions through the chemical evolution of metabolic network. Second, this method can incorporate the chemical information of enzymes and structural changes of compounds to offer new way defining reaction class and module, such as those in KEGG. Third, as a vertex-focused treatment, this method can supply novel structural and functional annotation for ill-defined molecules. The related source code is available on request.

Exploration, antifungal and antiaflatoxigenic activity of halophilic bacteria communities from saline soils of Howze-Soltan playa in Iran.

In the present study, halophilic bacteria communities were explored in saline soils of Howze-Soltan playa in Iran with special attention to their biological activity against an aflatoxigenic Aspergillus parasiticus NRRL 2999. Halophilic bacteria were isolated from a total of 20 saline soils using specific culture media and identified by 16S rRNA sequencing in neighbor-joining tree analysis. Antifungal and antiaflatoxigenic activities of the bacteria were screened by a nor-mutant A. parasiticus NRRL 2999 using visual agar plate assay and confirmed by high-performance liquid chromatography. Among a total of 177 halophilic bacteria belonging to 11 genera, 121 isolates (68.3%) inhibited A. parasiticus growth and/or aflatoxin production. The most potent inhibitory bacteria of the genera Bacillus, Paenibacillus and Staphylococcus were distributed in three main phylogenetic clusters as evidenced by 16S rRNA sequence analysis. A. parasiticus growth was inhibited by 0.7-92.7%, while AFB1 and AFG1 productions were suppressed by 15.1-98.9 and 57.0-99.6%, respectively. Taken together, halophilic bacteria identified in this study may be considered as potential sources of novel bioactive metabolites as well as promising candidates to develop new biocontrol agents for managing toxigenic fungi growth and subsequent aflatoxin contamination of food and feed in practice.

An Antarctic Extreme Halophile and Its Polyextremophilic Enzyme: Effects of Perchlorate Salts.

Effects of perchlorate salts prevalent on the surface of Mars are of significant interest to astrobiology from the perspective of potential life on the Red Planet. Halorubrum lacusprofundi, a cold-adapted halophilic Antarctic archaeon, was able to grow anaerobically on 0.04 M concentration of perchlorate. With increasing concentrations of perchlorate, growth was inhibited, with half-maximal growth rate in ca. 0.3 M NaClO4 and 0.1 M Mg(ClO4)2 under aerobic conditions. Magnesium ions were also inhibitory for growth, but at considerably higher concentrations, with half-maximal growth rate above 1 M. For a purified halophilic ß-galactosidase enzyme of H. lacusprofundi expressed in Halobacterium sp. NRC-1, 50% inhibition of catalytic activity was observed at 0.88 M NaClO4 and 0.13 M Mg(ClO4)2. Magnesium ions were a more potent inhibitor of the enzyme than of cell growth. Steady-state kinetic analysis showed that Mg(ClO4)2 acts as a mixed inhibitor (KI = 0.04 M), with magnesium alone being a competitive inhibitor (KI = 0.3 M) and perchlorate alone acting as a very weak noncompetitive inhibitor (KI = 2 M). Based on the estimated concentrations of perchlorate salts on the surface of Mars, our results show that neither sodium nor magnesium perchlorates would significantly inhibit growth and enzyme activity of halophiles. This is the first study of perchlorate effects on a purified enzyme. Key Words: Halophilic archaea-Perchlorate-Enzyme inhibition-Magnesium. Astrobiology 18, 412-418.

Haloarchaea: worth exploring for their biotechnological potential.

Halophilic archaea are unique microorganisms adapted to survive under high salt conditions and biomolecules produced by them may possess unusual properties. Haloarchaeal metabolites are stable at high salt and temperature conditions that are useful for industrial applications. Proteins and enzymes of this group of archaea are functional under salt concentrations at which bacterial counterparts fail to be active. Such properties makes haloarchaeal enzymes suitable for salt-based applications and their use under dehydrating conditions. For example, bacteriorhodopsin or the purple membrane protein present in halophilic archaea has the most recognizable applications in photoelectric devices, artificial retinas, holograms etc. Haloarchaea are also useful for bioremediation of polluted hypersaline areas. Polyhydroxyalkanoates and exopolysccharides produced by these microorganisms are biodegradable and have the potential to replace commercial non-degradable plastics and polymers. Moreover, halophilic archaea have excellent potential to be used as drug delivery systems and for nanobiotechnology by virtue of their gas vesicles and S-layer glycoproteins. Despite of possible applications of halophilic archaea, laboratory-to-industrial transition of these potential candidates is yet to be established.

Transcriptome analysis of Haloquadratum walsbyi: vanity is but the surface.

BACKGROUND: Haloquadratum walsbyi dominates saturated thalassic lakes worldwide where they can constitute up to 80-90% of the total prokaryotic community. Despite the abundance of the enigmatic square-flattened cells, only 7 isolates are currently known with 2 genomes fully sequenced and annotated due to difficulties to grow them under laboratory conditions. We have performed a transcriptomic analysis of one of these isolates, the Spanish strain HBSQ001 in order to investigate gene transcription under light and dark conditions. RESULTS: Despite a potential advantage for light as additional source of energy, no significant differences were found between light and dark expressed genes. Constitutive high gene expression was observed in genes encoding surface glycoproteins, light mediated proton pumping by bacteriorhodopsin, several nutrient uptake systems, buoyancy and storage of excess carbon. Two low expressed regions of the genome were characterized by a lower codon adaptation index, low GC content and high incidence of hypothetical genes. CONCLUSIONS: Under the extant cultivation conditions, the square hyperhalophile devoted most of its transcriptome towards processes maintaining cell integrity and exploiting solar energy. Surface glycoproteins are essential for maintaining the large surface to volume ratio that facilitates light and organic nutrient harvesting whereas constitutive expression of bacteriorhodopsin warrants an immediate source of energy when light becomes available.

Discovery of anaerobic lithoheterotrophic haloarchaea, ubiquitous in hypersaline habitats.

Hypersaline anoxic habitats harbour numerous novel uncultured archaea whose metabolic and ecological roles remain to be elucidated. Until recently, it was believed that energy generation via dissimilatory reduction of sulfur compounds is not functional at salt saturation conditions. Recent discovery of the strictly anaerobic acetotrophic Halanaeroarchaeum compels to change both this assumption and the traditional view on haloarchaea as aerobic heterotrophs. Here we report on isolation and characterization of a novel group of strictly anaerobic lithoheterotrophic haloarchaea, which we propose to classify as a new genus Halodesulfurarchaeum. Members of this previously unknown physiological group are capable of utilising formate or hydrogen as electron donors and elemental sulfur, thiosulfate or dimethylsulfoxide as electron acceptors. Using genome-wide proteomic analysis we have detected the full set of enzymes required for anaerobic respiration and analysed their substrate-specific expression. Such advanced metabolic plasticity and type of respiration, never seen before in haloarchaea, empower the wide distribution of Halodesulfurarchaeum in hypersaline inland lakes, solar salterns, lagoons and deep submarine anoxic brines. The discovery of this novel functional group of sulfur-respiring haloarchaea strengthens the evidence of their possible role in biogeochemical sulfur cycling linked to the terminal anaerobic carbon mineralisation in so far overlooked hypersaline anoxic habitats.

Global transcriptome analysis of Halolamina sp. to decipher the salt tolerance in extremely halophilic archaea.

Extremely halophilic archaea survive in the hypersaline environments such as salt lakes or salt mines. Therefore, these microorganisms are good sources to investigate the molecular mechanisms underlying the tolerance to high salt concentrations. In this study, a global transcriptome analysis was conducted in an extremely halophilic archaeon, Halolamina sp. YKT1, isolated from a salt mine in Turkey. A comparative RNA-seq analysis was performed using YKT1 isolate grown either at 2.7M NaCl or 5.5M NaCl concentrations. A total of 2149 genes were predicted to be up-regulated and 1638 genes were down-regulated in the presence of 5.5M NaCl. The salt tolerance of Halolamina sp. YKT1 involves the up-regulation of genes related with membrane transporters, CRISPR-Cas systems, osmoprotectant solutes, oxidative stress proteins, and iron metabolism. On the other hand, the genes encoding the proteins involved in DNA replication, transcription, translation, mismatch and nucleotide excision repair were down-regulated. The RNA-seq data were verified for seven up-regulated genes as well as six down-regulated genes via qRT-PCR analysis. This comprehensive transcriptome analysis showed that the halophilic archaeon canalizes its energy towards keeping the intracellular osmotic balance minimizing the production of nucleic acids and peptides.

Salt-inducible Protein Splicing in cis and trans by Inteins from Extremely Halophilic Archaea as a Novel Protein-Engineering Tool.

Intervening protein sequences (inteins) from extremely halophilic haloarchaea can be inactive under low salinity but could be activated by increasing the salt content to a specific concentration for each intein. The halo-obligatory inteins confer high solubility under both low and high salinity conditions. We showed the broad utility of salt-dependent protein splicing in cis and trans by demonstrating backbone cyclization, self-cleavage for purification, and scarless protein ligation for segmental isotopic labeling. Artificially split MCM2 intein derived from Halorhabdus utahensis remained highly soluble and was capable of protein trans-splicing with excellent ligation kinetics by reassembly under high salinity conditions. Importantly, the MCM2 intein has the active site residue of Ser at the +1 position, which remains in the ligated product, instead of Cys as found in many other efficient split inteins. Since Ser is more abundant than Cys in proteins, the novel split intein could widen the applications of segmental labeling in protein NMR spectroscopy and traceless protein ligation by exploiting a Ser residue in the native sequences as the +1 position of the MCM2 intein. The split halo-obligatory intein was successfully used to demonstrate the utility in NMR investigation of intact proteins by producing segmentally isotope-labeled intact TonB protein from Helicobacter pylori.

Crystal structure of Halobacterium salinarum halorhodopsin with a partially depopulated primary chloride-binding site.

The transmembrane pump halorhodopsin in halophilic archaea translocates chloride ions from the extracellular to the cytoplasmic side upon illumination. In the ground state a tightly bound chloride ion occupies the primary chloride-binding site (CBS I) close to the protonated Schiff base that links the retinal chromophore to the protein. The light-triggered trans-cis isomerization of retinal causes structural changes in the protein associated with movement of the chloride ion. In reverse, chemical depletion of CBS I in Natronomonas pharaonis halorhodopsin (NpHR) through deprotonation of the Schiff base results in conformational changes of the protein: a state thought to mimic late stages of the photocycle. Here, crystals of Halobacterium salinarum halorhodopsin (HsHR) were soaked at high pH to provoke deprotonation of the Schiff base and loss of chloride. The crystals changed colour from purple to yellow and the occupancy of CBS I was reduced from 1 to about 0.5. In contrast to NpHR, this chloride depletion did not cause substantial conformational changes in the protein. Nevertheless, two observations indicate that chloride depletion could eventually result in structural changes similar to those found in NpHR. Firstly, the partially chloride-depleted form of HsHR has increased normalized B factors in the region of helix C that is close to CBS I and changes its conformation in NpHR. Secondly, prolonged soaking of HsHR crystals at high pH resulted in loss of diffraction. In conclusion, the conformation of the chloride-free protein may not be compatible with this crystal form of HsHR despite a packing arrangement that hardly restrains helices E and F that presumably move during ion transport.

Oil removal and effects of spilled oil on active microbial communities in close to salt-saturation brines.

Abiotic and biotic processes associated with the degradation of a light petroleum in brines close to the salt-saturation (~31 %) and the effect of labile organic matter (LOM) supply (casaminoacids/citrate; 0.2 and 0.1 % w/v, respectively) were followed during an incubation of 30 days. After 4-week incubation at 40 °C under light/dark cycles, a 24 % of abiotic degradation was observed in untreated brines. The stimulation of native brines community with LOM addition allowed an additional 12.8 % oil attenuation due to biodegradation processes. Successional changes in the active microbial community structure due to the oil contamination (16S rRNA DGGE approach) showed the selection of one phylotype affiliated to Salinibacter and the disappearance of Haloquadratum walsbyi in untreated brines. In LOM-amended microcosms, phylotypes related to Salinibacter, Haloarcula, Haloterrigena and Halorhabdus were selected. An effect of hydrocarbon contamination was only observed in the bacterial community with the inhibition of two dominant proteobacterial phylotypes. This study further confirms that short-term and moderate oil biodegradation is possible in LOM-stimulated brines. Biodegradation should be much more reduced under in situ conditions. Self-cleaning capacities of close to saturation hypersaline lakes appears, therefore very limited compared to non-extreme haline environments.

Lentibacillus kimchii sp. nov., an extremely halophilic bacterium isolated from kimchi, a Korean fermented vegetable.

A Gram-positive, aerobic, non-motile and extremely halophilic bacterial strain, designated K9(T), was isolated from kimchi, a Korean fermented food. The strain was observed as endospore-forming rod-shaped cells showing oxidase and catalase activity. It was found to grow at 10.0-30.0 % (w/v) NaCl (optimum, 15.0-20.0 %), pH 7.0-8.0 (optimum, pH 7.5) and 15-40 °C (optimum, 30 °C). The polar lipids of strain K9(T) were identified as phosphatidylglycerol, three unidentified phospholipids and an unidentified glycolipid. The isoprenoid quinone was identified as menaquinone-7. The major cellular fatty acids (>20 % of the total) were found to be anteisio-C15:0 and anteisio-C17:0. The cell wall peptidoglycan composition was determined to contain meso-diaminopimelic acid. The G + C content of genomic DNA was determined to be 48.2 mol %. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the isolated strain is closely related to Lentibacillus salinarum AHS-1(T) (96.7 % sequence similarity). Based on its phenotypic, chemotaxonomic and phylogenetic data, strain K9(T) is considered to represent a novel species of the genus Lentibacillus, for which the name Lentibacillus kimchii sp. nov., is proposed. The type strain is K9(T) (=KACC 18490(T) = JCM 30234(T)).