The parasitic lifestyle of an archaeal symbiont.

DPANN archaea are a diverse group of microorganisms characterised by small cells and reduced genomes. To date, all cultivated DPANN archaea are ectosymbionts that require direct cell contact with an archaeal host species for growth and survival. However, these interactions and their impact on the host species are poorly understood. Here, we show that a DPANN archaeon (Candidatus Nanohaloarchaeum antarcticus) engages in parasitic interactions with its host (Halorubrum lacusprofundi) that result in host cell lysis. During these interactions, the nanohaloarchaeon appears to enter, or be engulfed by, the host cell. Our results provide experimental evidence for a predatory-like lifestyle of an archaeon, suggesting that at least some DPANN archaea may have roles in controlling host populations and their ecology.

The biosynthesis mechanism of bacterioruberin in halophilic archaea revealed by genome and transcriptome analysis.

Halophilic archaea are promising microbial cell factories for bacterioruberin (BR) production. BR is a natural product with multi-bioactivities, allowing potential application in many fields. In the previous work, a haloarchaeon Halorubrum sp. HRM-150 with a high proportion of BR (about 85%) was isolated, but the low yield impeded its large-scale production. This work figured out BR synthesis characteristics and mechanisms, and proposed strategies for yield improvement. First, glucose (10 g/L) and tryptone (15 g/L) were tested to be better sources for BR production. Besides, the combination of glucose and starch achieved the diauxic growth, and the biomass and BR productivity increased by 85% and 54% than using glucose. Additionally, this work first proposed the BR synthesis pattern, which differs from that of other carotenoids. As a structural component of cell membranes, the BR synthesis is highly coupled with growth, which was most active in the logarithm phase. Meanwhile, the osmotic down shock at the logarithm phase could increase the BR productivity without sacrificing the biomass. Moreover, the de-novo pathway for BR synthesis with a key gene of lyeJ, and its competitive pathways (notably tetraether lipids and retinal) were revealed through genome, transcriptome, and osmotic down shock. Therefore, the BR yield is expected to be improved through mutant construction, such as the overexpression of key gene lyeJ and the knockout of competitive genes, which need to be further explored. The findings will contribute to a better understanding of the metabolism mechanism in haloarchaea and the development of haloarchaea as microbial cell factories. IMPORTANCE: Recent studies have revealed that halophilic microorganism is a promising microbial factory for the next-generation industrialization. Among them, halophilic archaea are advantageous as microbial factories due to their low contamination risk and low freshwater consumption. The halophilic archaea usually accumulate long chain C50 carotenoids, which are barely found in other organisms. Bacterioruberin (BR), the major C50 carotenoid, has multi-bioactivities, allowing potential application in food, cosmetic, and biomedical industries. However, the low yield impedes its large-scale application. This work figured out the BR synthesis characteristics and mechanism, and proposed several strategies for BR yield improvement, encouraging halophilic archaea to function as microbial factories for BR production. Meanwhile, the archaea have special evolutionary status and unique characteristics in taxonomy, the revelation of BR biosynthesis mechanism is beneficial for a better understanding of archaea.

Using the pan-genomic framework for the discovery of genomic islands in the haloarchaeon Halorubrum ezzemoulense.

In this study, we use pan-genomics to characterize the genomic variability of the widely dispersed halophilic archaeal species Halorubrum ezzemoulense (Hez). We include a multi-regional sampling of newly sequenced, high-quality draft genomes. The pan-genome graph of the species reveals 50 genomic islands that represent rare accessory genetic capabilities available to members. Most notably, we observe rearrangements that have led to the insertion/recombination/replacement of mutually exclusive genomic islands in equivalent genome positions ("homeocassettes"). These conflicting islands encode for similar functions, but homologs from islands located between the same core genes exhibit high divergence on the amino acid level, while the neighboring core genes are nearly identical. Both islands of a homeocassette often coexist in the same geographic location, suggesting that either island may be beyond the reach of selective sweeps and that these loci of divergence between Hez members are maintained and persist long term. This implies that subsections of the population have different niche preferences and rare metabolic capabilities. After an evaluation of the gene content in the homeocassettes, we speculate that these islands may play a role in the speciation, niche adaptability, and group selection dynamics in Hez. Though homeocassettes are first described in this study, similar replacements and divergence of genes on genomic islands have been previously reported in other Haloarchaea and distantly related Archaea, suggesting that homeocassettes may be a feature in a wide range of organisms outside of Hez.IMPORTANCEThis study catalogs the rare genes discovered in strains of the species Halorubrum ezzemoulense (Hez), an obligate halophilic archaeon, through the perspective of its pan-genome. These rare genes are often found to be arranged on islands that confer metabolic and transport functions and contain genes that have eluded previous studies. The discovery of divergent, but homologous islands occupying equivalent genome positions ("homeocassettes") in different genomes, reveals significant new information on genome evolution in Hez. Homeocassette pairs encode for similar functions, but their dissimilarity and distribution imply high rates of recombination, different specializations, and niche preferences in Hez. The coexistence of both islands of a homeocassette pair in multiple environments demonstrates that both islands are beyond the reach of selective sweeps and that these genome content differences between strains persist long term. The switch between islands through recombination under different environmental conditions may lead to a greater range of niche adaptability in Hez.

Selective lipid recruitment by an archaeal DPANN symbiont from its host.

The symbiont Ca. Nanohaloarchaeum antarcticus is obligately dependent on its host Halorubrum lacusprofundi for lipids and other metabolites due to its lack of certain biosynthetic genes. However, it remains unclear which specific lipids or metabolites are acquired from its host, and how the host responds to infection. Here, we explored the lipidome dynamics of the Ca. Nha. antarcticus - Hrr. lacusprofundi symbiotic relationship during co-cultivation. By using a comprehensive untargeted lipidomic methodology, our study reveals that Ca. Nha. antarcticus selectively recruits 110 lipid species from its host, i.e., nearly two-thirds of the total number of host lipids. Lipid profiles of co-cultures displayed shifts in abundances of bacterioruberins and menaquinones and changes in degree of bilayer-forming glycerolipid unsaturation. This likely results in increased membrane fluidity and improved resistance to membrane disruptions, consistent with compensation for higher metabolic load and mechanical stress on host membranes when in contact with Ca. Nha. antarcticus cells. Notably, our findings differ from previous observations of other DPANN symbiont-host systems, where no differences in lipidome composition were reported. Altogether, our work emphasizes the strength of employing untargeted lipidomics approaches to provide details into the dynamics underlying a DPANN symbiont-host system.

Optimization of bacterioruberin production from Halorubrum ruber and assessment of its antioxidant potential.

Haloarchaea produce bacterioruberin, a major C50 carotenoid with antioxidant properties that allow for its potential application in the food, cosmetic, and pharmaceutical industries. This study aimed to optimize culture conditions for total carotenoid, predominantly comprising bacterioruberin, production using Halorubrum ruber MBLA0099. A one-factor-at-a-time and statistically-based experimental design were applied to optimize the culture conditions. Culture in the optimized medium caused an increase in total carotenoid production from 0.496 to 1.966 mg L- 1 Maximal carotenoid productivity was achieved in a 7-L laboratory-scale fermentation and represented a 6.05-fold increase (0.492 mg L-1 d-1). The carotenoid extracts from strain MBLA0099 exhibited a 1.8-10.3-fold higher antioxidant activity in vitro, and allowed for a higher survival rate of Caenorhabditis elegans under oxidative stress conditions. These results demonstrated that Hrr. ruber MBLA0099 has significant potential as a haloarchaon for the commercial production of bacterioruberin.

Carotenoid characterization, fatty acid profiles, and antioxidant activities of haloarchaeal extracts.

Microorganisms that can survive in saline environments, known as halotolerant or halophilic organisms, have a wide range of current and potential uses in biotechnology. In this study, it was aimed to determine the carotenoids of halophilic archaea strains isolated from the brine samples taken from different points of Salt Lake (Turkey) and determine the antioxidant activities of their carotenoids. To identify the halophilic archaea strains, they were cultivated in MAM JCM 168 medium and subjected to antibiotic susceptibility, fatty acid, two-dimensional and three-dimensional imaging by scanning electron microscopy and atomic force microscopy, biochemical and phylogenetic assays. The findings show that five different halophilic archaea strains have been identified as Halorubrum lipolyticum, Halorubrum sodomense, Haloarcula salaria, Halorubrum chaoviator, and Haloarcula japonica with 98% and above similarity ratio. The main fatty acids of all haloarchaeal strains were octadecanoic acid (C18:0) and palmitic acid (C16:0). The major carotenoid of the species was determined as all-trans bacterioruberin, and different carotenoid types such as lycopene, ß-carotene, and 2-isopentenyl-3,4-dehydrorodopin were found as well as bacterioruberin isomers. The antioxidant activities of carotenoids extracted from the species were analyzed by the 2,2-diphenyl-1-picrylhydrazyl radical scavenging method and the extracts showed antioxidant activity statistically significantly higher than ascorbic acid and butylated hydroxytoluene as reference products (p < 0.05).

Halorubrum hochsteinianum sp. nov., an ancient haloarchaeon from a natural experiment.

A single extremely halophilic strain was isolated from salt brine produced when a fresh water lake flooded a large salt mine located beneath the lake. The water that entered this mine contained less than 0.34 M NaCl, but over time, this sealed brine became saturated by Cenozoic age salt (121-125 million-year BCE). The isolated strain requires at least 1.7 M NaCl for survival and grows optimally in 3.1 M NaCl. Therefore, it could not have survived or been present in the waters that flooded this salt mine. The strain grows at a pH range from 6.5 to 9.0 and has a wide tolerance to temperatures from 25 ℃ to at least 60 ℃. The comparison of 16S rRNA and rpoB' genes revealed that strain 1-13-28T is related to Halorubrum tebenquichense DSM 14210T showing 98.6% and 98.1% similarities, respectively. Phylogenetic analyses based on 16S rRNA, rpoB' genes and 122 concatenated archaeal genes show that the strain 1-13-28T consistently forms a cluster with Halorubrum tebenquichense of the genus Halorubrum. Strain 1-13-28T contained sulfated mannosyl glucosyl diether, and the polar lipid profile was identical to those of most Halorubrum species. Based on the overall combination of physiological, phylogenetic, polar lipids and phylogenomic characteristics, strain 1-13-28T (= ATCC 700083T = CGMCC 1.62627T) represents a newly identified species within the genus Halorubrum for which the name Halorubrum hochsteinianum is proposed.

Genomic analyses reveal a low-temperature adapted clade in Halorubrum, a widespread haloarchaeon across global hypersaline environments.

BACKGROUND: Cold-adapted archaea have diverse ecological roles in a wide range of low-temperature environments. Improving our knowledge of the genomic features that enable psychrophiles to grow in cold environments helps us to understand their adaptive responses. However, samples from typical cold regions such as the remote Arctic and Antarctic are rare, and the limited number of high-quality genomes available leaves us with little data on genomic traits that are statistically associated with cold environmental conditions. RESULTS: In this study, we examined the haloarchaeal genus Halorubrum and defined a new clade that represents six isolates from polar and deep earth environments ('PD group' hereafter). The genomic G + C content and amino acid composition of this group distinguishes it from other Halorubrum and the trends are consistent with the established genomic optimization of psychrophiles. The cold adaptation of the PD group was further supported by observations of increased flexibility of proteins encoded across the genome and the findings of a growth test. CONCLUSIONS: The PD group Halorubrum exhibited denser genome packing, which confers higher metabolic potential with constant genome size, relative to the reference group, resulting in significant differences in carbon, nitrogen and sulfur metabolic patterns. The most marked feature was the enrichment of genes involved in sulfur cycling, especially the production of sulfite from organic sulfur-containing compounds. Our study provides an updated view of the genomic traits and metabolic potential of Halorubrum and expands the range of sources of cold-adapted haloarchaea.

Influence of N-Glycosylation on Virus-Host Interactions in Halorubrum lacusprofundi.

N-glycosylation is a post-translational modification of proteins that occurs across all three domains of life. In Archaea, N-glycosylation is crucial for cell stability and motility, but importantly also has significant implications for virus-host interactions. While some archaeal viruses present glycosylated proteins or interact with glycosylated host proteins, the direct influence of N-glycosylation on archaeal virus-host interactions remains to be elucidated. In this study, we generated an N-glycosylation-deficient mutant of Halorubrum lacusprofundi, a halophilic archaeon commonly used to study cold adaptation, and examined the impact of compromised N-glycosylation on the infection dynamics of two very diverse viruses. While compromised N-glycosylation had no influence on the life cycle of the head-tailed virus HRTV-DL1, we observed a significant effect on membrane-containing virus HFPV-1. Both intracellular genome numbers and extracellular virus particle numbers of HFPV-1 were increased in the mutant strain, which we attribute to instability of the surface-layer which builds the protein envelope of the cell. When testing the impact of compromised N-glycosylation on the life cycle of plasmid vesicles, specialized membrane vesicles that transfer a plasmid between host cells, we determined that plasmid vesicle stability is strongly dependent on the host glycosylation machinery. Our study thus provides important insight into the role of N-glycosylation in virus-host interactions in Archaea, while pointing to how this influence strongly differs amongst various viruses and virus-like elements.

Optimization of C50 Carotenoids Production by Open Fermentation of Halorubrum sp. HRM-150.

C50 carotenoids, as unique bioactive molecules, have many biological properties, including antioxidant, anticancer, and antibacterial activity, and have a wide range of potential uses in the food, cosmetic, and biomedical industries. The majority of C50 carotenoids are produced by the sterile fermentation of halophilic archaea. This study aims to look at more cost-effective and manageable ways of producing C50 carotenoids. The basic medium, carbon source supplementation, and optimal culture conditions for Halorubrum sp. HRM-150 C50 carotenoids production by open fermentation were examined in this work. The results indicated that Halorubrum sp. HRM-150 grown in natural brine medium grew faster than artificial brine medium. The addition of glucose, sucrose, and lactose (10 g/L) enhanced both biomass and carotenoids productivity, with the highest level reaching 4.53 ± 0.32 µg/mL when glucose was added. According to the findings of orthogonal studies based on the OD600 and carotenoids productivity, the best conditions for open fermentation were salinity 20-25%, rotation speed 150-200 rpm, and pH 7.0-8.2. The up-scaled open fermentation was carried out in a 7 L medium under optimum culture conditions. At 96 h, the OD600 and carotenoids productivity were 9.86 ± 0.51 (dry weight 10.40 ± 1.27 g/L) and 7.31 ± 0.65 µg/mL (701.40 ± 21.51 µg/g dry weight, respectively). When amplified with both universal bacterial primer and archaeal primer in the open fermentation, Halorubrum remained the dominating species, indicating that contamination was kept within an acceptable level. To summarize, open fermentation of Halorubrum is a promising method for producing C50 carotenoids.

Genomic and physiological analysis of C50 carotenoid-producing novel Halorubrum ruber sp. nov.

A novel haloarchaeal species designated as MBLA0099T was isolated from seawater near Yeongheung Island. Cells were Gram-negative, non-motile, red-pigmented, and rod-shaped. They grew at 10-45°C, within pH 5.5-9.0, and between 7.5% and 30% NaCl concentrations. Cells were able to grow without Mg2+ and were lysed in distilled water. The size of the whole-genome and G + C content of DNA was 3.02 Mb and 68.9 mol%, respectively. Phylogenetic analysis shows that the strain MBLA0099T belongs to the genus Halorubrum. The average nucleotide and amino acid identity, and in silico DNA-DNA hybridization values were below the species delineation threshold. Pan-genomic analysis revealed that 3.2% of all genes present in strain MBLA0099T were unique to the strain. The red carotenoid produced by strain MBLA0099T was subjected to spectrometric and chromatographic analyses and confirmed to be bacterioruberin as C50 carotenoid. Mevalonic acid, terpenoid backbone, and carotenoid biosynthesis pathway were annotated for strain MBLA0099T. The C50 carotenoid production by strain MBLA0099T was also enhanced under various stress conditions including relatively netural pH, high oxidative and salinity conditions. Additionally, the strain MBLA0099T-derived bacterioruberin showed the antioxidant activity with EC50 value of 12.29 µg/ml, based on the evaluation of DPPH free radical scavenging activity. The present study would be the first report on the identification of C50 carotenoid from the strain MBLA0099T representing a novel species of the genus Halorubrum, for which the name Halorubrum ruber sp. nov. is proposed. The typestrain used was MBLA0099T (= KCTC 4296T = JCM 34701T).

Genomic features of a new head-tail halovirus VOLN27B infecting a Halorubrum strain.

Relatively few viruses infecting haloarchaea (haloviruses) have been reported. In this study, the genome sequence of VOLN27B, a recently described archaeal tailed virus (arTV) with a myovirus morphotype was described, along with the sequence of its host, Halorubrum spp. LN27. Halovirus VOLN27B contains a linear, dsDNA genome of 76,891 bp which is predicted to encode 109 proteins and four tRNAs (tRNAThr, tRNAArg, tRNAGly and tRNAAsn). The DNA G + C content of VOLN27B genome is 56.1 mol%, nearly 10% lower than that of its host strain. A 315 bp LTR (long terminal repeat) was detected in the genome. The genome of its host strain LN27 was 3,301,211 bp (chromosome and 1 plasmid) with a DNA G + C content of 68.3 mol% and 3142 annotated protein coding genes. At least two hypothetical proviruses were detected in the genome. It lacked a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) locus. Sequence similarity and phylogenetic tree reconstructions placed it within the genus Halorubrum as a potential new species. VOLN27B exhibits a distinct difference in the frequency of codon usage against its host strain Halorubrum sp. LN27. The organization of VOLN27B genome shows remarkable synteny and amino acid sequence similarity to the genomes and predicted proteins of HF1-like haloviruses (genus Haloferacalesvirus) and a provirus in the genome of Halorubrum depositum Y78. VOLN27B and its host Halorubrum sp. LN27 comprise a new virus-host system from a hypersaline ecosystem and can be used to further understand the novel biology at extreme salt concentration.

The Osmoprotectant Switch of Potassium to Compatible Solutes in an Extremely Halophilic Archaea Halorubrum kocurii 2020YC7.

The main osmoadaptive mechanisms of extremely halophilic archaea include the "salt-in" strategy and the "compatible solutes" strategy. Here we report the osmoadaptive mechanism of an extremely halophilic archaea H. kocurii 2020YC7, isolated from a high salt environment sample. Genomic data revealed that strain 2020YC7 harbors genes trkA, trkH, kch for K+ uptake, kefB for K+ output, treS for trehalose production from polysaccharide, and betaine/carnitine/choline transporter family gene for glycine betaine uptake. Strain 2020YC7 could accumulate 8.17 to 28.67 µmol/mg protein K+ in a defined medium, with its content increasing along with the increasing salinity from 100 to 200 g/L. When exogenous glycine betaine was added, glycine betaine functioned as the primary osmotic solute between 200 and 250 g/L NaCl, which was accumulated up to 15.27 mg/mg protein in 2020YC7 cells. RT-qPCR results completely confirmed these results. Notably, the concentrations of intracellular trehalose decreased from 5.26 to 2.61 mg/mg protein as the NaCl increased from 50 to 250 g/L. In combination with this result, the transcript level of gene treS, which catalyzes the production of trehalose from polysaccharide, was significantly up-regulated at 50-100 g/L NaCl. Therefore, trehalose does not act as an osmotic solute at high NaCl concentrations (more than 100 g/L) but at relatively low NaCl concentrations (50-100 g/L). And we propose that the degradation of cell wall polysaccharide, as a source of trehalose in a low-salt environment, may be one of the reasons for the obligate halophilic characteristics of strain 2020YC7.

Halorubrum pleomorphic virus-6 Membrane Fusion Is Triggered by an S-Layer Component of Its Haloarchaeal Host.

(1) Background: Haloarchaea comprise extremely halophilic organisms of the Archaea domain. They are single-cell organisms with distinctive membrane lipids and a protein-based cell wall or surface layer (S-layer) formed by a glycoprotein array. Pleolipoviruses, which infect haloarchaeal cells, have an envelope analogous to eukaryotic enveloped viruses. One such member, Halorubrum pleomorphic virus 6 (HRPV-6), has been shown to enter host cells through virus-cell membrane fusion. The HRPV-6 fusion activity was attributed to its VP4-like spike protein, but the physiological trigger required to induce membrane fusion remains yet unknown. (2) Methods: We used SDS-PAGE mass spectroscopy to characterize the S-layer extract, established a proteoliposome system, and used R18-fluorescence dequenching to measure membrane fusion. (3) Results: We show that the S-layer extraction by Mg2+ chelating from the HRPV-6 host, Halorubrum sp. SS7-4, abrogates HRPV-6 membrane fusion. When we in turn reconstituted the S-layer extract from Hrr. sp. SS7-4 onto liposomes in the presence of Mg2+, HRPV-6 membrane fusion with the proteoliposomes could be readily observed. This was not the case with liposomes alone or with proteoliposomes carrying the S-layer extract from other haloarchaea, such as Haloferax volcanii. (4) Conclusions: The S-layer extract from the host, Hrr. sp. SS7-4, corresponds to the physiological fusion trigger of HRPV-6.

Halorubrum salinarum sp. nov., an extremely halophilic archaeon isolated from a saturated brine pond of a saltern.

A novel extremely halophilic archaeon, strain RHB-CT, was isolated from a saturated brine pond of a solar saltern in Bolinao, Pangasinan, Philippines. Colonies were orange-red-pigmented, smooth, convex and round on a solid modified growth medium containing 25 % (w/v) of total salts. Cells of strain RHB-CT on the solid modified growth medium were ovoid-shaped (0.89-2.66 µm long), while the cells in a liquid modified growth medium were rod-shaped (1.53-5.65 µm long and 0.45-1.03 µm wide). The strain was Gram-stain-negative, motile and strictly aerobic. Strain RHB-CT grew with NaCl concentrations ranging from 10 to 30 % (w/v; optimum, 20-25 %), at pH 6.5-8.5 (optimum, pH 7.0-7.5) and at 20-55 °C (optimum, 40-45 °C). Furthermore, the strain grew even in the absence of Mg2+; however, when supplemented with Mg2+, growth was observed optimally at 0.2-0.4 M Mg2+. The 16S rRNA gene phylogeny inferred that the strain is a member of the genus Halorubrum and was related to Halorubrum xinjiangense CGMCC 1.3527T (99.0 %), Halorubrum sodomense DSM 3755T (98.8 %), Halorubrum coriense Ch2T (98.8 %), Halorubrum trapanicum NRC 34021T (98.4 %) and Halorubrum distributum JCM 9100T (98.1 %). The rpoB' gene sequences also showed that strain RHB-CT is related to Hrr. xinjiangense JCM 12388T (97.1 %), Hrr. distributum JCM 9100T (97.1 %), Hrr. coriense JCM 9275T (96.5 %), Hrr. californiense JCM 14715T (96.5 %), Hrr. trapanicum JCM 10477T (96.3%), Hrr. sodomense JCM 8880T (96.2%) and Hrr. tebenquichense DSM 14210T (95.6 %). The DNA G+C content of strain RHB-CT was 68.7 mol% (genome). Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strain RHB-CT and the closely related species of Halorubrum were below 40 and 90 %, respectively, which are far below the thresholds to delineate a new species. The polar lipids of strain RHB-CT were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulphate and sulfated mannosyl glycosyl diether. Based on dDDH and ANI values, and the significant morphological and physiological differences from known taxa, it is hereby suggested that strain RHB-CT represents a novel species of the genus Halorubrum, for which the name Halorubrum salinarum sp. nov. is proposed. The type strain is RHB-CT (=KCTC 4274T=CMS 2103T).

Two states of a light-sensitive membrane protein captured at room temperature using thin-film sample mounts.

Room-temperature diffraction methods are highly desirable for dynamic studies of biological macromolecules, since they allow high-resolution structural data to be collected as proteins undergo conformational changes. For crystals grown in lipidic cubic phase (LCP), an extruder is commonly used to pass a stream of microcrystals through the X-ray beam; however, the sample quantities required for this method may be difficult to produce for many membrane proteins. A more sample-efficient environment was created using two layers of low X-ray transmittance polymer films to mount crystals of the archaerhodopsin-3 (AR3) photoreceptor and room-temperature diffraction data were acquired. By using transparent and opaque polymer films, two structures, one corresponding to the desensitized, dark-adapted (DA) state and the other to the ground or light-adapted (LA) state, were solved to better than 1.9 Šresolution. All of the key structural features of AR3 were resolved, including the retinal chromophore, which is present as the 13-cis isomer in the DA state and as the all-trans isomer in the LA state. The film-sandwich sample environment enables diffraction data to be recorded at room temperature in both illuminated and dark conditions, which more closely approximate those in vivo. This simple approach is applicable to a wide range of membrane proteins crystallized in LCP and light-sensitive samples in general at synchrotron and laboratory X-ray sources.

Extremophile enzyme optimization for low temperature and high salinity are fundamentally incompatible.

The evolutionary mechanisms behind cold and high-saline co-adaptation of proteins are not thoroughly understood. To explore how enzymes evolve in response to multiple environmental pressures we developed a novel in silico method to model the directed evolution of proteins, the Protein Evolution Parameter Calculator (PEPC). PEPC carries out single amino acid substitutions that lead to improvements in the selected user-defined parameters. To investigate the evolutionary relationship between increased flexibility and decreased isoelectric point, which are presumed indicators of cold and saline adaptation in proteins, we applied PEPC to a subset of core haloarchaea orthologous group (cHOG) proteins from the mesophilic Halobacterium salinarum NRC-1 and cold-tolerant Halorubrum lacusprofundi strain ATCC 49239. The results suggest that mutations that increase flexibility will also generally increase isoelectric point. These findings suggest that enzyme adaptation to low temperature and high salinity might be evolutionarily counterposed based on the structural characteristics of probable amino acid mutations. This may help to explain the apparent lack of truly psychrophilic halophiles in nature, and why microbes adapted to polar hypersaline environments typically have mesophilic temperature optima. A better understanding of protein evolution to extremely cold and salty conditions will aid in our understanding of where and how life is distributed on Earth and in our solar system.

Halorubrum salipaludis sp. nov., isolated from the saline-alkaline soil.

A novel extremely halophilic archaeon, designated WN019T, was isolated from the natural saline-alkali wetland soil of Binhai new district, Tianjin, China. Cells of WN019T were aerobic, motile, and pleomorphic rod-shaped, 0.5-0.8 µm in width and 2.0-2.5 µm in length, and the growth occurred optimally at 33-37 °C, pH 7.5-8.0, and in the presence of 15.0-20.0% (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequence comparison showed that the isolate belonged to the genus Halorubrum and exhibited moderate sequence similarity of 97.8% to Halorubrum saccharovorum JCM 8865T. The major respiratory quinones of strain WN019T were MK-8 and MK-8 (H2), and the major polar lipids were glycolipid (GL), phospholipid (PL), phosphatidylglycerol-sulphate (PGS), phosphatidylglycerol (PG) and phosphatidylglycerol-phosphate-methyl ester (Me-PGP). The DNA G + C content of the strain was 67.4 mol%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) value based on whole genome sequences of strain WN019T and Halorubrum saccharovorum JCM 8865T were 87.5% and 35.4%, respectively. Phenotypic, chemotaxonomic, phylogenetic, and genomic analyses suggested that strain WN019T represents a novel species of the genus Halorubrum, for which the name Halorubrum salipaludis sp. nov. is proposed. The type strain is WN019T (= KCTC 4269T = ACCC 19977T).

Photocycle dynamics of the Archaerhodopsin 3 based fluorescent voltage sensor Archon2.

The retinal photocycle dynamics of the fluorescent voltage sensor Archon2 in pH 8 Tris buffer was studied. Archon2 is a mutant of Archaerhodopsin 3 (Arch) from Halorubrum sodomense obtained by a robotic multidimensional directed evolution approach (Archon2 = Arch T56P-P60S-T80P-D95H-T99S-T116I-F161V-T183I-L197I-A225C). The samples were photo-excited to the first absorption band of the protonated retinal Schiff base (PRSB) Ret_586 (absorption maximum at λmax = 586 nm, excitation wavelengths λexc = 590 nm and 632.8 nm). The photocycle dynamics were studied by recording absorption spectra during light exposure and after light exposure. Ret_586 photoisomerized to Ret_535 (main component) and Ret_485 (minor component). Ret_535 backward photoisomerized to Ret_586 in light-adapted state (named Ret_586la) and partly deprotonated to neutral retinal Schiff base (RSB) Ret_372 in light adapted state (named Ret_372la, same isomer form as Ret_535). After excitation light switch-off Ret_372la recovered to Ret_372 in dark-adapted state (Ret_372da) which slowly re-protonated to Ret_535, and Ret_535 slowly isomerized back to Ret_586 in dark-adapted state (Ret_586da). Photocycle schemes and reaction coordinate diagrams are developed and photocycle parameters are determined.

Hydrogel composite of lanthanum and Halorubrum ejinoor sp. cell lysate as an adsorbing material.

OBJECTIVES: Although halophilic archaea are rich in natural environments, their biotechnological applications are not as prevalent as those of other extremophiles, such as thermophiles and alkaliphiles. This study presents an simple method to prepare a hydrogel composite using crude cell lysate of a halophilic archaea, Halorubrum ejinoor sp. (H.e.) which was isolated from a saline lake in Inner Mongolia, China. Furthermore, formation mechanism and potential applications of the hydrogel as an adsorbing material are discussed. RESULTS: Halorubrum ejinoor sp. (H.e.) cell lysate was firstly prepared by adding pure water onto the H.e. cell pellet, followed by a short incubation at 60 °C. The cell lysate was injected into different metal ion (or H+) solutions to obtain the hydrogel composite. It was observed that H+, Fe3+, La3+, Cu2+, and Ca2+ induced gelation of the cell lysate, while Fe2+, Co2+, Ni2+, Mg2+, Na+, and K+ did not. DNA and extracellular polysaccharides (EPS) in the H.e. cell lysate were found to be responsible for the gelation reaction. These results suggest that DNA and EPS should be crosslinked by metal ions (or H+) and form a networked structure in which the metal ion (or H+) serves as an anchor point. Potential application of the hydrogel as an adsorbing material was explored using La3+-induced H.e. hydrogel composite. The hydrogel composite can adsorb the fluoride, phosphate and DNA-binding carcinogenic agents, such as acridine orange. CONCLUSIONS: The simplicity and cost effectiveness of the preparation method might make H.e. hydrogel a promising adsorbing material. This work is expected to expand the technical applications of haloarchaea.