Halorubrum Research Articles

Amino Acid Substitutions in Cold-Adapted Proteins from Halorubrum lacusprofundi, an Extremely Halophilic Microbe from Antarctica

The halophilic Archaeon Halorubrum lacusprofundi, isolated from the perennially cold and hypersaline Deep Lake in Antarctica, was recently sequenced and compared to 12 Haloarchaea from temperate climates by comparative genomics. Amino acid substitutions for 604 H. lacusprofundi proteins belonging to conserved haloarchaeal orthologous groups (cHOGs) were determined and found to occur at 7.85% of positions invariant in proteins from mesophilic Haloarchaea. The following substitutions were observed most frequently: (a) glutamic acid with aspartic acid or alanine; (b) small polar residues with other small polar or non-polar amino acids; (c) small non-polar residues with other small non-polar residues; (d) aromatic residues, especially tryptophan, with other aromatic residues; and (e) some larger polar residues with other similar residues. Amino acid substitutions for a cold-active H. lacusprofundi β-galactosidase were then examined in the context of a homology modeled structure at residues invariant in homologous enzymes from mesophilic Haloarchaea. Similar substitutions were observed as in the genome-wide approach, with the surface accessible regions of β-galactosidase displaying reduced acidity and increased hydrophobicity, and internal regions displaying mainly subtle changes among smaller non-polar and polar residues. These findings are consistent with H. lacusprofundi proteins displaying amino acid substitutions that increase structural flexibility and protein function at low temperature. We discuss the likely mechanisms of protein adaptation to a cold, hypersaline environment on Earth, with possible relevance to life elsewhere.

Bacterioruberin and salinixanthin carotenoids of extremely halophilic Archaea and Bacteria: A Raman spectroscopic study

Laboratory cultures of a number of red extremely halophilic Archaea (Halobacterium salinarum strains NRC-1 and R1, Halorubrum sodomense, Haloarcula valismortis) and of Salinibacter ruber, a red extremely halophilic member of the Bacteria, have been investigated by Raman spectroscopy using 514.5nm excitation to characterize their carotenoids. The 50-carbon carotenoid α-bacterioruberin was detected as the major carotenoid in all archaeal strains. Raman spectroscopy also detected bacterioruberin as the main pigment in a red pellet of cells collected from a saltern crystallizer pond. Salinibacter contains the C40-carotenoid acyl glycoside salinixanthin (all-E, 2′S)-2′-hydroxy-1′-[6-O-(methyltetradecanoyl)-β-d-glycopyranosyloxy]-3′,4′-didehydro-1′,2′-dihydro-β,ψ-carotene-4-one), for which the Raman bands assignments of are given here for the first time.

The function of gas vesicles in halophilic archaea and bacteria: theories and experimental evidence.

A few extremely halophilic Archaea (Halobacterium salinarum, Haloquadratum walsbyi, Haloferax mediterranei, Halorubrum vacuolatum, Halogeometricum borinquense, Haloplanus spp.) possess gas vesicles that bestow buoyancy on the cells. Gas vesicles are also produced by the anaerobic endospore-forming halophilic Bacteria Sporohalobacter lortetii and Orenia sivashensis. We have extensive information on the properties of gas vesicles in Hbt. salinarum and Hfx. mediterranei and the regulation of their formation. Different functions were suggested for gas vesicle synthesis: buoying cells towards oxygen-rich surface layers in hypersaline water bodies to prevent oxygen limitation, reaching higher light intensities for the light-driven proton pump bacteriorhodopsin, positioning the cells optimally for light absorption, light shielding, reducing the cytoplasmic volume leading to a higher surface-area-to-volume ratio (for the Archaea) and dispersal of endospores (for the anaerobic spore-forming Bacteria). Except for Hqr. walsbyi which abounds in saltern crystallizer brines, gas-vacuolate halophiles are not among the dominant life forms in hypersaline environments. There only has been little research on gas vesicles in natural communities of halophilic microorganisms, and the few existing studies failed to provide clear evidence for their possible function. This paper summarizes the current status of the different theories why gas vesicles may provide a selective advantage to some halophilic microorganisms.

Near-IR Resonance Raman Spectroscopy of Archaerhodopsin 3: Effects of Transmembrane Potential

Archaerhodopsin 3 (AR3) is a light driven proton pump from Halorubrum sodomense that has been used as a genetically targetable neuronal silencer and an effective fluorescent sensor of transmembrane potential. Unlike the more extensively studied bacteriorhodopsin (BR) from Halobacterium salinarum, AR3 readily incorporates into the plasma membrane of both E. coli and mammalian cells. Here, we used near-IR resonance Raman confocal microscopy to study the effects of pH and membrane potential on the AR3 retinal chromophore structure. Measurements were performed both on AR3 reconstituted into E. coli polar lipids and in vivo in E. coli expressing AR3 in the absence and presence of a negative transmembrane potential. The retinal chromophore structure of AR3 is in an all-trans configuration almost identical to BR over the entire pH range from 3 to 11. Small changes are detected in the retinal ethylenic stretching frequency and Schiff Base (SB) hydrogen bonding strength relative to BR which may be related to a different water structure near the SB. In the case of the AR3 mutant D95N, at neutral pH an all-trans retinal O-like species (O(all-trans)) is found. At higher pH a second 13-cis retinal N-like species (N(13-cis)) is detected which is attributed to a slowly decaying intermediate in the red-light photocycle of D95N. However, the amount of N(13-cis) detected is less in E. coli cells but is restored upon addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or sonication, both of which dissipate the normal negative membrane potential. We postulate that these changes are due to the effect of membrane potential on the N(13-cis) to M(13-cis) levels accumulated in the D95N red-light photocycle and on a molecular level by the effects of the electric field on the protonation/deprotonation of the cytoplasmic accessible SB. This mechanism also provides a possible explanation for the observed fluorescence dependence of AR3 and other microbial rhodopsins on transmembrane potential.

Biofilm formation by haloarchaea

A fluorescence-based live-cell adhesion assay was used to examine biofilm formation by 20 different haloarchaea, including species of Halobacterium, Haloferax and Halorubrum, as well as novel natural isolates from an Antarctic salt lake. Thirteen of the 20 tested strains significantly adhered (P-value < 0.05) to a plastic surface. Examination of adherent cell layers on glass surfaces by differential interference contrast, fluorescence and confocal microscopy showed two types of biofilm structures. Carpet-like, multi-layered biofilms containing micro- and macrocolonies (up to 50 μm in height) were formed by strains of Halobacterium salinarum and the Antarctic isolate t-ADL strain DL24. The second type of biofilm, characterized by large aggregates of cells adhering to surfaces, was formed by Haloferax volcanii DSM 3757T and Halorubrum lacusprofundi DL28. Staining of the biofilms formed by the strongly adhesive haloarchaeal strains revealed the presence of extracellular polymers, such as eDNA and glycoconjugates, substances previously shown to stabilize bacterial biofilms. For Hbt. salinarum DSM 3754T and Hfx. volcanii DSM 3757T , cells adhered within 1 day of culture and remained viable for at least 2 months in mature biofilms. Adherent cells of Hbt. salinarum DSM 3754T showed several types of cellular appendages that could be involved in the initial attachment. Our results show that biofilm formation occurs in a surprisingly wide variety of haloarchaeal species.

Microbial Light-Activatable Proton Pumps as Neuronal Inhibitors to Functionally Dissect Neuronal Networks in C. elegans

Essentially any behavior in simple and complex animals depends on neuronal network function. Currently, the best-defined system to study neuronal circuits is the nematode Caenorhabditis elegans, as the connectivity of its 302 neurons is exactly known. Individual neurons can be activated by photostimulation of Channelrhodopsin-2 (ChR2) using blue light, allowing to directly probe the importance of a particular neuron for the respective behavioral output of the network under study. In analogy, other excitable cells can be inhibited by expressing Halorhodopsin from Natronomonas pharaonis (NpHR) and subsequent illumination with yellow light. However, inhibiting C. elegans neurons using NpHR is difficult. Recently, proton pumps from various sources were established as valuable alternative hyperpolarizers. Here we show that archaerhodopsin-3 (Arch) from Halorubrum sodomense and a proton pump from the fungus Leptosphaeria maculans (Mac) can be utilized to effectively inhibit excitable cells in C. elegans. Arch is the most powerful hyperpolarizer when illuminated with yellow or green light while the action spectrum of Mac is more blue-shifted, as analyzed by light-evoked behaviors and electrophysiology. This allows these tools to be combined in various ways with ChR2 to analyze different subsets of neurons within a circuit. We exemplify this by means of the polymodal aversive sensory ASH neurons, and the downstream command interneurons to which ASH neurons signal to trigger a reversal followed by a directional turn. Photostimulating ASH and subsequently inhibiting command interneurons using two-color illumination of different body segments, allows investigating temporal aspects of signaling downstream of ASH.

Isolation and identification of selenite reducing archaea from Tuz (salt) Lake In Turkey

In this study, Tuz lake brine samples were investigated for isolation and identification of selenite resistant halophilic prokaryotes. Among the 20 strains of extremely halophilic Bacteria and Archaea, a Gram negative rod designated as strain 106, showed high capacity in the resistance to selenite (25 mM) under aerobic conditions. Phenotypic characterizations and phylogenetic analyses based on 16S rDNA sequence comparison indicated that strain 106 was Halorubrum xinjiangense. The ability of strain 106 to deposite selenium-containing particles were investigated by Transmission Electron Microscopy (TEM). Electron micrographs shows intact cells after selenite reduction and large amounts of selenium-containing particles are present in the culture medium indicating that strain 106 is able to efficiently transport elemental selenium out of the cell.

Ultradeformable archaeosomes as new topical adjuvants

Ultradeformable archaeosomes (UDA) are vesicles made of soybean phosphatidylcholine (SPC), sodium cholate (NaChol) and polar lipids from Halorubrum tebenquichense (3:1:3 wt/wt). Although ultradeformable liposomes (UDL, made of SPC and NaChol at 6:1 wt/wt) and UDA were neither captured nor caused cytotoxicity on keratinocytes, UDA was avidly captured by macrophages, their viability being reduced by 0.4-1.6 mg/mL phospholipids by 25 to 60%. Instead, UDL were poorly captured and caused no toxicity. Balb/C mice immunized by the topical route with four doses of ovalbumin (OVA)-loaded UDA, at 75 μg OVA/600 μg phospholipids (125 nm mean size and -42 mV zeta potential), induced IgG titers tenfold to 100-fold higher than those immunized with OVA-loaded UDL at the same dosage. Both matrices penetrate to the same skin depth (nearly 10 μm after 1 hour on excised human skin), being the higher topical adjuvancy and higher phagocytic uptake of UDA related to its glycolipid content. From the Clinical EditorThis work summarizes key findings related to the development of ultradeformable archaeosomes as vehicles utilized in transdermal delivery systems with improved skin penetration.

Isolation, Characterization and Phylogenetic Analysis of Halophilic Archaea from a Salt Mine in Central Anatolia (Turkey)

The haloarchaeal diversity of a salt mine, a natural cave in central Anatolia, was investigated using convential microbiological and molecular biology methods. Eight halophilic archaeal isolates selected based on their colony morphology and whole cell protein profiles were taxonomically classified on the basis of their morphological, physiological, biochemical properties, polar lipid and protein profiles and 16S rDNA sequences. From the 16S rDNA sequences comparisons it was established that the isolates CH2, CH3 and CHC resembled Halorubrum saccharovorum by 98.8%, 98.9% and 99.5%, respectively. There was a 99.7% similarity between the isolate CH11 and Halobacterium noricense and 99.2% between the isolate CHA1 and Haloarcula argentinensis. The isolate CH8K and CH8B revealed a similarity rate of 99.8% and 99.3% to Halococcus dombrowskii, respectively. It was concluded that the isolates named CH2, CH3 and CHC were clustered in the genus Halorubrum and that CHA1 and CH7 in the genus Haloarcula, CH8K and CH8B in the genus Halococcus and CH11 in the genus Halobacterium.

Conformational changes in the archaerhodopsin-3 proton pump: detection of conserved strongly hydrogen bonded water networks

Archaerhodopsin-3 (AR3) is a light-driven proton pump from Halorubrum sodomense, but little is known about its photocycle. Recent interest has focused on AR3 because of its ability to serve both as a high-performance, genetically-targetable optical silencer of neuronal activity and as a membrane voltage sensor. We examined light-activated structural changes of the protein, retinal chromophore, and internal water molecules during the photocycle of AR3. Low-temperature and rapid-scan time-resolved FTIR-difference spectroscopy revealed that conformational changes during formation of the K, M, and N photocycle intermediates are similar, although not identical, to bacteriorhodopsin (BR). Positive/negative bands in the region above 3,600cm( - 1), which have previously been assigned to structural changes of weakly hydrogen bonded internal water molecules, were substantially different between AR3 and BR. This included the absence of positive bands recently associated with a chain of proton transporting water molecules in the cytoplasmic channel and a weakly hydrogen bonded water (W401), which is part of a hydrogen-bonded pentagonal cluster located near the retinal Schiff base. However, many of the broad IR continuum absorption changes below 3,000cm( - 1) assigned to networks of water molecules involved in proton transport through cytoplasmic and extracellular portions in BR were very similar in AR3. This work and subsequent studies comparing BR and AR3 structural changes will help identify conserved elements in BR-like proton pumps as well as bioengineer AR3 to optimize neural silencing and voltage sensing.

Optical recording of action potentials in mammalian neurons using a microbial rhodopsin

Reliable optical detection of single action potentials in mammalian neurons has been one of the longest-standing challenges in neuroscience. Here we achieve this goal by using the endogenous fluorescence of a microbial rhodopsin protein, Archaerhodopsin 3 (Arch) from Halorubrum sodomense, expressed in cultured rat hippocampal neurons. This genetically encoded voltage indicator exhibited an approximately 10-fold improvement in sensitivity and speed over existing protein-based voltage indicators, with a roughly linear two-fold increase in brightness between −150 mV and +150 mV and a sub-millisecond response time. Arch detected single electrically triggered action potentials with an optical signal-to-noise ratio > 10. The mutant Arch(D95N) lacked endogenous proton pumping and showed 50% greater sensitivity than wild-type, but had a slower response (41 ms). Nonetheless, Arch(D95N) also resolved individual action potentials. Microbial rhodopsin-based voltage indicators promise to enable optical interrogation of complex neural circuits, and electrophysiology in systems for which electrode-based techniques are challenging.

A High-Light Sensitivity Optical Neural Silencer: Development and Application to Optogenetic Control of Non-Human Primate Cortex

Technologies for silencing the electrical activity of genetically targeted neurons in the brain are important for assessing the contribution of specific cell types and pathways toward behaviors and pathologies. Recently we found that archaerhodopsin-3 from Halorubrum sodomense (Arch), a light-driven outward proton pump, when genetically expressed in neurons, enables them to be powerfully, transiently, and repeatedly silenced in response to pulses of light. Because of the impressive characteristics of Arch, we explored the optogenetic utility of opsins with high sequence homology to Arch, from archaea of the Halorubrum genus. We found that the archaerhodopsin from Halorubrum strain TP009, which we named ArchT, could mediate photocurrents of similar maximum amplitude to those of Arch (∼900 pA in vitro), but with a >3-fold improvement in light sensitivity over Arch, most notably in the optogenetic range of 1–10 mW/mm2, equating to >2× increase in brain tissue volume addressed by a typical single optical fiber. Upon expression in mouse or rhesus macaque cortical neurons, ArchT expressed well on neuronal membranes, including excellent trafficking for long distances down neuronal axons. The high light sensitivity prompted us to explore ArchT use in the cortex of the rhesus macaque. Optical perturbation of ArchT-expressing neurons in the brain of an awake rhesus macaque resulted in a rapid and complete (∼100%) silencing of most recorded cells, with suppressed cells achieving a median firing rate of 0 spikes/s upon illumination. A small population of neurons showed increased firing rates at long latencies following the onset of light stimulation, suggesting the existence of a mechanism of network-level neural activity balancing. The powerful net suppression of activity suggests that ArchT silencing technology might be of great use not only in the causal analysis of neural circuits, but may have therapeutic applications.

The dying Dead Sea: The microbiology of an increasingly extreme environment

The Dead Sea, on the border between Israel and Jordan, currently contains around 348 g salt L−1. Divalent cations (Mg2+, Ca2+) dominate over monovalent cations (Na+, K+), while Cl− and Br− are the main anions. The pH of the Sea is about 6. The water balance of the lake is negative, having dropped over 1 m year−1 over the past decade. The water is supersaturated with Na+, with massive quantities of halite precipitating to the bottom of the lake. Biological monitoring since 1980 has indicated that blooms of the unicellular green alga Dunaliella and halophilic Archaea of the family Halobacteriaceae only develop following significant dilution of the upper water layers in the lake after very rainy winters. Such events occurred in 1980, and even more dramatically in 1992, when up to 3.5 × 107 Archaea mL−1 in the diluted upper 5–10 m of the water column coloured the lake red. Species isolated from the lake include Haloferax volcanii, Haloarcula marismortui, Halorubrum sodomense and Halobaculum gomorrense. Dunaliella was no longer observed in the lake after 1996, with prokaryote numbers remaining low. To characterize the residual microbial community in the lake, biomass was collected in February 2007 for environmental genomic analyses. The results were compared with the metagenome of microbial bloom material collected in 1992. The 16S rRNA archaeal phylotypes recovered from the 2007 sample were diverse, with phylotypes distantly related to the genera Halorhabdus, Haloplanus, Natronomonas and others. Halorhabdus sp. also was recovered in culture. The 1992 bloom sample was very homogeneous, however, with a single cluster remotely related to Halobacterium salinarum. These results illustrate that the microbial communities are dynamic, even in one of the most extreme environments on Earth, and exhibit strong shifts in species composition as conditions for life become increasingly adverse.

High-performance genetically targetable optical neural silencing by light-driven proton pumps

The ability to silence the activity of genetically specified neurons in a temporally precise fashion would open up the ability to investigate the causal role of specific cell classes in neural computations, behaviors, and pathologies. Here we show that members of the class of light-driven outward proton pumps can mediate very powerful, safe, multiple-color silencing of neural activity. The gene archaerhodopsin-31 (Arch) from Halorubrum sodomense enables near-100% silencing of neurons in the awake brain when virally expressed in mouse cortex and illuminated with yellow light. Arch mediates currents of several hundred picoamps at low light powers, and supports neural silencing currents approaching 900 pA at light powers easily achievable in vivo. In addition, Arch spontaneously recovers from light-dependent inactivation, unlike light-driven chloride pumps that enter long-lasting inactive states in response to light. These properties of Arch are appropriate to mediate the optical silencing of significant brain volumes over behaviourally-relevant timescales. Arch function in neurons is well tolerated because pH excursions created by Arch illumination are minimized by self-limiting mechanisms to levels comparable to those mediated by channelrhodopsins2,3 or natural spike firing. To highlight how proton pump ecological and genomic diversity may support new innovation, we show that the blue-green light-drivable proton pump from the fungus Leptosphaeria maculans4 (Mac) can, when expressed in neurons, enable neural silencing by blue light, thus enabling alongside other developed reagents the potential for independent silencing of two neural populations by blue vs. red light. Light-driven proton pumps thus represent a high-performance and extremely versatile class of “optogenetic” voltage and ion modulator, which will broadly empower new neuroscientific, biological, neurological, and psychiatric investigations.

Archaeosomes made of Halorubrum tebenquichensetotal polar lipids: a new source of adjuvancy

BackgroundArchaeosomes (ARC), vesicles prepared from total polar lipids (TPL) extracted from selected genera and species from the Archaea domain, elicit both antibody and cell-mediated immunity to the entrapped antigen, as well as efficient cross priming of exogenous antigens, evoking a profound memory response. Screening for unexplored Archaea genus as new sources of adjuvancy, here we report the presence of two new Halorubrum tebenquichense strains isolated from grey crystals (GC) and black mood (BM) strata from a littoral Argentinean Patagonia salt flat. Cytotoxicity, intracellular transit and immune response induced by two subcutaneous (sc) administrations (days 0 and 21) with BSA entrapped in ARC made of TPL either form BM (ARC-BM) and from GC (ARC-GC) at 2% w/w (BSA/lipids), to C3H/HeN mice (25 μg BSA, 1.3 mg of archaeal lipids per mouse) and boosted on day 180 with 25 μg of bare BSA, were determined.ResultsDNA G+C content (59.5 and 61.7% mol BM and GC, respectively), 16S rDNA sequentiation, DNA-DNA hybridization, arbitrarily primed fingerprint assay and biochemical data confirmed that BM and GC isolates were two non-previously described strains of H. tebenquichense. Both multilamellar ARC mean size were 564 ± 22 nm, with -50 mV zeta-potential, and were not cytotoxic on Vero cells up to 1 mg/ml and up to 0.1 mg/ml of lipids on J-774 macrophages (XTT method). ARC inner aqueous content remained inside the phago-lysosomal system of J-774 cells beyond the first incubation hour at 37°C, as revealed by pyranine loaded in ARC. Upon subcutaneous immunization of C3H/HeN mice, BSA entrapped in ARC-BM or ARC-GC elicited a strong and sustained primary antibody response, as well as improved specific humoral immunity after boosting with the bare antigen. Both IgG1 and IgG2a enhanced antibody titers could be demonstrated in long-term (200 days) recall suggesting induction of a mixed Th1/Th2 response.ConclusionWe herein report the finding of new H. tebenquichense non alkaliphilic strains in Argentinean Patagonia together with the adjuvant properties of ARC after sc administration in mice. Our results indicate that archaeosomes prepared with TPL from these two strains could be successfully used as vaccine delivery vehicles.

Halonotius pteroides gen. nov., sp. nov., an extremely halophilic archaeon recovered from a saltern crystallizer

Strains 1.15.5(T), 2.27.5, 5.24.4 and 6.14.5 were isolated from a solar saltern. They have flattened, rod-shaped cells and are aerobic, extremely halophilic members of the domain Archaea and family Halobacteriaceae. Cells stained Gram-negative and grew optimally in media around neutral pH and containing 20-24 % (w/v) (strains 1.15.5(T) and 2.27.5) or 22-24 % (w/v) (5.24.4 and 6.14.5) salts. Mg(2+) was not required. The DNA G+C contents of these isolates were all close to 58 mol%, and DNA-DNA cross-hybridization showed a mean relatedness of 77 %. Their 16S rRNA gene sequences differed by no more than 1.6 % from each other. Phylogenetic tree reconstructions with other recognized members of the Halobacteriaceae indicated that they formed a distinct clade, with the closest relative being Halorubrum saccharovorum (86.6-87.6 % 16S rRNA gene sequence similarity to the type strain). The only major polar lipid of all four isolates was the sulfated diglycosyl diether lipid S-DGD-1. By phase-contrast microscopy, the long, flattened cells of these strains often displayed a 'wing-like' shape. The phenotypic and phylogenetic data support the placement of these isolates into a novel species in a new genus within the Halobacteriaceae, for which we propose the name Halonotius pteroides gen. nov., sp. nov. The type strain of Halonotius pteroides is 1.15.5(T) (=JCM 14355(T) =CECT 7525(T) =DSM 18729(T)), with the additional reference strains 2.27.5 (=JCM 14356 =DSM 18671), 5.24.4 (=JCM 14357 =DSM 18673) and 6.14.5 (=JCM 14358 =DSM 18692).

An evidence of laccases in archaea.

Laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) are a diverse group of multicopper oxidases that catalyze the oxidation of a variety of aromatic compounds. Here we present evidence for distribution of laccases among archaea and their probable functions. Putative laccase genes have been found in different archaeal groups that might have branched off early during evolution, e.g. Haloarcula marismortui ATCC 43049, Natronomonas pharaonis DSM2160, Pyrobaculum aerophilum IM2, Candidatus Nitrosopumilus maritimus SCM1, Halorubrum lacusprofundi ATCC 49239. Most of the archaeal multicopper oxidases reported here are of Type 1 and Type 2 whereas type 3 copper-binding domain could be found in Pyrobaculum aerophilum IM2 and Halorubrum lacusprofundi ATCC49239. An analysis of the genome sequence database revealed the presence of novel types of two-domain laccases in archaea. ed using this method. CyMVin the positive samples of Phalaenopsis sp. and Arachnis sp. was confirmed by DNA sequencing and cp gene homeology blast. The results showed that CyMV extracted from the leaves of orchid in Hangzhou, Zhejiang Province, China, could be derived from Kunming city (KM), Yunnan Province, China. This method characterized by high sensitivity, specificity, and precision is suitable for early diagnosis and quantitative detection of CyMV.

Halorubrum californiense sp. nov., an extreme archaeal halophile isolated from a crystallizer pond at a solar salt plant in California, USA

A motile, rod-shaped, pink-pigmented, extremely halophilic archaeon, strain SF3-213(T), was isolated from a crystallizer pond at the Cargill Solar Salt Plant, Newark, California (USA). Analysis of the almost-complete 16S rRNA gene sequence showed that the isolate was phylogenetically related to species of the genus Halorubrum, with a close relationship to Halorubrum trapanicum NRC 34021(T) (98.6 % similarity), Halorubrum sodomense ATCC 33755(T) (98.3 %) and Halorubrum xinjiangense AS 1.3527(T) (98.2 %). The polar lipids of strain SF3-213(T) were C(20)C(20) derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and a sulfated diglycosyl-diether. Strain SF3-213(T) grew in 2.5-5.0 M NaCl. The temperature and pH ranges for growth were 25-42 degrees C and 6.8-8.5, respectively. Optimal growth occurred at 3.5-4.5 M NaCl, 37 degrees C and pH 7.3. Mg(2+) was required for growth. The DNA G+C content was 69.4 mol%. DNA-DNA hybridization values lower than 70 % were obtained between strain SF3-213(T) and the closely related species of the genus Halorubrum. Based on the data presented in this study, strain SF3-213(T) represents a novel species for which the name Halorubrum californiense sp. nov. is proposed; the type strain is SF3-213(T) (=CECT 7256(T)=DSM 19288(T)=JCM 14715(T)).

Halorubrum kocurii sp. nov., an archaeon isolated from a saline lake

A Gram-negative, non-motile, neutrophilic, rod-shaped, extremely halophilic archaeon, designated strain BG-1(T), was isolated from a salt lake, Lake Bagaejinnor, in Inner Mongolia, China. Strain BG-1(T) was able to grow at 25-55 degrees C, required at least 2.5 M NaCl for growth (with an optimum at 3.4 M NaCl) and grew at pH 6.0-9.0 (with an optimum at pH 7.5). Hypotonic treatment with less than 2.0 M NaCl caused cell lysis. Phylogenetic analysis of the almost-complete 16S rRNA gene sequence positioned the isolate within the genus Halorubrum in the family Halobacteriaceae. Strain BG-1(T) was most closely related to Halorubrum aidingense 31-hong(T) (98.8% sequence similarity), Halorubrum saccharovorum NCIMB 2081(T) (98.6%), Halorubrum lacusprofundi ACAM 34(T) (98.6%) and Halorubrum lipolyticum 9-3(T) (98.4%). However, values for DNA-DNA hybridization between strain BG-1(T) and the most closely related members of the genus Halorubrum were below 40%. Analysis of the polar lipids of strain BG-1(T) revealed the presence of mannosyl-2-sulfate-(1-4)-glycosyl-archaeol, the main glycolipid found in neutrophilic species of the genus Halorubrum. The G+C content of the genomic DNA was 69.4 mol% (T(m)). Comparison of the phenotypic characteristics of the strain with those of Halorubrum species supported the conclusion that BG-1(T) represents a novel species within this genus, for which the name Halorubrum kocurii sp. nov. is proposed. The type strain is BG-1(T) (=CECT 7322(T) =CGMCC 1.7018(T) =JCM 14978(T)).

Sequence analysis of an Archaeal virus isolated from a hypersaline lake in Inner Mongolia, China

BackgroundWe are profoundly ignorant about the diversity of viruses that infect the domain Archaea. Less than 100 have been identified and described and very few of these have had their genomic sequences determined. Here we report the genomic sequence of a previously undescribed archaeal virus.ResultsHaloarchaeal strains with 16S rRNA gene sequences 98% identical to Halorubrum saccharovorum were isolated from a hypersaline lake in Inner Mongolia. Two lytic viruses infecting these were isolated from the lake water. The BJ1 virus is described in this paper. It has an icosahedral head and tail morphology and most likely a linear double stranded DNA genome exhibiting terminal redundancy. Its genome sequence has 42,271 base pairs with a GC content of ~65 mol%. The genome of BJ1 is predicted to encode 70 ORFs, including one for a tRNA. Fifty of the seventy ORFs had no identity to data base entries; twenty showed sequence identity matches to archaeal viruses and to haloarchaea. ORFs possibly coding for an origin of replication complex, integrase, helicase and structural capsid proteins were identified. Evidence for viral integration was obtained.ConclusionThe virus described here has a very low sequence identity to any previously described virus. Fifty of the seventy ORFs could not be annotated in any way based on amino acid identities with sequences already present in the databases. Determining functions for ORFs such as these is probably easier using a simple virus as a model system.