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.

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.

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.

Absorption and Emission Spectroscopic Investigation of the Thermal Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor Archon2.

Archon2 is a fluorescent voltage sensor derived from Archaerhodopsin 3 (Arch) of Halorubrum sodomense using robotic multidimensional directed evolution approach. Here we report absorption and emission spectroscopic studies of Archon2 in Tris buffer at pH 8. Absorption cross-section spectra, fluorescence quantum distributions, fluorescence quantum yields, and fluorescence excitation spectra were determined. The thermal stability of Archon2 was studied by long-time attenuation coefficient measurements at room temperature (21 ± 1 °C) and at refrigerator temperature (3 ± 1 °C). The apparent melting temperature was determined by stepwise sample heating up and cooling down (obtained apparent melting temperature: 63 ± 3 °C). In the protein melting process protonated retinal Schiff base (PRSB) with absorption maximum at 586 nm converted to de-protonated retinal Schiff base (RSB) with absorption maximum at 380 nm. Storage of Archon2 at room temperature and refrigerator temperature caused absorption coefficient decrease because of partial protein clustering to aggregates at condensation nuclei and sedimentation. At room temperature an onset of light scattering was observed after two days because of the beginning of protein unfolding. During the period of observation (18 days at 21 °C, 22 days at 3 °C) no change of retinal isomer composition was observed indicating a high potential energy barrier of S0 ground-state isomerization.

Fast Biofilm Penetration and Anti-PAO1 Activity of Nebulized Azithromycin in Nanoarchaeosomes.

Azithromycin (AZ) is a broad-spectrum antibiotic with anti-inflammatory and antiquorum sensing activity against biofilm forming bacteria such as Pseudomonas aeruginosa. AZ administered by oral or parenteral routes, however, neither efficiently accesses nor remains in therapeutic doses inside pulmonary biofilm depths. Instead, inhaled nanocarriers loaded with AZ may revert the problem of low accessibility and permanence of AZ into biofilms, enhancing its antimicrobial activity. The first inhalable nanovesicle formulation of AZ, nanoarchaeosome-AZ (nanoARC-AZ), is here presented. NanoARC prepared with total polar archaeolipids (TPAs), rich in 2,3-di-O-phytanyl-sn-glycero-1-phospho-(3'-sn-glycerol-1'-methylphosphate) (PGP-Me) from Halorubrum tebenquichense archaebacteria, consisted of ∼180 nm-diameter nanovesicles, loaded with 0.28 w/w AZ/TPA. NanoARC-AZ displayed lower minimal inhibitory concentration and minimal bactericidal concentration, higher preformed biofilm disruptive, and anti-PAO1 activity in biofilms than AZ. NanoARC penetrated and disrupted the structure of the PAO1 biofilm within only 1 h. Two milliliters of 15 µg/mL AZ nanoARC-AZ nebulized for 5 min rendered AZ doses compatible with in vitro antibacterial activity. The strong association between AZ and the nanoARC bilayer, combined with electrostatic attraction and trapping into perpendicular methyl groups of archaeolipids, as determined by Laurdan fluorescence anisotropy, generalized polarization, and small-angle X-ray scattering, was critical to stabilize during storage and endure shear forces of nebulization. NanoARC-AZ was noncytotoxic on A549 cells and human THP-1-derived macrophages, deserving further preclinical exploration as enhancers of AZ anti-PAO1 activity.

The anti MRSA biofilm activity of Thymus vulgaris essential oil in nanovesicles.

BACKGROUND: Thymus vulgaris essential oil (T) could be an alternative to classical antibiotics against bacterial biofilms, which show increased tolerance to antibiotics and host defence systems and contribute to the persistence of chronic bacterial infections. HYPOTHESIS: A nanovesicular formulation of T may chemically protect the structure and relative composition of its multiple components, potentially improving its antibacterial and antibiofilm activity. STUDY DESIGN: We prepared and structurally characterized T in two types of nanovesicles: nanoliposomes (L80-T) made of Soybean phosphatidylcholine (SPC) and Polysorbate 80 (P80) [SPC:P80:T 1:0.75:0.3 w:w], and nanoarchaeosomes (A80-T) made of SPC, P80 and total polar archaeolipids (TPA) extracted from archaebacteria Halorubrum tebenquichense [SPC:TPA:P80:T 0.5:0.50.75:0.7 w:w]. We determined the macrophage cytotoxicity and the antibacterial activity against Staphylococcus aureus ATCC 25,923 and four MRSA clinical strains. RESULTS: L80-T (Z potential -4.1 ±â€¯0.6 mV, ∼ 115 nm, ∼ 22 mg/ml T) and A80-T (Z potential -6.6 ±â€¯1.5 mV, ∼ 130 nm, ∼ 42 mg/ml T) were colloidally and chemically stable, maintaining size, PDI, Z potential and T concentration for at least 90 days. While MIC90 of L80-T was > 4 mg/ml T, MIC90 of A80-T was 2 mg/ml T for all S. aureus strains. The antibiofilm formation activity was maximal for A80-T, while L80-T did not inhibit biofilm formation compared to untreated control. A80-T significantly decreased the biomass of preformed biofilms of S. aureus ATCC 25,923 strain and of 3 of the 4 clinical MRSA isolates at 4 mg/ml T. It was found that the viability of J774A.1 macrophages was decreased significantly upon 24 h incubation with A80-T, L80-T and T emulsion at 0.4 mg/ml T. These results show that from 0.4 mg/ml T, a value lower than MIC90 and the one displaying antibiofilm activity, with independence of its formulation, T significantly decreased the macrophages viability. CONCLUSION: Overall, because of its lower MIC90 against planktonic bacteria, higher antibiofilm formation capacity and stability during storage, A80-T resulted better antibacterial agent than T emulsion and L80-T. These results open new avenues to explode the A80-T antimicrobial intracellular activity.

Using a portable Raman spectrometer to detect carotenoids of halophilic prokaryotes in synthetic inclusions in NaCl, KCl, and sulfates.

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

Halorubrum salsamenti sp. nov., a Novel Halophilic Archaeon Isolated from a Brine of Salt Mine.

A non-motile, spherical or oval extremely halophilic archaeon, strain Y69T, was isolated from a brine of the Yunnan salt mine, China. Colonies on JCM 168 agar plate were round (1-2 mm in diameter), moist, and orange-pigmented. Phylogenetic analysis of the almost-complete 16S rRNA gene sequence showed that the isolate belonged to the species of the genus Halorubrum, with a close relationship to Halorubrum aidingense 31-hongT (98.5%), Halorubrum lacusprofundi ATCC 49239T (98.2%), and Halorubrum kocurii BG-1T (98.0%). The major polar lipids of strain Y69T were phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and a sulfated diglycosyl diether. Strain Y69T grew in 15-30% (w/v) NaCl. The temperature and pH ranges for growth were 25-50 °C and 6.5-9.0, respectively. Optimal growth occurred at 20% (w/v) NaCl, 42 °C, and pH 8.0. Mg2+ was required for growth. The DNA G+C content was determined to be 65.1 mol% by the thermal denaturation method. DNA-DNA hybridization values between strain Y69T and the closely related species were lower than 70%. Based on the data presented in this study, strain Y69T represents a novel species for which the name Halorubrum salsamenti sp. nov. is proposed. The type of the strain is Y69T (=CGMCC 1.15455T = JCM 31270T).

Ultra-small solid archaeolipid nanoparticles for active targeting to macrophages of the inflamed mucosa.

AIM: Develop nanoparticulate agents for oral targeted delivery of dexamethasone (Dex) to macrophages of inflamed mucosa. MATERIALS & METHODS: Solid archaeolipid nanoparticles (SAN-Dex) (compritol/Halorubrum tebenquichense polar archaeolipids/soybean phosphatidylcholine/Tween-80 4; 0.9; 0.3; 3% w/w) loaded with Dex were prepared. Their mucopenetration, stability under digestion and in vitro anti-inflammatory activity, were determined. RESULTS: Ultra-small SAN-Dex strongly reduced the levels of TNF-α, IL-6 and IL-12 on J774A1 cells stimulated with lipopolysaccharides as compared with free Dex or loaded in ordinary solid lipid nanoparticles-Dex. After in vitro digestion, the anti-inflammatory activity of SAN-Dex was retained, while that of solid lipid nanoparticles-Dex was lost. CONCLUSION: Because of their structural and pharmacodynamic features, SAN-Dex may be suitable for oral targeted delivery to inflamed mucosa.

Important roles for membrane lipids in haloarchaeal bioenergetics.

Recent advances in lipidomic analysis in combination with various physiological experiments set the stage for deciphering the structure-function of haloarchaeal membrane lipids. Here we focused primarily on changes in lipid composition of Haloferax volcanii, but also performed a comparative analysis with four other haloarchaeal species (Halobacterium salinarum, Halorubrum lacusprofundi, Halorubrum sodomense and Haloplanus natans) all representing distinctive cell morphologies and behaviors (i.e., rod shape vs. pleomorphic behavior). Common to all five haloarchaea, our data reveal an extraordinary high level of menaquinone, reaching up to 72% of the total lipids. This ubiquity suggests that menaquinones may function beyond their ordinary role as electron and proton transporter, acting simultaneously as ion permeability barriers and as powerful shield against oxidative stress. In addition, we aimed at understanding the role of cations interacting with the characteristic negatively charged surface of haloarchaeal membranes. We propose for instance that by bridging the negative charges of adjacent anionic phospholipids, Mg2+ acts as surrogate for cardiolipin, a molecule that is known to control curvature stress of membranes. This study further provides a bioenergetic perspective as to how haloarchaea evolved following oxygenation of Earth's atmosphere. The success of the aerobic lifestyle of haloarchaea includes multiple membrane-based strategies that successfully balance the need for a robust bilayer structure with the need for high rates of electron transport - collectively representing the molecular basis to inhabit hypersaline water bodies around the planet.

Surviving nebulization-induced stress: dexamethasone in pH-sensitive archaeosomes.

AIM: To increase the subcellular delivery of dexamethasone phosphate (DP) and stability to nebulization stress, pH-sensitive nanoliposomes (LpH) exhibiting archaeolipids, acting as ligands for scavenger receptors (pH-sensitive archaeosomes [ApH]), were prepared. MATERIALS & METHODS: The anti-inflammatory effect of 0.18 mg DP/mg total lipid, 100-150 nm DP-containing ApH (dioleylphosphatidylethanolamine: Halorubrum tebenquichense total polar archaeolipids:cholesteryl hemisuccinate 4.2:2.8:3 w:w) was tested on different cell lines. Size and HPTS retention of ApH and conventional LpH (dioleylphosphatidylethanolamine:cholesteryl hemisuccinate 7:3 w:w) before and after nebulization were determined. RESULTS & CONCLUSION: DP-ApH suppressed IL-6 and TNF-α on phagocytic cells. Nebulized after 6-month storage, LpH increased size and completely lost its HPTS while ApH3 conserved size and polydispersity, fully retaining its original HPTS content.

Ultradeformable Archaeosomes for Needle Free Nanovaccination with Leishmania braziliensis Antigens.

Total antigens from Leishmania braziliensis promastigotes, solubilized with sodium cholate (dsLp), were formulated within ultradeformable nanovesicles (dsLp-ultradeformable archaeosomes, (dsLp-UDA), and dsLp-ultradeformable liposomes (dsLp-UDL)) and topically administered to Balb/c mice. Ultradeformable nanovesicles can penetrate the intact stratum corneum up to the viable epidermis, with no aid of classical permeation enhancers that can damage the barrier function of the skin. Briefly, 100 nm unilamellar dsLp-UDA (soybean phosphatidylcholine: Halorubrum tebenquichense total polar lipids (TPL): sodium cholate, 3:3:1 w:w) of -31.45 mV Z potential, containing 4.84 ± 0.53% w/w protein/lipid dsLp, 235 KPa Young modulus were prepared. In vitro, dsLp-UDA was extensively taken up by J774A1 and bone marrow derive cells, and the only that induced an immediate secretion of IL-6, IL-12p40 and TNF-α, followed by IL-1ß, by J774A1 cells. Such extensive uptake is a key feature of UDA ascribed to the highly negatively charged archaeolipids of the TPL, which are recognized by a receptor specialized in uptake and not involved in downstream signaling. Despite dsLp alone was also immunostimulatory on J774A1 cells, applied twice a week on consecutive days along 7 weeks on Balb/c mice, it raised no measurable response unless associated to UDL or UDA. The highest systemic response, IgGa2 mediated, 1 log lower than im dsLp Al2O3, was elicited by dsLp-UDA. Such findings suggest that in vivo, UDL and UDA acted as penetration enhancers for dsLp, but only dsLp-UDA, owed to its pronounced uptake by APC, succeeded as topical adjuvants. The actual TPL composition, fully made of sn2,3 ether linked saturated archaeolipids, gives the UDA bilayer resistance against chemical, physical and enzymatic attacks that destroy ordinary phospholipids bilayers. Together, these properties make UDA a promising platform for topical drug targeted delivery and vaccination, that may be of help for countries with a deficient healthcare system.

Characterization of a bacterioruberin-producing Haloarchaea isolated from the marshlands of the Odiel river in the southwest of Spain.

In this work, we describe the isolation, identification, pigment characterization, and optimization of the culture conditions for a haloarchaea strain isolated from salt evaporation ponds in the Odiel river, at Southwest of Spain. The haloarchaea belongs to the genus Halorobrum, as deduced from the analysis of its 16S rRNA encoding gene and has been designated as Halorubrum sp. SH1. The growth conditions for the new strain were optimized studying temperature, NaCl concentration, agitation rate and light intensity. The C50-carotenoids, bacterioruberin, and its derivatives bisanhydrobacterioruberin and trisanhydrobacterioruberin, were found to be the predominant pigments produced by this strain of Halorubrum, as determined using HPLC-DAD and UHPLC-ESI-MS/MS techniques. This extremely halophilic archaeon could be a good candidate for the production of bacterioruberins of high added-value due to their coloring, antioxidant, and possible anticancer properties. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:592-600, 2016.

Microbial rhodopsins of Halorubrum species isolated from Ejinoor salt lake in Inner Mongolia of China.

Microbial rhodopsins are photoactive proteins that use a retinal molecule as the photoactive center. Because of structural simplicity and functional diversity, microbial rhodopsins have been an excellent model system for structural biology. In this study, a halophilic archaea that has three microbial rhodopsin-type genes in its genome was isolated from Ejinoor salt lake in Inner Mongolia of China. A sequence of 16S rRNA showed that the strain belongs to Halorubrum genus and named Halorubrum sp. ejinoor (He). The translated amino acid sequences of its microbial rhodopsin-type genes suggest that they are homologs of archaerhodopsin (HeAR), halorhodopsin (HeHR) and sensory rhodopsin II (HeSRII). The mRNAs of three types of genes were detected by RT-PCR and their amounts were investigated by Real-Time RT-PCR. The amount of mRNA of HeSRII was the smallest and the amounts of of HeAR and HeHR were 30 times and 10 times greater than that of HeSRII. The results of light-induced pH changes suggested the presence of a light-driven proton pump and a light-driven chloride ion pump in the membrane vesicles of He. Flash induced absorbance changes of the He membrane fraction indicated that HeAR and HeHR are photoactive and undergo their own photocycles. This study revealed that three microbial rhodopsin-type genes are all expressed in the strain and at least two of them, HeAR and HeHR, are photochemically and physiologically active like BR and HR of Halobacterium salinarum, respectively. To our knowledge, this is the first report of physiological activity of HR-homolog of Halorubrum species.

Structural features of ultradeformable archaeosomes for topical delivery of ovalbumin.

The ultradeformable archaeosomes (UDA, made of total polar archaeolipids (TPA) extracted from the extreme halophile archaea Halorubrum tebenquichense:soybean phosphatidylcholine (SPC):sodium cholate (NaChol), 3:3:1 w:w), are promising topical adjuvants showing high deformability, an essential property for intact skin penetration up to the viable epidermis/dermis. To gain insights on UDA structure, the interactions between TPA, SPC and the edge activator NaChol, were assessed by electrospray ionization mass spectroscopy (ESI-MS) and confocal fluorescence microscopy of giant unilamellar vesicles (GUV). The non covalent heterodimers NaChol-SPC, NaChol-phosphatidylglycerophosphate methyl ether (PGPMe), NaChol-sulfated diglycosyl diphytanyl-glycerol diether (SDGD5) and SPC-PGPMe detected in the gas phase by ESI-MS after direct infusion of UDA, together with the homogeneous partition of FASTDiO and DiIC18 in GUV suggested that in these proportions, lipids and NaChol were miscible. We propose therefore, a model where in UDA the SPC diluted sufficient enough in the rich PGPMe TPA, so as to the low lateral mobility of molecules (typical of rich in PGPMe bilayers) was no longer experienced. We also found that 50µm deep within in vitro human skin canyons, the fluorescence of Alexa fluor 647-ovalbumin in UDL was ∼1.5 folds higher than in UDA, indicating a potential steric hindrance of the voluminous structure of PGPMe UDA bilayer, to the penetration of a particulate cargo such as the 7nm diameter ovalbumin. According to these observations, a further reduction in PGPMe - a lipid playing no immune role - content could help to improve the performance of UDA as topical adjuvants.

Halorubrum salinum sp. nov., isolated from a marine solar saltern.

The halophilic archaeal strain GX71(T) was isolated from the Gangxi marine solar saltern near the Weihai city of Shandong Province, China. Cells of the strain were pleomorphic and lysed in distilled water, stained Gram-negative and formed red-pigmented colonies. Strain GX71(T) was able to grow at 25-45 °C (optimum 30 °C), in the presence of 1.7-4.8 M NaCl (optimum 2.6 M NaCl), with 0.005-0.7 M MgCl2 (optimum 0.05 M MgCl2) and at pH 5.5-9.5 (optimum pH 7.0-7.5). Cells lysed in distilled water and the minimal NaCl concentration to prevent cell lysis was 10 % (w/v). The major polar lipids of the strain were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, one major glycolipid chromatographically identical to sulfated mannosyl glucosyl diether (S-DGD-3) and an unidentified lipid was also detected. The 16S rRNA gene sequence of strain GX71(T) showed 94.0-97.0 % similarity to members of the genus Halorubrum of the family Halobacteriaceae. The rpoB' gene sequence of strain GX71(T) was 87.3-93.4 % similarity to current members of the genus Halorubrum. The DNA G+C content of GX71(T) was 67.1 mol%. Strain GX71(T) showed low DNA-DNA relatedness with Halorubrum lipolyticum CGMCC 1.5332(T), Halorubrum saccharovorum CGMCC 1.2147(T), Halorubrum kocurii CGMCC 1.7018(T) and Halorubrum arcis CGMCC 1.5343(T), the most closely related members of the genus Halorubrum. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain GX71(T) represents a novel species of the genus Halorubrum, for which the name Halorubrum salinum sp. nov. is proposed. The type strain is GX71(T) (= CGMCC 1.10458(T) = JCM 17093(T)).

Halorubrum halophilum sp. nov., an extremely halophilic archaeon isolated from a salt-fermented seafood.

A novel, red-pigmented, pleomorphic and short rod-shaped haloarchaeon, designated B8(T), was isolated from a salt-fermented seafood. Strain B8(T) was found to be able to grow at 20-45 °C, in the presence of 15-30 % (w/v) NaCl and at pH 7.0-9.0. The optimum requirements were found to be a temperature range of 35-40 °C, pH 8.0 and the presence of 25 % NaCl. The cells of strain B8(T) were observed to be Gram-staining negative and lysed in distilled water. Anaerobic growth did not occur in the presence of nitrate, L-arginine, dimethyl sulfoxide or trimethylamine N-oxide. The catalase and oxidase activities were found to be positive and nitrate was reduced in aerobic conditions. Tween 20, 40 and 80 were found to be hydrolyzed, whereas casein, gelatin and starch were not hydrolyzed. Indole or H2S was not formed and urease activity was not detected. A phylogenetic analysis based on the 16S rRNA gene sequences indicated that strain B8(T) is most closely related to members of the genus Halorubrum in the family Halobacteriaceae. Strain B8(T) was found to have three 16S rRNA genes, rrnA, rrnB and rrnC; similarities between the 16S rRNA gene sequences are 99.0-99.8 %. Strain B8(T) shared 99.0 % 16S rRNA gene sequence similarity with Halorubrum (Hrr.) lipolyticum JCM 13559(T) and Hrr. saccharovorum DSM 1137(T), 98.8 % with Hrr. kocurii JCM 14978(T), 98.3 % with Hrr. lacusprofundi DSM 5036(T), 98.0 % with Hrr. arcis JCM 13916(T), 97.7 % with Hrr. aidingense JCM 13560(T) and 97.0 % with Hrr. aquaticum JCM 14031(T), as well as 93.7-96.5 % with other type strains in the genus Halorubrum. The RNA polymerase subunit B' gene sequence similarity of strain B8(T) with Hrr. kocurii JCM 14978(T) is 97.2 % and lower with other members of the genus Halorubrum. DNA-DNA hybridization experiments showed that strain B8(T) shared equal or lower than 50 % relatedness with reference species in the genus Halorubrum. The genomic DNA G+C content of strain B8(T) was determined to be 64.6 mol%. The major isoprenoid quinone of strain B8(T) was identified as menaquinone-8 and the major polar lipids as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, sulfated mannosyl glucosyl diether and an unidentified phospholipid. Based on this polyphasic taxonomic study, strain B8(T) is considered to represent a new species in the genus Halorubrum, for which the name Hrr. halophilum sp. nov. is proposed. The type strain is B8(T) (=JCM 18963(T) = CECT 8278(T)).

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)-ß-d-glycopyranosyloxy]-3',4'-didehydro-1',2'-dihydro-ß,ψ-carotene-4-one), for which the Raman bands assignments of are given here for the first time.

Archaeosomes display immunoadjuvant potential for a vaccine against Chagas disease.

Archaeosomes (ARC), vesicles made from lipids extracted from Archaea, display strong adjuvant properties. In this study, we evaluated the ability of the highly stable ARC formulated from total polar lipids of a new Halorubrum tebenquichense strain found in Argentinean Patagonia, to act as adjuvant for soluble parasite antigens in developing prophylactic vaccine against the intracellular protozoan T. cruzi, the etiologic agent of Chagas disease. We demonstrated for the first time that C3H/HeN mice subcutaneously immunized with trypanosomal antigens entrapped in these ARC (ARC-TcAg) rapidly developed higher levels of circulating T. cruzi antibodies than those measured in the sera from animals receiving the antigen alone. Enhanced humoral responses elicited by ARC-TcAg presented a dominant IgG2a antibody isotype, usually associated with Th1-type immunity and resistance against T. cruzi. More importantly, ARC-TcAg-vaccinated mice displayed reduced parasitemia during early infection and were protected against an otherwise lethal challenge with the virulent Tulahuén strain of the parasite. Our findings suggest that, as an adjuvant, H. tebenquichense-derived ARC may hold great potential to develop a safe and helpful vaccine against this relevant human pathogen.

Enhancement of the long-wavelength sensitivity of optogenetic microbial rhodopsins by 3,4-dehydroretinal.

Electrogenic microbial rhodopsins (ion pumps and channelrhodopsins) are widely used to control the activity of neurons and other cells by light (optogenetics). Long-wavelength absorption by optogenetic tools is desirable for increasing the penetration depth of the stimulus light by minimizing tissue scattering and absorption by hemoglobin. A2 retinal (3,4-dehydroretinal) is a natural retinoid that serves as the chromophore in red-shifted visual pigments of several lower aquatic animals. Here we show that A2 retinal reconstitutes a fully functional archaerhodopsin-3 (AR-3) proton pump and four channelrhodopsin variants (CrChR1, CrChR2, CaChR1, and MvChR1). Substitution of A1 with A2 retinal significantly shifted the spectral sensitivity of all tested rhodopsins to longer wavelengths without altering other aspects of their function. The spectral shift upon substitution of A1 with A2 in AR-3 was close to that measured in other archaeal rhodopsins. Notably, the shifts in channelrhodopsins were larger than those measured in archaeal rhodopsins and close to those in animal visual pigments with similar absorption maxima of their A1-bound forms. Our results show that chromophore substitution provides a complementary strategy for improving the efficiency of optogenetic tools.