VOLN27B: A New Head-Tailed Halovirus Isolated from an Underground Salt Crystal and Infecting Halorubrum.

In the present study, we aimed to extract, purify, analyze monosaccharide composition of exopolysaccharide (EPS) produced by Halorubrum sp. TBZ112 (KCTC 4203 and IBRC-M 10773) and also to evaluate its possible antiproliferative activity against human gastric cancer (MKN-45) cell line and its biocompatibility effect on normal cells using human dermal fibroblast (HDF) cell line. Average molecular weight and monosaccharide composition were determined by high-pressure size exclusion chromatography (HPSEC) with multi-angle laser light scattering (MALLS) and high-pressure anion exchange chromatography (HPAEC), respectively. Fourier transform infrared (FTIR) spectroscopy was used for the partial characterization of the EPS. The EPS effect on the cell proliferation and viability of MKN-45 and HDF cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue dye exclusion, respectively. Strain TBZ112 excreted 480mg.l-1 of the EPS under optimal growth conditions. The EPS had a molecular weight of 5.052kDa and was a heteropolysaccharide containing ten moieties mainly composed of mannose (19.95%), glucosamine (15.55%), galacturonic acid (15.43%), arabinose (12.24%), and glucuronic acid (12.05%). No significant difference of the EPS treatments on the proliferation activity of MKN-45 and HDF cells were observed (P > 0.05). For the first time, the EPS from Halorubrum sp. TBZ112, an extremely halophilic archaeon related to Halorubrum genus, was isolated and chemically characterized. The EPS from Halorubrum sp. TBZ112 possesses a relatively low molecular weight and might be applied as a biocompatible compound. More investigations are needed to determine other biological activities of the EPS along with further details of its chemical structure.

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

Carotenoids are one of the most diverse and broadly distributed classes of pigments in nature with a high number of biotechnological applications. Carotenoids have a broad range of functions, especially in relation to human health and their role as biological antioxidants. The increasing demand for consumption of natural carotenoids has raised interest in their bio-production. The objective of the present study was the analysis of environmental factors (temperature, pH and salinity) through response surface methodology (RSM) on the total carotenoid production of Halorubrum sp. TBZ126. In addition the effect of light was evaluated. Five levels of temperature, pH, and salinity were selected based on central composite design (CCD) and RSM to reach the optimum values for the cell growth and carotenoid production. Bio-production was carried out in an orbital shaker using a 10% (v/v) inoculum, and agitation at 120 rpm for 9 days in a non-illuminated environment. Dry cell weight was determined and total carotenoid was estimated by spectrophotometer. The production of biomass ranged from 0.04 to 0.84 g/l and the total carotenoid from 0.15 to 10.78 mg/l. The optimum conditions for cell growth and total carotenoid production in Halorubrum sp. TBZ126 cultures, were temperature 31oC and 32oC, pH 7.51 and 7.94 and NaCl (w/v) 18.33% and 20.55%, respectively. In conclusion, employing RSM design and under the light as an inducing factor, carotenoid production by Halorubrum sp. TBZ126 was elevated to about 145%. Additionally, TBZ126 could produce carotenoids at lower concentrations of NaCl (as low as 2.5%), in the absence of sodium acetate without elevating magnesium sulfate concentration.

Halovirus is a major force that affects the evolution of extreme halophiles and the biogeochemistry of hypersaline environments. However, until now, the systematic studies on the halovirus ecology and the effects of salt concentration on virus-host systems are lacking. To provide more valuable information for understanding ecological strategies of a virus-host system in the hypersaline ecosystem, we studied the interaction between halovirus SNJ1 and its host Natrinema sp.J7-2 under various NaCl concentrations. We found that the adsorption rate and lytic rate increased with salt concentration, demonstrating that a higher salt concentration promoted viral adsorption and proliferation. Contrary to the lytic rate, the lysogenic rate decreased as the salt concentration increased. Our results also demonstrated that cells incubated at a high salt concentration prior to infection increased the ability of the virus to adsorb and lyse its host cells; therefore, the physiological status of host cells also affected the virus-host interaction. In conclusion, SNJ1 acted as a predator, lysing host cells and releasing progeny viruses in hypersaline environments; in low salt environments, viruses lysogenized host cells to escape the damage from low salinity.

Environments containing significant concentration of NaCl such as salt lakes harbor extremophiles microorganisms which have a great biotechnology interest. To explore the diversity of Bacteria in Chott Tinsilt (Algeria), an isolation program was performed. Water samples were collected from the saltern during the pre-salt harvesting phase. This Chott is high in salt (22.47% (w/v). Seven halophiles Bacteria were selected for further characterization. The isolated strains were able to grow optimally in media with 10–25% (w/v) total salts. Molecular identification of the isolates was performed by sequencing the 16S rRNA gene. It showed that these cultured isolates included members belonging to the Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus genera with less than 98% of similarity with their closest phylogenetic relative. The halophilic bacterial isolates were also characterized for the production of biosurfactant and industrially important enzymes. Most isolates produced hydrolases and biosurfactants at high salt concentration. In fact, this is the first report on bacterial strains (A4 and B4) which were a good biosurfactant and coagulase producer at 20% and 25% ((w/v)) NaCl. In addition, the biosurfactant produced by the strain B4 at high salinity (25%) was also stable at high temperature (30-100°C) and high alkalinity (pH 11).

A set of 110 extremely halophilic archaeal strains were isolated from seven distinct saline habitats located in different regions of Algeria. The physicochemical characterization of the samples showed that these habitats were thalassohaline. The carotenoid production from isolated strains varied from 0.1 to 3.68 µg/ml. Based on their physiological characteristics and pigment production, 43 strains were selected and identified by means of phenotypic tests and 16S ribosomal RNA gene sequencing. Phylogenetic analysis indicated that the isolates corresponded to the class Halobacteria and were closely related to genera Halorubrum, Haloarcula, Haloferax, Natrinema, Halogeometricum, Haloterrigena, and Halopiger. Carotenoids of the highest producer, strain Halorubrum sp. BS2 were identified using high-performance liquid chromatography-diode array detector and liquid chromatography-mass spectrometry. Bacterioruberin and bisanhydrobacterioruberin were the predominant carotenoids. The scavenging activity of these carotenoids reached 99% at a concentration of 18 μg/ml, which was much higher than that of ascorbic acid used as a reference compound. These carotenoids also exhibited significant antibacterial activities against four human-pathogenic strains and four fish-pathogenic strains. Variations in salinity, agitation rate, temperature, and light intensity were found to influence growth and carotenoid production of Halorubrum sp. BS2. Our results suggest that halophilic archaea represent a potential source for carotenoids, which are characterized by high antioxidant and antibacterial activities.

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.

A novel haloarchaeal plasmid, pHRDV1 (13,053bp), was isolated from the haloarchaeal isolate Halorubrum sp. T3. Molecular and bioinformatics analyses showed that this element is a double-stranded circular DNA molecule containing two putative transcripts with opposite directions. The amino acid sequences of six of the nineteen predicted open reading frames were similar to those found in haloarchaeal pleomorphic viruses, such as Halorubrum pleomorphic virus 3 and Halogeometricum pleomorphic virus 1. There was also a strong conservation in gene order between the plasmid and these viruses. All three conserved viral proteins (VPs), which are characteristic of haloarchaeal pleomorphic viruses VP3, VP4 and VP8, were found in pHRDV1. Furthermore, a typical repressor-operator system similar to haloarchaeal myovirus φCh1, was found on the genome of pHRDV1. However, no viral particles were detected in the supernatants of Halorubrum sp. T3, either in the presence or absence of mitomycin C. These results imply that plasmid pHRDV1 is a distinctive virus-like mobile genetic element that harbors some unique properties that make it different from all of the known haloarchaeal plasmids or viruses.

The morphology and dimensions of bacteriophage K particles were determined by electron microscopy. This virus had an icosahedral head (approx. 70 nm diam.) and a long (210 nm) thin (15 nm) contractile tail which terminated in a complex basal appendage. The precise dimensions of the particles were dependent on the negative stain employed. The buoyant densities of the K virus particle and its DNA were 1 X 479 g/ml and 1 X 689 g/ml respectively. The DNA had a base composition of 30% G + C, a contour length of 16 X 1 micron and a calculated mol. wt. of 33 X 10(6). With Staphylococcus aureus (NCTC 9318) as host, the latent period was 25 min, the eclipse period 14 min and the average burst size 60 p.f.u./bacterium. Infection resulted in inhibition of host DNA synthesis and degradation of the bacterial DNA: the products were used for tee synthesis of phage DNA. The kinetics of DNA synthesis is infected and uninfected bacteria were examined. There was no initial cessation of DNA synthesis in the infected bacteria.

Carotenoids are of great interest in many scientific disciplines because of their wide distribution, diverse functions and interesting properties. The present report describes a new natural source for carotenoid production. Halorubrum sp., TBZ126, an extremely halophilic archaeon, was isolated from Urmia Lack following culture of water sample on marine agar medium and incubation at 30 °C. Then single colonies were cultivated in broth media. After that the cells were collected and carotenoids were extracted with acetone-methanol (7:3 v/v). The identification of carotenoids was performed by UV-VIS spectroscopy and confirmed by thin layer chromatography (TLC) in the presence of antimony pentachloride (SbCl5). The production profile was analyzed using liquid-chromatography mass spectroscopy (LC-MS) techniques. Phenotypic characteristics of the isolate were carried out and the 16S rRNA gene was amplified using polymerase chain reaction (PCR). LC-MS analytical results revealed that produced carotenoids are bacterioruberin, lycopene and β-carotene. Bacterioruberin was found to be the predominant produced carotenoid. 16S rRNA analysis showed that TBZ126 has 100% similarity with Halorubrum chaoviator Halo-G*T (AM048786). Halorubrum sp. TBZ126, isolated from Urmia Lake has high capacity in the production of carotenoids. This extremely halophilic archaeon could be considered as a prokaryotic candidate for carotenoid production source for future studies.

This work aims to characterize the haloarchaeal diversity of unexplored environmental salty samples from a hypersaline environment on the southern coast of Jeddah, Saudi Arabia, looking for new isolates able to produce polyhydroxyalkanoates (PHAs). Thus, the list of PHA producers has been extended by describing two species of Halolamina; Halolamina sediminis sp. strain NRS_35 and unclassified Halolamina sp. strain NRS_38. The growth and PHA-production were investigated in the presence of different carbon sources, (glucose, sucrose, starch, carboxymethyl cellulose (CMC), and glycerol), pH values, (5-9), temperature ranges (4-65 °C), and NaCl concentrations (100-350 g L-1). Fourier-transform infra-red analysis (FT-IR) and Liquid chromatography-mass spectrometry (LC-MS) were used for qualitative identification of the biopolymer. The highest yield of PHB was 33.4% and 27.29% by NRS_35 and NRS_38, respectively, using starch as a carbon source at 37 °C, pH 7, and 25% NaCl (w/v). The FT-IR pattern indicated sharp peaks formed around 1628.98 and 1629.28 cm-1, which confirmed the presence of the carbonyl group (C=O) on amides and related to proteins, which is typical of PHB. LC-MS/MS analysis displayed peaks at retention times of 5.2, 7.3, and 8.1. This peak range indicates the occurrence of PHB and its synthetic products: Acetoacetyl-CoA and PHB synthase (PhaC). In summary, the two newly isolated Halolamina species showed a high capacity to produce PHB using different sources of carbon. Further research using other low-cost feedstocks is needed to improve both the quality and quantity of PHB production. With these results, the use of haloarchaea as cell factories to produce PHAs is reinforced, and light is shed on the global concern about replacing plastics with biodegradable polymers.

Bioaccumulation of hexavalent chromium by enriched microbial cultures obtained from molasses and NaCl containing media

Coupled phase and aqueous species equilibrium of the H 2O–CO 2–NaCl–CaCO 3 system from 0 to 250 °C, 1 to 1000 bar with NaCl concentrations up to saturation of halite

Metastable and equilibrium phase diagrams for unconjugated bilirubin IXα (UCB) in bile are yet to be determined for understanding the physical chemistry of pigment gallstone formation. Also, UCB is a molecule of considerable biomedical importance because it is a potent antioxidant and an inhibitor of atherogenesis. We employed principally a titrimetric approach to obtain metastable and equilibrium UCB solubilities in model bile systems composed of taurine-conjugated bile salts, egg yolk lecithin (mixed long-chain phosphatidylcholines), and cholesterol as functions of total lipid concentration, biliary pH values, and CaCl2 plus NaCl concentrations. Metastable and equilibrium precipitation pH values were obtained, and average pKa values of the two carboxyl groups of UCB were calculated. Added lecithin and increased temperature decreased UCB solubility markedly, whereas increases in bile salt concentrations and molar levels of urea augmented solubility. A wide range of NaCl and cholesterol concentrations resulted in no specific effects, whereas added CaCl2 produced large decreases in UCB solubilities at alkaline pH values only. UV-visible absorption spectra were consistent with both hydrophobic and hydrophilic interactions between UCB and bile salts that were strongly influenced by pH. Reliable literature values for UCB compositions of native gallbladder biles revealed that biles from hemolytic mice and humans with black pigment gallstones are markedly supersaturated with UCB and exhibit more acidic pH values, whereas biles from nonstone control animals and patients with cholesterol gallstone are unsaturated with UCB.

Biogenic stromatolites are sources of significant information on the evolution of microbial life. Despite their evolutionary significance, little is known about the mechanisms of osmoadaptation by microorganisms that comprise living stromatolites thriving in hypersaline environments. Osmoadaptive strategies for Halococcus hamelinensis, a novel halophilic archaeon recently isolated from living stromatolites in the hypersaline reaches of Shark Bay, were thus a particular interest in this study. To investigate the possibility of "salt-in-cytoplasm"-associated osmoadaptation for this archaeon, flame photometry studies were performed. From the results, it was evident that this halophilic archaeon did not accumulate intracellular K(+) ions when cells were exposed to either osmotic shock or conditions with gradual increments in salinity. These results were further supported by polymerase chain reaction (PCR) analyses where there was no evidence for the existence of homologous genes to an ATP-driven, high-affinity potassium uptake system in Halococcus hamelinensis. To identify an alternative salt adaptation mechanism associated with accumulation of compatible solutes for this archaeon, (1)H nuclear magnetic resonance (NMR) spectroscopy experiments were carried out. Results indicate that glycine betaine, trehalose, and glutamate are solutes likely to be involved in osmoregulation in this archeaon. Subsequent (1)H NMR analysis of cell extracts from this microorganism grown under various NaCl concentrations revealed that intracellular levels of glycine betaine increased with increasing concentrations of NaCl. This behavior of increasing glycine betaine concentration with increasing external NaCl is consistent with its identity as an osmolyte. In contrast, intracellular levels of trehalose were decreased in high concentrations of NaCl. This provides evidence that compatible solute accumulation appears to be the preferential salt regulation mechanism for this haloarchaeon, in contrast to the salt-in-cytoplasm strategy employed by many other halophilic archaea.