Development of a genetic system for Haloferax gibbonsii LR2-5, model host for haloarchaeal viruses.

Archaeal viruses are among the most enigmatic members of the virosphere, and their diverse morphologies raise many questions about their infection mechanisms. The study of molecular mechanisms underlying virus-host interactions hinges upon robust model organisms with a system for gene expression and deletion. Currently, there are only a limited number of archaea that have associated viruses and have a well-developed genetic system. Here, we report the development of a genetic system for the euryarchaeon Haloferax gibbonsii LR2-5. This strain can be infected by multiple viruses and is a model for the study of virus-host interactions. We created a Hfx. gibbonsii LR2-5 ∆pyrE strain, resulting in uracil auxotrophy, which could be used as a selection marker. An expression plasmid carrying a pyrE gene from the well-established Haloferax volcanii system was tested for functionality. Expression of a GFP-MinD fusion under a tryptophan inducible promoter was fully functional and showed similar cellular localization as in Hfx. volcanii. Thus, the plasmids of the Hfx. volcanii system can be used directly for the Hfx. gibbonsii LR2-5 genetic system, facilitating the transfer of tools between the two. Finally, we tested for the functionality of gene deletions by knocking out two genes of the archaeal motility structure, the archaellum. These deletion mutants were as expected non-motile and the phenotype of one deletion could be rescued by the expression of the deleted archaellum gene from a plasmid. Thus, we developed a functional genetic toolbox for the euryarchaeal virus host Hfx. gibbonsii LR2-5, which will propel future studies on archaeal viruses. IMPORTANCE: Species from all domains of life are infected by viruses. In some environments, viruses outnumber their microbial hosts by a factor of 10, and viruses are the most important predators of microorganisms. While much has been discovered about the infection mechanisms of bacterial and eukaryotic viruses, archaeal viruses remain understudied. Good model systems are needed to study their virus-host interactions in detail. The salt-loving archaeon Haloferax gibbonsii LR2-5 has been shown to be infected by a variety of different viruses and, thus, is an excellent model to study archaeal viruses. By establishing a genetic system, we have significantly expanded the toolbox for this model organism, which will fuel our understanding of infection strategies of the underexplored archaeal viruses.

Halocin H4 is activated through cleavage by halolysin HlyR4.

Halocins are antimicrobial peptides secreted by haloarchaea capable of inhibiting the growth of other haloarchaea or bacteria. Halocin H4 (HalH4) is secreted by the model halophilic archaeon Haloferax mediterranei ATCC 33500. Despite attempts to express halH4 heterologously in Escherichia coli and subsequent careful renaturation procedures commonly employed for haloarchaeal proteins, no active halocin was obtained. However, it was discovered that the antihaloarchaeal activity of this halocin could be activated through cleavage by halolysin R4 (HlyR4), a serine protease also secreted by Hfx. mediterranei ATCC 33500. Replacement of the cysteine at the number 115 amino acid with glycine and deletion of the internal trans-membrane region (15 aa) markedly abolished HalH4's antihaloarchaeal activity. Compared to the N-terminus, the C-terminal amino acid sequence was found to be more crucial for HalH4 to exert its antihaloarchaeal activity. Mass spectrometry analysis revealed that the biologically active antihaloarchaeal peptide produced after hydrolytic cleavage by HlyR4 was the C-terminus of HalH4, suggesting a potential mechanism of action involving pore formation within competitor species' cell membranes. Taken together, this study offers novel insights into the interplay between halocins and secreted proteases, as well as their contribution to antagonistic interaction within haloarchaea. IMPORTANCE: The antihaloarchaeal function of halocin H4 (HalH4) can be activated by extracellular proteases from haloarchaea, as demonstrated in this study. Notably, we report the first instance of halocin activation through proteolytic cleavage, highlighting its significance in the field. The C-terminus of HalH4 (CTH4) has been identified as the antihaloarchaeal peptide present in hydrolysates generated by HlyR4. The CTH4 exhibited inhibitory activity against a range of haloarchaeal species (Haloarchaeobius spp., Haloarcula spp., Haloferax spp., Halorubellus spp., and Halorubrum spp.), as well as selected bacterial species (Aliifodinibius spp. and Salicola spp.), indicating its broad-spectrum inhibitory potential across domains. The encoding gene of halocin HalH4, halH4, from the model halophilic archaeon Haloferax mediterranei ATCC 33500 can be expressed in Escherichia coli without codon optimization.

Genome-wide transcriptional response to silver stress in extremely halophilic archaeon Haloferax alexandrinus DSM 27206 T.

BACKGROUND: The extremely halophilic archaeon Haloferax (Hfx.) alexandrinus DSM 27206 T was previously documented for the ability to biosynthesize silver nanoparticles while mechanisms underlying its silver tolerance were overlooked. In the current study, we aimed to assess the transcriptional response of this haloarchaeon to varying concentrations of silver, seeking a comprehensive understanding of the molecular determinants underpinning its heavy metal tolerance. RESULTS: The growth curves confirmed the capacity of Hfx. alexandrinus to surmount silver stress, while the SEM-EDS analysis illustrated the presence of silver nanoparticles in cultures exposed to 0.5 mM silver nitrate. The RNA-Seq based transcriptomic analysis of Hfx. alexandrinus cells exposed to 0.1, 0.25, and 0.5 mM silver nitrate revealed the differential expression of multiple sets of genes potentially employed in heavy-metal stress response, genes mostly related to metal transporters, basic metabolism, oxidative stress response and cellular motility. The RT-qPCR analysis of selected transcripts was conducted to verify and validate the generated RNA-Seq data. CONCLUSIONS: Our results indicated that copA, encoding the copper ATPase, is essential for the survival of Hfx. alexandrinus cells in silver-containing saline media. The silver-exposed cultures underwent several metabolic adjustments that enabled the activation of enzymes involved in the oxidative stress response and impairment of the cellular movement capacity. To our knowledge, this study represents the first comprehensive analysis of gene expression in halophillic archaea facing increased levels of heavy metals.

Nanohaloarchaea as beneficiaries of xylan degradation by haloarchaea.

Climate change, desertification, salinisation of soils and the changing hydrology of the Earth are creating or modifying microbial habitats at all scales including the oceans, saline groundwaters and brine lakes. In environments that are saline or hypersaline, the biodegradation of recalcitrant plant and animal polysaccharides can be inhibited by salt-induced microbial stress and/or by limitation of the metabolic capabilities of halophilic microbes. We recently demonstrated that the chitinolytic haloarchaeon Halomicrobium can serve as the host for an ectosymbiont, nanohaloarchaeon 'Candidatus Nanohalobium constans'. Here, we consider whether nanohaloarchaea can benefit from the haloarchaea-mediated degradation of xylan, a major hemicellulose component of wood. Using samples of natural evaporitic brines and anthropogenic solar salterns, we describe genome-inferred trophic relations in two extremely halophilic xylan-degrading three-member consortia. We succeeded in genome assembly and closure for all members of both xylan-degrading cultures and elucidated the respective food chains within these consortia. We provide evidence that ectosymbiontic nanohaloarchaea is an active ecophysiological component of extremely halophilic xylan-degrading communities (although by proxy) in hypersaline environments. In each consortium, nanohaloarchaea occur as ectosymbionts of Haloferax, which in turn act as scavenger of oligosaccharides produced by xylan-hydrolysing Halorhabdus. We further obtained and characterised the nanohaloarchaea-host associations using microscopy, multi-omics and cultivation approaches. The current study also doubled culturable nanohaloarchaeal symbionts and demonstrated that these enigmatic nano-sized archaea can be readily isolated in binary co-cultures using an appropriate enrichment strategy. We discuss the implications of xylan degradation by halophiles in biotechnology and for the United Nation's Sustainable Development Goals.

Archaeal Host Cell Recognition and Viral Binding of HFTV1 to Its Haloferax Host.

Viruses are highly abundant and the main predator of microorganisms. Microorganisms of each domain of life are infected by dedicated viruses. Viruses infecting archaea are genomically and structurally highly diverse. Archaea are undersampled for viruses in comparison with bacteria and eukaryotes. Consequently, the infection mechanisms of archaeal viruses are largely unknown, and most available knowledge stems from viruses infecting a select group of archaea, such as crenarchaea. We employed Haloferax tailed virus 1 (HFTV1) and its host, Haloferax gibbonsii LR2-5, to study viral infection in euryarchaea. We found that HFTV1, which has a siphovirus morphology, is virulent, and interestingly, viral particles adsorb to their host several orders of magnitude faster than most studied haloarchaeal viruses. As the binding site for infection, HFTV1 uses the cell wall component surface (S)-layer protein. Electron microscopy of infected cells revealed that viral particles often made direct contact with their heads to the cell surface, whereby the virion tails were perpendicular to the surface. This seemingly unfavorable orientation for genome delivery might represent a first reversible contact between virus and cell and could enhance viral adsorption rates. In a next irreversible step, the virion tail is orientated toward the cell surface for genome delivery. With these findings, we uncover parallels between entry mechanisms of archaeal viruses and those of bacterial jumbo phages and bacterial gene transfer agents. IMPORTANCE Archaeal viruses are the most enigmatic members of the virosphere. These viruses infect ubiquitous archaea and display an unusually high structural and genetic diversity. Unraveling their mechanisms of infection will shed light on the question if entry and egress mechanisms are highly conserved between viruses infecting a single domain of life or if these mechanisms are dependent on the morphology of the virus and the growth conditions of the host. We studied the entry mechanism of the tailed archaeal virus HFTV1. This showed that despite "typical" siphovirus morphology, the infection mechanism is different from standard laboratory models of tailed phages. We observed that particles bound first with their head to the host cell envelope, and, as such, we discovered parallels between archaeal viruses and nonmodel bacteriophages. This work contributes to a better understanding of entry mechanisms of archaeal viruses and a more complete view of microbial viruses in general.

Simultaneous purification and characterization of detergent-stable, solvent-tolerant haloextremozymes protease and lipase from Haloferax sp. strain GUBF 2.

Industrial important proteases and lipases are in increasing demand for various biotechnological applications. In the present study, the concomitantly produced protease and lipase by Haloferax sp. strain GUBF 2 were simultaneously purified as a heterogeneous lipase (45 and 66 kDa) and homogeneous protease (180 kDa); with 28.3 and 31.36 fold purity, respectively using Sephadex G-200. The aforementioned extremozymes were active at pH 3-13, 20-80 °C, 1-5 M NaCl, with optimal activity at pH 6, 70 °C, and 3 M NaCl, thus exhibiting attributes of true haloextremozymes. The Km and Vmax of purified lipase were 3.47 mM and 16.2 U/mL, while protease were 3.29 mg/mL and 28.5 U/mL, respectively. FTIR bands corresponding to the vibrations of amide II and amide III were detected in haloextremozymes which could perhaps be used to determine the secondary structure of the purified proteins. Furthermore, the activity of both enzymes was stimulated by Ca2+ and inhibited by 10 mM Hg2+ and phenylmethyl sulphonyl fluoride (PMSF). Additionally, these haloextremozymes are stable in the presence of detergent additives and organic solvents. In addition, purified protease displayed 74.3 ± 4.85% in-vitro blood clot dissolution activity. Conclusively this study revealed the key features, unusual properties, and possible biomedical applications of detergent-stable and organic solvent-tolerant haloextremozymes from Haloferax sp. strain GUBF 2 to date unexplored.

Genetic Manipulation of Haloferax Species.

In this chapter, we describe the reverse genetics methodology behind generating a targeted gene deletion or replacement in archaeal species of the genus Haloferax, which are renowned for their ease of manipulation. Individual steps in the method include the design of a gene-targeting vector, its use in transforming Haloferax to yield "pop-in" and "pop-out" clones, and techniques for validating the genetically manipulated strain. The vector carries DNA fragments of 500-1000 bp that flank the gene of interest (or a mutant allele), in addition to the pyrE2 gene for uracil biosynthesis (Bitan-Banin et al. J Bacteriol 185:772-778, 2003). The latter is used as a selectable marker for the transformation of Haloferax, wherein the vector integrates by homologous recombination at the genomic locus to generate the "pop-in" strain; this is also known as allele-coupled exchange. Culturing of these transformants in nonselective broth and subsequent plating on 5-fluoroorotic acid (5-FOA)-containing media selects for excision of the vector, yielding either wild type or mutant "pop-out" clones. These 5-FOA-resistant clones are screened to confirm the desired mutation, using a combination of phenotypic assays, colony hybridization and Southern blotting. The pop-in/pop-out method allows for the recycling of the pyrE2 marker to enable multiple gene deletions to be carried out in a single strain, thereby providing insights into the function of multiple proteins and how they interact in their respective cellular pathways.

The Viral Susceptibility of the Haloferax Species.

Viruses can infect members of all three domains of life. However, little is known about viruses infecting archaea and the mechanisms that determine their host interactions are poorly understood. Investigations of molecular mechanisms of viral infection rely on genetically accessible virus-host model systems. Euryarchaea belonging to the genus Haloferax are interesting models, as a reliable genetic system and versatile microscopy methods are available. However, only one virus infecting the Haloferax species is currently available. In this study, we tested ~100 haloarchaeal virus isolates for their infectivity on 14 Haloferax strains. From this, we identified 10 virus isolates in total capable of infecting Haloferax strains, which represented myovirus or siphovirus morphotypes. Surprisingly, the only susceptible strain of all 14 tested was Haloferax gibbonsii LR2-5, which serves as an auspicious host for all of these 10 viruses. By applying comparative genomics, we shed light on factors determining the host range of haloarchaeal viruses on Haloferax. We anticipate our study to be a starting point in the study of haloarchaeal virus-host interactions.

Conversion of selenite by Haloferax alexandrinus GUSF-1 (KF796625) to pentagonal selenium nanoforms which in vitro modulates the formation of calcium oxalate crystals.

AIM: To investigate the ability of Haloferax alexandrinus GUSF-1 (KF796625) to biosynthesize non-toxic elemental selenium (Se0 ) and check their capacity in in vitro crystal structure modulation of calcium oxalate, which are implicated in the development of renal calculi. METHODS AND RESULTS: Haloferax alexandrinus GUSF-1 (KF796625) during growth in the presence of 5 mmol L-1 of selenite formed insoluble brick-red particles. Se0 formed was monitored spectrophotometrically using a combination of two assays; the ascorbic acid reduction and sodium sulphide solubilization assay. After 168 h of growth, 2.89 mmol L-1 of Se0 was formed from 4.9 mmol L-1 of selenite. Absorption bands at 1.5, 11.2 and 12.5 keV in EDX spectroscopy confirmed that the brick-red particulate matter was Se0 . Furthermore, these selenium nanoparticles (SeNPs) were pentagonal in shape in transmission electron microscopy imaging. The peak positions in X-ray diffractogram at 2θ values of 23.40°, 29.66°, 41.26°, 43.68°, 45.24°, 51.62°, 55.93° and 61.47° and the relative intensities further confirmed the formation of Se0 . In vitro addition of 50 and 100 µg ml-1 of these SeNPs to the mixture of sodium chloride, calcium chloride and sodium oxalate affected and modulated the shape and size of rectangular-shaped calcium oxalate crystals (average area of 1.23 ± 0.2 µm2 ) to smaller rectangular-shaped crystals (average area of 0.54 ± 0.2 µm2 ) and spherical-shaped crystals (average area 0.13 ± 0.005 µm2 ). CONCLUSION: Haloferax alexandrinus GUSF-1 (KF796625) transformed selenite to Se0 pentagonal nanoforms that modulated in vitro the formation of crystal shape and size of calcium oxalate. SIGNIFICANCE AND IMPACT OF STUDY: There are no reports on conversion of selenite to Se0 among the Haloferax genera, and this study involving the formation of pentagonal SeNPs with capacity to modulate the formation of calcium oxalate crystals in haloarchaea is recorded as the first report and of significance in pharmaceutical research related to formulations abetting urinary calculi.

Haloferax litoreum sp. nov., Haloferax marinisediminis sp. nov., and Haloferax marinum sp. nov., low salt-tolerant haloarchaea isolated from seawater and sediment.

Three novel halophilic archaea were isolated from seawater and sediment near Yeoungheungdo Island, Republic of Korea. The genome size and G + C content of the isolates MBLA0076T, MBLA0077T, and MBLA0078T were 3.56, 3.48, and 3.48 Mb and 61.7, 60.8, and 61.1 mol%, respectively. The three strains shared 98.5-99.5 % sequence similarity of the 16 S rRNA gene, whereas their sequence similarity to the 16 S rRNA gene of type strains was below 98.5 %. Phylogenetic analysis based on sequences of the 16 S rRNA and RNA polymerase subunit beta genes indicated that the isolates belonged to the genus Haloferax. The orthologous average nucleotide identity, average amino-acid identity, and in silico DNA-DNA hybridization values were below species delineation thresholds. Pan-genomic analysis indicated that the three novel strains and 11 reference strains had 8981 pan-orthologous groups in total. Fourteen Haloferax strains shared 1766 core pan-genome orthologous groups, which were mainly related to amino acid transport and metabolism. Cells of the three isolates were gram-negative, motile, red-pink pigmented, and pleomorphic. The strains grew optimally at 30 °C (MBLA0076T) and 40 °C (MBLA0077T, MBLA0078T) in the presence of 1.28 M (MBLA0077T) and 1.7 M (MBLA0076T, MBLA0078T) NaCl and 0.1 M (MBLA0077T), 0.2 M (MBLA0076T), and 0.3 M (MBLA0078T) MgCl2·6H2O at pH 7.0-8.0. Cells of all isolates lysed in distilled water; the minimum NaCl concentration necessary to prevent lysis was 0.43 M. The major polar lipids of the three strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, and sulphated diglycosyl archaeol-1. Based on their phenotypic and genotypic properties, MBLA0076T, MBLA0077T, and MBLA0078T were described as novel species of Haloferax, for which we propose the names Haloferax litoreum sp. nov., Haloferax marinisediminis sp. nov., and Haloferax marinum sp. nov., respectively. The respective type strains of these species are MBLA0076T (= KCTC 4288T = JCM 34,169T), MBLA0077T (= KCTC 4289T = JCM 34,170T), and MBLA0078T (= KCTC 4290T = JCM 34,171T).

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

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

Purification and Characterization of a New Halocin HA4 from Haloferax larsenii HA4 Isolated from a Salt Lake.

Halocins are antimicrobial peptides secreted by different members of haloarchaea. Halocin HA4 was purified from Haloferax larsenii HA4 using combination of ultrafiltration and chromatographic techniques. It was found to be ~ 14 kDa with unique N-terminal sequence, H2N-AEEEIFXPDX, which did not show homology with the known sequence suggesting a new/novel compound. It was found to be heat resistant up to 100 °C, stable at pH 2.0-10.0, and retained complete activity in the presence of different organic compounds such as methanol, ethanol, acetone, isopropanol, ethyl acetate, Tween 80, acetonitrile, SDS, Triton X-100, and urea. However, complete activity was reduced after the treatment with trypsin, papain, and proteinase K suggesting proteinaceous nature of the compound. The cytocidal nature of halocin HA4 was evidenced with complete loss of viable count of indicator strain, H. larsenii HA10. The change in FTIR spectrum of halocin-treated cells suggested halocin HA4 interacts with cell membrane and nucleic acids of the target cells. Thus, we report a new halocin inhibitory to related strains and may be applied in the preservation of salted foods and leather hides in the respective industries.

Anti-Pseudomonas aeruginosa biofilm activity of tellurium nanorods biosynthesized by cell lysate of Haloferax alexandrinus GUSF-1(KF796625).

Pseudomonas aeruginosa, an opportunistic human pathogen, is a major health concern as it grows as a biofilm and evades the host's immune defenses. Formation of biofilms on catheter and endotracheal tubes demands the development of biofilm-preventive (anti-biofilm) approaches and evaluation of nanomaterials as alternatives to antibiotics. The present study reports the successful biosynthesis of tellurium nanorods using cell lysate of Haloferax alexandrinus GUSF-1 (KF796625). The black particulate matter had absorption bands at 0.5 and 3.6 keV suggestive of elemental tellurium; showed x-ray diffraction peaks at 2θ values 24.50°, 28.74°, 38.99°, 43.13°, 50.23° and displayed a crystallite size of 36.99 nm. The black nanorods of tellurium were an average size of 40 nm × 7 nm, as observed in transmission electron microscopy. To our knowledge, the use of cell lysate of Haloferax alexandrinus GUSF-1 (KF796625) as a green route for the biosynthesis of tellurium nanorods with a Pseudomonas aeruginosa biofilm inhibiting capacity is novel to haloarchaea. At 50 µg mL-1, these tellurium nanorods exhibited 75.03% in-vitro reduction of biofilms of Pseudomonas aeruginosa ATCC 9027, comparable to that of ciprofloxacin, which is used in treatment of Pseudomonas infections. Further, the observed ability of these nanoparticles to inhibit the formation of Pseudomonas biofilms is worthy of future research perusal.

The Novel Halovirus Hardycor1, and the Presence of Active (Induced) Proviruses in Four Haloarchaea.

The virus Hardycor1 was isolated in 1998 and infects the haloarchaeon Halorubrum coriense. DNA from a frozen stock (HC1) was sequenced and the viral genome found to be 45,142 bp of dsDNA, probably having redundant, circularly permuted termini. The genome showed little similarity (BLASTn) to known viruses. Only twenty-two of the 53 (41%) predicted proteins were significantly similar to sequences in the NCBI nr protein database (E-value ≤ 10-15). Six caudovirus-like proteins were encoded, including large subunit terminase (TerL), major capsid protein (Mcp) and tape measure protein (Tmp). Hardycor1 was predicted to be a siphovirus (VIRFAM). No close relationship to other viruses was found using phylogenetic tree reconstructions based on TerL and Mcp. Unexpectedly, the sequenced virus stock HC1 also revealed two induced proviruses of the host: a siphovirus (Humcor1) and a pleolipovirus (Humcor2). A re-examination of other similarly sequenced, archival virus stocks revealed induced proviruses of Haloferax volcanii, Haloferax gibbonsii and Haloarcula hispanica, three of which were pleolipoviruses. One provirus (Halfvol2) of Hfx. volcanii showed little similarity (BLASTn) to known viruses and probably represents a novel virus group. The attP sequences of many pleolipoproviruses were found to be embedded in a newly detected coding sequence, split in the provirus state, that spans between genes for integrase and a downstream CxxC-motif protein. This gene might play an important role in regulation of the temperate state.

Production and characterization of a bioactive extracellular homopolysaccharide produced by Haloferax sp. BKW301.

The production of extracellular polysaccharides (EPS) by haloarchaeal members, with novel and unusual physicochemical properties, is of special importance and has the potential for extensive biotechnological exploitation. An extremely halophilic archaeon, Haloferax sp. BKW301 (GenBank Accession No. KT240044) isolated from a solar saltern of Baksal, West Bengal, India has been optimized for the production of EPS under batch culture. It produced a considerable amount (5.95 g/L) of EPS in the medium for halophiles with 15% NaCl, 3% glucose, 0.5% yeast extract, and 6% inoculum under shake flask culture at 120 rpm. The purified EPS, a homopolymer of galactose as revealed by chromatographic methods and Fourier-transform infrared spectroscopy, is noncrystalline (CIxrd , 0.82), amorphous, and could emulsify hydrocarbons like kerosene, petrol, xylene, and so forth. Moreover, the polymer is highly thermostable (up to 420°C) and displayed pseudoplastic rheology. Biologically, the EPS was able to scavenge DPPH (2,2-diphenyl-1-picrylhydrazyl) radical efficiently and inhibit the proliferation of the Huh-7 cell line at an IC50 value of 6.25 µg/ml with a Hill coefficient of 0.844. Large-scale production of this thermostable, pseudoplastic homopolysaccharide, therefore, could find suitable applications in industry and biotechnology.

Haloarchaea swim slowly for optimal chemotactic efficiency in low nutrient environments.

Archaea have evolved to survive in some of the most extreme environments on earth. Life in extreme, nutrient-poor conditions gives the opportunity to probe fundamental energy limitations on movement and response to stimuli, two essential markers of living systems. Here we use three-dimensional holographic microscopy and computer simulations to reveal that halophilic archaea achieve chemotaxis with power requirements one hundred-fold lower than common eubacterial model systems. Their swimming direction is stabilised by their flagella (archaella), enhancing directional persistence in a manner similar to that displayed by eubacteria, albeit with a different motility apparatus. Our experiments and simulations reveal that the cells are capable of slow but deterministic chemotaxis up a chemical gradient, in a biased random walk at the thermodynamic limit.

Physicochemical characterization and emulsifying properties of a novel exopolysaccharide produced by haloarchaeon Haloferax mucosum.

The physicochemical characterization and emulsifying functional properties of a novel exopolysaccharide (EPS) produced by haloarchaea Haloferax mucosum (DSM 27191) were investigated. This biopolymer has a high molecular weight of 152 kDa and important protein content of 10%. Different culture media compositions were investigated taking the ATCC 2185 medium as a base and supplementing with varying concentrations of yeast extract and glucose or sucrose as carbon sources to produce the EPS in a liquid medium. The highest EPS production (7.15 ±â€¯0.44 g/L) was obtained at 96 h. EPS aqueous dispersions showed a non-Newtonian rheological behavior which was well fitted to the Cross equation. The EPS (at 0.32% w/w) was capable of stabilizing water-in-oil emulsions with different nonpolar solvents, including n-hexane, kerosene, chloroform, castor oil and mineral oil. EPS retained its emulsifying activity after to be incubated for one hour in a wide range of temperatures (25, 40, 70 and 100 °C), pH (4, 6.5, 7 and 12) and NaCl concentrations (0, 2.0 and 4.0 M). The viscoelastic behavior and stability of hexane-in-water emulsion were examined through oscillatory shear measurements.

Specificity of protein-DNA interactions in hypersaline environment: structural studies on complexes of Halobacterium salinarum oxidative stress-dependent protein hsRosR.

Interactions between proteins and DNA are crucial for all biological systems. Many studies have shown the dependence of protein-DNA interactions on the surrounding salt concentration. How these interactions are maintained in the hypersaline environments that halophiles inhabit remains puzzling. Towards solving this enigma, we identified the DNA motif recognized by the Halobactrium salinarum ROS-dependent transcription factor (hsRosR), determined the structure of several hsRosR-DNA complexes and investigated the DNA-binding process under extreme high-salt conditions. The picture that emerges from this work contributes to our understanding of the principles underlying the interplay between electrostatic interactions and salt-mediated protein-DNA interactions in an ionic environment characterized by molar salt concentrations.

Division plane placement in pleomorphic archaea is dynamically coupled to cell shape.

One mechanism for achieving accurate placement of the cell division machinery is via Turing patterns, where nonlinear molecular interactions spontaneously produce spatiotemporal concentration gradients. The resulting patterns are dictated by cell shape. For example, the Min system of Escherichia coli shows spatiotemporal oscillation between cell poles, leaving a mid-cell zone for division. The universality of pattern-forming mechanisms in divisome placement is currently unclear. We examined the location of the division plane in two pleomorphic archaea, Haloferax volcanii and Haloarcula japonica, and showed that it correlates with the predictions of Turing patterning. Time-lapse analysis of H. volcanii shows that divisome locations after successive rounds of division are dynamically determined by daughter cell shape. For H. volcanii, we show that the location of DNA does not influence division plane location, ruling out nucleoid occlusion. Triangular cells provide a stringent test for Turing patterning, where there is a bifurcation in division plane orientation. For the two archaea examined, most triangular cells divide as predicted by a Turing mechanism; however, in some cases multiple division planes are observed resulting in cells dividing into three viable progeny. Our results suggest that the division site placement is consistent with a Turing patterning system in these archaea.

Haloferax sulfurifontis GUMFAZ2 producing xylanase-free cellulase retrieved from Haliclona sp. inhabiting rocky shore of Anjuna, Goa-India.

Salt stable cellulases are implicated in detritic food webs of marine invertebrates for their role in the degradation of cellulosic material. A haloarchaeon, Haloferax sulfurifontis GUMFAZ2 producing cellulase was successfully isolated from marine Haliclona sp., a sponge inhabiting the rocky intertidal region of Anjuna, Goa. The culture produced extracellular xylanase-free cellulase with a maximum activity of 11.7 U/ml, using carboxymethylcellulose-Na (CMC-Na), as a sole source of carbon in 3.5 M NaCl containing medium, pH 7 at 40°C and produced cellobiose and glucose, detectable by thin-layer chromatography. Nondenaturing polyacrylamide gel electrophoresis of the crude enzyme, revealed a single protein band of 19.6 kDa which on zymographic analysis exhibited cellulase activity while corresponding sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a molecular weight of 46 kDa. Unlike conventional cellulases, this enzyme is active in presence of 5 M NaCl and does not have accompanying xylanase activity, hence can be considered as xylanase-free cellulase. Such enzymes from haloarchaea offer great potential for biotechnological application because of their stability at high salinity and is therefore worth pursuing.