Discovery of bacteriorhodopsins in Haloarchaeal species isolated from Indian solar salterns: deciphering the role of the N-terminal residues in protein folding and functional expression.

Interesting optical and photochemical properties make microbial rhodopsin a promising biological material suitable for various applications, but the cost-prohibitive nature of production has limited its commercialization. The aim of this study was to explore the natural biodiversity of Indian solar salterns to isolate natural bacteriorhodopsin (BR) variants that can be functionally expressed in Escherichia coli. In this study, we report the isolation, functional expression and purification of BRs from three pigmented haloarchaea, wsp3 (water sample Pondicherry), wsp5 and K1T isolated from two Indian solar salterns. The results of the 16S rRNA data analysis suggest that wsp3, wsp5 and K1T are novel strains belonging to the genera Halogeometricum, Haloferax and Haloarcula respectively. Overall, the results of our study suggest that 17 N-terminal residues, that were not included in the gene annotation of the close sequence homologues, are essential for functional expression of BRs. The primary sequence, secondary structural content, thermal stability and absorbance spectral properties of these recombinant BRs are similar to those of the previously reported Haloarcula marismortui HmBRI. This study demonstrates the cost-effective, functional expression of BRs isolated from haloarchaeal species using E. coli as an expression host and paves the way for feasibility studies for future applications.

Genome sequence and description of Haloferax massiliense sp. nov., a new halophilic archaeon isolated from the human gut.

By applying the culturomics concept and using culture conditions containing a high salt concentration, we herein isolated the first known halophilic archaeon colonizing the human gut. Here we described its phenotypic and biochemical characterization as well as its genome annotation. Strain Arc-HrT (= CSUR P0974 = CECT 9307) was mesophile and grew optimally at 37 °C and pH 7. Strain Arc-HrT was also extremely halophilic with an optimal growth observed at 15% NaCl. It showed gram-negative cocci, was strictly aerobic, non-motile and non-spore-forming, and exhibited catalase and oxidase activities. The 4,015,175 bp long genome exhibits a G + C% content of 65.36% and contains 3911 protein-coding and 64 predicted RNA genes. PCR-amplified 16S rRNA gene of strain Arc-HrT yielded a 99.2% sequence similarity with Haloferax prahovense, the phylogenetically closest validly published species in the Haloferax genus. The DDH was of 50.70 ± 5.2% with H. prahovense, 53.70 ± 2.69% with H. volcanii, 50.90 ± 2.64% with H. alexandrinus, 52.90 ± 2.67% with H. gibbonsii and 54.30 ± 2.70% with H. lucentense. The data herein represented confirm strain Arc-HrT as a unique species and consequently we propose its classification as representative of a novel species belonging to the genus Haloferax, as Haloferax massiliense sp. nov.

Isolation and characterization from solar salterns of North Algeria of a haloarchaeon producing a new halocin.

Halophilic archaea, thriving in hypersaline environments, synthesize antimicrobial substances with an unknown role, called halocins. It has been suggested that halocin production gives transient competitive advantages to the producer strains and represents one of the environmental factors influencing the microbial community composition. Herein, we report on the antibacterial activity of a new haloarchaeon selected from solar salterns of the northern coast of Algeria. A total of 81 halophilic strains, isolated from the microbial consortia, were screened for the production of antimicrobial compounds by interspecies competition test and against a collection of commercial haloarchaea. On the basis of the partial 16S rRNA sequencing, the most efficient halocin producer was recognized as belonging to Haloferax (Hfx) sp., while the best indicator microorganism, showing high sensitivity toward halocin, was related to Haloarcula genus. The main morphological, physiological and biochemical properties of Hfx were investigated and a partial purification of the produced halocin was allowed to identify it as a surface membrane protein with a molecular mass between 30 and 40 kDa. Therefore, in this study, we isolated a new strain belonging to Haloferax genus and producing a promising antimicrobial compound useful for applications in health and food industries.

Isolation and characterization of extremely halophilic CO-oxidizing Euryarchaeota from hypersaline cinders, sediments and soils and description of a novel CO oxidizer, Haloferax namakaokahaiae Mke2.3T, sp. nov.

The phylogenetic affiliations of organisms responsible for aerobic CO oxidation in hypersaline soils and sediments were assessed using media containing 3.8 M NaCl. CO-oxidizing strains of the euryarchaeotes, Haloarcula, Halorubrum, Haloterrigena and Natronorubrum, were isolated from the Bonneville Salt Flats (UT) and Atacama Desert salterns (Chile). A halophilic euryarchaeote, Haloferax strain Mke2.3(T), was isolated from Hawai'i Island saline cinders. Haloferax strain Mke2.3(T) was most closely related to Haloferax larsenii JCM 13917(T) (97.0% 16S rRNA sequence identity). It grew with a limited range of substrates, and oxidized CO at a headspace concentration of 0.1%. However, it did not grow with CO as a sole carbon and energy source. Its ability to oxidize CO, its polar lipid composition, substrate utilization and numerous other traits distinguished it from H. larsenii JCM 13917(T), and supported designation of the novel isolate as Haloferax namakaokahaiae Mke2.3(T), sp. nov (= DSM 29988, = LMG 29162). CO oxidation was also documented for 'Natronorubrum thiooxidans' HG1 (Sorokin, Tourova and Muyzer 2005), N. bangense (Xu, Zhou and Tian 1999) and N. sulfidifaciens AD2(T) (Cui et al. 2007). Collectively, these results established a previously unsuspected capacity for extremely halophilic aerobic CO oxidation, and indicated that the trait might be widespread among the Halobacteriaceae, and occur in a wide range of hypersaline habitats.

Description of a halocin-producing Haloferax larsenii HA1 isolated from Pachpadra salt lake in Rajasthan.

Haloarchaea grow in the extreme environment, such as high salt concentration, and secrete antimicrobial peptides known as halocins. Identification of Haloferax larsenii strain HA1 was carried out using biochemical and molecular methods. Strain HA1 was found as a strict aerobe, catalase positive and Gram negative. It was able to grow optimally at 15 % NaCl (w/v), 42 °C and pH 7.2. Strain HA1 was sensitive to bile acid, was resistant to chloramphenicol and could not utilize arginine. Halocin, produced by strain HA1, was stable up to 100 °C and in a pH range of 5.0-9.0. Antimicrobial activity was not affected by organic solvents, surfactants and detergents, but it was completely lost in the presence of proteinase K, suggesting proteinaceous nature of the compound. It was halocidal against indicator strain Hfx. larsenii HA10. The molecular weight of halocin HA1 was found to be ~14 kDa. These properties of halocin HA1 may be applicable to the preservation of salted foods.

Complete genome sequence of Haloferax gibbonsii strain ARA6, a potential producer of polyhydroxyalkanoates and halocins isolated from Araruama, Rio de Janeiro, Brasil.

Haloferax gibbonsii strain ARA6 is a haloarchaea isolated from saline saltern samples from Vermelha lake, located in Araruama region, Rio de Janeiro, Brazil. Its genome displays 66,2% G+C content and is composed by one circular chromosome of 2,945,391 bp and four circular plasmids comprising 993,063 bp. This genomic information shows H. gibbonsii's potential for biotechnological applications and can also contribute to assign evolutionary traits in the genus Haloferax.

Accumulation of polyhydroxyalkanoates by halophilic archaea isolated from traditional solar salterns of India.

Extremely halophilic archaeal isolates obtained from brine and sediment samples of solar salterns of Goa and Tamil Nadu, India were screened for accumulation of polyhydroxyalkanoates (PHA). Seven polymer accumulating haloarchaeal strains (TN4, TN5, TN6, TN7, TN9, TN10 and BBK2) were selected based on their growth and intensity of fluorescence when grown on 20 % NaCl synthetic medium supplemented with 2 % glucose and incorporated with Nile red dye. The polymer was quantified by conversion of PHA to crotonic acid which gave a characteristic absorption maxima at 235 nm. On the basis of phenotypic and genotypic characterization the cultures TN4, TN5, TN6, TN7, TN10 and BBK2 were grouped under genus Haloferax whereas isolate TN9 was grouped under the genus Halogeometricum. Growth kinetics and polymer accumulation studies revealed that the culture Halogeometricum borinquense strain TN9 accumulates PHA maximally at the mid-log phase, i.e. 5th day of growth (approx. 14 wt% PHA of CDW). Analysis of the polymer by IR, (1)H NMR and (13)C NMR confirmed it to be a homopolymer of 3-hydroxybutyrate.

Haloferax chudinovii sp. nov., a halophilic archaeon from Permian potassium salt deposits.

Three pigmented strains of halophilic archaea (RS75, RS77, RS79) were isolated from the monoliths of mottled sylvinite from the Verkhnekamsk salt deposit (Solikamsk, Russia). The cells were nonmotile, gram-negative, pleomorphic, disk-shaped or ovoid, 0.8-1.0 × 1.5-2.5 µm. The organism was a chemoorganotrophic obligate aerobe producing catalase and oxidase. A number of carbohydrates and carboxylic acids were used as growth substrates. Growth occurred in the presence of 7-27% NaCl (with the optimum at 15-18%), 0.02-20% KCl (0.2-1%), 0.2-16% MgCl2 (2-3%), in the temperature range from 23 to 51 °C (40-45 °C), and pH 5.5-8.0 (6.8-7.0). The membranes contained carotenoids of the bacterioruberin series. Phosphatidylglyceromethylphosphate (PGP-Me), phosphatidylglycerol (PG), sulfated diglycosyl diether (S-DGD-1) predominated among the polar lipids. The DNA G + C content was 64.0-65.0 mol %. Phylogenetic analysis of the 16S rRNA gene sequences showed high similarity of the new strains to Haloferax species: H. denitrificans (99.2%) and H. volcanii (99.1%), H. larsenii (96.9%) and H. elongans (96.6%). DNA-DNA hybridization revealed 93-95% similarity between strain RS75 and strains RS77 and RS79; the similarity levels between strain RS75 and the type strains of Haloferax denitrificans VKM B-1754(T) and Halobacterium salinarum VKM B-1769(T) were 50 and 10%, respectively. According to its phenotypic and genotypic characteristics, the organism was classified as a member of the genus Haloferax, forming a new species with the proposed name Haloferax chudinovii sp. nov. type strain is RS75(T) (=VKPM B-11279(T)).

Isolation of hydrocarbon-degrading extremely halophilic archaea from an uncontaminated hypersaline pond (Camargue, France).

Little information exists about the ability of halophilic archaea present in hypersaline environments to degrade hydrocarbons. In order to identify the potential actors of hydrocarbon degradation in these environments, enrichment cultures were prepared using samples collected from a shallow crystallizer pond with no known contamination history in Camargue, France, with n-alkanes provided as source of carbon and energy. Five alkane-degrading halophilic archaeal strains were isolated: one (strain MSNC 2) was closely related to Haloarcula and three (strains MSNC 4, MSNC 14, and MSNC 16) to Haloferax. Biodegradation assays showed that depending on the strain, 32 to 95% (0.5 g/l) of heptadecane was degraded after 30 days of incubation at 40 degrees C in 225 g/l NaCl artificial medium. One of the strains (MSNC 14) was also able to degrade phenanthrene. This work clearly shows for the first time the potential role of halophilic archaea belonging to the genera Haloarcula and Haloferax in the degradation of hydrocarbons in both pristine and hydrocarbon-contaminated hypersaline environments.

Halogranum rubrum gen. nov., sp. nov., a halophilic archaeon isolated from a marine solar saltern.

Two extremely halophilic archaea, strains RO2-11(T) and HO2-1, were isolated from two Chinese marine solar salterns, Rudong solar saltern and Haimen solar saltern, respectively. Cells of the two strains were polymorphic and Gram-stain-negative; colonies were red-pigmented. The two strains grew at NaCl concentrations of 2.6-4.3 M (optimum 3.9 M) and required at least 0.1 M Mg2+ for growth. They were able to grow over a pH range of 6.0-8.0 and a temperature range of 20-50 degrees C, with optimal pH of 7.5 and optimal temperature of 37 degrees C. The major polar lipids of strain RO2-11(T) and strain HO2-1 were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and three glycolipids, two of them chromatographically identical to S-DGD-1 and DGD-1, the third unidentified. The 16S rRNA gene sequence similarity of strain RO2-11(T) and strain HO2-1 was 99.3 % and highest sequence similarity with the closest relative (Haloferax larsenii) was 91.4 %. Based on the data obtained, the two isolates could not be classified in any recognized genus of the family Halobacteriaceae. Strain RO2-11(T) and strain HO2-1 are thus considered to represent a novel species of a new genus within the family Halobacteriaceae, for which the name Halogranum rubrum gen. nov., sp. nov. is proposed. The type strain is RO2-11(T) (=CGMCC 1.7738(T) =JCM 15772(T)).

Haloferax elongans sp. nov. and Haloferax mucosum sp. nov., isolated from microbial mats from Hamelin Pool, Shark Bay, Australia.

Extremely halophilic archaea were cultivated from smooth and pustular microbial mats collected from Hamelin Pool, Shark Bay, Western Australia. On the basis of morphology, two phenotypes were present and 16S rRNA gene sequence analysis indicated that all strains were most closely related to members of the genus Haloferax (98.1-99.4 % similarity). One representative strain from each phenotype was selected for further taxonomic characterization. Strain SA5T, isolated from the smooth mat, formed small ( approximately 1 mm diameter), red, translucent colonies on agar medium and strain PA12T, isolated from the pustular mat, formed large (3-5 mm diameter), pink, mucoid, domed colonies. Both strains grew in media with 1.7-5.1 M NaCl, required at least 0.2 M Mg2+ for growth and had pH optima of 7.4. The 16S rRNA gene similarity between strains SA5T and PA12T was 97.1 %. Physiological properties, G+C content and polar lipid composition supported placement of both strains in the genus Haloferax. Phenotypic analysis indicated that the two strains were distinct from each other and from all other members of the genus. This was confirmed by the low DNA-DNA relatedness between strains SA5T and PA12T (18-30 %) and between both strains and all other recognized Haloferax species. Two novel species of the genus Haloferax are proposed to accommodate these novel isolates, Haloferax elongans sp. nov. (type strain SA5T=JCM 14791T=ATCC BAA-1513T=UNSW 104100T) and Haloferax mucosum sp. nov. (type strain PA12T=JCM 14792T=ATCC BAA-1512T=UNSW 104200T).

Haloferax larsenii sp. nov., an extremely halophilic archaeon from a solar saltern.

Three strains of Gram-negative, aerobic, neutrophilic, extremely halophilic archaea, designated ZJ206(T), ZJ203 and ZJ204, were isolated from a solar saltern in Zhe-Jiang Province, China. Phenotypically and on the basis of 16S rRNA gene sequences, the strains were very similar. Comparative 16S rRNA gene analysis revealed 96.4-97.4 % sequence similarity to members of the genus Haloferax. The major polar lipids were C(20)C(20) derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, diglycosyl glycerol diether and sulfated diglycosyl diether. The DNA G+C content of strain ZJ206(T) was 62.2 mol%. The results of DNA-DNA hybridizations and physiological and biochemical tests allowed genotypic and phenotypic differentiation of the isolates from closely related species. Therefore the isolates should be classified as members of a novel species, for which the name Haloferax larsenii sp. nov. is proposed. The type strain is ZJ206(T) (=CGMCC 1.5347(T)=JCM 13917(T)).

Haloferax prahovense sp. nov., an extremely halophilic archaeon isolated from a Romanian salt lake.

A novel halophilic archaeon, strain TL6T, was isolated from Telega Lake, a hypersaline environment in Prahova county, Romania. Strain TL6T was able to grow in media with a salt concentration of between 2.5 and 5.2 M, with optimum growth at a concentration of 3.5 M. The novel strain was able to grow at concentrations of 1 M MgCl2 or less, with an optimum of 0.4 M Mg2+. Growth of the novel strain occurred between pH 6.0 and 8.5, with an optimum of pH 7.0-7.5. The G+C content of the total DNA was 63.7 mol%. The 16S rRNA gene sequence of the novel strain was most closely related to species of the genus Haloferax (97.3-99.3 % sequence similarity). The lipid profile of the novel strain corresponded to that of other species belonging to the genus Haloferax. A comparative analysis of the phenotypic properties and DNA-DNA hybridization between the novel strain and other species of the genus Haloferax strongly supported the conclusion that strain TL6T represents a novel species within this genus, for which the name Haloferax prahovense sp. nov., is proposed. The type strain is TL6T (=JCM 13924T=DSM 18310T).

Haloferax sulfurifontis sp. nov., a halophilic archaeon isolated from a sulfide- and sulfur-rich spring.

A pleomorphic, extremely halophilic archaeon (strain M6(T)) was isolated from a sulfide- and sulfur-rich spring in south-western Oklahoma (USA). It formed small (0.8-1.0 mm), salmon pink, elevated colonies on agar medium. The strain grew in a wide range of NaCl concentrations (6 % to saturation) and required at least 1 mM Mg(2+) for growth. Strain M6(T) was able to reduce sulfur to sulfide anaerobically. 16S rRNA gene sequence analysis indicated that strain M6(T) belongs to the family Halobacteriaceae, genus Haloferax; it showed 96.7-98.0 % similarity to other members of the genus with validly published names and 89 % similarity to Halogeometricum borinquense, its closest relative outside the genus Haloferax. Polar lipid analysis and DNA G+C content further supported placement of strain M6(T) in the genus Haloferax. DNA-DNA hybridization values, as well as biochemical and physiological characterization, allowed strain M6(T) to be differentiated from other members of the genus Haloferax. A novel species, Haloferax sulfurifontis sp. nov., is therefore proposed to accommodate the strain. The type strain is M6(T) (=JCM 12327(T)=CCM 7217(T)=DSM 16227(T)=CIP 108334(T)).

Combined use of cultivation-dependent and cultivation-independent methods indicates that members of most haloarchaeal groups in an Australian crystallizer pond are cultivable.

Haloarchaea are the dominant microbial flora in hypersaline waters with near-saturating salt levels. The haloarchaeal diversity of an Australian saltern crystallizer pond was examined by use of a library of PCR-amplified 16S rRNA genes and by cultivation. High viable counts (10(6) CFU/ml) were obtained on solid media. Long incubation times (> or =8 weeks) appeared to be more important than the medium composition for maximizing viable counts and diversity. Of 66 isolates examined, all belonged to the family Halobacteriaceae, including members related to species of the genera Haloferax, Halorubrum, and Natronomonas. In addition, isolates belonging to a novel group (the ADL group), previously detected only as 16S rRNA genes in an Antarctic hypersaline lake (Deep Lake), were cultivated for the first time. The 16S rRNA gene library identified the following five main groups: Halorubrum groups 1 and 2 (49%), the SHOW (square haloarchaea of Walsby) group (33%), the ADL group (16%), and the Natronomonas group (2%). There were two significant differences between the organisms detected in cultivation and 16S rRNA sequence results. Firstly, Haloferax spp. were frequently isolated on plates (15% of all isolates) but were not detected in the 16S rRNA sequences. Control experiments indicated that a bias against Haloferax sequences in the generation of the 16S rRNA gene library was unlikely, suggesting that Haloferax spp. readily form colonies, even though they were not a dominant group. Secondly, while the 16S rRNA gene library identified the SHOW group as a major component of the microbial community, no isolates of this group were obtained. This inability to culture members of the SHOW group remains an outstanding problem in studying the ecology of hypersaline environments.

Microorganismos halófitos: potencial biotecnológico e interés farmacéutico / Biotechnological potential and pharmaceutical interest of Halophilic Microorganisms

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Fluorescence in situ hybridization analysis of the prokaryotic community inhabiting crystallizer ponds.

A fluorescence in situ hybridization (FISH) protocol suitable for the identification of prokaryotes inhabiting hypersaline environments was developed and applied to several crystallizer ponds with salinities above 36% from a multipond solar saltern in Alicante, Spain. Two morphotypes were abundant in these environments: rods and square or square-like prokaryotes that could be affiliated to Bacteria and Archaea, respectively, by FISH with domain-specific probes. FISH with a newly designed probe proved that the archaeal 16S rDNA sequence most frequently recovered from the crystallizers, SPhT, originated from the dominant square-like prokaryotes. These uncultured prokaryotes have the morphology of Walsby's square bacteria. Additionally, FISH with a probe targeted to the genus Haloarcula, members of which are frequently isolated from this environment, indicated that this genus accounts for less than 0.1% of the total prokaryotic community.