Taxonomic Reframe of Some Species of the Genera Haloferax and Halobellus.

Haloferax and Halobellus are the representatives of the family Haloferacaceae and they are dominant in hypersaline ecosystems. Some Haloferax and Halobellus species exhibit a close evolutionary relationship. Genomic, phylogenetic (based on 16S rRNA gene sequence), and phylogenomic analysis were performed to evaluate the taxonomic positions of the genera Haloferax and Halobellus. Based on the results we propose to reclassify Halobellus ramosii as a later heterotypic synonym of Halobellus inordinatus; Haloferax lucentense and Haloferax alexandrinum as later heterotypic synonyms of Haloferax volcanii.

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.

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.

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.

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.

Mapping of active replication origins in vivo in thaum- and euryarchaeal replicons.

We report mapping of active replication origins in thaum- and euryarchaeal replicons using high-throughput sequencing-based marker frequency analysis. The chromosome of the thaumarchaeon Nitrosopumilus maritimus is shown to contain a single origin of replication, whereas the main chromosome in the halophilic euryarchaea Haloferax mediterranei and Haloferax volcanii each contains two origins. All replication origins specified bidirectional replication, and the two origins in the halophiles were initiated in synchrony. The pHM500 plasmid of H. mediterranei is shown to contain a single origin, and the copy numbers of five plasmid replicons in the two halophiles were inferred to be close to that of the main chromosome. Origin recognition boxes (ORBs) that provide binding sites for Orc1/Cdc6 replication initiator proteins are identified at all chromosomal origins, as well as in a range of additional thaumarchaeal species. An annotation update is provided for all three species.

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.

Lateral gene transfer occurring in haloarchaea: an interpretative imitation study.

Lateral gene transfer (LGT) plays an important role in the molecular evolution of haloarchaea. Polyethylene glycol-mediated LGT in haloarchaea has been demonstrated in the laboratory, yet few explanations have been put forward for the apparently common, natural occurrence of plentiful plasmids within haloarchaeal cells. In this study, LGT was induced in two genera of haloarchaea, Haloferax and Halorubrum, by modification of salt concentration of media-a factor that may vary naturally in native haloarchaeal habitat. Minimal growth salt concentrations (MGSCs) of four strains of haloarchaea from these two genera were established, and transformations using two circular double-stranded DNAs (dsDNAs), pSY1 and pWL102, were then produced in media at strain-appropriate MGSCs. The four strains of haloarchaea were transformed successfully by both kinds of dsDNAs with an efficiency of 10(2)-10(3) transformants per microgram dsDNA. The transformation under reduced salt concentration may be an imitation of natural LGT of dsDNA into haloarchaea when salinity in normally hypersaline environments is altered by sudden introduction of fresh water--for example, by rainfall, snow-melt, or flooding--providing a reasonable interpretation for haloarchaea being naturally richer in plasmids than any other known organisms.

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.

Taxonomic characterization of Haloferax sp. (" H. alicantei") strain Aa 2.2: description of Haloferax lucentensis sp. nov.

An extremely halophilic archaeon, previously named as Haloferax sp. strain Aa 2.2 or "Haloferax alicantei" that has been extensively used for genetic studies with halobacteria, was taxonomically characterized by using phenotypic tests (including morphological, physiological, biochemical and nutritional features), DNA-DNA hybridization and 16S rRNA sequence phylogenetic analysis. This organism was isolated in 1986 by Torreblanca et al. from a pond of a Spanish saltern located in Alicante. The cells were pleomorphic, Gram negative and grew optimally at 25% NaCl. The polar lipid composition was similar to that of species of the genus Haloferax. The DNA G+C content of this strain was 64.5 mol%. Phylogenetic analysis based on 16S rRNA sequence comparison confirmed that this archaeon is a member of the genus Haloferax and was most closely related to Haloferax volcanii. DNA-DNA hybridization between strain Aa 2.2 and the type strain of all named species of the genus Haloferax revealed low levels of relatedness (25-2%), supporting the placement of this organism in a new species. On the basis of the phenotypic characteristics, molecular data and phylogenetic analysis we propose to name strain Aa 2.2 as a new species, Haloferax lucentensis sp. nov. The type strain is Aa 2.2 (=JCM 9276=NCIMB 13854=CIP 107410=DSM 14919=CECT 5871=CCM 7023).

PCR and blot hybridization for rapid identification of Haloferax species.

Based on the amplification of a 16S rDNA, a PCR assay for the identification of species of Haloferax to genus level was performed. Two variable regions of the 16S rDNA in Haloferax spp. were selected as genus-specific primers for the PCR assay and hybridization probe. Five genera of halophilic Archaea and Escherichia coli were examined as outside groups. Using this approach, all strains of Haloferax spp. were positive. In contrast, all species belonging to the most closely related genera, including Natrinema, Halorubrum, Halobacterium, and Haloarcula, were negative. In addition, the mass bloom of halophilic Archaea that develops in the El-Mallahet saltern of Alexandria City was positive using the same approach. This assay, which does not require pure cultures of microorganisms, is a specific and rapid method for identifying Haloferax spp. in hypersaline environments.

Haloferax alexandrinus sp. nov., an extremely halophilic canthaxanthin-producing archaeon from a solar saltern in Alexandria (Egypt).

An extremely halophilic red micro-organism designated strain TM(T) was isolated from a solar saltern in Alexandria, Egypt. The micro-organism stains gram-negative, is very pleomorphic, non-motile and strictly aerobic and requires at least 10 g NaCl l(-1) for growth. The growth optimum is 250 g NaCl l(-1). Growth is also observed over a wide range of MgSO4 concentrations (10-40 g l(-1)). Aerobic reduction of nitrate without gas production was detected. Cells grew aerobically in a minimal salts medium containing ammonium chloride and glucose. Strain TM(T) produced acid from fructose, glucose, rhamnose, maltose and glycerol. The G+C content of the DNA was 59.5+/-0.3 mol %. On the basis of polar lipid analysis, the isolate belonged to the genus Haloferax. Analysis of the 16S rDNA sequence showed the highest similarity (>99%) to be to the type strain Haloferax volcanii. Although the spectrum of antibiotic susceptibility was similar to that of validly described species of the genus Haloferax, the strain could be distinguished from them by its different response to josamycin and rifampicin. Strain TM(T) is unique within the genus Haloferax in producing canthaxanthin. Comparative analysis of phenotypic properties and DNA-DNA hybridization between strain TM(T) and Haloferax species supported the conclusion that TM(T) is a novel species within this genus, for which the name Haloferax alexandrinus sp. nov. is proposed. The type strain is TM(T) (= JCM 10717T = IFO 16590T).

Taxonomic analysis of extremely halophilic archaea isolated from 56-years-old dead sea brine samples.

A taxonomic study comprising both phenotypic and genotypic characterization, has been carried out on a total of 158 extremely halophilic aerobic archaeal strains. These strains were isolated from enrichments prepared from Dead Sea water samples dating from 1936 that were collected by B. E. Volcani for the demonstration of microbial life in the Dead Sea. The isolates were examined for 126 morphological, physiological, biochemical and nutritional tests. Numerical analysis of the data, by using the S(J) coefficient and UPGMA clustering method, showed that the isolates clustered into six phenons. Twenty-two out of the 158 strains used in this study were characterized previously (ARAHAL et al., 1996) and were placed into five phenotypic groups. The genotypic study included both the determination of the guanineplus-cytosine content of the DNA and DNA-DNA hybridization studies. For this purpose, representative strains from the six phenons were chosen. These groups were found to represent some members of three different genera - Haloarcula (phenons A, B, and C), Haloferax (phenons D and E) and Halobacterium (phenon F) - of the family Halobacteriaceae, some of them never reported to occur in the Dead Sea, such as Haloarcula hispanica, while Haloferax volcanii (phenons D and E) was described in the Dead Sea by studies carried out several decades later than Volcani's work.

Transfer of Halobacterium denitrificans (Tomlinson, Jahnke, and Hochstein) to the genus Haloferax as Haloferax denitrificans comb. nov.

Halobacterium denitrificans (Tomlinson, Jahnke, and Hochstein) was described at a time when the taxonomic subdivision of the family Halobacteriaceae was in a state of flux. On the basis of both biochemical and chemotaxonomic data, this organism exhibits features which indicate that it is more closely related to members of the genus Haloferax. On the basis of such criteria, we propose that Halobacterium denitrificans be reclassified as Haloferax denitrificans comb. nov. The type strain is strain ATCC 35960 (= DSM 4425).