Symbiosis between nanohaloarchaeon and haloarchaeon is based on utilization of different polysaccharides.

Nano-sized archaeota, with their small genomes and limited metabolic capabilities, are known to associate with other microbes, thereby compensating for their own auxotrophies. These diminutive and yet ubiquitous organisms thrive in hypersaline habitats that they share with haloarchaea. Here, we reveal the genetic and physiological nature of a nanohaloarchaeon-haloarchaeon association, with both microbes obtained from a solar saltern and reproducibly cultivated together in vitro. The nanohaloarchaeon Candidatus Nanohalobium constans LC1Nh is an aerotolerant, sugar-fermenting anaerobe, lacking key anabolic machinery and respiratory complexes. The nanohaloarchaeon cells are found physically connected to the chitinolytic haloarchaeon Halomicrobium sp. LC1Hm. Our experiments revealed that this haloarchaeon can hydrolyze chitin outside the cell (to produce the monosaccharide N-acetylglucosamine), using this beta-glucan to obtain carbon and energy for growth. However, LC1Hm could not metabolize either glycogen or starch (both alpha-glucans) or other polysaccharides tested. Remarkably, the nanohaloarchaeon's ability to hydrolyze glycogen and starch to glucose enabled growth of Halomicrobium sp. LC1Hm in the absence of a chitin. These findings indicated that the nanohaloarchaeon-haloarchaeon association is both mutualistic and symbiotic; in this case, each microbe relies on its partner's ability to degrade different polysaccharides. This suggests, in turn, that other nano-sized archaeota may also be beneficial for their hosts. Given that availability of carbon substrates can vary both spatially and temporarily, the susceptibility of Halomicrobium to colonization by Ca Nanohalobium can be interpreted as a strategy to maximize the long-term fitness of the host.

Survival of the Halophilic Archaeon Halovarius luteus after Desiccation, Simulated Martian UV Radiation and Vacuum in Comparison to Bacillus atrophaeus.

Extraterrestrial environments influence the biochemistry of organisms through a variety of factors, including high levels of radiation and vacuum, temperature extremes and a lack of water and nutrients. A wide variety of terrestrial microorganisms, including those counted amongst the most ancient inhabitants of Earth, can cope with high levels of salinity, extreme temperatures, desiccation and high levels of radiation. Key among these are the haloarchaea, considered particularly relevant for astrobiological studies due to their ability to thrive in hypersaline environments. In this study, a novel haloarchaea isolated from Urmia Salt Lake, Iran, Halovarius luteus strain DA50T, was exposed to varying levels of simulated extraterrestrial conditions and compared to that of the bacteria Bacillus atrophaeus. Bacillus atrophaeus was selected for comparison due to its well-described resistance to extreme conditions and its ability to produce strong spore structures. Thin films were produced to investigate viability without the protective influence of cell multi-layers. Late exponential phase cultures of Hvr. luteus and B. atrophaeus were placed in brine and phosphate buffered saline media, respectively. The solutions were allowed to evaporate and cells were encapsulated and exposed to radiation, desiccation and vacuum conditions, and their post-exposure viability was studied by the Most Probable Number method. The protein profile using High Performance Liquid Chromatography and Matrix Assisted Laser Desorption/Ionization bench top reflector time-of-flight are explored after vacuum and UV-radiation exposure. Results showed that the change in viability of the spore-forming bacteria B. atrophaeus was only minor whereas Hvr. luteus demonstrated a range of viability under different conditions. At the peak radiation flux of 105 J/m2 under nitrogen flow and after two weeks of desiccation, Hvr. luteus demonstrated the greatest decrease in viability. This study further expands our understanding of the boundary conditions of astrobiologically relevant organisms in the harsh space environment.

MALDI-TOF analysis of a novel extremophile peptide purified from Halarchaeum acidiphilum ASDL78 with antiarchaeal and antibacterial activities.

In hypersaline environments, halophilic archaea synthesize antimicrobial substances called halocins. There is a promise to make new drugs for antibiotic-resistant strains. Here, we report the antibacterial activity of a new haloarchaea selected from Lut Desert, Iran. A total of 38 isolated halophilic bacteria and archaea were screened for the antagonistic activity test of each strain against other bacterial and archaeal strains. Finally, a strain, recognized as Halarchaeum acidiphilum, with a fast grown strain and high antagonistic potential against different strains was identified by morphological, physiological, and molecular characteristics. The halocin was produced in a semisolid submerge medium and partially purified by heat treatments and molecular weight ultrafiltration cutoff (3, 50, and 10 kDa). It was a cell-free, heat-resistant (85°C for 2 h) protein with a molecular mass near to 20 kDa produced at the endpoint of logarithmic growth. The molecular weight of halocin was 17 kDa, and indicated no apparent homology with known halocins, suggesting that this might be a new halocin. Therefore, a new strain belonging to Halarchaeum genus was isolated and characterized here that produced an antimicrobial and anti-haloarchaea halocin.

Haloterrigena salifodinae sp. nov., an extremely halophilic archaeon isolated from a subterranean rock salt.

A novel extremely halophilic strain, designated ZY19T, was isolated from a rock salt sample from Yunnan salt mine, PR China. Strain ZY19T is neutrophilic, non-motile and requires at least 10% (w/v) NaCl for growth. Optimal growth is observed at 20-25% (w/v) NaCl, pH 7.5-8.0 and 42 °C. Mg2+ is not required for growth. The cells do not lyse in distilled water. On the basis of 16S rRNA gene sequence analysis, strain ZY19T belongs to the genus Haloterrigena (Htg.) and is closely related to Haloterrigena salina XH-65T (98.5% sequence similarity) and Haloterrigena turkmenica DSM 5511T (97.9%). Phylogenetic and phylogenomic analysis showed that strain ZY19T clusters with the species Htg. salina and Htg. turkmenica forming an independent clade separated from other members of the genus. The value of genomic average nucleotide identity (ANI) between strains ZY19T and its close relative, Htg. salina XH-65T was 94.2%. DNA-DNA relatedness between strains ZY19T and Htg. salina XH-65T revealed by in silico DNA-DNA hybridization (DDH) was 56.3%. Both the ANI value and the degree of in silico DDH are below the accepted threshold for members of the same species. The major polar lipids were found to consist of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, sulfated diglycosyl-diether-1 and mannose-2, 6-disulfate (1 → 2)-glucose glycerol diether. The genomic DNA G+C content was determined to be 64.5 mol%. Based on the results of the phenotypic, chemotaxonomic, genetic similarity and inferred phylogeny, strain ZY19T is distinct from other validly named species and thus represents a novel species within the genus Haloterrigena, for which the name Haloterrigena salifodinae sp. nov. is proposed. The type strain is ZY19T (=CGMCC 1.16114T=NBRC 112981T).

Haererehalobacter sp. JS1, a bioemulsifier producing halophilic bacterium isolated from Indian solar salt works.

Bioemulsifier (BE)-producing Haererehalobacter sp. JS1 was isolated and identified from the solar salt works in India. The BE was extracted, purified, and characterized by Gas Chromatography-Mass Spectrometry (GC-MS) analysis. Emulsification activity was performed against different oils and dye degradation potential against different dyes. The production of BE was optimized using different carbon sources (C), nitrogen sources (N), pH, and NaCl. BE screening methods revealed that, Haererehalobacter sp. JS1 was highly positive BE production. Identification by 16S rRNA sequencing and analyses was found that, the Haererehalobacter sp. JS1 was closely related to Salinicoccus halophilus and Haererehalobacter sp. The structural characterization analysis confirmed that the partially purified bioemulsifier belongs to siloxane-type. Emulsification activity (E24) revealed that the bioemulsifier significantly (p < = 0.001) emulsified the commercial oils including coconut oil, gingelly oil, olive oil, and palmolein oils. Haererehalobacter sp. JS1 also significantly (p < = 0.001) degraded the dyes such as orange MR, direct violet, cotton red, reactive yellow, nitro green, and azo dye. RSM regression co-efficient and contour plot analysis clearly indicated that the combination of pH and NaCl helped to increase BE production. Siloxane-type of BE obtained from Haererehalobacter sp. JS1 was able to emulsify different oils and commercial dyes.

Antagonistic interactions and production of halocin antimicrobial peptides among extremely halophilic prokaryotes isolated from the solar saltern of Sfax, Tunisia.

Thirty-five extremely halophilic microbial strains isolated from crystallizer (TS18) and non-crystallizer (M1) ponds in the Sfax solar saltern in Tunisia were examined for their ability to exert antimicrobial activity. Antagonistic assays resulted in the selection of eleven strains that displayed such antimicrobial activity and they were further characterized. Three cases of cross-domain inhibition (archaea/bacteria or bacteria/archaea) were observed. Four archaeal strains exerted antimicrobial activity against several other strains. Three strains, for which several lines of evidence suggested the antimicrobial activity was, at least in part, due to peptide/protein agents (Halobacterium salinarum ETD5, Hbt. salinarum ETD8, and Haloterrigena thermotolerans SS1R12), were studied further. Optimal culture conditions for growth and antimicrobial production were determined. Using DNA amplification with specific primers, sequencing and RT-PCR analysis, Hbt. salinarum ETD5 and Hbt. salinarum ETD8 were shown to encode and express halocin S8, a hydrophobic antimicrobial peptide targeting halophilic archaea. Although the gene encoding halocin H4 was amplified from the genome of Htg. thermotolerans SS1R12, no transcript could be detected and the antimicrobial activity was most likely due to multiple antimicrobial compounds. This is also the first report that points to four different strains isolated from different geographical locations with the capacity to produce identical halocin S8 proteins.

Halorussus ruber sp. nov., isolated from an inland salt lake of China.

Halophilic archaeal strain YC25(T) was isolated from Yuncheng salt lake in Shanxi, China. Cells of strain YC25(T) were observed to be pleomorphic rods, stained Gram-negative, and formed red-pigmented colonies on solid media. Strain YC25(T) was found to be able to grow at 25-50 °C (optimum 37 °C), at 1.4-4.8 M NaCl (optimum 1.7 M), at 0-1.0 M MgCl2 (optimum 0.01 M), and at pH 5.5-9.0 (optimum pH 6.5). The cells lysed in distilled water, and the minimal NaCl concentration to prevent cell lysis was found to be 8 % (w/v). The major polar lipids of the strain were phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol sulfate (PGS), sulfated galactosyl mannosyl glucosyl diether (S-TGD-1), sulfated mannosyl glucosyl diether (S-DGD-1), galactosyl mannosyl glucosyl diether (TGD-1), mannosyl glucosyl diether (DGD-1), and an unknown diglycosyl diether (DGD-2) chromatographically identical to those of Halorussus rarus CGMCC 1.10122(T). The 16S rRNA gene and rpoB' gene of strain YC25(T) were phylogenetically related to the corresponding genes of Halorussus rarus CGMCC 1.10122(T) (94.3-95.4 and 91.5 % nucleotide identity, respectively). The DNA G+C content of strain YC25(T) was determined to be 63.3 mol%. The phenotypic, chemotaxonomic, and phylogenetic properties suggested that strain YC25(T) (=CGMCC 1.12122(T) = JCM 18363(T)) represents a new species of Halorussus, for which the name Halorussus ruber sp. nov. is proposed.

Halosimplex litoreum sp. nov., isolated from a marine solar saltern.

A halophilic archaeal strain, YGH94(T), was isolated from the Yinggehai marine solar saltern near the Shanya city of Hainan Province, China. Cells of the strain were observed to be short rods, stain Gram-negative and to form red-pigmented colonies on solid media. Strain YGH94(T) was found to grow at 25-50 °C (optimum 40 °C), at 0.9-4.8 M NaCl (optimum 3.1 M), at 0-1.0 M MgCl2 (optimum 0.05 M) and at pH 5.0-9.0 (optimum pH 7.5). The cells were found to lyse in distilled water and the minimal NaCl concentration to prevent cell-lysis was determined to be 5 % (w/v). The major polar lipids were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and four major glycolipids (disulfated mannosyl glucosyl diether, sulfated mannosyl glucosyl diether and two unidentified glycolipids chromatographically identical to glycolipids in Halosimplex carlsbadense JCM 11222(T)). Strain YGH94(T) was found to possess two heterogeneous 16S rRNA genes (rrnA and rrnB) and both are related to those of Hsx. carlsbadense JCM 11222(T) (92.7-98.6 % similarities), Halosimplex pelagicum R2(T) (94.6-99.2 % similarities) and Halosimplex rubrum R27(T) (92.9-98.8 % similarities). The rpoB' gene similarity between strain YGH94(T) and Hsx. carlsbadense JCM 11222(T), Hsx. pelagicum R2(T) and Hsx. rubrum R27(T) are 95.4, 94.9 and 95.1 %, respectively. The DNA G+C content of strain YGH94(T) was determined to be 64.0 mol%. Strain YGH94(T) showed low DNA-DNA relatedness (35-39 %) with the current three members of the genus Halosimplex. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain YGH94(T) (=CGMCC 1.12235(T) = JCM 18647(T)) represents a new species of the genus Halosimplex, for which the name Halosimplex litoreum sp. nov. is proposed.

Taxonomy of halophilic Archaea: current status and future challenges.

Several groups of Archaea, all Euryarchaeota, develop in hypersaline environments (from >10% salt up to saturation). The cultured diversity of halophilic Archaea includes the family Halobacteriaceae of aerobic or facultative anaerobic, generally red-pigmented species (47 genera and 165 species as of February 2014) and seven representatives of four genera of methanogens, most of which obtain energy from methylated amines under anaerobic conditions. Metagenomic studies have identified an additional deep lineage of Archaea in salt lakes and ponds with brines approaching NaCl saturation. Genomic information is now available for representatives of these 'Nanohaloarchaea', but no members of this lineage have yet been cultured. Multilocus sequence analysis is becoming increasingly popular in taxonomic studies of the Halobacteriaceae, and such studies have demonstrated that recombination of genetic traits occurs at an extremely high frequency at least in some genera. Metagenomic studies in an Antarctic lake showed that large identical regions of up to 35 kb in length can be shared by members of different genera living together in the same environment. Such observations have important implications not only for the taxonomy of the Halobacteriaceae, but also for species concepts and questions on taxonomy and classification for prokaryotic microorganisms in general.

Haloarchaeobius litoreus sp. nov., isolated from a marine solar saltern.

Two extremely halophilic archaeal strains GX1(T) and GX60 were isolated from the Gangxi marine solar saltern, China. Cells from the two strains were observed to be rod-shaped and stained Gram-negative, with red-pigmented colonies. Strains GX1(T) and GX60 were found to be able to grow at 25-50 °C (optimum 37 °C), at 1.4-4.8 M NaCl (optimum 2.6 M), at pH 5.5-9.5 (optimum pH 7.0) and neither strain required Mg(2+) for growth. The cells lysed in distilled water and the minimal NaCl concentration to prevent cell-lysis was found to be 8 % (w/v). The major polar lipids of the two strains were identified as phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and three glycolipids chromatographically identical to those of Haloarchaeobius iranensis IBRC-M 10013(T). 16S rRNA gene analysis revealed that each strain had two dissimilar 16S rRNA genes and both strains were phylogenetically related to Hab. iranensis IBRC-M 10013(T) (94.9-98.9 % nucleotide identity). The rpoB' gene similarity between strains GX1(T) and GX60, and between these strains and Hab. iranensis IBRC-M 10013(T) were found to be 99.6, 96.0 and 95.8 %, respectively. The DNA G + C content of strain GX1(T) and GX60 were determined to be 67.7 and 67.8 mol %, respectively. The DNA-DNA hybridization value of strains GX1(T) and GX60 was 86 % and the two strains showed low DNA-DNA relatedness with Hab. iranensis IBRC-M 10013(T) (38 and 32 %). It was concluded that strain GX1(T) (= CGMCC 1.10390(T) = JCM 17114(T)) and strain GX60 (= CGMCC 1.10389 = JCM 17120) represent a new species of Haloarchaeobius, for which the name Haloarchaeobius litoreus sp. nov. is proposed.

Halolamina rubra sp. nov., a haloarchaeon isolated from non-purified solar salt.

Two Gram-stain negative, rod-shaped and motile extreme halophiles, designated CBA1107(T) and CBA1108, were isolated from non-purified solar salt. Based on the phylogenetic analysis, strains CBA1107(T) and CBA1108 were shown to belong to the genus Halolamina, with similarities for the 16S rRNA gene sequences between strains CBA1107(T) and Halolamina pelagica TBN21(T) , Halolamina salina WSY15-H3(T) and Halolamina salifodinae WSY15-H1(T) of 98.3, 97.6 and 97.3 %, respectively; the similarities for the rpoB' gene sequences between the same strains were 96.0, 95.3 and 94.6 %, respectively. The colonies of both strains were observed to be red pigmented on growth medium. Strain CBA1107(T) was observed to grow at 20-50 °C, in the presence of 15-30 % NaCl, at pH 6.0-9.0, and with 0.005-0.5 M Mg(2+). The cells of both strains lysed in distilled water. The DNA-DNA hybridization experiments showed that strain CBA1107(T) shared 97 % relatedness with CBA1108 and <50 % relatedness with H. pelagica JCM 16809(T), H. salina JCM 18549(T) and H. salifodinae JCM 18548(T). The genomic DNA G+C content of strain CBA1107(T) was determined to be 65.1 mol%. The major polar lipids of the two strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and glycolipids including sulfated mannosyl glucosyl diether and mannosyl glucosyl diether. Based on the polyphasic taxonomic analyses, the strains are considered to represent a new taxon for which the name Halolamina rubra sp. nov. is proposed, with the type strain CBA1107(T) (=CECT 8421(T) =JCM 19436(T)).

Haloplanus litoreus sp. nov. and Haloplanus ruber sp. nov., from a marine solar saltern and an aquaculture farm, respectively.

Two halophilic archaea, strains GX21(T) and R35(T), were isolated from a marine solar saltern and an aquaculture farm in China, respectively. Cells of the two strains were observed to be pleomorphic, flat, to contain gas vesicles, stain Gram-negative and produce red-pigmented colonies. Strain GX21(T) was found to be able to grow at 25-50 °C (optimum 37 °C), at 2.6-4.8 M NaCl (optimum 3.4 M NaCl), at 0.05-1.0 M MgCl2 (optimum 0.1 M MgCl2) and at pH 6.0-8.5 (optimum pH 6.5) while strain R35(T) was found to be able to grow at 25-45 °C (optimum 37 °C), at 2.1-4.8 M NaCl (optimum 3.1 M NaCl), at 0-0.7 M MgCl2 (optimum 0.03 M MgCl2) and at pH 5.5-9.5 (optimum pH 6.5-7.0). The cells of both isolates were observed to lyse in distilled water. The minimum NaCl concentrations that prevented cell lysis were determined to be 15 % (w/v) for strain GX21(T) and 12 % (w/v) for strain R35(T). The major polar lipids of the two strains were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, one major glycolipid and a minor lipid chromatographically identical to sulfated mannosyl glucosyl diether and mannosyl glucosyl diether, respectively. 16S rRNA gene sequence analysis revealed that strains GX21(T) and R35(T) show 97.1 % sequence similarity to each other and are closely related to Haloplanus aerogenes TBN37(T) (96.8 and 95.8 %), Haloplanus vescus RO5-8(T) (96.7 and 96.1 %), Haloplanus salinus YGH66(T) (96.4 and 95.8 %) and Haloplanus natans JCM 14081(T) (96.3 and 95.4 %). The rpoB' gene similarity between strains GX21(T) and R35(T) is 90.5 % and show 88.5-90.8 % similarity to the Haloplanus species with validly published names. The DNA G+C content of strain GX21(T) and R35(T) were determined to be 65.8 and 66.0 mol%, respectively. The DNA-DNA hybridization values between strain GX21(T) and strain R35(T), and the two strains with the Haloplanus species with validly published names, showed less than 50 % DNA-DNA relatedness. It was concluded that strain GX21(T) (=CGMCC 1.10456(T) = JCM 17092(T)) and strain R35(T) (=CGMCC 1.10594 (T) = JCM 17271(T)) represent two new species of Haloplanus, for which the names Haloplanus litoreus sp. nov. and Haloplanus ruber sp. nov. are proposed.

How could haloalkaliphilic microorganisms contribute to biotechnology?

Haloalkaliphiles are microorganisms requiring Na(+) concentrations of at least 0.5 mol·L(-1) and an alkaline pH of 9 for optimal growth. Their unique features enable them to make significant contributions to a wide array of biotechnological applications. Organic compatible solutes produced by haloalkaliphiles, such as ectoine and glycine betaine, are correlated with osmoadaptation and may serve as stabilizers of intracellular proteins, salt antagonists, osmoprotectants, and dermatological moisturizers. Haloalkaliphiles are an important source of secondary metabolites like rhodopsin, polyhydroxyalkanoates, and exopolysaccharides that play essential roles in biogeocycling organic compounds. These microorganisms also can secrete unique exoenzymes, including proteases, amylases, and cellulases, that are highly active and stable in extreme haloalkaline conditions and can be used for the production of laundry detergent. Furthermore, the unique metabolic pathways of haloalkaliphiles can be applied in the biodegradation and (or) biotransformation of a broad range of toxic industrial pollutants and heavy metals, in wastewater treatment, and in the biofuel industry.

Haloplanus salinus sp. nov., an extremely halophilic archaeon from a Chinese marine solar saltern.

Halophilic archaeal strain YGH66(T) was isolated from the Yinggehai marine solar saltern near the Sanya city of Hainan Province, China. Cells were pleomorphic, flat, stained Gram-negative, and produced pink-pigmented colonies. Strain YGH66(T) was able to grow at 20-50 °C (optimum 37 °C), at 0.9-4.8 M NaCl (optimum 3.1 M NaCl), at 0.005-1.0 M MgCl2 (optimum 0.05 M MgCl2), and at pH 6.0-8.0 (optimum pH 7.0). The cells of strain YGH66(T) were lysed in distilled water, and the minimum NaCl concentration that prevented cell lysis was 5 % (w/v). The major polar lipids of the strain were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, one major glycolipid (GL1) chromatographically identical to sulfated mannosyl glucosyl diether and a minor unidentified lipid (GL2), respectively. On the basis of 16S rRNA gene sequence analysis, strain YGH66(T) was closely related to Haloplanus natans JCM 14081(T), Haloplanus aerogenes TBN37(T), and Haloplanus vescus RO5-8(T) with the similarities of 98.0, 97.6, and 96.9 %, respectively. The rpoB' gene similarity between strain YGH66(T) and the current three members of Haloplanus were 90.4-92.8 %. The DNA G+C content of strain YGH66(T) was 67.2 mol %. The DNA-DNA hybridization values between strain YGH66(T) and three members of Haloplanus, H. natans JCM 14081(T), H. aerogenes TBN37(T), H. vescus RO5-8(T) were 50, 46 and 39 %, respectively. It was concluded that strain YGH66(T) represents a novel species of the genus Haloplanus, for which the name Haloplanus salinus sp. nov. is proposed. The type strain is YGH66(T) (=CGMCC 1.12127(T) = JCM 18368(T)).

Halalkalicoccus paucihalophilus sp. nov., a halophilic archaeon from Lop Nur region in Xinjiang, northwest of China.

Two extremely halophilic archaea, designated YIM 93701(T) and YIM 93664, were isolated from Lop Nur region in Xinjiang Province, northwest of China. The cells of the two strains were observed to be cocci, non-motile and Gram-negative. The organisms were determined to be aerobic and required at least 6 % NaCl for growth (optimum 20-25 % and maximum 35 %). Growth was found to occur in the ranges of 16-50 °C (optimum 37 °C) and pH 6.0-8.5 (optimum 6.5-7.5). Cells did not lyse in distilled water. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the two strains belongs to the genus Halalkalicoccus and possessed 99.3 and 99.5 % similarities with their closest phylogenetic relative Halalkalicoccus tibetensis JCM 11890(T). Major polar lipids of the two strains were determined to be phosphatidylglycerol(PG),phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol sulfate (PGS) and three unidentified glycolipids. The DNA G+C contents were determined to be 60.0-60.4 mol%. The DNA hybridization between the two strains was 92.0 %. In addition, the hybridizations of both strains to H. tibetensis were 49 and 52 %, respectively, and to Halalkalicoccus jeotali were 38 and 33 %, respectively. On the basis of physiological, biochemical tests and phylogenetic differentiations, strains YIM 93701(T) and YIM 93664 were classified as the same species which represent a novel species in the genus Halalkalicoccus, for which the name Halalkalicoccus paucihalophilus sp. nov. is proposed. The type strain is YIM 93701(T) (=JCM 17505(T) = CCTCC 2012803(T)).

Natronomonas gomsonensis sp. nov., isolated from a solar saltern.

A halophilic archaeal strain, SA3(T), was isolated from sediment of a solar saltern in Gomso Bay, Republic of Korea. Cells of strain SA3(T) were observed to be coccoid-shaped, to lyse in distilled water, Gram stain-negative and to form red-pigmented colonies. Strain SA3(T) was found to require at least 18 % (w/v) NaCl for growth. Optimal growth was observed at 24 % (w/v) NaCl and 6 % (w/v) MgCl2. The optimum pH and temperature for growth were determined to be pH 7.0 and 40 °C, respectively, while the strain was found to grow within pH and temperature ranges of 5.5-8.0 and 20-45 °C, respectively. The polar lipids were determined to consist of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, unidentified phosphoglycolipids and unidentified phospholipids. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain SA3(T) was most closely related to the members of the genus Natronomonas, Natronomonas moolapensis JCM 14361(T) (95.2 %) and Natronomonas pharaonis JCM 8858(T) (95.1 %). The genomic DNA G+C content (61.8 mol%) determined for strain SA3(T) was slightly lower than those of N. moolapensis JCM 14361(T) (63.4 mol%) and N. pharaonis JCM 8858(T) (64.3 mol%). DNA-DNA hybridization values between N. moolapensis JCM 14361(T) and N. pharaonis JCM 8858(T) and strain SA3(T) were <20 %. Based on phenotypic, chemotaxonomic and phylogenetic properties, we describe a new species of the genus Natronomonas, represented by strain SA3(T) (=JCM 17867(T) = KCTC 4088(T)), for which we propose the name Natronomonas gomsonensis sp. nov.

Halobellus rarus sp. nov., a halophilic archaeon from an inland salt lake of China.

Two halophilic archaeal strains, YC21(T) and YC77, were isolated from an inland salt lake of China. Both have pleomorphic rod-shaped cells that lyse in distilled water, stain Gram-negative and form red-pigmented colonies. They are neutrophilic, require at least 2.1 M NaCl for growth under the optimum growth temperature of 37 °C. The major polar lipids of the two strains were phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol sulfate (PGS), two major glycolipids (GL1 and GL2) chromatographically identical to sulfated mannosyl glucosyl diether (S-DGD-1) and mannosyl glucosyl diether (DGD-1), respectively. Trace amounts of two unidentified lipids (GL0-1 and GL0-2) were also detected. The 16S rRNA gene sequences of the two strains are 99.9 % identical, show 94.0-98.9 % similarity to the closest relative members of Halobellus of the family Halobacteriaceae. The rpoB' gene similarity between strains YC21(T) and YC77 is 99.8 % and show 90.3-95.3 % similarity to the closest relative members of Halobellus. The DNA G+C content of strains YC21(T) and YC77 were 66.1 and 66.2 mol%, respectively. The DNA-DNA hybridization value between strain YC20(T) and strain YC77 was 89 %, and the two strains showed low DNA-DNA relatedness with Halobellus limi TBN53(T), the most related member of Halobellus. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strains YC21(T) and YC77 represent a novel species of the genus Halobellus, for which the name Halobellus rarus sp. nov. is proposed. The type strain is YC21(T) (=CGMCC 1.12121(T) = JCM 18362(T)).

A microbial rhodopsin with a unique retinal composition shows both sensory rhodopsin II and bacteriorhodopsin-like properties.

Rhodopsins possess retinal chromophore surrounded by seven transmembrane α-helices, are widespread in prokaryotes and in eukaryotes, and can be utilized as optogenetic tools. Although rhodopsins work as distinctly different photoreceptors in various organisms, they can be roughly divided according to their two basic functions, light-energy conversion and light-signal transduction. In microbes, light-driven proton transporters functioning as light-energy converters have been modified by evolution to produce sensory receptors that relay signals to transducer proteins to control motility. In this study, we cloned and characterized two newly identified microbial rhodopsins from Haloquadratum walsbyi. One of them has photochemical properties and a proton pumping activity similar to the well known proton pump bacteriorhodopsin (BR). The other, named middle rhodopsin (MR), is evolutionarily transitional between BR and the phototactic sensory rhodopsin II (SRII), having an SRII-like absorption maximum, a BR-like photocycle, and a unique retinal composition. The wild-type MR does not have a light-induced proton pumping activity. On the other hand, a mutant MR with two key hydrogen-bonding residues located at the interaction surface with the transducer protein HtrII shows robust phototaxis responses similar to SRII, indicating that MR is potentially capable of the signaling. These results demonstrate that color tuning and insertion of the critical threonine residue occurred early in the evolution of sensory rhodopsins. MR may be a missing link in the evolution from type 1 rhodopsins (microorganisms) to type 2 rhodopsins (animals), because it is the first microbial rhodopsin known to have 11-cis-retinal similar to type 2 rhodopsins.

Biofilm formation by haloarchaea.

A fluorescence-based live-cell adhesion assay was used to examine biofilm formation by 20 different haloarchaea, including species of Halobacterium, Haloferax and Halorubrum, as well as novel natural isolates from an Antarctic salt lake. Thirteen of the 20 tested strains significantly adhered (P-value < 0.05) to a plastic surface. Examination of adherent cell layers on glass surfaces by differential interference contrast, fluorescence and confocal microscopy showed two types of biofilm structures. Carpet-like, multi-layered biofilms containing micro- and macrocolonies (up to 50 µm in height) were formed by strains of Halobacterium salinarum and the Antarctic isolate t-ADL strain DL24. The second type of biofilm, characterized by large aggregates of cells adhering to surfaces, was formed by Haloferax volcanii DSM 3757T and Halorubrum lacusprofundi DL28. Staining of the biofilms formed by the strongly adhesive haloarchaeal strains revealed the presence of extracellular polymers, such as eDNA and glycoconjugates, substances previously shown to stabilize bacterial biofilms. For Hbt. salinarum DSM 3754T and Hfx. volcanii DSM 3757T , cells adhered within 1 day of culture and remained viable for at least 2 months in mature biofilms. Adherent cells of Hbt. salinarum DSM 3754T showed several types of cellular appendages that could be involved in the initial attachment. Our results show that biofilm formation occurs in a surprisingly wide variety of haloarchaeal species.

Taxonomy of the family Halobacteriaceae: a paradigm for changing concepts in prokaryote systematics.

The halophilic Archaea of the family Halobacteriaceae (36 genera with 129 species with standing in nomenclature as of November 2011) provide an excellent example of how changing concepts on prokaryote taxonomy and the development of new methods have influenced the way in which the taxonomy of a single group of prokaryotes is treated. This review gives an overview of the taxonomy of the family Halobacteriaceae, showing the impact that methods of phenotypic characterization, numerical taxonomy, chemotaxonomy and especially polar lipid analysis, 16S rRNA gene sequence comparisons, multilocus type analysis and comparative genomics have had on their classification.