Halorubrum aquaticum sp. nov., an archaeon isolated from hypersaline lakes.

Two halophilic archaea, strains EN-2(T) and SH-4, were isolated from the saline lakes Erliannor and Shangmatala, respectively, in Inner Mongolia, China. Cells were strictly aerobic, motile rods. Colonies were red. Strains EN-2(T) and SH-4 were able to grow at 25-50 °C (optimum 35-40 °C), with 2.5-5.0 M NaCl (optimum 3.4 M NaCl) and at pH 6.0-9.0 (optimum pH 7.5). MgCl(2) was not required for growth. Cells lysed in distilled water and the lowest NaCl concentration that prevented cell lysis was 12 % (w/v). On the basis of 16S rRNA gene sequence analysis, strains EN-2(T) and SH-4 were closely related to Halorubrum cibi B31(T) (97.9 and 98.0 % similarity, respectively), Hrr. tibetense 8W8(T) (97.3 and 97.7 %), Hrr. alkaliphilum DZ-1(T) (96.8 and 97.1 %), Hrr. luteum CGSA15(T) (96.8 and 97.0 %) and Hrr. lipolyticum 9-3(T) (96.8 and 97.0 %). DNA-DNA hybridization showed that strains EN-2(T) and SH-4 did not belong to the same species as any of these strains (≤ 45 % DNA-DNA relatedness) but that they are members of the same species (>70 % DNA-DNA relatedness). Polar lipid analysis revealed that strains EN-2(T) and SH-4 contained phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, sulfated diglycosyl diethers and several unidentified glycolipids. The DNA G+C content of both isolates was 62.1 mol%. It was concluded that strains EN-2(T) and SH-4 represent a novel species of the genus Halorubrum, for which the name Halorubrum aquaticum sp. nov. is proposed. The type strain is EN-2(T) ( = CECT 7174(T)  = CGMCC 1.6377(T)  = JCM 14031(T)).

Halolysin R4, a serine proteinase from the halophilic archaeon Haloferax mediterranei; gene cloning, expression and structural studies.

A gene encoding a halophilic serine proteinase, halolysin R4, from a halophilic archaeon Haloferax mediterranei strain R4 was cloned, its nucleotide sequence determined, and expressed in Haloferax volcanii WFD11. The deduced amino-acid sequence (403 aa in length) showed the highest similarity to halolysin 172P1, produced by another halophilic archaeon, strain 172P1 (now designated as Natrialba asiatica). Both halolysins belong to the thermitase branch of class I subtilases, but show long C-terminal extensions of 117 and 123 amino acids, respectively. Removal of this "tail' region from halolysin R4 abolished proteinase activity, indicating it provides an essential (but as yet unknown) function. Substitution of the two cysteine residues in the C-terminal extension with serine decreased enzyme stability in hypotonic solutions, possibly owing to disruption of potential disulfide bonds or perturbation of calcium binding site(s).

Polar lipids of a non-alkaliphilic extremely halophilic archaebacterium strain 172: a novel bis-sulfated glycolipid.

Extremely halophilic archaebacteria which require high salt concentrations for growth and survival contain glycerol diether analogues of phospholipids and sulfated glycolipids as major membrane polar lipids. A non-alkaliphilic, non-pigmented rod-shaped extreme halophile, isolated from sea sand in Japan and designated 'strain 172', was found to contain two phospholipids, phosphatidylglycerol (PG) and phosphatidylglyceromethylphosphate (PGP-Me), derived from both C20-C20- and C20-C25-glycerol diethers, and a novel major glycolipid (designated SGL-X). This glycolipid has been identified as a bis-sulfated diglycosyl C20-C20- or C20-C25-glycerol diether, on the basis of its TLC mobility, positive-staining behavior with sugar and sulfate-staining reagents, its mole ratio sulfate/glycolipid = 2.2, and by spectrometric analysis (IR and FAB-MS) of the intact and the desulfated SGL-X. The sugars were identified as mannose and glucose, after acid hydrolysis of SGL-X, by paper chromatography of the free sugars and GC-MS of the derivatized sugars (alditol acetates). Permethylation analysis and 1H- and 13C-NMR analysis established the position and configuration of the sugar linkages and the positions of the sulfate groups. The final structure of SGL-X (now designated S2-DGD-1) is proposed to be: 2,3-diphytanyl- or phytanyl-sesterterpenyl-1-[2,6-(HSO3)2-alpha-Manp-1--> 2- Glcp]-sn-glycerol. This lipid is the first bis-sulfated glycolipid to be reported in extremely halophilic archaebacteria, and is the first in the biosphere that possesses two sulfate groups attached to the same monosaccaride.

Polyamines in photosynthetic eubacteria and extreme-halophilic archaebacteria.

Qualitative and quantitative determinations of polyamines have been done in 4 photosynthetic eubacteria and 6 extreme-halophilic archaebacteria. For comparison, 5 moderate-halophilic eubacteria were also analyzed to determine their polyamine contents. Not only putrescine and spermidine but also homospermidine were found in the photosynthetic eubacteria, especially in the N2-fixing species, Rhodospirillum and Chromatium. Norspermidine, norspermine, and spermine were not detected in the phototrophic eubacteria. No appreciable amount of any polyamine was found in extreme-halophilic archaebacteria, Halobacterium and Halococcus, while moderate-halophilic eubacteria contained quite high concentrations of putrescine and spermidine and cadaverine. When arginine was incubated with cell lysates of these two archaebacteria, appreciable amounts of agmatine were produced; neither putrescine nor cadaverine was formed in the presence of ornithine or lysine. No detectable amount of spermidine was produced by the lysates on incubation with putrescine.

Haloterrigena salina sp. nov., an extremely halophilic archaeon isolated from a salt lake.

A novel extremely halophilic strain, designated XH-65(T), isolated from the salt lake Xilinhot in Inner Mongolia, PR China, was subjected to a polyphasic taxonomic characterization. Strain XH-65(T) is neutrophilic, non-motile and requires at least 2.5 M NaCl for growth, with an optimum at 3.4 M NaCl, and grows at pH 6.0-9.0, with optimum growth at pH 7.5. Strain XH-65(T) grows at 25-50 degrees C, with optimal growth at 37 degrees C. Magnesium is not required for growth. On the basis of 16S rRNA gene sequence analysis, strain XH-65(T) was shown to belong to the genus Haloterrigena and was related to Haloterrigena turkmenica VKM B-1734(T) (98.1 % sequence similarity), Haloterrigena saccharevitans AB14(T) (96.9 %), Haloterrigena thermotolerans PR5(T) (96.3 %), Haloterrigena limicola AX-7(T) (95.8 %) and Haloterrigena hispanica FP1(T) (95.7 %). DNA-DNA hybridization revealed 37 % relatedness between strain XH-65(T) and Htg. turkmenica VKM B-1734(T). The polar lipid composition revealed the presence of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and mannose-2,6-disulfate (1-->2)-glucose glycerol diether (S(2)-DGD). The results of the DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain XH-65(T) from the six Haloterrigena species with validly published names. Therefore, strain XH-65(T) represents a novel species, for which the name Haloterrigena salina sp. nov. is proposed, with the type strain XH-65(T) (=CGMCC 1.6203(T) =JCM 13891(T)).

Halorubrum kocurii sp. nov., an archaeon isolated from a saline lake.

A Gram-negative, non-motile, neutrophilic, rod-shaped, extremely halophilic archaeon, designated strain BG-1(T), was isolated from a salt lake, Lake Bagaejinnor, in Inner Mongolia, China. Strain BG-1(T) was able to grow at 25-55 degrees C, required at least 2.5 M NaCl for growth (with an optimum at 3.4 M NaCl) and grew at pH 6.0-9.0 (with an optimum at pH 7.5). Hypotonic treatment with less than 2.0 M NaCl caused cell lysis. Phylogenetic analysis of the almost-complete 16S rRNA gene sequence positioned the isolate within the genus Halorubrum in the family Halobacteriaceae. Strain BG-1(T) was most closely related to Halorubrum aidingense 31-hong(T) (98.8% sequence similarity), Halorubrum saccharovorum NCIMB 2081(T) (98.6%), Halorubrum lacusprofundi ACAM 34(T) (98.6%) and Halorubrum lipolyticum 9-3(T) (98.4%). However, values for DNA-DNA hybridization between strain BG-1(T) and the most closely related members of the genus Halorubrum were below 40%. Analysis of the polar lipids of strain BG-1(T) revealed the presence of mannosyl-2-sulfate-(1-4)-glycosyl-archaeol, the main glycolipid found in neutrophilic species of the genus Halorubrum. The G+C content of the genomic DNA was 69.4 mol% (T(m)). Comparison of the phenotypic characteristics of the strain with those of Halorubrum species supported the conclusion that BG-1(T) represents a novel species within this genus, for which the name Halorubrum kocurii sp. nov. is proposed. The type strain is BG-1(T) (=CECT 7322(T) =CGMCC 1.7018(T) =JCM 14978(T)).

Halovivax ruber sp. nov., an extremely halophilic archaeon isolated from Lake Xilinhot, Inner Mongolia, China.

A Gram-negative, pleomorphic, extremely halophilic archaeon, designated strain XH-70(T), was isolated from the saline Lake Xilinhot, in Inner Mongolia, China. It formed small (0.9-1.5 mm), red-pigmented, elevated colonies on agar medium. The strain required at least 2.5 M NaCl and 5 mM Mg(2+) for growth. The 16S rRNA gene sequence analysis indicated that strain XH-70(T) belongs to the family Halobacteriaceae, showing 99.5 % similarity to the type strain of Halovivax asiaticus and 94.7 and 94.6 % similarity, respectively, to the type strains of Natronococcus amylolyticus and Natronococcus occultus. Polar lipid analysis supported the placement of strain XH-70(T) in the genus Halovivax. DNA-DNA hybridization studies (32 % with Halovivax asiaticus CGMCC 1.4248(T)), as well as biochemical and physiological characterization, allowed strain XH-70(T) to be differentiated from Halovivax asiaticus. A novel species, Halovivax ruber sp. nov., is therefore proposed to accommodate this strain. The type strain is XH-70(T) (=CGMCC 1.6204(T)=DSM 18193(T)=JCM 13892(T)).

Halorubrum ejinorense sp. nov., isolated from Lake Ejinor, Inner Mongolia, China.

A novel halophilic archaeon, strain EJ-32T, was isolated from water from Lake Ejinor in Inner Mongolia, China. The taxonomy of strain EJ-32T was studied by using a polyphasic approach. On the basis of 16S rRNA gene sequence similarities, strain EJ-32T was shown to be phylogenetically related to Halorubrum coriense (97.9%), Halorubrum trapanicum (97.9%), Halorubrum sodomense (97.8%), Halorubrum tebenquichense (97.8%), Halorubrum xinjiangense (97.6%), Halorubrum terrestre (97.4%), Halorubrum distributum (97.1%) and Halorubrum saccharovorum (96.4%). Strain EJ-32T was found to be neutrophilic, non-motile and Gram-negative. It grew in medium containing saturation concentrations of NaCl and did not require magnesium for optimal growth. The G+C content of the DNA is 64.0 mol%. Values for DNA-DNA hybridization with respect to phylogenetically related Halorubrum species were

Halopiger xanaduensis gen. nov., sp. nov., an extremely halophilic archaeon isolated from saline Lake Shangmatala in Inner Mongolia, China.

Strain SH-6(T) was isolated from the sediment of Lake Shangmatala, a saline lake in Inner Mongolia (China). Cells were pleomorphic. The organism was neutrophilic and required at least 2.5 M (15 %) NaCl, but not MgCl(2), for growth; optimal growth occurred at 4.3 M (25 %) NaCl. The G+C content of its DNA was 63.1 mol%. 16S rRNA gene sequence analysis revealed that strain SH-6(T) is a member of the family Halobacteriaceae, but there was a low level of similarity with other members of this family. Highest sequence similarity (94.6 %) was obtained with the 16S rRNA genes of the type strains of Natronolimnobius innermongolicus and Natronolimnobius baerhuensis. Polar lipid analyses revealed that strain SH-6(T) contains phosphatidylglycerol and phosphatidylglyceromethylphosphate, derived from both C(20)C(20) and C(20)C(25) glycerol diethers together with the glycolipid S(2)-DGD-1. On the basis of the data obtained, the new isolate could not be classified in any recognized genus. Strain SH-6(T) is thus considered to represent a novel species in a new genus within the family Halobacteriaceae, order Halobacteriales, for which the name Halopiger xanaduensis gen. nov., sp. nov. is proposed. The type strain of Halopiger xanaduensis is SH-6(T) (=CECT 7173(T)=CGMCC 1.6379(T)=JCM 14033(T)).

Halostagnicola larsenii gen. nov., sp. nov., an extremely halophilic archaeon from a saline lake in Inner Mongolia, China.

Strain XH-48(T) was isolated from the sediment of Lake Xilinhot, a saline lake in Inner Mongolia (China). The organism is pleomorphic, neutrophilic and requires at least 2.5 M (15 %) NaCl, but not MgCl(2), for growth; it exhibits optimal growth at 3.4 M (20 %) NaCl. The G+C content of its DNA is 61 mol%. 16S rRNA gene sequence analysis revealed that strain XH-48(T) is a member of the family Halobacteriaceae, but there were low levels of similarity with other members of this family. The highest sequence similarity values (94.5 and 93.3 %) were obtained with the 16S rRNA genes of Natrialba aegyptiaca and Natrialba asiatica, respectively. Polar lipid analyses revealed that strain XH-48(T) contains phosphatidylglycerol and phosphatidylglyceromethylphosphate, derived from both C(20)C(20) and C(20)C(25) glycerol diethers, and two unidentified glycolipids. On the basis of the data obtained, the novel isolate cannot be classified within any recognized genus. Strain XH-48(T) should be placed within a novel genus and species within the family Halobacteriaceae, order Halobacteriales, for which the name Halostagnicola larsenii gen. nov., sp. nov. is proposed. The type strain of Halostagnicola larsenii is strain XH-48(T) (=DSM 17691(T)=CGMCC 1.5338(T)=JCM 13463(T)=CECT 7116(T)).

Halovivax asiaticus gen. nov., sp. nov., a novel extremely halophilic archaeon isolated from Inner Mongolia, China.

Strain EJ-46T, a novel pleomorphic, aerobic, extremely halophilic member of the Archaea was isolated from sediment of the saline Lake Ejinor, in Inner Mongolia, China. This organism was neutrophilic and required at least 15 % (2.5 M) NaCl for growth. MgCl2 was not required. The isolate was able to grow at pH 6.0-9.0. Optimum growth occurred in media containing 20 % (3.4 M) NaCl at pH 7.0-7.5. Polar lipid analysis revealed the presence of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester, derived from both C20C20 and C20C25 glycerol diethers. Four glycolipids were detected, one of which may be novel. The DNA G+C content was 60.3 mol%. 16S rRNA gene analysis revealed that strain EJ-46T was a member of the phylogenetic group defined by the family Halobacteriaceae, and the highest 16S rRNA gene similarity values of 94.9 and 94.8 % were obtained with the haloalkaliphilic species of the genus Natronococcus, Natronococcus occultus and Natronococcus amylolyticus, respectively. Based on the phenotypic, genotypic and phylogenetic analyses, it is proposed that the novel isolate should be classified as representing a new genus and species, for which the name Halovivax asiaticus gen. nov., sp. nov. is proposed. The type strain is EJ-46T (=CGMCC 1.4248T = CECT 7098T).

Halorubrum orientale sp. nov., a halophilic archaeon isolated from Lake Ejinor, Inner Mongolia, China.

A motile, pleomorphic, red-pigmented archaeon, strain EJ-52T, was isolated from water from Lake Ejinor, a saline lake in Inner Mongolia, China. Analysis of the almost-complete 16S rRNA gene sequence showed that the isolate was phylogenetically related to species of the genus Halorubrum, being most closely related to Halorubrum saccharovorum ATCC 29252T (96.1% sequence similarity), Halorubrum lacusprofundi JCM 8891T (95.9%), Halorubrum tibetense AS 1.3239T (95.2%), Halorubrum alcaliphilum AS 1.3528T (95.2%) and Halorubrum vacuolatum JCM 9060T (95.1%). The polar lipids of strain EJ-52T were C20C20 derivatives of phosphatidylglycerol phosphate and phosphatidylglycerol phosphate methyl ester and a sulfated diglycosyl diether. Strain EJ-52T requires at least 2.5 M NaCl for growth and grows optimally at 3.4 M NaCl. The strain grows at 25-50 degrees C, with optimal growth occurring at 35-45 degrees C. Mg2+ is not required. The DNA G+C content is 64.2 mol%. On the basis of the data obtained in this study, strain EJ52T represents a novel species, for which the name Halorubrum orientale sp. nov. is proposed. The type strain is EJ-52T (=CECT 7145T=JCM 13889T=CGMCC 1.6295T).

Natrinema ejinorense sp. nov., isolated from a saline lake in Inner Mongolia, China.

A Gram-negative, non-motile, neutrophilic, pleomorphic and extremely halophilic archaeon, strain EJ-57T, was isolated from saline Lake Ejinor in Inner Mongolia, China. Strain EJ-57T was able to grow at 25-50 degrees C, required at least 1.8 M NaCl for growth (optimum at 3.4 M NaCl) and grew over a pH range from 6.0 to 8.5 (optimum at pH 7.0). Hypotonic treatment with less than 1.5 M NaCl caused cell lysis. Analysis of the almost complete 16S rRNA gene sequence indicated that the isolate represented a member of the genus Natrinema in the family Halobacteriaceae. Strain EJ-57T was most closely related to Natrinema versiforme JCM 10478T (96.2% sequence similarity), Natrinema pallidum NCIMB 777T (95.9% sequence similarity), Natrinema altunense JCM 12890T (95.8% sequence similarity) and Natrinema pellirubrum NCIMB 786T (95.5 % sequence similarity). However, DNA-DNA hybridization experiments showed that strain EJ-57T was not related to these species, with levels of DNA-DNA relatedness equal to or below 39%. The major polar lipids of the isolate were C20C20 and C20C25 derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and the disulfated glycolipid S2-DGA-1. The G+C content of the genomic DNA was 64.7 mol%. Comparative analysis of phenotypic characteristics between strain EJ-57T and recognized Natrinema species supported the conclusion that EJ-57T represents a novel species within this genus, for which the name Natrinema ejinorense sp. nov. is proposed. The type strain is EJ-57T (=CECT 7144T=JCM 13890T=CGMCC 1.6202T).

Diversity of extremely halophilic bacteria.

In this review, the history of the classification of the family Halobacteriaceae, the extremely halophilic aerobic Archaea, is reviewed with some emphasis on the recently described new genera Halobaculum, Halorubrum, Natrialba, Natronomonas, and "Haloterrigena." Speculation is made about the evolutionary relationship between members of the Halobacteriaceae and the extremely halophilic, anaerobic methanogens of the genera Methanohalobium and Methanohalophilus. Efforts to find missing links between the two groups are also reviewed.

Partial sequence of the gene for a serine protease from a halophilic archaeum Haloferax mediterranei R4, and nucleotide sequences of 16S rRNA encoding genes from several halophilic archaea.

A part of the gene coding for a halophilic serine protease from a halophilic archaeum Haloferax mediterranei R4 was amplified by PCR and its 672 nucleotide sequence was determined. Tentative translation to the amino acid sequence suggested that the enzyme was quite similar to halolysin produced by another halophilic archaeum strain 172P1. Nucleotide sequences of 16S rRNA encoding genes from 9 halophilic archaea were determined. Alignment of 19 sequences known so far showed that there are more than 20 positions carrying bases or deletions specific for each halobacterial genus: Halobacterium, Haloarcula, Haloferax, and Halococcus.

Structural diversity of membrane lipids in members of Halobacteriaceae.

This minireview gives an updated and consolidated summary of taxonomic classification correlated with membrane phospholipid, glycolipid, and core lipid structural diversity within the family Halobacteriaceae. We also point out that the recently reported diversity in the membrane core lipid structure of a putative strain of Halobacterium (Halobacterium halobium strain IAM 13167) (Morita et al., Biosci. Biotechnol. Biochem., 62, 596-598, 1998) is not correct since the strain used by the authors has for some time been recognized not to be a member of the genus Halobacterium but a member of halobacteria group 2 (Grant and Larsen, Bergey's Manual of Systematic Bacteriology, Vol.3, pp. 2216-2233, 1989), which has recently been designated as a new genus, Natrinema (McGenity et al., Int. J. Syst. Bacteriol. 48, 1187-1196, 1998).

Halophilic nuclease from a moderately halophilic Micrococcus varians.

The moderately halophilic bacterium Micrococcus varians, isolated from soy sauce mash, produced extracellular nuclease when cultivated aerobically in media containing 1 to 4 M NaCl or KCl. The enzyme, purified to an electrophoretically homogeneous state, had both ribonuclease and deoxyribonuclease activities. The nuclease had maximal activity in the presence of 2.9 M NaCl or 2.1 M KCl at 40 C. The enzymatic activity was lost by dialysis against low-salt buffer, whereas when the inactivated enzyme was dialyzed against 3.4 M NaCl buffer as much as 77% of the initial activity could be restored.

Protease formation by a moderately halophilic Bacillus strain.

A moderately halophilic strain of Bacillus, isolated from unrefined solar salt, was capable of growth in the presence of 4 M NaCl. Maximal growth was obtained in a medium containing 1 to 2 M NaCl. The organism produced protease when cultivated aerobically in media containing 0 to 3 M NaCl or 0 to 2 M KCl. The protease activity was optimal at 0.5 M NaCl and 0.75 M KCl.

A medium for commercial production of the halophilic Micrococcus nuclease.

A simple synthetic medium (glutamate-sucrose medium) was devised for production, during growth in shaken flasks, of extracellular halophilic nuclease (nuclease H) by a moderate halophile, Micrococcus varians subsp. halophilus. A simple medium consisting of 0.7% ammonium sulfate, 1.0% glucose, minerals, three vitamins, and 2 M NaCl gave good growth and excellent production of nuclease H in a jar fermentor when the pH was adjusted to 7.5 to 8.0 during cultivation.

Flocculation and adsorption of enzymes during growth of a moderate halophile, Micrococcus varians var. Halophilus.

Flocculation of a moderate halophile, Micrococcus varians ATCC 2197, occurred during growth in complex medium containing 3 M NaCl and a concentration of MgSO4 and KH2PO4 greater than 40 and 14 mM, respectively. Extracellular nuclease activity was absent in the flocculated cultures. Repeated washing of flocs by Mg2+-free Tris buffer containing 3 M NaCl, lowering of pH value of floc suspension below 6.3, or addition of ethylenediaminetetraacetic acid resulted in complete dissociation of the flocs and release of Mg2+ ions as well as nuclease and amylase. Inhibition of extracellular enzyme production accompanied by flocculation appeared to be the result of adsorption of enzyme proteins to surfaces of the flocs, but not of inhibition of biosynthesis. Floc formation could also occur in media containing 18 mM CaCl2 and 3.0 mM KH2PO4, but the Ca flocs were not deflocculated by washing with Ca2+-free buffer, suggesting that the affinity of Ca2+ for cell envelopes was stronger than that of Mg2+. It was also observed that most halophilic Planococcus and Micrococcus flocculated in the presence of MgSO4 and phosphate but halophilic Pseudomonas, Acinetobacter, and Bacillus did not.