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Int J Syst Evol Microbiol 54 (2004), 1213-1216; DOI  10.1099/ijs.0.03032-0
© 2004 International Union of Microbiological Societies

Halorubrum tibetense sp. nov., a novel haloalkaliphilic archaeon from Lake Zabuye in Tibet, China

Huapeng Fan1, Yanfen Xue1, Yanhe Ma1, Antonio Ventosa2 and William D. Grant3

1 Department of Microbial Physiology and Ecology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China
2 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain
3 Department of Microbiology and Immunology, University of Leicester, Leicester LE1 9HN, UK

Correspondence
Yanhe Ma
mayanhe{at}sun.im.ac.cn


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A novel haloalkaliphilic archaeon, strain 8W8T, was isolated from Lake Zabuye, on the Tibetan Plateau, China. On the basis of 16S rRNA gene sequence analysis, strain 8W8T was shown to belong to the genus Halorubrum and was related to Halorubrum vacuolatum (96·7 % sequence similarity), Halorubrum saccharovorum (96·0 %), Halorubrum lacusprofundi (95·4 %) and Halorubrum sodomense (95·3 %). The phylogenetic distance from any species within the other genera of Halobacteriales was lower than 90 %. The major polar lipids of strain 8W8T were C20C20 and C20C25 derivatives of phosphatidylglycerol phosphate and phosphatidylglycerol phosphate methyl ester. The results of DNA–DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain 8W8T from the eight Halorubrum species with validly published names. Therefore, strain 8W8T represents a novel species, for which the name Halorubrum tibetense sp. nov. is proposed, with the type strain 8W8T (=AS 1.3239T=JCM 11889T).

Abbreviations: PG, phosphatidylglycerol; PGP-Me, phosphatidylglycerol phosphate methyl ester; PGS, phosphatidylglycerol sulfate; S-DGD, sulfated diglycosyl diether

The GenBank accession number for the 16S rRNA gene sequence of strain 8W8T is AY149598.


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Based on the analysis of 16S rRNA gene sequences, the genus Halorubrum was formally proposed by McGenity & Grant (1995)Go to accommodate several species previously included in the genus Halobacterium: Halorubrum trapanicum NRC 34021T (Petter, 1931Go), Halorubrum saccharovorum (Tomlinson & Hochstein, 1976Go), Halorubrum sodomense (Oren, 1983Go) and Halorubrum lacusprofundi (Franzmann et al., 1988Go). Subsequently, Oren & Ventosa (1996)Go transferred Halorubrobacterium coriense (Kamekura & Dyall-Smith, 1995Go) and Halorubrobacterium distributum (Zvyagintseva & Tarasov, 1987Go) to the genus Halorubrum. Natronobacterium vacuolatum (Mwatha & Grant, 1993Go) was also reassigned into Halorubrum as Halorubrum vacuolatum by Kamekura et al. (1997)Go. More recently, a novel species of the genus Halorubrum, Halorubrum tebenquichense, has been described (Lizama et al., 2002Go). The aim of this study was to describe a novel haloalkaliphilic archaeon isolated from the Lake Zabuye in Tibet, China and we propose a novel species, Halorubrum tibetense sp. nov.

Lake Zabuye (31° 20' N 84° 05' E) is located on the Tibetan Plateau 4421 m above sea level. It is an alkaline chloride-sulfate salt lake (pH 9·4, 250 g salt l–1) and is especially rich in lithium ions (0·1 g l–1; Holland et al., 1991Go). During a broad study of characterization of haloarchaea isolated from Lake Zabuye, strain 8W8T was isolated using a complex medium containing (g l–1): Casamino acids (Difco), 7·5; yeast extract (Difco), 10·0; trisodium citrate, 3·0; MgSO4.7H2O, 1·0; KCl, 10·0; LiCl, 0·1; Fe2+ and Mn2+, trace; NaCl, 200; Na2CO3, 10·0. The method used for enrichment and isolation was described previously (Tindall et al., 1980Go). Cells of strain 8W8T were irregular, rod-shaped and non-motile, as determined by phase-contrast and electron microscopy (Fig. 1Go).



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  		Fig. 1. Electron micrograph showing the morphology of cells of strain 8W8T. Bar, 1 µm.

 
The methods used for genomic DNA preparation, PCR amplification of 16S rRNA gene and the gene sequencing were described previously (McGenity & Grant, 1993Go). The almost complete 16S rDNA sequence (1470 bp) of strain 8W8T was determined and compared to the sequences of members of the genus Halorubrum and other closely related haloarchaea. The phylogenetic analysis was performed by the neighbour-joining method with the Kimura two-parameter calculation model in TREECONW version 1.3b (Van de Peer & De Wachter, 1994Go) after multiple alignment of data by CLUSTAL W version 1.8 (Thompson et al., 1994Go). Positions with any gaps and alignment uncertainty were omitted from the analysis. The 1412 unambiguous nucleotides were used for computing evolutionary distance. The phylogenetic tree (Fig. 2Go) indicated that strain 8W8T clustered with the species of the genus Halorubrum and constituted a subgroup with one described haloalkaliphilic species (Halorubrum vacuolatum JCM 9060T). Sequence similarity calculations after a neighbour-joining analysis indicated that strain 8W8T was related to Halorubrum vacuolatum (96·7 % sequence similarity), Halorubrum saccharovorum (96·0 %), Halorubrum lacusprofundi (95·4 %), Halorubrum sodomense (95·3 %), and other species of Halorubrum (93·5–95·0 %). Lower sequence similarities (<90·0 %) were found with all species of the other genera of Halobacteriales. Moreover, the 16S rDNA sequence of strain 8W8T possessed signature sequences of the genus Halorubrum: 199–229, 656–672 and 1242–1259 (positions based on the sequence of Escherichia coli) and 11 of 14 signature bases specific for Halorubrum (Grant et al., 2001Go).



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  		Fig. 2. Phylogenetic analysis based on 16S rRNA gene sequences available from GenBank databases (accession numbers in parentheses). Bootstrap values based on 100 replications are listed as percentages at the branching points. Scale bar, 0·05 expected changes per site.

 
Results of the chemotaxonomic analyses are given in the species description and Table 1Go. The polar lipid profile of strain 8W8T (Fig. 3Go), analysed by thin-layer chromatography as previously described (Ross et al., 1981Go), was similar to that of Halorubrum vacuolatum (Mwatha & Grant, 1993Go). Glycolipid and phosphatidylglycerol sulfate (PGS) were not present in these two species, whereas PGS was present in other species of Halorubrum. This feature would imply that the genus Halorubrum constitutes a heterogeneous group. As shown in Table 1Go, the genus Halorubrum contains non-alkaliphilic, facultatively alkaliphilic and obligately alkaliphilic species and they show different polar lipid compositions. Non-alkaliphilic members contained sulfated diglycosyl diether (S-DGD) and PGS besides phosphatidylglycerol (PG) and phosphatidylglycerol phosphate methyl ester (PGP-Me); facultatively alkaliphilic members had S-DGD besides PG and PGP-Me and obligately alkaliphilic members contained only PG and PGP-Me.


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  		Table 1. Phenotypic characteristics of strain 8W8T and some related Halorubrum species

Species: 1, Halorubrum vacuolatum; 2, Halorubrum saccharovorum; 3, Halorubrum lacusprofundi; 4, Halorubrum sodomense; 5, Halorubrum coriense; 6, Halorubrum distributum; 7, Halorubrum trapanicum NRC 34021T; 8, Halorubrum tebenquichense. +, Positive; –, negative; ND, not determined.

 


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  		Fig. 3. Polar lipid pattern of strain 8W8T by two-dimensional TLC. PG, Phosphatidylglycerol; PGP-Me, phosphatidylglycerol phosphate methyl ester; PL, unidentified phospholipids. The origin is in the bottom left corner.

 
The results of the physiological characterization are given in the species description. The methods used were in accordance with the recommended minimal standards for description of new taxa in the haloarchaea (Oren et al., 1997Go). DNA–DNA reassociation values were determined with strain 8W8T and the type strains of closely related Halorubrum species using the spectrophotometric renaturation rate method (Huß et al., 1983Go). Strain 8W8T showed relatively low DNA–DNA hybridization percentages to the type strain of Halorubrum saccharovorum ATCC 29252T (22 %), Halorubrum vacuolatum JCM 9060T (19 %), Halorubrum sodomense NCIMB 2197T (14 %) and Halorubrum lacusprofundi JCM 8891T (14 %).

Overall, our data show that strain 8W8T represents a novel organism of the genus Halorubrum and we propose to place it in a novel species with the name Halorubrum tibetense sp. nov.

Description of Halorubrum tibetense sp. nov.
Halorubrum tibetense (ti.be.ten'se. N.L. neut. adj. tibetense from Tibet).

Cells are irregular rod-shaped (0·5–1x1·5–2·5 µm). Gram-negative. Cells lyse in medium with less than 1·5 M NaCl. Colonies are 0·5–1 mm in diameter, red-pigmented, smooth, circular and convex. Haloalkaliphilic. Growth occurs optimally at 3·0–3·4 M NaCl. The pH range for growth is 8·0–10·5, with optimum pH 9·0–9·5. Optimal temperature for growth is 37–40 °C. Magnesium is not required for growth. Chemo-organotrophic and strictly aerobic. Catalase- and oxidase-positive. Gelatin, starch and casein not hydrolysed. Tweens 20, 60 and 80 hydrolysed. Nitrate reduced to nitrite. Indole not produced. Amino acids are utilized. Glucose, lactose, mannose, sucrose, maltose, sorbose, mannitol, starch, succinate and acetate are used as carbon sources. Sensitive to polymycin and novobiocin and insensitive to penicillin, chloramphenicol, streptomycin, tetracycline, ampicillin, rifampicin, erythromycin, bacitracin, neomycin and sulphafurazole. The polar lipids are C20C20 and C20C25 diether derivatives of PG and PGP-Me with five uncharacterized minor phospholipids. The G+C content of the DNA is 63·3 mol% (Tm). The type strain, 8W8T (=AS 1.3239T=JCM 11889T) was isolated from Lake Zabuye (soda lake). The GenBank accession number of the 16S rRNA is AY149598.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Chinese Academy of Sciences (Knowledge Innovation Program), the Ministry of Science and Technology of China (863 Programs) and the European Commission (‘Biotechnology of Extremophiles’, contract no. BIO2-CT93-0274 and ‘Multigenome Access Technology for Industrial Catalysts’, contract no. QLK3-CT-2002-01972).


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