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Halorubrum alkaliphilum sp. nov., a novel haloalkaliphile isolated from a soda lake in Xinjiang, China

¹ State Key Laboratory for Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, ZhongGuanCun, Haidian, Beijing 100080, PR China
² Ecosystem Sciences Division, Department of Environmental Science, Policy and Management, University of California at Berkeley, CA 94720, USA

Correspondence
Shuang-Jiang Liu
shuangjiang{at}hotmail.com

	ABSTRACT

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A novel haloalkaliphilic archaeon, strain DZ-1^T, was isolated from a soda lake in Xinjiang, China. The taxonomy of strain DZ-1^T was studied by polyphasic methods. According to 16S rRNA gene sequence similarity, strain DZ-1^T was phylogenetically related to Halorubrum tibetense (97·5 %), Halorubrum vacuolatum (95·7 %) and Halorubrum saccharovorum (95·9 %). Strain DZ-1^T was able to grow at 20–44 °C and was also physiologically different from the above-mentioned species with respect to assimilation of sugars and utilization of organic acids. The DNA G+C content of strain DZ-1^T was 62·1 mol% (T_m). The DNA–DNA relatedness of strain DZ-1^T to H. tibetense and H. vacuolatum was 22 and 13 %, respectively. It was concluded that strain DZ-1^T represents a novel species of the genus Halorubrum, for which the name Halorubrum alkaliphilum (type strain, DZ-1^T=AS 1.3528^T=JCM 12358^T) is proposed.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain DZ-1^T is AY510708.

	MAIN TEXT

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Species of the genus Halorubrum (McGenity & Grant, 1995) can be classified into two groups according to their growth pH ranges and origins. Group 1 includes species that cannot grow at pH 10·5, such as Halorubrum sodomense (Oren, 1983), Halorubrum lacusprofundi (Franzmann et al., 1988), Halorubrum saccharovorum (Tomlinson & Hochstein, 1976), Halorubrum trapanicum (McGenity & Grant, 1995), Halorubrum coriense (Kamekura & Dyall-Smith, 1995), Halorubrum distributum (Oren et al., 1997b), Halorubrum tebenquichense (Lizama et al., 2002), Halorubrum terrestre (Ventosa et al., 2004) and Halorubrum xinjiangense (Feng et al., 2004). Group 2 contains the haloalkaliphiles that are able to grow at pH 10·5 and are from soda lakes, currently with only two species, Halorubrum vacuolatum (Kamekura et al., 1997) and Halorubrum tibetense (Fan et al., 2004). This grouping is supported by phylogenetic analysis based on 16S rRNA gene sequences (Fig. 1). 16S rRNA gene sequence similarities among the recognized members of groups 1 and 2 are less than 96·5 %. During our screening for archaerhodopsin-producers, a haloalkaliphilic archaeon, strain DZ-1^T, was obtained. In this report, we present the identification and characterization of this strain.

View larger version (22K): [in this window] [in a new window]   Fig. 1. Phylogenetic tree showing the position of strain DZ-1T among the species representing the genus Halorubrum and other genera of Halobacteriales based on their 16S rRNA gene sequences (GenBank/EMBL/DDBJ accession numbers are shown in parentheses). The 16S rRNA gene sequence of Methanospirillum hungatei DSM 864T was used as outgroup. The numbers at branch points indicate the level of bootstrap support, based on 1000 resamplings; values >60 % are shown. Bar, 0·1 expected changes per site.

Phenotypic tests were performed according to the proposed minimal standards for the description of new taxa in the order Halobacteriales (Oren et al., 1997a). Cell motility and morphology were examined by phase-contrast and transmission electron microscopy of exponentially growing liquid cultures. Gram staining was carried out as described by Dussault (1955). Colony morphology was observed on optimal growth agar medium after incubation at 38 °C for 4 days. Anaerobic growth was tested in the presence of 5 g nitrate, L-arginine or DMSO l^–1 in filled, stoppered tubes. Tests for catalase and oxidase activities, and hydrolysis of starch, casein, gelatin and Tween 80 were performed as described previously (Gonzalez et al., 1978). Tests for nitrate reduction, H₂S formation, indole formation and the utilization of sugars, alcohols, amino acids and organic acids were carried out as described by Oren et al. (1997a). Antibiotic sensitivity tests were performed by spreading bacterial suspensions on culture plates and applying discs impregnated with the tested antibiotics. Phospholipids and glycolipids were separated on silica gel plates (10 cmx10 cm) by TLC and were analysed according to the method of Xin et al. (2001).

The DNA base composition was determined by thermal denaturation (T_m) (Marmur & Doty, 1962). The 16S rRNA gene was amplified under conditions described previously (Zhang et al., 2003), except that the following pair of archaeal primers was used: 5'-ATTCCGGTTGATCCTGCCGG-3' (nt 6–25, according to Escherichia coli numbering) and 5'-AGGAGGTGATCCAGCCGCAG-3' (nt 1540–1521). The PCR product was sequenced by GeneCore Biotechnologies (Shanghai, China) using the BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems) and an automated DNA sequencer (model ABI3700; Applied Biosystems). A BLAST search was performed to identify the nearest taxonomically identified relatives of strain DZ-1^T, the sequences of which were retrieved from GenBank (AY149498, H. tibetense; U17364, H. saccharovorum). Multiple sequence alignments of the 16S rRNA gene sequences were performed using CLUSTAL W version 1.8 (Thompson et al., 1994). A phylogenetic tree (Fig. 1) was constructed using the neighbour-joining method with Kimura's two-parameter calculation model in TREECON W version 1.3b (Van de Peer & De Wachter, 1994). DNA–DNA hybridization of strain DZ-1^T to the type strains of H. vacuolatum and H. tibetense was performed by the thermal denaturation and renaturation method of De Ley et al. (1970) as modified by Huss et al. (1983).

16S rRNA gene sequence similarity searches indicated that strain DZ-1^T was phylogenetically related to the haloalkaliphilic species of the genus Halorubrum, i.e. H. tibetense (97·5 %) and H. vacuolatum (95·7 %). Strain DZ-1^T contained C₂₀C₂₀ and C₂₀C₂₅ derivatives of phosphatidyl glycerol and phosphatidyl glycerol phosphate methyl ester, but lacked phosphatidyl glycerol sulfate and sulfated glycolipid, similar to H. tibetense and H. vacuolatum. Phylogenetic tree analysis showed that strain DZ-1^T and H. tibetense and H. vacuolatum were in the same cluster (Fig. 1). The lipid composition, physiological properties (see the following species description) and characteristics of the DNA molecule indicated that strain DZ-1^T was a member of the genus Halorubrum and was phylogenetically close to the haloalkaliphilic species. However, strain DZ-1^T was distinguished from H. tibetense and H. vacuolatum in phenotypic and physiological properties: strain DZ-1^T was motile, while H. tibetense and H. vacuolatum are non-motile. Strain DZ-1^T assimilated fructose but not sucrose, lactose or galactose. Strain DZ-1^T utilized lactate but not acetate or succinate. The characteristics of strain DZ-1^T that differentiate it from other Halorubrum species are listed in Table 1. The DNA–DNA relatedness of strain DZ-1^T to H. vacuolatum and H. tibetense was 13 and 22 %, respectively. The G+C content of strain DZ-1^T was 62·1 mol%. Thus, strain DZ-1^T represents a novel species of the genus Halorubrum, for which the name Halorubrum alkaliphilum sp. nov. is proposed.

Cells are short rods (0·8–1·0x1·8–2·0 µm) and Gram-negative. Colonies on agar plates containing 20 % (w/v) total salts are red, elevated and round. Growth is chemo-organotrophic, aerobic and occurs at concentrations of 1·8–5·2 M NaCl, at pH 8·0–10·5 and at 20–44 °C. Optimal NaCl concentration, pH and temperature for growth are 3·9–4·3 M, pH 9·0–10·0 and 38 °C, respectively. Magnesium is not required for growth. Catalase- and oxidase-positive. Anaerobic growth with nitrate, arginine or DMSO does not occur. Nitrate reduction to nitrite is observed and H₂S is produced from cystein. Indole formation is positive. Tween 80, casein and starch are not hydrolysed. Gelatinase-negative. Amino acids are required for growth. Fructose, glucose, mannose and maltose are utilized with the production of acids. Glycerol is utilized without acid production. No growth or acid production is observed on sucrose, lactose, raffinose, rhamnose, galactose, D-ribose, mannitol or sorbitol. Lactate and glutamic acid are utilized. Resistant to penicillin G, rifampicin, chloramphenicol, neomycin and bacitracin. Cells contain C₂₀C₂₀ and C₂₀C₂₅ derivatives of phosphatidyl glycerol and phosphatidyl glycerol phosphate methyl ester, but lack phosphatidyl glycerol sulfate and sulfated glycolipid. The DNA G+C content is 62·1 mol% (T_m). Isolated from a soda lake in Xinjiang, China.

The type strain is DZ-1^T and has been deposited in the China General Microbiological Culture Collection Center (CGMCC) as AS 1.3528^T and in the Japanese Collection of Microorganisms as JCM 12358^T.

	ACKNOWLEDGEMENTS

 
This work was supported by grants from the Ministry of Science and Technology (973 project) and the Chinese Academy of Sciences (KJCX1-SW-07). We express our thanks to Professor Dr H. G. Trüper at Bonn Univerisity and Professor R.-Y. Zheng at the Institute of Microbiology (Beijing) for their help with the nomenclature.

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