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Halobacterium jilantaiense sp. nov., a halophilic archaeon isolated from a saline lake in Inner Mongolia, China

¹ State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, ZhongGuanCun, Haidian, Beijing 100080, PR China
² Graduate School of Chinese Academy of Sciences, Beijing 100049, China

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

	ABSTRACT

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A novel halophilic archaeon, NG4^T, was isolated from Jilantai salt lake in Inner Mongolia, China. The taxonomy of strain NG4^T was studied by polyphasic methods. Strain NG4^T grew at pH 5.5–8.5 and at a temperature of 22–55 °C. It was chemo-organotrophic, aerobic and required concentrations of 2.7–5.2 M NaCl and 0.05–0.3 M Mg²⁺ for growth. Cells were Gram-negative, slender rods. Colonies on agar plates containing 25 % (w/v) total salts were red, elevated and round. According to 16S rRNA gene sequence similarity, strain NG4^T was phylogenetically related to Halobacterium salinarum DSM 3754^T (98.2 %) and Halobacterium noricense A1^T (97.3 %). The DNA G+C content was 64.2 mol%. DNA–DNA relatedness values with Hbt. salinarum DSM 3754^T and Hbt. noricense A1^T were 47 and 35 %, respectively. The polar lipids of strain NG4^T consisted of phosphatidylglycerol, methylated phosphatidylglycerol phosphate, phosphatidylglycerol sulfate, triglycosyl diether, sulfated triglycosyl diether and sulfated tetraglycosyl diether. It was concluded that strain NG4^T represents a novel species of the genus Halobacterium, for which the name Halobacterium jilantaiense sp. nov. is proposed. The type strain is NG4^T (=CGMCC 1.5337^T=JCM 13558^T).

A two-dimensional thin-layer chromatograph of polar lipids extracted from strain NG4^T is available as supplementary material in IJSEM Online.

	MAIN TEXT

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Members of the genus Halobacterium (Grant, 2001) are obligately aerobic and heterotrophic and live in highly saline environments such as salt lakes and salterns. Several former members of the genus Halobacterium have been excluded from the genus, based on distant relationships as determined by phylogenetic analysis (McGenity & Grant, 1995). Currently, the genus Halobacterium contains two recognized species: Halobacterium salinarum (Ventosa & Oren, 1996; Grant, 2001) and Halobacterium noricense (Gruber et al., 2004).

Strain NG4^T was isolated from the salt lake of Jilantai (105° 42' E 39° 45' N) in Inner Mongolia, China, by enrichment and subsequent plating on agar medium containing the following ingredients (g l^–1): Casamino acids, 7.5; yeast extract, 10.0; MgSO₄.7H₂O, 20.0; trisodium citrate, 3.0; KCl, 2.0; FeSO₄.7H₂O, 0.05; NaCl, 200; agar, 15 (pH adjusted to 7.4 with 1 M NaOH). The strain grew at a temperature range of 22–55 °C (optimum 40 °C) and at a pH range of 5.5–8.5 (optimum 7.0–7.5). Routine cultivation was conducted at 40 °C and pH 7.4. Strain NG4^T required 2.7–5.2 M NaCl (optimum 3.1–3.5 M) and 0.05–0.3 M MgSO₄ (optimum 0.1–0.2 M) for growth.

Phenotypic tests were performed according to the proposed minimal standards for the description of new taxa in the order Halobacteriales (Oren et al., 1997). 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 by growth on agar medium after incubation at 40 °C for 7 days. Anaerobic growth was tested in the presence of 5 g potassium nitrate l^–1, L-arginine and DMSO in filled, stoppered tubes. Tests for catalase and oxidase activity and hydrolysis of starch and of Tweens 20, 40, 60 and 80 were performed as described previously (Gonzalez et al., 1978). Nitrate reduction, H₂S formation, indole formation and the utilization of sugars, alcohols, amino acids and organic acids were examined as described by Oren et al. (1997). Phospholipids and glycolipids were separated by thin layer chromatography on silica gel plates (10x10 cm) and were analysed according to the method of Xin et al. (2001).

DNA base composition was determined by thermal denaturation (T_m) (Marmur & Doty, 1962). The 16S rRNA gene was amplified by PCR using the halophilic archaea-specific primers 5'-TTCCGGTTGATCCTGCC-3' and 5'-AAGGAGGTGATCCAGCC-3'. The PCR product was sequenced using an ABI BigDye 3.1 sequencing kit (Applied Biosystems) and an automated DNA sequencer (model ABI 3730; Applied Biosystems). DNA–DNA hybridization of strain NG4^T to the type strains of Hbt. salinarum and Hbt. noricense was performed using the method of De Ley et al. (1970), as modified by Huß et al. (1983). A phylogenetic tree (Fig. 1) was constructed by the neighbour-joining method with Kimura's two-parameter calculation model in MEGA version 3.1 (Kumar et al., 2004).

View larger version (15K): [in this window] [in a new window]   Fig. 1. Phylogenetic tree based on 16S rRNA gene sequences showing the relationship between strain NG4T, other members of the genus Halobacterium and members ofother selected genera of the family Halobacteriaceae. Bootstrap values (%) are based on 1000 replicates and are shown for branches with more than 70 % bootstrap support. Bar, 0.02 expected changes per site.

Description of Halobacterium jilantaiense sp. nov.
Halobacterium jilantaiense (ji.lan.tai.en'se. N.L. neut. adj. jilantaiense pertaining to Jilantai salt lake, where the type strain was isolated).

Cells are slender rods (0.5–1.0x1.0–3.0 µm) and Gram-negative. Colonies on agar plates containing 25 % (w/v) total salts are red, elevated and round. Growth is chemo-organotrophic and aerobic; it occurs at 2.7–5.2 M NaCl and 0.05–0.3 M Mg²⁺ at pH 5.5–8.5 and at 22–55 °C. Optimal NaCl concentration, Mg²⁺ concentration, pH and temperature for growth are 3.1–3.5 M, 0.1–0.2 M, pH 7.0–7.5 and 40 °C, respectively. Catalase- and oxidase-positive. Grows anaerobically in the presence of L-arginine, nitrate and DMSO. Nitrate reduction to nitrite is observed and H₂S is produced from cysteine. Indole formation is positive. Tween 80 and starch are not hydrolysed. Casein and gelatin are hydrolysed. Amino acids are required for growth. Starch is not hydrolysed. Glucose, galactose, sucrose, maltose, fructose, sorbose, xylose, mannose, lactose, D-ribose, mannitol and sorbitol are not utilized in 7–10 days. Glycerol is utilized without acid production. Susceptible to anisomycin and novobiocin. Resistant to penicillin, ampicillin, rifampicin, chloramphenicol, gentamicin, kanamycin, streptomycin, tetracycline, bacitracin and erythromycin. Cells contain C₂₀C₂₀ derivatives of PG, PGP-Me, PGS, S-TGD-1, S-TeGD and TGD-1. The DNA G+C content is 64.2 mol%.

The type strain, NG4^T (=CGMCC 1.5337^T=JCM 13558^T), was isolated from Jilantai salt lake in Inner Mongolia, China.

	ACKNOWLEDGEMENTS

 
This work was supported by a grant from the Ministry of Science and Technology (2004CB719601). We are grateful to Ms Claudia Gruber for kindly providing the type strain of Hbt. noricense. We express our thanks to Professor W.-Y. Zhuang at the Institute of Microbiology (Beijing) for help with nomenclature.

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