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1 College of Life Sciences, Zhejiang University, 38 Zheda Road, Hangzhou 310027, P. R. China
2 Institute of Microbiology, Chinese Academy of Sciences, ZhongGuanCun, Haidian, Beijing 100080, P. R. China
Correspondence
Pei-Jin Zhou
zhou{at}sun.im.ac.cn
Min Wu
wumin{at}cls.zju.edu.cn
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain AJ2T is AY208972.
Thin-layer chromatograms showing the lipid content of strain AJ2T and other Natrinema species, along with a table of some characteristics distinguishing this strain from Natrinema versiforme AS 1.2365T, are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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). In a phylogenetic tree based on 16S rRNA gene sequences, Natrinema species formed an independent cluster with respect to Halobacterium species. Natrinema species could be cultured at low salt concentrations, and possessed a specific protein profile and polar lipid composition. Subsequently, a novel species of this genus, Natrinema versiforme, was described (Xin et al., 2000). Thus, to date there are three species in the genus Natrinema. In this study, we describe a novel extremely halophilic archaeon isolated from Ayakekum salt lake (37° 37' N, 89° 29' E; 3884 m altitude) located in the Altun Mountain National Nature Reserve in Xinjiang, China, and propose a novel species, Natrinema altunense sp. nov.
The low temperature, low nutrient levels, abundant sunlight and remote geographical location of Ayakekum salt lake make it a relatively isolated ecosystem. A water sample (approx. 400 ml) was collected from the edge of Ayakekum salt lake in summer. The pH of the water (determined using a pH meter) was slightly alkali, at approximately pH 7·8. The isolate was routinely grown aerobically at 37 °C in rich medium (Oesterhelt & Stoeckenius, 1974). Pure cultures were obtained by restreaking several times. The organism was grown and maintained on S-G medium (Sehgal & Gibbons, 1960).
The phenotypic tests were performed according to the proposed minimal standards for the description of new taxon of the order Halobacteriales (Oren et al., 1997). The optimal conditions for growth were determined in S-G medium modified with 0·85–5·1 M NaCl or 0–1·0 M Mg2+. To determine the pH required for growth (using increments of 0·5 pH units, from pH 5·0 to pH 9·5), 50 mM MES (pH 5·0–6·0), 50 mM PIPES (pH 6·5–7·0), 50 mM Tricine (pH 7·5–8·5) and 50 mM CHES (pH 9·0–9·5) were employed as buffers. Cell morphology and motility were examined by using light microscopy (BX40; Olympus) and transmission electron microscopy (S-570; Hitachi). Gram staining was performed using samples fixed with acetic acid, as described by Dussault (1955). Anaerobic growth was tested in the presence of nitrate, L-arginine or DMSO (each at 5 g l–1) in filled, stoppered tubes. Gelatin hydrolysis was determined as described by Oren et al. (2002). Hydrolysis of starch, casein and Tweens 20, 40 and 80, reduction of nitrate and nitrite, production of indole and H2S, activities of catalase and oxidase, and utilization of sugars, alcohols, amino acids and organic acids were tested according to Xin et al. (2000), as described by Oren et al. (1997).
Total lipids were extracted by using the modified method of Kamekura & Kates (1988). Phospholipids and glycolipids were separated on silica-gel plates (10x10 cm) by TLC and were analysed according to Xin et al. (2000). Genomic DNA was prepared by the method of Marmur (1961) and the purity was checked spectrometrically. The G+C content of the DNA was determined by thermal denaturation (Tm) (Marmur & Doty, 1962). The 16S rRNA gene sequence was amplified under conditions like those described by Gupta et al. (1983). The sequence was analysed along with sequences of closely related reference organisms from the FASTA network service. Sequence data were aligned with CLUSTAL W software, version 1.8 (Thompson et al., 1994). Phylogenetic trees were constructed by using neighbour-joining methods (Saitou & Nei, 1987) with the MEGA 3 program package (Kumar et al., 2004). DNA–DNA hybridization was performed by using the thermal denaturation and renaturation method of De Ley et al. (1970) as modified by Huß et al. (1983).
The profile of the major polar lipids of strain AJ2T, comprising C20C20 and C20C25 derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol sulfate, was similar to that of Natrinema species (see Supplementary Fig. A in IJSEM Online). In a phylogenetic tree based on 16S rRNA gene sequences (Fig. 1), strain AJ2T clustered with Natrinema species with validly published names. The results indicated that strain AJ2T belongs to the genus Natrinema.
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) (additional distinguishing characteristics are available in a Supplementary Table in IJSEM Online). Two-dimensional TLC revealed that strain AJ2T possessed a major glycolipid, which ran very slowly. The glycolipid spot was also found in N. pellirubrum JCM 10476T. The amount of this glycolipid in AJ2T was observably less than that in N. pellirubrum JCM 10476T, and the amount of phosphatidylglycerol sulfate in AJ2T was more than that in N. pellirubrum JCM 10476T (McGenity et al., 1998). Moreover, strain AJ2T did not contain glycolipids found in N. pallidum JCM 8980T and N. versiforme AS 1.2365T. Therefore, the polar lipid profiles among Natrinema species also served to distinguish them (see Supplementary Fig. B in IJSEM Online). The 16S rRNA gene sequence similarities between strain AJ2T and N. pallidum JCM 8980T, N. pellirubrum JCM 10476T and N. versiforme AS 1.2365T were 97·06, 96·64 and 97·71 %, respectively. Furthermore, the DNA–DNA relatedness values for strain AJ2T with respect to N. pallidum JCM 8980T, N. pellirubrum JCM 10476T and N. versiforme AS 1.2365T were 49·3 % (SD=4·3 %), 35·5 % (SD=2·6 %) and 51·4 % (SD=1·3 %), respectively. The values were based on three independent determinations.
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Description of Natrinema altunense sp. nov.
Natrinema altunense (al.tu.nen'se. N.L. neut. adj. altunense of Altun, referring to isolation of the organism from Altun Mountain, China).
Cells are rods that measure 0·8–1·2x3–7 µm and become pleomorphic under unfavourable conditions. Cells are motile and Gram-negative. Colonies are orange or red, smooth, circular and elevated. Growth requires at least 1·7 M NaCl, optimally 3·0–4·3 M NaCl. Growth occurs at 0·005–1 M MgCl2, optimally at around 0·05–0·2 M MgCl2. The pH range for growth is 6·0–8·0, with an optimum at pH 7·0–7·7. Chemo-organotrophic. Grows anaerobically in the presence of nitrate. Oxidase- and catalase-positive. Nitrate and nitrite are reduced, and gas is produced. Indole formation is negative. Starch and casein are not hydrolysed. Gelatin and Tweens 20, 40 and 80 are hydrolysed. H2S is produced from thiosulfate. The following substrates are utilized for growth: glucose, glycerol, maltose, glutamate, alanine, arginine, lysine, ornithine, acetate, fumarate, malate, propionate, pyruvate and succinate. Acid is produced from glucose, glycerol, maltose and mannose. Sensitive to norfloxacin, but not to erythromycin, neomycin, ciprofloxacin, streptomycin, kanamycin, ampicillin or vancomycin. The major polar lipids are C20C20 and C20C25 derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and some unidentified glycolipids. The G+C content of the DNA is 65·6 mol% (Tm).
The type strain, AJ2T (=AS 1.3731T=JCM 12890T), was isolated from a salt lake in Altun Mountain in China.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Dussault, H. P. (1955). An improved technique for staining red halophilic bacteria. J Bacteriol 70, 484–485.
Gupta, R., Lanter, J. M. & Woese, C. R. (1983). Sequence of the 16S ribosomal RNA from Halobacterium volcanii, an archaebacterium. Science 221, 656–659.
Huß, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. Syst Appl Microbiol 4, 184–192.
Kamekura, M. & Kates, M. (1988). Lipids of halophilic archaebacteria. In Halophilic Bacteria II, pp. 25–54. Edited by F. Rodriguez-Valera. Boca Raton, FL: CRC Press.
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform 5, 150–163.
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5, 109–118.[Medline]
McGenity, T. J., Gemmell, R. T. & Grant, W. D. (1998). Proposal of a new halobacterial genus Natrinema gen. nov., with two species Natrinema pellirubrum nom. nov. and Natrinema pallidum nom. nov. Int J Syst Bacteriol 48, 1187–1196.[CrossRef][Medline]
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Xin, H., Itoh, T., Zhou, P., Suzuki, K., Kamekura, M. & Nakase, T. (2000). Natrinema versiforme sp. nov., an extremely halophilic archaeon from Aibi salt lake, Xinjiang, China. Int J Syst Evol Microbiol 50, 1297–1303.[Abstract]
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