HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Supplementary material Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Xu, X.-W. Articles by Zhou, P.-J. Search for Related Content PubMed PubMed Citation Articles by Xu, X.-W. Articles by Zhou, P.-J. Agricola Articles by Xu, X.-W. Articles by Zhou, P.-J.

Natrinema altunense sp. nov., an extremely halophilic archaeon isolated from a salt lake in Altun Mountain in Xinjiang, China

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

	ABSTRACT

TOP ABSTRACT MAIN TEXT REFERENCES

 
A novel extremely halophilic strain, AJ2^T, was isolated from Ayakekum salt lake located in the Altun Mountain National Nature Reserve in Xinjiang, China. This isolate was neutrophilic, motile and grew in a wide range of MgCl₂ concentrations (0·005–1·0 M). The major polar lipids of the isolate were C₂₀C₂₀ and C₂₀C₂₅ derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol sulfate. A comprehensive 16S rRNA gene sequence analysis revealed that the isolate shared 96·6–97·7 % sequence identity with Natrinema species. The isolate, however, could be genetically differentiated from these species by DNA–DNA hybridization analysis and on the basis of its physiological properties. On the basis of the polyphasic evidence, strain AJ2^T (=AS 1.3731^T=JCM 12890^T) represents the type strain of a novel species, for which the name Natrinema altunense sp. nov. is proposed.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain AJ2^T is AY208972.

Thin-layer chromatograms showing the lipid content of strain AJ2^T and other Natrinema species, along with a table of some characteristics distinguishing this strain from Natrinema versiforme AS 1.2365^T, are available as supplementary material in IJSEM Online.

	MAIN TEXT

TOP ABSTRACT MAIN TEXT REFERENCES

 
On the basis of 16S rRNA gene sequences, salt tolerance and chemotaxonomic and physiological characteristics, the genus Natrinema was created in 1998 to accommodate Natrinema pellirubrum (formerly Halobacterium salinarum NCIMB 786) and Natrinema pallidum (formerly Halobacterium halobium NCIMB 777) (McGenity et al., 1998). 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 (T_m) (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 AJ2^T, 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 AJ2^T clustered with Natrinema species with validly published names. The results indicated that strain AJ2^T belongs to the genus Natrinema.

View larger version (33K): [in this window] [in a new window]   Fig. 1. Phylogenetic tree based on 16S rRNA gene sequences from strain AJ2T and other related organisms. The tree was constructed using the neighbour-joining method, with bootstrap values calculated from 1000 resamplings. The number at each branch point represents the percentage bootstrap support. Bar, 10 substitutions at any nucleotide position per 100 nucleotide positions.

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% (T_m).

The type strain, AJ2^T (=AS 1.3731^T=JCM 12890^T), was isolated from a salt lake in Altun Mountain in China.

	ACKNOWLEDGEMENTS

 
We thank M. Zhi-Pei Liu, Bao-Jun Wang and Mrs Jie Feng for their help and comments. This work was partially supported by a grant from the National Natural Science Foundation of China (grant no. 30370029) and the Major State Basic Research Development Program of China (973 Program) (grant no. 2004cb719601).

	REFERENCES

TOP ABSTRACT MAIN TEXT REFERENCES

 
De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[Medline]

Dussault, H. P. (1955). An improved technique for staining red halophilic bacteria. J Bacteriol 70, 484–485.[Free Full Text]

Gupta, R., Lanter, J. M. & Woese, C. R. (1983). Sequence of the 16S ribosomal RNA from Halobacterium volcanii, an archaebacterium. Science 221, 656–659.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

Oesterhelt, D. & Stoeckenius, W. (1974). Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane. Methods Enzymol 31, 667–678.[Medline]

Oren, A., Ventosa, A. & Grant, W. D. (1997). Proposed minimal standards for description of new taxa in the order Halobacteriales. Int J Syst Bacteriol 47, 233–238.[Abstract/Free Full Text]

Oren, A., Elevi, R., Watanabe, S., Ihara, K. & Corcelli, A. (2002). Halomicrobium mukohataei gen. nov., comb. nov., and emended description of Halomicrobium mukohataei. Int J Syst Evol Microbiol 52, 1831–1835.[Abstract]

Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]

Sehgal, S. N. & Gibbons, N. E. (1960). Effect of metal ions on the growth of Halobacterium cutirubrum. Can J Microbiol 6, 165–169.[Medline]

Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.[Abstract/Free Full Text]

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]

This article has been cited by other articles:

				S. W. Roh, Y.-D. Nam, H.-W. Chang, K.-H. Kim, Y. Sung, M.-S. Kim, H.-M. Oh, and J.-W. Bae Haloterrigena jeotgali sp. nov., an extremely halophilic archaeon from salt-fermented food Int J Syst Evol Microbiol, September 1, 2009; 59(9): 2359 - 2363. [Abstract] [Full Text] [PDF]

				W. Tapingkae, S. Tanasupawat, T. Itoh, K. L. Parkin, S. Benjakul, W. Visessanguan, and R. Valyasevi Natrinema gari sp. nov., a halophilic archaeon isolated from fish sauce in Thailand Int J Syst Evol Microbiol, October 1, 2008; 58(10): 2378 - 2383. [Abstract] [Full Text] [PDF]

				S. Mei, C. Sun, X. Liu, Q. Lu, L. Cai, Y. Li, and H. Xiang The Helix-Loop-Helix Motif at the N Terminus of HalI Is Essential for Its Immunity Function against Halocin C8 J. Bacteriol., October 1, 2008; 190(19): 6501 - 6508. [Abstract] [Full Text] [PDF]

				Y.-H. Wu, X.-W. Xu, Y.-Y. Huo, P. Zhou, X.-F. Zhu, H.-B. Zhang, and M. Wu Halomonas caseinilytica sp. nov., a halophilic bacterium isolated from a saline lake on the Qinghai-Tibet Plateau, China Int J Syst Evol Microbiol, May 1, 2008; 58(5): 1259 - 1262. [Abstract] [Full Text] [PDF]

				I. Romano, A. Poli, I. Finore, F. J. Huertas, A. Gambacorta, S. Pelliccione, G. Nicolaus, L. Lama, and B. Nicolaus Haloterrigena hispanica sp. nov., an extremely halophilic archaeon from Fuente de Piedra, southern Spain Int J Syst Evol Microbiol, July 1, 2007; 57(7): 1499 - 1503. [Abstract] [Full Text] [PDF]

				X.-W. Xu, Y.-H. Wu, H.-b. Zhang, and M. Wu Halorubrum arcis sp. nov., an extremely halophilic archaeon isolated from a saline lake on the Qinghai-Tibet Plateau, China Int J Syst Evol Microbiol, May 1, 2007; 57(5): 1069 - 1072. [Abstract] [Full Text] [PDF]

				A. M. Castillo, M. C. Gutierrez, M. Kamekura, Y. Xue, Y. Ma, D. A. Cowan, B. E. Jones, W. D. Grant, and A. Ventosa Natrinema ejinorense sp. nov., isolated from a saline lake in Inner Mongolia, China. Int J Syst Evol Microbiol, November 1, 2006; 56(Pt 11): 2683 - 2687. [Abstract] [Full Text] [PDF]

				A. M. Castillo, M. C. Gutierrez, M. Kamekura, Y. Xue, Y. Ma, D. A. Cowan, B. E. Jones, W. D. Grant, and A. Ventosa Halostagnicola larsenii gen. nov., sp. nov., an extremely halophilic archaeon from a saline lake in Inner Mongolia, China. Int J Syst Evol Microbiol, July 1, 2006; 56(Pt 7): 1519 - 1524. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplementary material Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Xu, X.-W. Articles by Zhou, P.-J. Search for Related Content PubMed PubMed Citation Articles by Xu, X.-W. Articles by Zhou, P.-J. Agricola Articles by Xu, X.-W. Articles by Zhou, P.-J.