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1 College of Life Sciences, Zhejiang University, Hangzhou 310027, P. R. China
2 Institute of Microbiology, Chinese Academy of Sciences, ZhongGuanCun, Haidian, Beijing 100080, P. R. China
3 Department of Biology, Xinjiang Normal University, Urumqi 830054, P. R. China
4 Institute of Life Sciences and the Moshe Shilo Minerva Center for Marine Biogeochemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
Correspondence
Pei-Jin Zhou
zhou{at}sun.im.ac.cn
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ABSTRACT |
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2 glucose-glycerol diether. The genomic DNA G+C content is 66·6 mol%. The 16S rRNA gene sequence similarity values of strain AB14T with its nearest phylogenetic neighbours (Haloterrigena thermotolerans and Haloterrigena turkmenica) are 98·6 and 96·0 %, respectively. DNA–DNA hybridization revealed 54 % relatedness between strain AB14T and Haloterrigena thermotolerans JCM 11050T and 21 % between strain AB14T and Haloterrigena turkmenica JCM 9101T. It is therefore proposed that strain AB14T represents a novel species, for which the name Haloterrigena saccharevitans sp. nov. is proposed. The type strain is AB14T (=AS 1.3730T=JCM 12889T).
2 glucose-glycerol dietherThe GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain AB14T is AY820137.
A thin-layer chromatogram of lipids from strain AB14T and Haloterrigena thermotolerans JCM 11050T is available as supplementary material in IJSEM Online.
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) and Haloterrigena thermotolerans (Montalvo-Rodríguez et al., 2000). In phylogenetic trees based on 16S rRNA gene sequences, species of the genera Haloterrigena and Natrinema sometimes cluster together (Montalvo-Rodríguez et al., 2000; Xin et al., 2000; Tindall, 2003). However, there are striking differences in the polar lipid composition among species of these two genera. The type species of Haloterrigena possesses mannose-2,6 disulfate 12 glucose-glycerol diether (S2-DGD), but lacks phosphatidylglycerol sulfate (Ventosa et al., 1999; Montalvo-Rodríguez et al., 2000). The opposite is true for the type species of Natrinema (McGenity et al., 1998; Xin et al., 2000). Based on a combination of other morphological and chemotaxonomic characters, species of these two genera can thus be distinguished from each other.
Strain AB14T was isolated from a soil sample collected from the near-edge floor of Aibi salt lake located in Xin-Jiang, China. The isolate was grown and maintained aerobically at 37 °C in S-G medium (Sehgal & Gibbons, 1960). A pure culture was obtained by repeated restreaking. Phenotypic tests were performed according to the proposed minimal standards for the description of new taxa in the order Halobacteriales (Oren et al., 1997). The optimal conditions for growth were determined in S-G medium with 0·85–5·10 M NaCl and 0–1·0 M Mg2+, respectively. The pH range for growth (assayed from pH 5·0 to 9·5 at intervals of 0·5) was determined by adding MES (pH 5·0–6·0), PIPES (pH 6·5–7·0), Tricine (pH 7·5–8·5) and CHES (pH 9·0–9·5) to S-G medium at a concentration of 50 mM. The temperature range for growth of strain AB14T in S-G medium (pH 7·5) with optimal NaCl and Mg2+ concentrations was determined using a TN3F temperature gradient incubator (ADVANTEC). Cell morphology and motility were examined by optical and transmission electron microscopy (H-600; Hitachi). Gram staining was performed using acetic acid-fixed samples, 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). The following characteristics were tested according to Xin et al. (2000) as described previously (Oren et al., 1997): hydrolysis of starch, casein, Tween 40 and Tween 80; nitrate reduction; production of indole and H2S; catalase and oxidase activities; and utilization of sugars, alcohols, amino acids and organic acids. Halorubrum sodomense JCM 8880T and Haloterrigena thermotolerans JCM 11050T were used as controls in tests.
Total lipids were extracted by the modified method of Kamekura & Kates (1988). Phospholipids and glycolipids were separated by TLC on silica gel plates (10x10 cm) and analysed according to Xin et al. (2000). Genomic DNA was prepared by the method of Marmur (1961) and the purity was checked spectrophotometrically. The DNA G+C content was determined by thermal denaturation (Tm) (Marmur & Doty, 1962) using Escherichia coli K-12 DNA as calibration standard. The 16S rRNA gene sequence was amplified under conditions described by Feng et al. (2005) with the following primers (position given according to E. coli 16S rRNA gene): primer 1, 5'-ATTCCGGTTGATCCTGC-3' (positions 6–22); and primer 2, 5'-AGGAGGTGATCCAGCCGCAG-3' (positions 1540–1521).
The sequence was compared with closely related sequences of reference organisms from the FASTA network service. Sequence data were aligned with CLUSTAL_W 1.8 (Thompson et al., 1994). Phylogenetic trees were constructed by the neighbour-joining method with the MEGA3 program package (Kumar et al., 2004). DNA–DNA hybridizations were performed by the thermal denaturation and renaturation method of De Ley et al. (1970), as modified by Huß et al. (1983), using a Beckman DU 800 spectrophotometer.
The 16S rRNA gene sequence similarity values between strain AB14T and the type strains of Haloterrigena thermotolerans JCM 11050T and Haloterrigena turkmenica JCM 9101T were 98·6 and 96·0 %, respectively. Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that strain AB14T formed a coherent cluster with Haloterrigena thermotolerans with a bootstrap resampling value of 99 % (Fig. 1). The polar lipid profile of strain AB14T, which possesses the C20C20 and C20C25 derivatives of phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me) and S2-DGD, was consistent with that of Haloterrigena species. The contents of PG and S2-DGD of strain AB14T, however, were different from those in Haloterrigena thermotolerans JCM 11050T (see the supplementary figure in IJSEM Online). S2-DGD was present in strain AB14T in greatest abundance, whereas the amount of PG was lower than that in Haloterrigena thermotolerans JCM 11050T. Strain AB14T and Haloterrigena thermotolerans JCM 11050T were incubated at 37 °C for 6 days and the polar lipids were extracted under identical conditions. The DNA G+C content of strain AB14T (66·6 mol%) was notably higher than that of Haloterrigena thermotolerans (63·3 mol%) (Montalvo-Rodríguez et al., 2000) and Haloterrigena turkmenica (59·2–60·2 mol%) (Ventosa et al., 1999). The DNA–DNA relatedness levels of strain AB14T to Haloterrigena thermotolerans JCM 11050T and Haloterrigena turkmenica JCM 9101T were 54±2 % and 21±2 %, respectively (mean values of two determinations). Comparison of phenotypic properties (Table 1) also indicated differences between strain AB14T and Haloterrigena thermotolerans. The optimal growth temperature of strain AB14T is 42–45 °C, which is lower than that of Haloterrigena thermotolerans (50 °C). Strain AB14T could reduce nitrate under anaerobic conditions and some cells deposited under the tube, whereas Haloterrigena thermotolerans was strictly aerobic. In addition, strain AB14T could be distinguished from Haloterrigena thermotolerans by its hydrolysis of gelatin and its sensitivity to tetracycline (Table 1); results were observed after 14 days, with weakly hydrolysable or sensitive being defined as the appearance of a zone of hydrolysis or inhibition of approximately 0·5–1·0 mm.
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Description of Haloterrigena saccharevitans sp. nov.
Haloterrigena saccharevitans (sac.char.e.vi'tans. L. neut. n. saccharon, -i a kind of sugar; L. part. adj. evitans shunning, avoiding; N.L. part. adj. saccharevitans sugar-avoiding, because it uses very few sugars).
Cells are Gram-negative, motile, rod-shaped (3–10x0·4–1·0 µm) and become coccoid in stationary cultures. Colonies on complex agar medium are 0·5–1·0 mm in diameter, smooth, circular, elevated and light red. At least 1·7 M NaCl is required for growth and growth is optimal at 3·0–3·4 M NaCl. Mg2+ range for growth is 0–1·0 M, with an optimum around 0–0·2 M. The pH and temperature ranges for growth are 6·5–8·5 (optimum at pH 7·5) and 24–58 °C (optimum at 42–45 °C), respectively. Chemo-organotrophic. Grows anaerobically in the presence of nitrate. Oxidase- and catalase-positive. Indole formation is negative. Nitrate is reduced without production of gas. H2S is produced from thiosulfate. Tweens 40 and 80 are hydrolysed. Gelatin, starch and casein are not hydrolysed. The following substrates are utilized for growth: glycerol, arginine, ornithine, acetate, fumarate, malate, propionate, pyruvate, succinate and lactate. Fructose, glucose, mannose, starch, arabinose, lactose, mannitol, rhamnose, sorbitol, maltose, galactose, D-ribose, sucrose, D-xylose, glutamate, lysine, aspartate, glycine, alanine and citrate are not utilized for growth. Acid is only produced from glycerol. Sensitive to tetracycline, but not to ciprofloxacin, streptomycin, norfloxacin, kanamycin, ampicillin or vancomycin. The major polar lipids are the C20C20 and C20C25 derivatives of PG, PGP-Me and S2-DGD. The DNA G+C content of the type strain is 66·6 mol% (Tm).
The type strain, AB14T (=AS 1.3730T=JCM 12889T), was isolated from Aibi salt lake, Xin-Jiang, China.
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ACKNOWLEDGEMENTS |
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Dussault, H. P. (1955). An improved technique for staining red halophilic bacteria. J Bacteriol 70, 484–485.
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Tindall, B. J. (2003). Taxonomic problems arising in the genera Haloterrigena and Natrinema. Int J Syst Evol Microbiol 53, 1697–1698.
Ventosa, A., Gutierrez, M. C., Kamekura, M. & Dyall-Smith, M. L. (1999). Proposal to transfer Halococcus turkmenicus, Halobacterium trapanicum JCM 9743 and strain GSL-11 to Haloterrigena turkmenica gen. nov., comb. nov. Int J Syst Bacteriol 49, 131–136.[CrossRef][Medline]
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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