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1 College of Life Sciences, Zhejiang University, Hangzhou 310058, People's Republic of China
2 Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China
3 Second Institute of Oceanography, State Oceanic Administration, Hangzhou 310012, People's Republic of China
4 Institute of Life Sciences and the Moshe Shilo Minerva Centre for Marine Biogeochemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
Correspondence
Min Wu
wumin{at}zju.edu.cn
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ABSTRACT |
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Phase-contrast photomicrographs and transmission electron photomicrographs of strain ZJ206T are available as supplementary figures in IJSEM Online.
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as a member of the family Halobacteriaceae. At present, the genus comprises seven species: Haloferax volcanii (Mullakhanbhai & Larsen, 1975), Hfx. mediterranei (Rodriguez-Valera et al., 1983), Hfx. denitrificans (Tomlinson et al., 1986; Tindall et al., 1989), Hfx. gibbonsii (Juez et al., 1986), Hfx. alexandrinus (Asker & Ohta, 2002), Hfx. lucentense (Gutierrez et al., 2002) and Hfx. sulfurifontis (Elshahed et al., 2004).
The current taxonomic classification of the Halobacteriaceae is mainly based on 16S rRNA gene sequence comparison, polar lipid composition and phenotypic characteristics (Oren et al., 1997; Grant et al., 2001; Castillo et al., 2006). All Haloferax species described so far possess diphytanyl ether derivatives of sulfated diglycosyl diether but do not contain phosphatidylglycerol sulfate (Grant et al., 2001). In phylogenetic trees based on 16S rRNA gene sequences, they form an independent cluster, the nearest taxon being Halogeometricum borinquense. Additionally, most members of the genus Haloferax are extremely pleomorphic and require relatively low salt levels for growth in comparison with other members of the Halobacteriaceae.
We have isolated three strains of extremely halophilic archaea from a mixture of mud and brine obtained from a solar saltern (122° 17' N 29° 55' E) located in the Zhoushan archipelago, Zhe-Jiang Province, China. The water sample (approx. 50 ml) was filtered through 0.45 and 0.22 µm filters in sequence and then the 0.22 µm membrane was added to DSMZ medium 823 and plated by using a 10-fold dilution-series method. The plates were incubated aerobically at 25 and 37 °C. After 1–2 weeks incubation, representative colonies were picked and maintained on S-G medium (Sehgal & Gibbons, 1960) at 37 °C. Strains were purified by repeated restreaking; purity was confirmed from the uniformity of the cell morphology. Cell morphology and motility were examined by using phase-contrast microscopy (BX40; Olympus/Axiovert 135TV; Zeiss) and transmission electron microscopy (H-600; Hitachi). The cells of all three isolates were very pleomorphic; in cultures grown at the highest salinities, elongated cells were common (see Supplementary Fig. S1 available in IJSEM Online). The cells showed motility, but we did not observe flagella by electron microscopy (Supplementary Fig. S2). The cultures were pink in colour as a result of the presence of bacterioruberin carotenoids. Phenotypic characteristics were tested according to Xu et al. (2005), as mentioned previously in the minimal standards for the description of new taxa in the order Halobacteriales (Oren et al., 1997). Parallel tests were performed with the strains Hfx. sulfurifontis JCM 12327T and Hfx. volcanii CGMCC 1.2350T.
Total lipids were extracted by using the modified method of Kamekura & Kates (1988). Polar lipids were separated by two-dimensional silica-gel TLC. Phospholipids were detected with Zinzadze reagent. Glycolipids were analysed by spraying the plate with 0.5 % 1-naphthol in methanol/water (1 : 1) and then with sulfuric acid/ethanol (1 : 1) before heating it at 120 °C for 10 min (Xin et al., 2000). The glycolipid analysis indicated the presence of sulfated diglycosyl diether and the absence of phosphatidylglycerol sulfate.
The 16S rRNA genes were analysed as described previously (Xu et al., 2005). Phylogenetic trees were constructed by using the neighbour-joining method with the MEGA3 program package (Kumar et al., 2004), after multiple alignment of the data by CLUSTAL_X (Thompson et al., 1997). The 16S rRNA gene sequences of the three isolates showed 99.8–100 % similarity with respect to each other. Sequence similarity analysis by the FASTA network service revealed that the strains shared 96.4–97.4 % similarity with the type strains of previously described Haloferax species, and 92.3–92.5 % similarity with Hgm. borinquense PR3T, its closest relative outside the genus Haloferax. Both the neighbour-joining tree (Fig. 1) and the maximum-parsimony tree (not shown) indicated that the isolates are separate from Haloferax species and cluster most closely with Hfx. sulfurifontis M6T.
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) was found to be 62.2 mol%, which is within the range reported for Haloferax species (Grant et al., 2001). DNA–DNA hybridizations were performed by using the thermal denaturation and renaturation method of De Ley et al. (1970), as modified by Huß et al. (1983), with a Beckman DU 800 spectrophotometer. DNA–DNA relatedness levels for strain ZJ206T with respect to Hfx. sulfurifontis JCM 12327T and Hfx. volcanii CGMCC 1.2350T were 48 and 44 %, respectively. Comparisons of phenotypic properties (Table 1) also indicated differences (such as motility, hydrolysis of substrates and acid production from sugars) between strain ZJ206T and Haloferax species.
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) and 16S rRNA gene sequences, together with the DNA–DNA hybridization data, justify the creation of a novel species. We propose to name the novel species in honour of Professor Helge Larsen, who has contributed much to our understanding of halophilic micro-organisms, and who originally described the type species of the genus Haloferax. Thus, strain ZJ206T represents a novel species within the genus Haloferax, for which the name Haloferax larsenii sp. nov. is proposed.
Description of Haloferax larsenii sp. nov.
Haloferax larsenii (lar.se'ni.i. N.L. gen. n. larsenii of Larsen, named in honour of Professor Helge Larsen, one of the pioneers of halophile research).
Gram-negative. Extremely pleomorphic, motile, occurring mainly as irregularly shaped cells (0.8–1.5 µm in diameter). Cells grown at the highest salinities are often elongated. Colonies on complex agar medium are 1–2 mm in diameter, smooth, circular, elevated and orange–red. Halophilic. Cells lyse immediately in distilled water after 2 h with 10–20 g NaCl l–1. The NaCl concentration for growth is between 1 and 4.8 M, with an optimum at 2.2–3.4 M. Saturated NaCl inhibits growth in liquid medium. Growth occurs in media containing more than 5 mM Mg2+, the optimum Mg2+ concentration being between 20 and 500 mM. The pH range for growth is 6.0–8.5, with an optimum at pH 6.5–7.0; the temperature range for growth is 25–55 °C, with an optimum at 42–45 °C. Chemoorganotrophic. Oxidase- and catalase-positive. Anaerobic growth with arginine or DMSO does not occur. Positive for indole formation. Nitrate is reduced to nitrite. Grows anaerobically on nitrate, with the production of gas. H2S is produced from thiosulfate. Starch and Tweens 40 and 80 are hydrolysed. Gelatin is hydrolysed; casein is not hydrolysed. The following substrates are utilized for growth: glucose, glycerol, mannose, starch, maltose, sucrose, glutamate, alanine, ornithine, fumarate, malate, pyruvate, succinate and lactate. Arabinose, lactose, mannitol, rhamnose, sorbitol, galactose, ribose, xylose, arginine, lysine, aspartate, glycine, acetate, propionate and citrate are not utilized for growth. Acid is produced from glycerol and maltose and, to a lesser extent, on glucose, fructose and sucrose. Sensitive to novobiocin, bacitracin, anisomycin, aphidicolin and rifampicin, but not to ampicillin, chloramphenicol, erythromycin, nalidixic acid, neomycin, nystatin, penicillin, tetracycline, streptomycin or kanamycin. The major polar lipids are the C20C20 derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, diglycosyl glycerol diether and sulfated diglycosyl diether. The DNA G+C content of DNA of the type strain is 62.2±0.8 mol% (Tm).
The type strain, ZJ206T (=CGMCC 1.5347T=JCM 13917T), was isolated from a solar saltern in Zhe-Jiang Province, China.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Castillo, A. M., Gutiérrez, M. C., Kamekura, M., Ma, Y.-H., Cowan, D. A., Jones, B. E., Grant, W. D. & Ventosa, A. (2006). Halovivax asiaticus gen. nov., sp. nov., a novel extremely halophilic archaeon isolated from Inner Mongolia, China. Int J Syst Evol Microbiol 56, 765–770.
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Grant, W. D., Kamekura, M., McGenity, T. J. & Ventosa, A. (2001). Class III. Halobacteria class. nov. In Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 1, pp. 294–301. Edited by D. R. Boone, R. W. Castenholz & G. M. Garrity. New York: Springer.
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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.
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Tindall, B. J., Tomlinson, G. A. & Hochstein, L. I. (1989). Transfer of Halobacterium denitrificans (Tomlinson, Jahnke, and Hochstein) to the genus Haloferax as Haloferax denitrificans comb. nov. Int J Syst Bacteriol 39, 359–360.[Medline]
Tomlinson, G. A., Jahnke, L. L. & Hochstein, L. I. (1986). Halobacterium denitrificans sp. nov., an extremely halophilic denitrifying bacterium. Int J Syst Bacteriol 36, 66–70.[Medline]
Torreblanca, M., Rodriguez-Valera, F., Juez, G., Ventosa, A., Kamekura, M. & Kates, M. (1986). Classification of non-alkaliphilic halobacteria based on numerical taxonomy and polar lipid composition, and description of Haloarcula gen. nov. and Haloferax gen. nov. Syst Appl Microbiol 8, 89–99.
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]
Xu, X.-W., Liu, S.-J., Tohty, D., Oren, A., Wu, M. & Zhou, P.-J. (2005). Haloterrigena saccharevitans sp. nov., an extremely halophilic archaeon from Xin-Jiang, China. Int J Syst Evol Microbiol 55, 2539–2542.
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