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1 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, 41012 Seville, Spain
2 Noda Institute for Scientific Research, 399 Noda, Noda-shi, Chiba-ken 278-0037, Japan
3 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100080 Beijing, P. R. China
4 Department of Biotechnology, University of the Western Cape, Bellville 7535, Cape Town, South Africa
5 Genencor International BV, Archimedesweg 30, 2333 CN Leiden, The Netherlands
6 Department of Infection, Immunity and Inflammation, University of Leicester, Leicester LE1 9HN, UK
Correspondence
A. Ventosa
ventosa{at}us.es
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A thin-layer chromatogram of the polar lipids of strain XH-48T and some haloarchaea is available as a supplementary figure in IJSEM Online.
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). Prior to the 1970s, haloarchaeal taxonomy was based mainly on standard biochemical tests and cell morphology (Gibbons, 1974). Recent sequence comparisons of haloarchaeal 16S rRNA genes as a basis for phylogeny as well as chemotaxonomic criteria, particularly polar lipid composition, have resulted in the recognition of great taxonomic diversity at the genus level, and 21 genera in this family have now been described (Tindall et al., 1984; Torreblanca et al., 1986; Oren et al., 1995; McGenity & Grant, 1995; Kamekura & Dyall-Smith, 1995; Kamekura et al., 1997; McGenity et al., 1998; Montalvo-Rodríguez et al., 1998; Y. Xu et al., 1999; Ventosa et al., 1999; Wainø et al., 2000; Grant 2001a, b; Oren et al., 2002; Hezayen et al., 2002; Vreeland et al., 2002; Itoh et al., 2005; Xue et al., 2005; Castillo et al., 2006).
The recent isolation of micro-organisms from hypersaline environments in different parts of China has led to the discovery of several novel species and genera belonging to the archaea (Y. Xu et al., 1999, 2001; Xin et al., 2000, 2001; Fan et al., 2004; Feng et al., 2004, 2005; Itoh et al., 2005; X.-W. Xu et al., 2005a, b, c; Xue et al., 2005; Castillo et al., 2006). In this paper, the almost complete 16S rRNA gene sequence from the halophilic strain XH-48T, isolated from Lake Xilinhot in Inner Mongolia (China), is reported, along with its polar lipid composition and physiological and biochemical characteristics. On the basis of the data presented, we have concluded that strain XH-48T is not closely related to any of the currently described haloarchaeal taxa and is sufficiently different to justify its classification as a novel species within a novel genus.
Strain XH-48T was isolated from a sediment sample of the saline Lake Xilinhot (43° 55' N 115° 37' E). Samples were diluted in a 20 % salt solution and spread on nutrient agar plates containing the following (l–1): NaCl, 195 g; MgCl2.6H2O, 32.5 g; MgSO4.7H2O, 50.8 g; CaCl2, 0.8 g; KCl, 5 g; NaHCO3, 0.16 g; NaBr, 0.6 g; yeast extract, 5 g. The pH of the medium was adjusted to 7.5 with 1 M NaOH. Sampling was carried out in September 2003. The water from Lake Xilinhot was at 23.6 °C and pH 8.5 and had a conductivity of 185 mS cm–1. Strain XH-48T grew at temperatures in the range 25–50 °C (optimum, 37 °C) and at pH values in the range 6.0–9.0 (optimum, pH 7.0–8.0). Routine cultivation was performed at 37 °C and pH 7.5. The requirements for NaCl and magnesium for growth were determined in media with 1.0–5.2 M NaCl or 0–0.5 M MgCl2. Strain XH-48T was capable of growing at a wide range of NaCl concentrations, from 2.5 (15 %) to 5.0 M (30 %). It grew optimally in the presence of 3.4 M (20 %) NaCl, as has been shown for most extremely halophilic archaea (Grant et al., 2001). MgCl2 was not required 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). Tests for catalase and oxidase activities and for the hydrolysis of starch and Tween 80 were performed as described previously (Gonzalez et al., 1978). Nitrate reduction, H2S formation, indole production and the utilization of sugars, alcohols, amino acids and organic acids were assessed as described by Oren et al. (1997). Strain XH-48T was oxidase- and catalase-positive. The results of methyl red, Voges–Proskauer, nitrate reduction, indole production from tryptone and Simmons' citrate tests were negative. Casein, aesculin and Tween 80 were not hydrolysed. Gelatin was not liquefied. The urease test was positive. Starch and DNA were hydrolysed. Susceptibility to antibiotics was determined on agar-medium plates by using absorbent paper discs impregnated with antibiotics at the following concentrations: ampicillin (10 µg), bacitracin (10 U), chloramphenicol (30 µg), erythromycin (15 µg); gentamicin (10 µg), nalidixic acid (30 µg), neomycin (10 µg), novobiocin (30 µg), penicillin G (10 U), rifampicin (30 µg), streptomycin (10 µg) and tetracycline (30 µg). The isolation medium, containing yeast extract at 0.05 % (w/v) and supplemented with 1 % (w/v) of the substrate being tested, was used to determine the utilization of different organic substrates as carbon and energy sources or as carbon, nitrogen and energy sources (Torreblanca et al., 1986). The results of the tests for antibiotic susceptibility and utilization of different substrates are included in the species description. The formation of acid from different sugars was assessed in media (in which the yeast extract concentration was 0.05 %, w/v) supplemented with 1 % (w/v) of the sugar being tested (sterilized separately).
Cell morphology and motility were examined using an Olympus BX41 microscope equipped with phase-contrast optics. For photography, drops of exponentially growing liquid cultures were mixed on a microscope slide with an equal volume of melted 1 % agarose containing 20 % NaCl and then covered with a coverslip. The cells were non-motile and pleomorphic, exhibiting rod-shaped, square or disc-shaped morphologies (Fig. 1). Colony morphology was observed on agar medium under optimal growth conditions after incubation at 37 °C for 10 days. Colonies of strain XH-48T that formed on agar plates were circular, smooth, entire, opaque and pink-pigmented.
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). TLC was performed using Merck HPTLC silica gel 60 plates (art. 5641) with the solvent system chloroform/methanol/acetic acid/water (85 : 22.5 : 10 : 4, by vol.). Glycolipids were detected as purple spots by means of spraying with 0.5 % 
-naphthol in methanol/water (1 : 1) and then with sulphuric acid/ethanol (1 : 1), followed by brief heating at 160 °C. TLC of the polar lipids (see Supplementary Fig. S1 available in IJSEM Online) suggested that strain XH-48T contained phosphatidylglycerol and phosphatidylglyceromethylphosphate derived from both C20C20 and C20C25 glycerol diethers, as is shown by the two spots (Xin et al., 2000). Two glycolipid spots (C20C20 and C20C25 moieties, marked with pencil) were detected between S-TGA-1 and S-TeGA, which showed mobilities different from that of S2-DGA-1, the characteristic glycolipid for species of the genus Natrinema.
Chromosomal DNA of strain XH-48T was isolated and purified according to the methods described by Wilson (1987) and Marmur (1961). The G+C content of the genomic DNA was determined from the mid-point (Tm) of the thermal denaturation profile (Marmur & Doty, 1962), using the equation of Owen & Hill (1979). The DNA G+C content of strain XH-48T is 61.1 mol%. The 16S rRNA gene of strain XH-48T was amplified by a PCR using three universal primers as described previously (Lopez-Garcia et al., 2001; Arahal et al., 1996), and an almost-complete nucleotide sequence (approx. 1400 bp) was determined. The ARB software package (Ludwig et al., 2004) was used for 16S rRNA gene sequence analysis. Base-frequency filters were applied in the sequence-comparison analysis and the effects on the results were evaluated. The 16S rRNA gene phylogenetic analysis, performed using the neighbour-joining method (Saitou & Nei, 1987), showed the position of strain XH-48T as a branch in the Natrialba clade (Fig. 2). Similar topologies were obtained when other treeing methods (maximum parsimony and maximum likelihood) were used. In consequence, it was concluded that strain XH-48T formed a new distinct branch related to Natrialba aegyptiaca (94.5 % 16S rRNA gene sequence similarity to the type strain) and Natrialba asiatica (93.3 %). The phenotypic characteristics of these two species of the genus Natrialba are very different from those of strain XH-48T, e.g. in terms of indole production, hydrolysis of gelatin, casein and Tween 80, acid production from carbon sources and susceptibility to antibiotics. In addition, the alignment of the 16S rRNA gene sequence with all published sequences of haloarchaea clearly shows that strain XH-48T does not belong to the genus Natrialba, since its sequence does not share the two signature bases, 403G and 560G, of those of members of the genus Natrialba (Kamekura et al., 2004).
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shows the characteristics that differentiate the novel genus from other related haloarchaeal genera.
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Gram-negative. Cells are pleomorphic although most are rod-shaped. Colonies are pink-pigmented. Strictly aerobic; oxygen is used as the terminal electron acceptor. Growth occurs at pH values between 6.0 and 9.0, at temperatures between 25 and 50 °C and at salinities between 15 and 30 % (2.5–5.0 M) NaCl. Optimal growth occurs at pH 7.0–8.0, 37 °C and 20 % (3.4 M) NaCl. The DNA G+C content of the type strain of the only species in the genus is 61.0 mol% (Tm method). Polar lipids include phosphatidylglycerol and phosphatidylglyceromethylphosphate, derived from both C20C20 and C20C25 glycerol diethers, and two unidentified glycolipids. Phylogenetically affiliated to the Halobacteriaceae. The type species is Halostagnicola larsenii, which was first isolated from a salt lake.
Description of Halostagnicola larsenii sp. nov.
Halostagnicola larsenii (lar.se'ni.i. N.L. gen. n. larsenii of Larsen, named for the Norwegian microbiologist H. Larsen, one of the pioneers in the study of haloarchaea).
Exhibits the following properties in addition to those given in the genus description. Cells are 0.5–1.0 µm wide and 1.0–3.0 µm long (Fig. 1
). Colonies are circular and 1–2 mm in diameter after incubation for 10 days at 37 °C. Extremely halophilic and the cells lyse in water. Magnesium is not required for growth. Growth does not occur above 50 °C. The pH range for growth is 6.0–9.0. Amino acids are not required for growth. Catalase- and oxidase-positive. Negative for indole production and in methyl red, Voges–Proskauer and Simmons' citrate tests. H2S is not produced from cysteine. Acid is produced from sucrose. Does not produce arginine dihydrolase, lysine decarboxylase or ornithine decarboxylase. Produces urease and phosphatase. Anaerobic growth with nitrate or arginine does not occur. Gelatin, Tween 80, casein and aesculin are not hydrolysed. Starch and DNA are hydrolysed. Nitrate is reduced to nitrite. The following substrates are utilized for growth: glycerol, fructose, maltose, trehalose, lactose, glucose, arabinose, galactose, mannitol, ribose, starch, xylose, propionate, glutamate and acetate. No growth occurs on mannitol, sorbitol, raffinose, succinate, malate or fumarate. Susceptible to bacitracin and novobiocin. Resistant to ampicillin, chloramphenicol, erythromycin, gentamicin, nalidixic acid, neomycin, penicillin G, rifampicin, streptomycin and tetracycline.
The type strain is strain XH-48T(=DSM 17691T=CGMCC 1.5338T=JCM 13463T=CECT 7116T), isolated from the saline Lake Xilinhot in Inner Mongolia, China.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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