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1 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, 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, 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 chromatograph of the polar lipids of strain SH-6T and related haloarchaea is available as a supplementary figure with the online version of this paper.
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). Classically, they were easily differentiated microscopically as rods or cocci that were, respectively, included within the genera Halobacterium or Halococcus (Gibbons, 1974). In recent years, the numbers of halobacterial genera and species have increased due to the use of different isolation media and culture conditions, combined with the study of a wide variety of hypersaline environments. The extremely halophilic archaea are the dominant microbial populations of hypersaline environments (Ventosa, 2006). The current classification of halophilic archaea is based on three kinds of data: phenotypic features, chemical data (polar lipid composition) and genetic data (16S rRNA gene sequence information and DNA–DNA hybridization) (Oren et al., 1997; Grant et al., 2001). At the time of writing, the aerobic, extremely halophilic archaea are classified within 22 different genera with a large number of species. These genera are Halalkalicoccus, Haloarcula, Halobacterium, Halobaculum, Halobiforma, Halococcus, Haloferax, Halogeometricum, Halomicrobium, Halorhabdus, Halorubrum, Halosimplex, Halostagnicola, Haloterrigena, Halovivax, Natrialba, Natrinema, Natronobacterium, Natronococcus, Natronolimnobius, Natronomonas and Natronorubrum (Tindall et al., 1984; Torreblanca et al., 1986; Oren et al., 1995, 2002; Kamekura & Dyall-Smith, 1995; Kamekura et al., 1997; McGenity et al., 1998; Montalvo-Rodriguez et al., 1998; Ventosa et al., 1999; Xu et al., 1999; Wainø et al., 2000; Grant, 2001a, b; Hezayen et al., 2002; Vreeland et al., 2002; Itoh et al., 2005; Xue et al., 2005; Castillo et al., 2006a, b). Here we describe a halophilic archaeal strain, designated SH-6T, which was isolated from Lake Shangmatala, located in Inner Mongolia Autonomous Region, China. Preliminary 16S rRNA gene sequence comparisons indicated that the isolate was a member of the family Halobacteriaceae. The aim of the present work was to determine the exact taxonomic position of strain SH-6T by using a polyphasic taxonomic characterization that combined phenotypic, chemotaxonomic and phylogenetic analyses.
Strain SH-6T was isolated from a sediment sample from Shangmatala salt lake (43° 12' N 114° 01' E) by enrichment in liquid medium after 15 days incubation and subsequent plating of the enriched culture until purity was obtained on the same medium but with 2 % agar added. The medium contained (per litre distilled water): 174 g NaCl, 30 g MgCl2.6H2O, 45.4 g MgSO4.7H2O, 0.8 g CaCl2, 4.5 g KCl, 0.16 g NaHCO3, 0.5 g NaBr and 5 g yeast extract; the pH was adjusted to 8 with 1 M NaOH solution. At the time of sampling, the water in the lake had a salinity of 16.7 %, a temperature of 21.8 °C and a pH of 8.5. Strain SH-6T grew at a temperature range of 28–45 °C (optimum 37 °C) and a pH range of 6.0–11 (optimum pH 7.5–8). Routine cultivation was conducted at 37 °C and pH 8. Growth ranges and optima for NaCl and MgCl2 were determined by using the growth medium containing various concentrations of NaCl (0.9–5.2 M) and MgCl2 (0–0.5 M), respectively. Strain SH-6T was capable of growing over a wide range of NaCl concentrations: 2.5 M (15 %) to 5 M (30 %). It grew optimally in the presence of 4.3 M (25 %) NaCl, as has been shown for most extremely halophilic archaea. 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). Cell motility and morphology were observed under a phase-contrast light microscope (Olympus BX41) from an early exponential phase liquid culture. Cells of strain SH-6T were non-motile and pleomorphic, although long rod-shaped cells were most common (Fig. 1). Rod-shaped cells were 3–13x0.5–1 µm in size. Colony morphology was observed on agar medium under optimal growth conditions after incubation at 37 °C for 10 days. Anaerobic growth was tested in the presence of 5 g nitrate or L-arginine l–1 in filled, stoppered tubes. Tests for catalase and oxidase activities and hydrolysis of starch, casein, gelatin and Tween 80 were performed as described by Gonzalez et al. (1978). Nitrate reduction, H2S formation, indole formation and the utilization of sugars, alcohols, amino acids and organic acids were carried out as described by Oren et al. (1997). Antibiotic sensitivity tests were performed by spreading bacterial suspensions on culture plates and applying discs impregnated with the following antibiotics (amounts in parentheses): ampicillin (10 µg), bacitracin (10 U), cephalothin (30 µg), chloramphenicol (30 µg), erythromycin (15 µg), gentamicin (10 µg), kanamycin (30 µg), nalidixic acid (30 µg), neomycin (10 µg), novobiocin (30 µg), penicillin G (10 U), rifampicin (30 µg), polymyxin (300 U), streptomycin (10 µg), sulfamethoxazole (25 µg), tetracycline (30 µg) and vancomycin (30 µg). The physiological and biochemical characteristics and antibiotic susceptibility of strain SH-6T are given in the species description below.
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. TLC was performed by using Merck HPTLC silica gel 60 plates (Art. 5641) in the solvent system, which comprised chloroform/methanol/acetic acid/water (85 : 22.5 : 10 : 4, by vol.). Glycolipids were detected as purple spots by spraying with 0.5 % 
-naphthol in methanol/water (1 : 1) and then with sulfuric acid/ethanol (1 : 1), followed by heating at 160 °C. TLC of polar lipids (see Supplementary Fig. S1 available in IJSEM Online) suggested that strain SH-6T contained phosphatidylglycerol and phosphatidylglyceromethylphosphate derived from both C20C20 and C20C25 glycerol diethers as shown from the two spots (Xin et al., 2000). The glycolipid S2-DGD-1 was also detected.
Chromosomal DNA of strain SH-6T 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) by using the equation of Owen & Hill (1979). The DNA G+C content of strain SH-6T was 63.1 mol%. The 16S rRNA gene of strain SH-6T was amplified by PCR by using three universal primers as described by Lopez-Garcia et al. (2001) and Arahal et al. (1996) and the near-full-length nucleotide sequence (approximately 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. Comparison of the sequence with members of the family Halobacteriaceae based on the neighbour-joining method (Saitou & Nei, 1987) revealed that strain SH-6T was distantly related to the other haloarchaeal genera investigated (Fig. 2). The type strains of Natronolimnobius innermongolicus and Natronolimnobius baerhuensis were shown to be the closest relatives of strain SH-6T, having a 16S rRNA sequence similarity value of 94.6 %. However, the similarity values were almost identical for several aerobic, extremely halophilic members of the Archaea representing various genera, e.g. the type strains of Natrialba aegyptiaca (94.5 %), Natronorubrum tibetense (93.5 %) and Natronococcus occultus (94.1 %). Similar topologies were obtained when other treeing methods (maximum-parsimony and maximum-likelihood) were used. In consequence, it was concluded that strain SH-6T formed a new distinct branch related to Natronolimnobius innermongolicus and Natronolimnobius baerhuensis. The phenotypic characteristics of these two species of the genus Natronolimnobius are very different from those of strain SH-6T, i.e. their morphology (Fig. 1
), optimum salinity and pH for growth, indole production, their ability to use carbon sources such as D-raffinose, D-arabinose, glycerol, fumarate and L-glutamate and their susceptibility to antibiotics (Table 1). Additionally, members of Natronolimnobius do not have detectable amounts of glycolipids, while strain SH-6T contains the glycolipid S2-DGD-1. It is worth noting that polar lipid composition has been found to be an excellent taxonomic marker for the delineation of haloarchaeal genera (Oren et al., 1997; Grant et al., 2001).
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details the characteristics that differentiate the new genus from other related haloarchaeal genera.
Description of Halopiger gen. nov.
Halopiger (Ha.lo.pi'ger. Gr. n. hals, halos salt; L. masc. adj. piger lazy; N.L. masc. n. Halopiger lazy halophile, referring to the slow growth under laboratory conditions).
Gram-negative. Cells are pleomorphic although most are long rods. Colonies are red pigmented. Strictly aerobic; oxygen is used as the terminal electron acceptor. Growth occurs at pH 6.0–11.0, at 28–45 °C and at 2.5–5.0 M (15–30 %) NaCl. Optimal growth occurs at pH 7.5–8.0, 37 °C and 4.3 M (25 %) NaCl. The DNA G+C content of the only species in the genus is 63.1 mol% (Tm method). Polar lipids include phosphatidylglycerol and phosphatidylglyceromethylphosphate derived from both C20C20 and C20C25 glycerol diethers and the glycolipid S2-DGD-1. Isolated from salt lakes. Phylogenetically affiliated to the Halobacteriaceae. The type species is Halopiger xanaduensis. Recommended three-letter abbreviation of the genus: Hpg.
Description of Halopiger xanaduensis sp. nov.
Halopiger xanaduensis (xa.na.du.en'sis. N.L. masc. adj. xanaduensis referring to Xanadu, the lost city of Kublai Khan, located in Inner Mongolia, from where the type strain was isolated).
Exhibits the following properties in addition to those given in the genus description. Cells are 0.5–1.0 µm wide and 3.0–13.0 µm long (Fig. 1). Colonies are circular, 1–2 mm in diameter after incubation for 10 days at 37 °C. Extremely halophilic. Cells lyse in water. Magnesium is not required. Growth does not occur above 45 °C. Amino acids are not required for growth. Catalase- and oxidase-positive. Anaerobic growth with arginine does not occur. Production of indole and methyl red, Voges–Proskauer and Simmons' citrate tests are negative. H2S is not produced from cysteine. Acid is produced from D-arabinose, D-glucose and D-xylose, but not from D-fructose, D-galactose, glycerol, lactose, maltose, D-mannitol, sucrose or D-trehalose. Arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase are not produced. Urea, Tween 80, gelatin and aesculin are hydrolysed, whereas starch, casein, DNA and phosphatase are not. Nitrate and nitrite are reduced with gas production. The following substrates are utilized as sole carbon and energy sources: D-galactose, D-glucose, D-xylose, L-asparagine, L-serine, acetate and L-glutamate. No growth on D-arabinose, D-fructose, lactose, maltose, D-mannose, D-raffinose, D-ribose, starch, D-trehalose, glycerol, D-sorbitol, D-mannitol, glycine, isoleucine, L-lysine, L-threonine, fumarate, malate, propionate or succinate. Susceptible to bacitracin (10 U), novobiocin (30 µg) and sulfamethoxazole (25 µg). Resistant to ampicillin (10 µg), cephalothin (30 µg), chloramphenicol (30 µg), erythromycin (15 µg), gentamicin (10 µg), kanamycin (30 µg), nalidixic acid (30 µg), neomycin (10 µg), penicillin G (10 U), rifampicin (30 µg), polymyxin (300 U), streptomycin (10 µg), tetracycline (30 µg) and vancomycin (30 µg). The G+C content of the DNA is 63.1 mol% (Tm).
The type strain, SH-6T (=CECT 7173T=CGMCC 1.6379T=JCM 14033T), was isolated from Shangmatala salt lake, Inner Mongolia, 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., Cowan, D. A., Jones, B. E., Grant, W. D. & Ventosa, A. (2006a). 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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