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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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; Oren, 2001; Ventosa, 2006). In November 2003, we isolated the halophilic strain EJ-57T from a sediment sample recovered from saline Lake Ejinor, Inner Mongolia, China. Based on 16S rRNA gene sequence analysis, strain EJ-57T was found to be closely related to members of the genus Natrinema. This genus was proposed by McGenity et al. (1998) to accommodate Natrinema pellirubrum (formerly Halobacterium salinarum NCIMB 786T) and Natrinema pallidum (formerly Halobacterium halobium NCIMB 777T). On the basis of 16S rRNA gene sequence analysis, phenotypic properties and polar lipid composition, two novel species, Natrinema versiforme (Xin et al., 2000) and Natrinema altunense (Xu et al., 2005), were also included in the genus. Here we describe the taxonomic properties of strain EJ-57T and propose that it represents a novel species of the genus Natrinema. Strain EJ-57T was isolated from a sediment sample of a saltern beside saline Lake Ejinor (45° 14' N 116° 31' E). At the time of sampling (September 2003), the water of this saltern was red in colour with a temperature of 27.6 °C, pH of 7.5 and a conductivity of 161 mS cm–1. Samples were diluted in 20 % salt solution and spread on nutrient agar plates containing (per litre): 137.3 g NaCl, 22.8 g MgCl2.6H2O, 35.5 g MgSO4.7H2O, 0.5 g CaCl2, 3.5 g KCl, 0.16 g NaHCO3, 0.4 g NaBr and 5 g yeast extract; pH was adjusted to 7.5 with 1 M NaOH.
Strain EJ-57T grew at a temperature range of 25–50 °C (optimum 37 °C) and a pH range of 6.0–8.5 (optimum pH 7.0). Routine cultivation was conducted at 37 °C and pH 7.0. 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, respectively. Strain EJ-57T was capable of growth over a wide range of NaCl concentrations, ranging from 1.8 M (approximately 10 %) to 5.0 M (approximately 30 %). It grew optimally in the presence of 3.4 M (20 %) NaCl, similar to results for most extremely halophilic archaea (Grant et al., 2001). MgCl2 was not required for growth.
Phenotypic characterization was carried out in accordance with the recommended minimal standards for the description of new taxa in the order Halobacteriales (Oren et al., 1997). Anaerobic growth was tested in the presence of 5 % nitrate and 3 % L-arginine in filled stoppered tubes (Oren et al., 1997). Formation of acid from different sugars was tested in media with 0.05 % (w/v) yeast extract supplemented with 1 % (w/v) of the sugar tested (sterilized separately). Strain EJ-57T was oxidase- and catalase-positive. Methyl red, Voges–Proskauer, indole production from tryptone and Simmons' citrate tests were negative. Reduction of nitrite with gas production was positive. Casein, aesculin and DNA were not hydrolysed. Gelatin was liquefied. Starch and Tween 80 were hydrolysed. The urease test was negative. Other phenotypic characteristics of strain EJ-57T are summarized in Table 1 and in the species description below.
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). Susceptibility to antibiotics was determined on agar medium plates by using discs with the following antibiotics: 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). Results from the utilization of different substrates and antibiotic susceptibility tests are included in the species description.
Cell morphology and motility were examined with an Olympus BX41 microscope equipped with phase-contrast optics. Cells were non-motile, pleomorphic and flat (triangular, square, rod, disc and other polygonal shapes) (Fig. 1). Colony morphology was observed on agar media under optimal growth conditions after incubation at 37 °C for 10 days. Colonies of strain EJ-57T formed on agar plates were circular (0.5–1.5 mm in diameter), smooth, entire, opaque and pale red pigmented.
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. TLC was performed using Merck HPTLC silica gel 60 plates (Art. 5641) in the solvent system, chloroform/methanol/acetic acid/water (85 : 22.5 : 10 : 4, by volume). Glycolipids were detected as purple spots by spraying with 0.5 % alpha-naphthol in methanol/water (1 : 1) and then with sulfuric acid/ethanol (1 : 1), followed by heating at 160 °C. The polar lipid profile of strain EJ-57T comprised C20C20 and C20C25 derivates of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and the sulfated diglycolipid, S2-DGA-1.
Chromosomal DNA of strain EJ-57T was isolated and purified according to the methods described by 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 EJ-57T was 64.7 mol%. The 16S rRNA gene of strain EJ-57T was amplified by PCR using three universal primer sets as described by Lopez-Garcia et al. (2001) and Arahal et al. (1996), and the almost-complete nucleotide sequence (1418 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. Fig. 2 shows the phylogenetic tree constructed by the neighbour-joining method (Saitou & Nei, 1987) in which strain EJ-57T clustered with the type strains of species of the genus Natrinema. Sequence similarities between strain EJ-57T and N. versiforme JCM 10478T, N. pallidum NCIMB 777T, N. altunense JCM 12890T and N. pellirubrum NCIMB 786T were 96.2, 95.9, 95.8 and 95.5 %, respectively. Similar tree topologies were obtained when other tree construction methods (maximum-parsimony and maximum-likelihood) were used. Several phenotypic differences were observed between strain EJ-57T and its most closely related neighbour, N. versiforme, such as hydrolysis of gelatin, indole production, H2S production, and the utilization of galactose, D-ribose and D-xylose.
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, as described in detail by Gutierrez et al. (2002). These experiments were carried out under optimal conditions, at a temperature of 58.4 °C, which is within the limits of validity for the filter method (De Ley & Tijtgat, 1970). The percentage of hybridization was calculated as described by Johnson (1994). DNA–DNA relatedness between strain EJ-57T and recognized Natrinema species was: 39 % with N. versiforme JCM 10478T, 24 % with N. pallidum NCIMB 777T and 25 % with N. pellirubrum NCIMB 786T. By contrast, DNA–DNA hybridization values between strain EJ-57T and species of the genus Haloterrigena were 4 % with Haloterrigena thermotolerans DSM 11552T and 2 % with Haloterrigena turkmenica VKM B-1734T. Our results show that, genotypically, strain EJ-57T is not related to species of the genera Natrinema or Haloterrigena, but is more closely related to species of the genus Natrinema. These data indicate that strain EJ-57T represents a novel species of the genus Natrinema, with DNA–DNA hybridization values lower than 70 % with recognized species (Wayne et al., 1987; Stackebrandt & Goebel, 1994).
Therefore, on the basis of these polyphasic taxonomic data, we propose that strain EJ-57T represents a novel species of the genus Natrinema (Stackebrandt & Goebel, 1994; Vandamme et al., 1996), for which the name Natrinema ejinorense sp. nov. is proposed. Table 1
details differential characteristics between N. ejinorense and other species of the genus Natrinema.
Description of Natrinema ejinorense sp. nov.
Natrinema ejinorense (e.ji.no.ren'se. N.L. neut. adj. ejinorense pertaining to the saline lake Ejinor, Inner Mongolia, China).
Cells are non-motile and pleomorphic, with triangular, square, rod, disc and other polygonal shapes, and are 1.5–4.0x0.8–2.0 µm in size. Colonies are circular, smooth, entire, opaque, pale red pigmented and 0.5–1.5 mm in diameter after 5 days at 37 °C on plates containing 20 % (w/v) total salts. Extremely halophilic; cells lyse in water. At least 1.8 M NaCl is required for growth (optimal growth at 3.4 M NaCl). MgCl2 is not required for growth. The pH and temperature ranges for growth are 6.0–8.5 (optimum at pH 7.0) and 25–50 °C (optimum at 37 °C), respectively. Chemo-organotrophic. Aerobic. Oxidase- and catalase-positive. Indole is not produced from tryptophan. Methyl red, Voges–Proskauer and Simmons' citrate tests are negative. Acid is not produced from lactose, glycerol, glucose, sucrose, fructose, arabinose, maltose, D-xylose, galactose, trehalose or mannose. Does not grow anaerobically in the presence of nitrate or L-arginine. Starch, gelatin and Tween 80 are hydrolysed, but DNA and casein are not. Does not produce arginine dihydrolase, lysine decarboxylase or ornithine decarboxylase. Nitrate and nitrite are reduced and gas is produced from nitrite. Utilizes glucose, fructose, glycerol, maltose, trehalose, starch, propionate, fumarate, acetate, threonine, asparagine and lysine as single carbon and energy sources for growth. No growth is observed on mannitol, sorbitol, lactose, arabinose, galactose, mannose, raffinose, D-ribose, D-xylose, malate, succinate or glutamate. The following compounds are not used as sole carbon, nitrogen or energy sources: isoleucine, L-serine and glycine. Sensitive to bacitracin and novobiocin, but resistant to ampicillin, chloramphenicol, erythromycin, gentamicin, nalidixic acid, neomycin, penicillin G, rifampicin, streptomycin and tetracycline. The major polar lipids are C20C20 and C20C25 derivates of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and the disulfated glycolipid, S2-DGA-1. The G+C content of the DNA is 64.7 mol% (Tm).
The type strain, EJ-57T (=CECT 7144T=JCM 13890T=CGMCC 1.6202T), was isolated from saline Lake Ejinor, Inner Mongolia, China.
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ACKNOWLEDGEMENTS |
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