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1 Japan Collection of Micro-organisms RIKEN BioResource Center, Wako-shi, Saitama-ken 351-0198, Japan
2 Institute of Biology of the Romanian Academy, Splaiul Independentei 296, PO Box 56-53, Bucharest 060031, Romania
3 Halophiles Research Institute, 677-1 Shimizu, Noda-shi, Chiba-ken 278-0043, Japan
Correspondence
Madalin Enache
madalin.enache{at}ibiol.ro
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; Grant et al., 2001; Hezayen et al., 2002; Oren et al., 2002; Vreeland et al., 2002; Itoh et al., 2005; Xue et al., 2005; Castillo et al., 2006a, b). Hypersaline environments, such as salt mines and salt lakes, are commonly found in Romania. Samples taken from five salt lakes (Telega, Bride Cave, Red Bath, Green Bath and Shepherd Bath) located in Prahova county (near Slanic Prahova city) and from one (Techirghiol Lake) close to the Black Sea coast, have led to the isolation of a number of halobacteria in media with a high Mg2+ concentration and taxonomic investigations have been partially conducted for 19 strains (Enache et al., 2000). Analysis of the partial 16S rRNA gene sequences of these strains suggested that many of them belonged to the genus Haloferax. The genus Haloferax includes species in which growth occurs in media containing 1.5–4.5 M NaCl and high Mg2+ concentrations and species display a characteristic polar lipid composition (Ventosa, 2001; Kamekura et al., 2004). The species currently recognized within this genus are Haloferax volcanii (Mullakhanbhai & Larsen, 1975; Torreblanca et al., 1986), Haloferax denitrificans (Tomlinson et al., 1986; Tindall et al., 1989), Haloferax gibbonsii (Juez et al., 1986), Haloferax mediterranei (Rodriguez-Valera et al., 1983), Haloferax alexandrinus (Asker & Ohta, 2002), Haloferax lucentense (Gutierrez et al., 2002) and Haloferax sulfurifontis (Elshahed et al., 2004). In this paper, we describe the detailed characterization of one representative strain from our salt lake isolates and a novel species is proposed.
Strain TL6T was isolated from a surface water sample from Telega Lake, a saline continental lake in Prahova County, around 100 km north of Bucharest, Romania, with a salinity of around 161 g l–1. The water sample was collected in an autoclaved glass bottle and kept in a refrigerator until plating. The novel strain was isolated in a medium with the following composition (l–1): 125 g NaCl, 160 g MgCl2.6H2O, 5 g K2SO4, 0.1 g CaCl2.2H2O, 1 g yeast extract, 1 g peptone (Difco), 2 g soluble starch and 20 g agar. The pH of the medium was 7.0–7.2 before sterilization. For further experiments, the novel strain was either grown in this medium or in JCM 168 medium which contained (l–1): 5 g Casamino acids, 5 g yeast extract, 1 g sodium glutamate, 3 g trisodium citrate, 29.5 g MgSO4.7H2O, 2 g KCl, 175.5 g NaCl, 0.036 g FeCl2.4H2O and 0.36 mg MnCl2.4H2O. Before sterilization, the pH of the medium was 7.0–7.2. Cultures were incubated at 38 °C with shaking at 180 r.p.m. Haloferax gibbonsii JCM 8863T, H. denitrificans JCM 8864T, H. mediterranei JCM 8866T, H. volcanii JCM 8879T, H. lucentense JCM 9276T, H. alexandrinus JCM 10717T and H. sulfurifontis JCM 12327T were used as reference strains and cultivated in JCM 168 medium.
Cell lysis was observed by diluting cell suspensions with distilled water. Growth ranges and optimum NaCl and Mg2+ concentrations were determined using media containing 0–5.2 M NaCl and 0–1 M MgCl2.6H2O. For the determination of the optimum pH for growth, the following buffer systems (50 mM of each) were used: MES (pH 5.5–6.5), PIPES (pH 6.5–7.5), HEPES (pH 7.0–8.0), Tricine (pH 7.5–9.0) and CHES (pH 9.0–10.0). Magnesium salts were eliminated from media with a pH value exceeding 8.5. Growth temperature was determined in the range 4–60 °C by using a temperature gradient incubator. Growth rate was determined by measuring OD660 using a spectrophotometer. Anaerobic growth in the presence of arginine, DMSO and nitrate was tested in JCM 168 medium supplemented with 5 g l–1 of each of the test compounds as described by Xin et al. (2000). Reduction of nitrate and formation of gas from nitrate were detected as described by Holding & Collee (1971). To estimate the utilization of various carbohydrates and other compounds as carbon and energy sources, medium JCM 168 (without Casamino acids but with 0.1 g yeast extract l–1, pH 7.0 with 50 mM HEPES) was supplemented with 10 g l–1 test compounds. Production of acids from carbohydrate was tested in the same medium but without the buffer and supplemented with 0.5 g l–1 of the test compounds. The pH was determined at the end of growth. Thin layer chromatography of membrane lipids was conducted using the method described by Kamekura (1993). DNA G+C content was determined by the HPLC method of Tamaoka (1994). DNA–DNA hybridization was assessed by the fluorometric method (Ezaki et al., 1989). During these experiments, the DNA isolated from strain TL6T was labelled. For 16S rRNA gene sequence analysis, total DNA was extracted and purified using the method of Tamaoka (1994) adapted for halophilic archaea. The 16S rRNA genes were amplified by a PCR method with the following forward and reverse primers: 5'-TCCGGTTGATCCTGCCG (position 8–24 according to the Escherichia coli numbering system) and 5'–GGAGGTGATCCAGCCG (E. coli position 1540–1525). PCR products were sequenced using a BigDye Terminator Cycle sequencing kit (Pharmacia Biotech) and a DNA sequencer (ABI Prism; Applied Biosystems). The sequences obtained were aligned with other reported halobacterial 16S rRNA gene sequences by using the CLUSTAL 1.7 program (Thompson et al., 1997). The phylogenetic tree was reconstructed by the neighbour-joining method (Saitou & Nei, 1987) and was evaluated by bootstrap sampling. Tests for catalase and oxidase activities, formation of indole and starch hydrolysis were performed according to standard procedures (Holding & Collee, 1971). The formation of sulfide was determined as described by Xin et al. (2000). The production of halocins was evaluated according to the procedure of Meseguer & Rodriguez-Valera (1985). Hydrolysis of Tween 80 and gelatin was detected according to the method of Gutierrez & Gonzalez (1972). Casein hydrolysis was tested on solidified JCM 168 medium (without Casamino acids but containing 1 g yeast extract l–1) supplemented with 1 % skimmed milk. Sensitivity to antimicrobial agents was determined in growth medium containing 50 µg ml–1 of each of the following antimicrobial agents: novobiocin, bacitracin, anisomycin, aphidicolin, erythromycin, penicillin, ampicillin, rifampicin, chloramphenicol and neomycin. Tests were conducted in liquid culture and growth with or without antibiotics was compared.
Cells of strain TL6T growing exponentially under optimal conditions were rod-shaped. Cells lysed in distilled water or culture medium without NaCl. The novel strain stained Gram-negative. Colonies formed on agar plates were elevated, transparent and circular with whole margins. The colonies were beige–orange in colour. The range of NaCl concentration for growth was affected by the presence or absence of sodium glutamate and trisodium citrate in the JCM 168 culture medium. In the presence of these compounds, strain TL6T was able to grow at 1.0–5.2 M NaCl, but a higher concentration of 2.5 M NaCl was required for growth in the absence of these sodium salts. The optimum NaCl concentration for growth, 3.5 M, was not affected by the presence of additional sodium salts. The strain was able to grow at a concentration of 1 M MgCl2 or less, with an optimum of 0.4 M. Strain TL6T grew between pH 6.0 and pH 8.5, with an optimum of pH 7.0–7.5. Pigmentation of the strain was affected by temperature but not by the NaCl concentration. A high intensity of pigmentation was observed at high temperatures, up to 45 °C.
The novel strain was aerobic and unable to grow anaerobically even in the presence of DMSO, arginine or nitrate. Strain TL6T formed sulfide from sulfur and sodium thiosulfate, formed indole from tryptophan and exhibited positive catalase and oxidase reactions. No hydrolysis of gelatin or casein was observed, but starch and Tween 80 were hydrolysed. The novel strain assimilated some carbohydrates and amino acids and formed acids from only a few carbohydrates as given in the species description. Strain TL6T was sensitive to novobiocin, anisomycin, aphidicolin and rifampicin, but resistant to bacitracin, erythromycin, penicillin, ampicillin, chloramphenicol and neomycin. The differences between strain TL6T and other members of the genus Haloferax are highlighted in Table 1.
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), supported the classification of the novel strain to this genus.
The 16S rRNA gene sequence of strain TL6T showed a high degree of similarity (Table 1
) to those of recognized species of the genus Haloferax. The reconstructed phylogenetic tree (Fig. 1) supported the classification of the novel strain to the genus Haloferax.
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). DNA–DNA hybridization experiments (Table 1) showed that the relatedness between strain TL6T and other species of the genus Haloferax ranged from 12 to 53 % suggesting that this strain represents a novel species of the genus Haloferax. The phenotypic data and phylogenetic data based on 16S rRNA gene sequence comparisons clearly support the placement of strain TL6T as representing a novel species of the genus Haloferax, for which we propose the name Haloferax prahovense sp. nov.
Description of Haloferax prahovense sp. nov.
Haloferax prahovense (pra.ho.ven'se. N.L. neut. adj. prahovense pertaining to Prahova county, Romania, from where the type strain was isolated).
Cells are lysed in distilled water. Colonies are pigmented beige–orange and are circular, convex, translucent, entire and smooth. Growth occurs in the range of 2.5 to 5.2 M NaCl with an optimum at 3.5 M. Growth occurs in a concentration of 1 M Mg2+ or less, with optimum growth at 0.4 M Mg2+. The pH range for growth is 6.0–8.5, with an optimum at pH 7.0–7.5. The temperature range for growth is 23–51 °C, with an optimum at 38–48 °C. Chemo-organotrophic. Aerobic. Oxidase- and catalase-positive. Nitrate is not reduced to nitrite. Does not produce gas from nitrate. Indole is produced from tryptophan and H2S is produced from sulfur and sodium thiosulfate. Starch and Tween 80 are hydrolysed, but gelatin and casein are not hydrolysed. Halocins are produced. The following substrates are used as carbon sources: glucose, arabinose, galactose, fructose, rhamnose, raffinose, D-xylose, maltose, sucrose, starch, lactose, glycerol, acetate, propionate, pyruvate, L-lactate, succinate, L-malate, fumarate, citrate, L-alanine, L-ornithine. Acid is produced from fructose and lactose. The following substrates are not used as carbon sources: mannose, D-ribose, mannitol, sorbitol, glycine, L-arginine, L-aspartate, L-glutamate and L-lysine. Sensitive to novobiocin, anisomycin, aphidicolin and rifampicin. Resistant to bacitracin, erythromycin, penicillin, ampicillin, chloramphenicol and neomycin. Lipids S-DGA-1, PGP-Me and PG are present, but phosphatidylglycerol sulfate is not present. The G+C content of the total DNA of the type strain is 63.7 mol%.
The type strain, TL6T (=JCM 13924T=DSM 18310T), was isolated from the saline Telega Lake, Prahova county, Romania.
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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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50 % (100 replicates) are shown. Bar, 0.005 substitutions per nucleotide position.