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1 Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
2 Department of Applied Biology, Faculty of Science, King Mongkut's Institute of Technology Ladkrabang, Bangkok 10520, Thailand
3 Thailand Institute of Scientific and Technological Research, Pathumthani 12120, Thailand
4 Japan Collection of Microorganisms, RIKEN BioResource Center, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
Correspondence
Somboon Tanasupawat
Somboon.T{at}chula.ac.th
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76.6 %) but showed low DNA–DNA relatedness to S. roseus JCM 14630T (21.7 %). On the basis of the phenotypic and molecular data presented, the 15 novel strains are suggested to represent a single novel species of the genus Salinicoccus, for which the name Salinicoccus siamensis sp. nov. is proposed. The type strain is PN1-2T (=JCM 12822T =PCU 242T =TISTR 1562T).
A scanning electron micrograph of cells of strain PN1-2T, an image showing the colonial appearance of strain PN1-2T, a 16S rRNA gene sequence phylogenetic tree showing the relationships between strains PN1-2T and PN7-1 and related species and a table giving DNA G+C contents and levels of DNA–DNA relatedness among the novel strains and S. roseus JCM 14630T are available as supplementary material in IJSEM Online.
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; Li et al., 2005), Salinicoccus roseus, S. hispanicus and S. alkaliphilus (Ventosa et al., 1990, 1992; Zhang et al., 2002), Nesterenkonia halobia (Stackebrandt et al., 1995), Halobacillus halophilus (Spring et al., 1996), Tetragenococcus muriaticus (Satomi et al., 1997), and Jeotgalicoccus halotolerans and J. psychrophilus (Yoon et al., 2003). In this study, a novel orange-pigmented species isolated from ‘ka-pi’ was identified as belonging to the genus Salinicoccus on the basis of phenotypic and chemotaxonomic characterization and 16S rRNA gene sequence analysis.
The halophilic bacteria investigated herein were isolated from samples of fermented shrimp paste, ‘ka-pi’, collected from a market in Nakhonsrithammarat Province in southern Thailand. The cultures were isolated by using the spread-plate technique on agar plates of JCM medium no. 377 (per litre distilled water: 100 g NaCl, 5 g Casamino acids, 5 g yeast extract, 1 g glutamic acid, 2 g KCl, 3 g trisodium citrate, 20 g MgSO4 . 7H2O, 36 mg FeCl2 . 4H2O, 0.36 mg MnCl2 . 4H2O, 20 g agar; pH 7.2) after incubation at 37 °C for 7 days. Liquid cultures were cultivated in Erlenmeyer flasks containing the same medium and incubated on a rotary shaker. All media contained 10 % (w/v) NaCl, except for tests of NaCl tolerance. Cell shape, size and arrangement and colony size were examined by using cells grown on JCM medium no. 377 agar at 37 °C for 5 days. The Hucker–Conn modification was used for Gram staining (Hucker & Conn, 1923). Spore formation was examined with Gram-stained specimens. Critical-point-dried cells were observed under a scanning electron microscope. The presence of flagella was examined as described by Forbes (1981) and observed by transmission electron microscopy. Physiological and biochemical characteristics were determined as described by Thornley (1960), Leifson (1963) and Barrow & Feltham (1993), by using medium supplemented with 10 % (w/v) NaCl. Growth under anaerobic conditions on agar plates with or without nitrate (1 %, w/v) was investigated with a GasPak (BBL) anaerobic jar. Growth at various temperatures (10–50 °C), pH values (5.0, 6.0, 7.0, 7.5, 8.0, 8.5 and 9.0) and NaCl concentrations (0–30 %, w/v) was assessed. At the lower NaCl concentrations (0.0–2.0 %, w/v), MgSO4.7H2O was omitted from the test medium. Growth was monitored by measuring culture turbidity at 660 nm. The presence of diaminopimelic acid in the cell-wall peptidoglycan and menaquinone profiles were analysed as described by Komagata & Suzuki (1987). A TLC plate (Merck no. 5577) developed with solvent systems of methanol/pyridine/12 M HCl/water (32 : 4 : 1 : 7, v/v) was used to determine lysine in the cell wall (Tanasupawat et al., 1993). Quantitative analysis of the cellular fatty acids was performed as described by Sasser (1990) and Kämpfer & Kroppenstedt (1996). DNA was isolated from cells grown in JCM medium no. 377 broth and purified according to the method of Saito & Miura (1963). The DNA G+C content was determined by the method of Tamaoka & Komagata (1984), by using reversed-phase HPLC. DNA–DNA hybridization was conducted in microdilution-well plates, as described by Ezaki et al. (1989), and was detected by using the colorimetric method reported by Tanasupawat et al. (2000). 16S rRNA genes were amplified, purified and sequenced as described by Seearunruangchai et al. (2004). The sequences determined (1489–1503 bases) were aligned with selected sequences obtained from the GenBank database by using CLUSTAL W, version 1.81 (Thompson et al., 1994). The alignment was edited manually to remove gaps and ambiguous nucleotides prior to the construction of the phylogenetic tree. The phylogenetic tree was constructed by using the neighbour-joining method (Saitou & Nei, 1987) in MEGA, version 2.1 (Kumar et al., 2001). The confidence values of branches of the phylogenetic tree were determined by using bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings.
We isolated 15 strains of moderately halophilic, strictly aerobic, Gram-positive cocci, approximately 0.6–0.8 µm in diameter, occurring singly and in pairs or in tetrads (see Supplementary Fig. S1 in IJSEM Online). Endospores and flagella were not observed. Colonies were smooth, circular, of low convexity and orange in colour (Supplementary Fig. S2). Anaerobic growth was not observed. They grew in JCM medium no. 377 with 1.5–25 % NaCl, at pH 6–9 and at 15–45 °C, but no growth was observed at 10 or 50 °C. The phenotypic and chemotaxonomic characteristics of the 15 novel strains are detailed in the species description below and in Tables 1 and 2. Representative strain PN1-2T contained L-Lys in the cell-wall peptidoglycan. This strain had isoprenoid quinone with six isoprene units (MK-6) as a predominant component, and had a polar lipid profile of phosphatidylglycerol, diphosphatidylglycerol and an unidentified glycolipid. The dominant cellular fatty acids of strains PN1-2T, PN1-8, PN2-2 and PN7-1 were anteiso-C15 : 0 (38.7–49.8 %) and iso-C15 : 0 (10.4–24.5 %) (Table 2). The DNA G+C contents of the novel strains ranged from 44.5 to 47.5 mol%. On the basis of 16S rRNA gene sequence analyses with the neighbour-joining algorithm, strains PN1-2T and PN7-1 were included in a monophyletic cluster consisting of species of the genus Salinicoccus, as shown in Fig. 1 (the maximum-parsimony tree is shown in Supplementary Fig. S3) (Ventosa et al., 1990, 1992; Zhang et al., 2002). Levels of 16S rRNA gene sequence similarity between strains PN1-2T and PN7-1 and S. roseus DSM 5351T, S. hispanicus DSM 5352T, S. alkaliphilus T8T and J. halotolerans YKJ-101T were 97.3–97.7, 96.5, 95.9 and 93.5 %, respectively. The novel strains were considered to represent the same species based on levels of DNA–DNA relatedness of over 76.6 % with strain PN1-2T (Wayne et al., 1987) (Supplementary Table S1). Strain PN1-2T showed low DNA–DNA relatedness to S. roseus JCM 14630T (21.7 %) and, reciprocally, S. roseus JCM 14630T showed low DNA–DNA relatedness to strain PN1-2T and the other 14 related strains (2.0–29.0 %). In addition, all 15 novel strains could be differentiated from S. roseus JCM 14630T and related species based on pigmentation, maximum growth temperature, NaCl tolerance, hydrolysis of casein, gelatin, Tween 80 and starch, acid production from fructose and D-glucose and DNA G+C content, as detailed in Table 1. Furthermore, they could be differentiated from Salinicoccus salsiraiae LMG 22840T based on pigmentation, maximum growth temperature, NaCl tolerance, hydrolysis of casein and gelatin and acid production from maltose and sucrose (França et al., 2006). Thus, the 15 novel strains isolated herein are considered to represent a single novel species of the genus Salinicoccus, for which we propose the name Salinicoccus siamensis sp. nov.
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Cells are Gram-positive, strictly aerobic cocci, approximately 0.6–0.8 µm in diameter, occurring singly and in pairs or in tetrads. Non-motile. Endospores are not formed. Colonies are smooth, circular, of low convexity and orange in colour. Growth occurs at pH 6 and 9 (optimally at pH 8.5), at temperatures of 15–45 °C (optimally at 37 °C) and in media with 1.5–25 % (w/v) NaCl (optimally at 10 % NaCl). Catalase- and oxidase-positive, but urease-negative. Unable to reduce nitrate. Negative for the indole, methyl red and Voges–Proskauer tests and for utilization of citrate. Aesculin, casein, arginine, gelatin, starch and Tween 80 are not hydrolysed. Acid is produced from D-fructose, D-glucose, glycerol, D-ribose and trehalose, but not from amygdalin, L-arabinose, cellobiose, aesculin, D-galactose, gluconate, myo-inositol, inulin, lactose, maltose, D-mannitol, D-mannose, melibiose, melezitose, methyl 
-D-glucoside, raffinose, L-rhamnose, salicin, sorbitol, sucrose or D-xylose. The cell-wall peptidoglycan is L-Lys type. The predominant isoprenoid quinone is MK-6. The major cellular fatty acids are anteiso-C15 : 0 and iso-C15 : 0. Phosphatidylglycerol, diphosphatidylglycerol and an unidentified glycolipid are predominant in the polar lipid profile. The DNA G+C content ranges from 44.5 to 47.5 mol%.
The type strain, PN1-2T (=JCM 12822T =PCU 242T =TISTR 1562T), was isolated from fermented shrimp paste (‘ka-pi’) in Thailand.
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ACKNOWLEDGEMENTS |
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