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1 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea
2 Department of Microbiology, Gyeongsang National University, 900 Gazwa, Jinju 660-701, Republic of Korea
Correspondence
Sung-Taik Lee
e_stlee{at}kaist.ac.kr
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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Supplementary tables comparing additional characteristics and the fatty acid content of strain S2R53-5T with members of the genus Salinicoccus and other related genera are available in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Salt-resistant aerobic and anaerobic bacteria exist in most jeotgals (Lee, 1993). Recent studies have reported the isolation of many novel species and genera from jeotgal: Bacillus jeotgali (Yoon et al., 2001a), Jeotgalibacillus alimentarius (Yoon et al., 2001b), Planomicrobium koreense (Yoon et al., 2001c), Halomonas alimentaria (Yoon et al., 2002), Jeotgalicoccus halotolerans and Jeotgalicoccus psychrophilus (Yoon et al., 2003a), Psychrobacter jeotgali (Yoon et al., 2003b), Bacillus cibi (Yoon et al., 2005a), Psychrobacter alimentarius (Yoon et al., 2005b) and Psychrobacter cibarius (Jung et al., 2005).
The genus Salinicoccus was first proposed by Ventosa et al. (1990) based on a single species, Salinicoccus roseus DSM 5351T. Marinococcus hispanicus was later transferred to the genus Salinicoccus as Salinicoccus hispanicus DSM 5352T (Ventosa et al., 1992). At the time of writing, there were three recognized species in the genus Salinicoccus; S. roseus DSM 5351T, S. hispanicus DSM 5352T and Salinicoccus alkaliphilus T8T (Zhang et al., 2002). The genus Salinicoccus is characterized chemotaxonomically by having menaquinone-6 as the predominant isoprenoid quinone, a cell-wall peptidoglycan type based on L-Lys–Gly5 and a DNA G+C content of 46–51 mol% (Ventosa et al., 1992).
In this study, a polyphasic approach, including phylogenetic analysis based on 16S rRNA gene sequences, chemotaxonomic and phenotypic properties, was adopted to determine the precise taxonomic position of strain S2R53-5T. The results obtained in this study indicate that strain S2R53-5T can be assigned to the genus Salinicoccus and the novel strain could be clearly distinguished from the other Salinicoccus species with validly published names. Here, we propose that strain S2R53-5T represents a novel species.
During the study of the bacterial diversity of jeotgal, it has been identified as a source not only of lactic acid bacteria and yeast, but of many other bacteria belonging to the phyla Actinobacteria, Firmicutes and Proteobacteria. To investigate the bacterial diversity of jeotgal, we took fish- (final pH 5.4) and shrimp-jeotgal (final pH 7.5) samples from the Daejeon and Suwon fish markets in Korea. One sample of shrimp-jeotgal (final pH 7.5) packed in a glass jar (net weight 160 g, www.han-sung.co.kr) was also tested. A small portion of each of these samples was blended with a homogenizer (Ace homogenizer; Nihonseiki Kaisha) for 2 min, serially diluted and then applied to R2A (Difco) medium containing 0.5 g yeast extract, 0.5 g proteose peptone, 0.5 g Casamino acids, 0.5 g glucose, 0.5 g soluble starch, 0.3 g sodium pyruvate, 0.3 g dipotassium phosphate, 0.05 g magnesium sulphate and 15.0 g agar dissolved in 1 l of distilled water with different concentrations of NaCl (0, 5, 10 and 20 %, w/v) at pH 7.0 and incubated at 30 °C. After one week of incubation, many pigmented and non-pigmented colonies appeared on the R2A agar with 5 % NaCl (w/v). One orange-pigmented strain, strain S2R53-5T, was transferred to new saline medium [R2A agar +5 % NaCl (w/v)]. All procedures were conducted in an aerobic atmosphere. This strain was derived from a sample of the shrimp-jeotgal packed in a glass jar. This novel strain was grown on saline medium at pH 7.0 and at 30 °C and was also preserved as a glycerol suspension (20 %, w/v) at –70 °C.
The Gram reaction was performed by the non-staining method as described by Buck (1982). Cell morphology was observed under a Nikon light microscope at x1000, with cells grown for 3 days at 30 °C on saline agar. Catalase and oxidase tests were performed according to Cappuccino & Sherman (2002). Substrate utilization as a sole carbon and energy source, the fermentation/oxidation profile and some other physiological characteristics were determined with API 32GN, API 20E and API 50CH (with API 50 CHB/E medium) galleries according to the manufacturer's instructions (bioMérieux). Growth at 4, 10, 15, 20, 25, 30, 37 and 45 °C was tested in saline agar. Salt tolerance was tested in R2A broth medium supplemented with 1–20 % (w/v) NaCl. Growth at different temperatures, pH values and NaCl concentrations was assessed after one week of incubation. Degradation of DNA [using DNA agar (Difco) flooded with 1 M HCl], casein, chitin, Tween 80, starch (Atlas, 1993), xylan and cellulose (Ten et al., 2004) were tested using 5 % NaCl (w/v) and 1.5 % agar (w/v). These tests were evaluated after two weeks of incubation.
Duplicate antibiotic sensitivity tests were performed using filter-paper disks containing the following: ampicillin, tetracycline, kanamycin (Sigma) and rifampicin, each containing 5, 10, 50 and 100 µg ml–1. Disks were placed on saline agar plates spread with strain S2R53-5T which were then incubated at 30 °C for 7 days. The physiological and biochemical characteristics of the novel strain and some related type strains are summarized in Table 1.
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). The nearly complete 16S rRNA gene sequences were compiled using SeqMan software (DNASTAR). The 16S rRNA gene sequences of related taxa were obtained from the GenBank database. Multiple alignments were performed using the CLUSTAL_X program (Thompson et al., 1997). Gaps were edited using the BioEdit program (Hall, 1999). Evolutionary distances were calculated by the method of Jukes & Cantor (1969). The phylogenetic tree was constructed by using a neighbour-joining method (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) using MEGA3 software (Kumar et al., 2004), with bootstrap values based on 1000 replications (Felsenstein, 1985).
In order to confirm whether strain S2R53-5T represented a novel species, DNA–DNA hybridizations were carried out with S. roseus DSM 5351T, S. hispanicus DSM 5352T and S. alkaliphilus KCTC 13928T using photobiotin-labelled probes in microplate wells, as described by Ezaki et al. (1989). A microplate fluorescence reader (FLX 800; Bio-Tek) was employed for fluorescence measurements. For the measurement of the G+C content of the chromosomal DNA, genomic DNA of the strains was extracted and purified as described by Ausubel et al. (1995). The DNA was then enzymically degraded into nucleosides and the G+C content was determined as described by Mesbah et al. (1989) using reversed-phase HPLC. Isoprenoid quinones were extracted with chloroform/methanol (2 : 1, v/v), evaporated under vacuum conditions and reextracted in n-hexane-water (1 : 1, v/v). The crude n-hexane–quinone solution was then purified using Sep-Pak Vac silica cartridges (Waters) and analysed by HPLC as described previously (Hiraishi et al., 1996). Cellular fatty acids were analysed from micro-organisms grown on saline agar for 2 days. Cellular fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI). Fatty acids analysed by GC (6890; Hewlett Packard) were identified by the microbial identification software package (Sasser, 1990). The peptidoglycan structure of the cell wall was determined by one-dimensional TLC (Schleifer & Kandler, 1972) followed by derivatization (MacKenzie, 1987).
Cells of strain S2R53-5T were strictly aerobic, Gram-positive cocci, 1.0–2.0 µm in diameter (Fig. 1). Colonies grown on saline agar plates (Difco) for 3 days were smooth, circular, orange-coloured with transparent edges and 1–2 mm in diameter. On saline agar, strain S2R53-5T was able to grow at 20–30 °C, but not at 15 or 37 °C. The physiological characteristics of strain S2R53-5T are summarized in the species description and a comparison of selected characteristics with those of related type strains is presented in Table 1
. Further characteristics that differentiate strain S2R53-5T from other similar Gram-positive, coccus strains are given in Supplementary Table S1 (available in IJSEM Online).
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; Stackebrandt & Goebel, 1994). In previous studies, we have found corresponding trends (Aslam et al., 2005a, b). Thus, based on the 16S rRNA gene sequence analysis and DNA–DNA hybridization data, a novel taxon was detected.
Chemotaxonomic data obtained from strain S2R53-5T are consistent with those expected for members of the genus Salinicoccus (Ventosa et al., 1990, 1992). The G+C content of the genomic DNA of strain S2R53-5T was 47.0 mol% and the novel strain had MK-6 as its major menaquinone and MK-7 present in trace amounts. The major fatty acids of strain S2R53-5T were iso-C15 : 0 (22.8 %) and anteiso-C15 : 0 (32.4 %). The fatty acid profiles of strain S2R53-5T and related type strains are given in Supplementary Table S2, available in IJSEM Online. The cell-wall murein type of the novel strain was L-Lys and Gly5. These data are also compatible with the assignment of strain S2R53-5T to the genus Salinicoccus.
In a previous study (Ventosa et al., 1992), Marinococcus hispanicus was transferred to the genus Salinicoccus as S. hispanicus on the basis that it contains MK-6 as a major menaquinone, contains straight and branched chain fatty acids and has L-Lys and Gly5 as the cell-wall murein type along with some other phenotypic characteristics. It is clear that strain S2R53-5T belongs to the genus Salinicoccus on the basis of chemotaxonomic data (major menaquinone, MK-6; major fatty acids, iso-C15 : 0 and anteiso-C15 : 0; cell-wall murein type, L-Lys and Gly5), phylogenetic analysis and some other morphological and biochemical characteristics. However, strain S2R53-5T is also different to the other recognized species of the genus Salinicoccus. Strain S2R53-5T has a low 16S rRNA gene sequence similarity with S. roseus DSM 5351T (96.8 %), S. hispanicus DSM 5352T (96.1 %) and S. alkaliphilus T8T (95.2 %), low levels of DNA–DNA relatedness and a separate position in the phylogenetic analysis (Fig. 2). In addition, the G+C content of genomic DNA (47.0 %), fatty acid profile and growth factors, such as pH and temperature range and NaCl tolerance, and some biochemical characteristics, such as acid production from maltose, glycerol, glucose and fructose (Table 1), clearly support the recognition of strain S2R53-5T as a novel species within the genus Salinicoccus, for which the name Salinicoccus jeotgali sp. nov. is proposed.
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Cells are strictly aerobic, Gram-positive, coccus-shaped, non-motile and 1.0–2.0 µm in diameter. Colonies on R2A supplemented with 5 % NaCl for 2–4 days are smooth, circular, orange-coloured with transparent edges and 1–2 mm in diameter. Growth occurs at pH 6.5–11.0 (optimum, pH 7.0), at temperatures of 20–30 °C (optimum, 30 °C) and in R2A media supplemented with 0.5–15.0 % (w/v) NaCl [optimum, 5.0 % (w/v) NaCl]. Tests for catalase and oxidase activity are positive. H2S and indole are produced. Nitrate is reduced to nitrite, but not to nitrogen gas. Aesculin is hydrolysed. Urease, 
-glucosidase, cellulase, xylanase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, amylase, lipase, chitinase, caseinase, DNase and gelatinase are not produced. Acetoin is produced. Tryptophan deaminase activity is positive. Acid is produced from D-glucose, D-fructose, D-maltose, D-trehalose, N-acetyl-D-glucosamine and glycerol, but not from D-mannitol, sucrose, D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-adonitol, D-galactose, D-mannose, L-sorbose, L-rhamnose, D-sorbitol, D-cellobiose, D-lactose, D-melibiose, D-melezitose, D-raffinose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, erythritol, methyl -D-xylopyranoside, dulcitol, inositol, methyl 
-D-mannopyranoside, methyl -D-glucopyranoside, amygdalin, arbutin, salicin, inulin, starch, glycogen, xylitol, gentiobiose, potassium gluconate, potassium 2-ketogluconate or potassium 5-ketogluconate. The following are utilized as sole carbon and energy sources: salicin, L-arabinose, D-glucose, citrate, histidine, L-rhamnose, N-acetyl-D-glucosamine, D-maltose, D-lactate, L-alanine and 5-ketogluconate. The following are not utilized as sole carbon and energy sources: mannitol, D-melibiose, L-fucose, D-sorbitol, propionate, caprate, valerate, 2-ketogluconate, 3-hydroxybutyrate, L-proline, D-ribose, inositol, D-sucrose, itaconate, suberate, malonate, acetate, glycogen, 3-hydroxybenzoate, 4-hydroxybenzoate and L-serine. The type strain is resistant to 100 µg ampicillin ml–1, 100 µg tetracycline ml–1, 100 µg rifampicin ml–1 and 100 µg kanamycin ml–1. The major cellular fatty acids are iso-C15 : 0 (22.8 %) and anteiso-C15 : 0 (32.4 %). The G+C content of the genomic DNA of the type strain is 47.0 mol% (as determined by HPLC). The predominant lipoquinone is MK-6 and MK-7 is present in trace amounts. The assumed cell-wall murein type is L-Lys–Gly5.
The type strain S2R53-5T (=KCTC 13030T=LMG 23640T), was isolated from jeotgal, a traditional Korean fermented seafood.
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ACKNOWLEDGEMENTS |
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