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Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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9c. The DNA G+C content was 53.9 mol%. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain ISL-52T fell within the genus Idiomarina, joining the type strain of Idiomarina homiensis at a bootstrap resampling value of 100 %. Strain ISL-52T exhibited 16S rRNA gene sequence similarity values of 94.9–96.7 % with respect to the type strains of eight recognized Idiomarina species. The differential phenotypic properties of ISL-52T, together with its phylogenetic distinctiveness, demonstrated that this strain is distinguishable from the recognized Idiomarina species. On the basis of phenotypic, phylogenetic and genetic data, therefore, strain ISL-52T represents a novel species of the genus Idiomarina, for which the name Idiomarina salinarum sp. nov. is proposed. The type strain is ISL-52T (=KCTC 12971T=CCUG 54359T).
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with the description of Idiomarina abyssalis and Idiomarina zobellii. Subsequently, six further Idiomarina species, Idiomarina baltica (Brettar et al., 2003), Idiomarina loihiensis (Donachie et al., 2003), Idiomarina fontislapidosi and Idiomarina ramblicola (Martínez-Cánovas et al., 2004), Idiomarina seosinensis (Choi & Cho, 2005) and Idiomarina homiensis (Kwon et al., 2006), have been described. In this study, we report on the taxonomic characterization of an Idiomarina-like bacterial strain, ISL-52T, isolated from a marine solar saltern of the Yellow Sea in Korea.
Strain ISL-52T was isolated by means of the dilution plating technique at 30 °C on marine agar 2216 (MA; Difco) supplemented with 8 % (w/v) NaCl. The morphological, physiological and biochemical characteristics of strain ISL-52T were investigated using routine cultivation on MA at 37 °C. The cell morphology was examined by light microscopy (E600; Nikon) and transmission electron microscopy. Flagellation was determined by using a Philips CM-20 transmission electron microscope with cells from exponentially growing cultures: for this purpose, the cells were negatively stained with 1 % (w/v) phosphotungstic acid and the grids were examined after being air-dried. Growth under anaerobic conditions was determined after incubation in a Forma anaerobic chamber on MA and on MA supplemented with nitrate, both of which had been prepared anaerobically using nitrogen. The pH range for growth was determined in marine broth 2216 (MB; Difco) that had been adjusted to various pH values (pH 4.5–9.5, using increments of 0.5 pH units). Growth in the absence of NaCl was investigated using trypticase soy broth prepared according to the formula of the Difco medium except that NaCl was excluded. Growth at various NaCl concentrations was investigated in MB or trypticase soy broth (Difco). Growth at various temperatures (4–45 °C) was measured on MA. Catalase and oxidase activities and hydrolysis of casein, starch and Tweens 20, 40, 60 and 80 were determined as described by Cowan & Steel (1965). DNase activity was examined by using DNase test agar with methyl green (Difco) supplemented with 3 % (w/v) NaCl. Hydrolysis of hypoxanthine, tyrosine and xanthine was tested on MA using the substrate concentrations described by Cowan & Steel (1965). Hydrolysis of aesculin, gelatin and urea and reduction of nitrate were investigated as described previously (Lanyi, 1987) with the modification that artificial seawater was used for preparation of the media. The artificial seawater contained the following (l–1 distilled water): 23.6 g NaCl, 0.64 g KCl, 4.53 g MgCl2 . 6H2O, 5.94 g MgSO4 . 7H2O and 1.3 g CaCl2 . 2H2O (Bruns et al., 2001). H2S production was tested as described previously (Bruns et al., 2001). Susceptibility to antibiotics was investigated on MA plates by using antibiotic discs with the following concentrations (µg unless indicated otherwise): polymyxin B, 100 U; streptomycin, 50; penicillin G, 20 U; chloramphenicol, 100; ampicillin, 10; cephalothin, 30; gentamicin, 30; novobiocin, 5; tetracycline, 30; kanamycin, 30; lincomycin, 15; oleandomycin, 15; neomycin, 30; carbenicillin, 100. Acid production from carbohydrates was tested as described by Leifson (1963). The utilization of various substrates for growth was determined as described by Baumann & Baumann (1981), using supplementation with 2 % (v/v) Hutner's mineral salts solution (Cohen-Bazire et al., 1957) and a 1 % (v/v) vitamin solution (Staley, 1968), and by Yurkov et al. (1994). Enzyme activities were determined by using the API ZYM system (bioMérieux).
Cell biomass for DNA extraction and for isoprenoid quinone analysis was obtained from cultivation in MB at 37 °C. Chromosomal DNA was isolated and purified according to the method described by Yoon et al. (1996), except that RNase T1 was used in combination with RNase A to minimize contamination of the RNA. The 16S rRNA gene was amplified by using a PCR with two universal primers, as described previously (Yoon et al., 1998). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed as described by Yoon et al. (2003). Isoprenoid quinones were analysed as described by Komagata & Suzuki (1987), using reversed-phase HPLC. For cellular fatty acid analysis, cell mass of strain ISL-52T was harvested from MA plates after cultivation for 3 days at 37 °C. The fatty acids were extracted and fatty acid methyl esters were prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). The DNA G+C content was determined by using the method of Tamaoka & Komagata (1984), with the modification that the DNA was hydrolysed using nuclease P1 (Sigma) and the resultant nucleotides were analysed by reversed-phase HPLC.
Morphological, cultural, physiological and biochemical characteristics of strain ISL-52T are given in the species description (see later) and in Table 1. The almost-complete 16S rRNA gene sequence of strain ISL-52T determined in this study comprised 1497 nt, representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence. A comparative 16S rRNA gene sequence analysis revealed that strain ISL-52T was phylogenetically most closely affiliated to members of the genus Idiomarina. In the phylogenetic tree based on the neighbour-joining algorithm, strain ISL-52T joined I. homiensis PO-M2T at a bootstrap confidence level of 100 % (Fig. 1). The same tree topology was also found in trees based on the maximum-likelihood and maximum-parsimony algorithms. Strain ISL-52T exhibited 16S rRNA gene sequence similarities of 96.7 % and 94.9–95.8 %, respectively, with I. homiensis PO-M2T and the type strains of other Idiomarina species. The sequence similarities with respect to other species used in the phylogenetic analysis were less than 90.7 %.
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9c (11.8 %), iso-C11 : 0 (4.2 %), iso-C15 : 1 (3.9 %), iso-C13 : 0 (1.6 %), iso-C16 : 0 (1.3 %) and anteiso-C17 : 0 (0.5 %); hydroxyl fatty acids iso-C13 : 0 3-OH (5.0 %) and iso-C11 : 0 3-OH (5.0 %); straight-chain fatty acids C16 : 0 (4.3 %) and C18 : 0 (0.8 %); cyclo fatty acids cyclo-C17 : 0 (1.7 %) and cyclo-C19 : 08c (0.6 %); unsaturated fatty acid C18 : 17c (0.9 %). This fatty acid profile was similar to those of Idiomarina species, although there are differences in the proportions of some fatty acids, perhaps because of differences in cultivation conditions and extraction procedures (Kwon et al., 2006). The DNA G+C content of strain ISL-52T was 53.9 mol%. There were no distinct phenotypic, particularly chemotaxonomic, properties to differentiate strain ISL-52T from the genus Idiomarina. Accordingly, it seems reasonable to allocate strain ISL-52T to the genus Idiomarina. Strain ISL-52T differs from eight recognized Idiomarina species in several phenotypic characteristics, as shown in Table 1
. The phylogenetic distinctiveness and differential phenotypic properties are sufficient to allocate strain ISL-52T to a species that is separate from the recognized Idiomarina species (Stackebrandt & Goebel, 1994; Table 1). Therefore, on the basis of the data presented, strain ISL-52T represents a novel species of the genus Idiomarina, for which the name Idiomarina salinarum sp. nov. is proposed.
Description of Idiomarina salinarum sp. nov.
Idiomarina salinarum (sa.li.na'rum. L. gen. pl. n. salinarum of salt-works).
Cells are Gram-negative and rod-shaped (0.3–0.6x0.8–3.5 µm). Colonies on MA are circular to slightly irregular, raised, smooth, glistening, pale yellow in colour and 1.5–2.0 mm in diameter after 3 days incubation at 37 °C. Growth occurs at 4 and 42 °C, but not at 43 °C. The optimal pH for growth is between 7.0 and 8.0; growth occurs at pH 6.0, but not at pH 5.5. Growth occurs in the presence of 14 % (w/v) NaCl, but not in the absence of NaCl or in the presence of more than 15 % (w/v) NaCl. Anaerobic growth does not occur on MA or on MA supplemented with nitrate. Tweens 20, 40 and 60 are hydrolysed, but hypoxanthine, xanthine and urea are not. D-Glucose, D-fructose, D-galactose, D-cellobiose, D-mannose, trehalose, D-xylose, sucrose, benzoate, L-malate, pyruvate, salicin, formate and L-glutamate are not utilized. Acid is not produced from the following substrates: L-arabinose, D-cellobiose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannose, D-melezitose, melibiose, D-raffinose, L-rhamnose, D-ribose, sucrose, trehalose, D-xylose, D-sorbitol, D-mannitol and myo-inositol. Susceptible to chloramphenicol and polymyxin B and weakly susceptible to gentamicin; resistant to cephalothin, lincomycin, neomycin, novobiocin, oleandomycin, penicillin G and tetracycline. The predominant ubiquinone is Q-8. The major fatty acids (>10 % of total fatty acids) are iso-C15 : 0, iso-C17 : 0 and iso-C17 : 19c. The DNA G+C content is 53.9 mol% (determined by HPLC). Other phenotypic characteristics are given in Table 1.
The type strain, ISL-52T (=KCTC 12971T=CCUG 54359T), was isolated from a marine solar saltern of the Yellow Sea, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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-galactosidase, 
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