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Department of Science Education, Cheju National University, Jeju 690-756, Republic of Korea
Correspondence
Soon Dong Lee
sdlee{at}cheju.ac.kr
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to accommodate aerobic, Gram-positive, coccus-shaped bacteria, which often reproduce by budding and multiple fission. The only (and type) species of the genus Blastococcus, Blastococcus aggregatus (Ahrens & Moll, 1970), was isolated from the Baltic Sea. Although the genus name was included on the Approved Lists of Bacterial Names (Skerman et al., 1980), it has not been described in detail taxonomically for many years. Its description was recently emended after a detailed taxonomic investigation that included morphological, chemotaxonomic and phylogenetic analyses; this resulted in the addition of the second species, Blastococcus saxobsidens (Urzì et al., 2004). The genus is characterized chemotaxonomically by the following features: the presence of meso-diaminopimelic acid in the cell wall; the presence of MK-9(H4) as the major menaquinone; cellular fatty acids consisting mainly of unsaturated and iso-branched acids; a phospholipid profile that includes diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylethanolamine; and a DNA G+C content of 74 mol%. Phylogenetically, members of this genus are related to members of the family Geodermatophilaceae and were isolated from somewhat restricted environments, such as the Baltic Sea and rock surfaces.
During the study of marine bacteria recovered from the coast of Jeju in the Republic of Korea, strain KST3-10T was isolated from sand sediment from Gwakji beach; the taxonomic status of this organism was investigated by means of polyphasic characterization. Sediment samples were taken, at a depth of 1 m, from surface water of the beach and were placed into sterilized 50 ml Falcon tubes containing seawater. For bacterial isolation, 1 g sand sediment was placed into a sterile plastic tube containing 9 ml sterile distilled water and then mixed in a tube rotator for 30 min at a moderate speed. Aliquots (100 µl) of the serial diluent of the samples were transferred onto SC-SW agar plates supplemented with 60 % (v/v) sterilized natural seawater. The isolation medium (SC-SW agar) consisted of 1 % soluble starch, 0.03 % casein, 0.2 % KNO3, 0.2 % NaCl, 0.002 % CaCO3, 0.005 % MgSO4.7H2O, 0.001 % FeSO4.7H2O and 1.8 % agar in 60 % sterilized natural seawater and 40 % distilled water (pH 7.2). The isolate was maintained on ISP 2 medium (Shirling & Gottlieb, 1966) supplemented with 60 % sterilized natural seawater (YE-SW agar), and in a 20 % (v/v) glycerol suspension supplemented with 60 % (v/v) sterilized natural seawater, at –20 and –80 °C.
Chromosomal DNA was extracted and purified by using the Wizard genomic DNA purification kit (Promega) according to the instructions of the manufacturer. The 16S rRNA gene of strain KST3-10T was amplified by a PCR (Lee et al., 2000a) and was subjected to direct sequence determination using the ABI PRISM BigDye Terminator cycle sequencing kit (Applied Biosystems) and an automatic DNA sequencer (model 3730xl; Applied Biosystems). The sequence determined in this study was aligned with corresponding sequences (retrieved from GenBank) by using the CLUSTAL_X program (Thompson et al., 1997) and then manually optimized according to the secondary structure of bacterial 16S rRNA. Phylogenetic analyses were performed using three treeing algorithms, namely the neighbour-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Fitch, 1971) methods. A phylogenetic tree was reconstructed, using the neighbour-joining method, from evolutionary distances calculated by the method described by Jukes & Cantor (1969). The reliability of the tree topology was evaluated by bootstrap analysis (Felsenstein, 1985) of 1000 replicated datasets.
An almost-complete 16S rRNA gene sequence for strain KST3-10T was determined in this study (1404 nt) and was used for an initial BLAST search against GenBank: the organism was found to belong to the family Geodermatophilaceae. The sequence was aligned with the corresponding sequences of related taxa in the family Geodermatophilaceae. A total of 1387 unambiguously aligned positions present in all strains between positions 50 and 1477 (Escherichia coli numbering) were used for phylogenetic analyses. Glycomyces harbinensis was used as an outgroup for tree construction. A neighbour-joining tree (Fig. 1) showed that strain KST3-10T occupied a unique position outside the Blastococcus cluster (74 % bootstrap support). Members of the genus Blastococcus formed a monophyletic clade with 84 % bootstrap support. This relationship was supported by all tree-making methods used in this study. The organism revealed 16S rRNA gene sequence similarity values of 98.1 and 98.2 % with respect to the type strains of B. aggregatus and B. saxobsidens, respectively. The type strains of B. aggregatus and B. saxobsidens shared 98.2 % sequence similarity with respect to each other, whereas the levels of 16S rRNA gene sequence similarity between the novel isolate and the type strains of the loosely associated relatives Modestobacter multiseptatus and Geodermatophilus obscurus were 96.9 and 96.2 %, respectively.
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), peptidoglycan acyl type (Uchida & Aida, 1984), whole-cell sugars (Saddler et al., 1991), mycolic acids (Minnikin et al., 1980), polar lipids (Minnikin et al., 1977) and lipoquinones (Kroppenstedt, 1985) were performed as described previously (Lee et al., 2000b). The cellular fatty acid methyl esters were prepared and analysed according to the instructions of the Microbial Identification System (MIDI), using cells grown on TSA for 3 days at 30 °C. The diaminopimelic acid isomer of the peptidoglycan was identified as the meso- form. Whole-cell hydrolysates contained arabinose and galactose as the characteristic sugars. The glycan moiety of the murein structure was acetylated. Mycolic acids were not detected. The predominant menaquinone was MK-9(H4). The polar lipid profile contained significant amounts of phosphatidylcholine, diphosphatidylglycerol and phosphatidylinositol. Small amounts of phosphatidylethanolamine and phosphatidylmethylethanolamine were also detected. Strain KST3-10T could be readily differentiated from the type strains of B. aggregatus and B. saxobsidens by the absence of phosphatidylglycerol and the presence of phosphatidylcholine and phosphatidylmethylethanolamine (Urzì et al., 2004). It was shown that the presence of phosphatidylcholine, in particular, could be a chemotaxonomic marker useful for species identification. The fatty acid profile was characterized by significant amounts of iso-C16 : 0 (32.59 %), C17 : 1
8c (11.15 %) and iso-C15 : 0 (10.58 %) acids. Small amounts (>1 % of total) of iso-C16 : 1 H (7.20 %), C18 : 0 (5.09 %), C16 : 0 (4.35 %), C17 : 16c (3.89 %), C15 : 0 (2.68 %), C17 : 0 (2.63 %), iso-C14 : 0 (2.46 %), C15 : 16c (1.98 %), anteiso-C15 : 0 (1.78 %), C18 : 19c (1.63 %), anteiso-C17 : 0 (1.48 %), iso-C17 : 0 (1.38 %), C12 : 0 (1.28 %) and iso-C17 : 19c (1.05 %) were present, along with summed feature 4 (C16 : 17c and/or iso-C15 : 0 2-OH, 2.64 %). Strain KST3-10T differed from B. saxobsidens in possessing C18 : 0 and C12 : 0, and in terms of the relative amounts of C16 : 0, C17 : 18c and C18 : 19c, and could be distinguished from B. aggregatus in terms of the relative amounts of iso-C16 : 1, C17 : 18c, C18 : 19c and C18 : 0 acids (Urzì et al., 2004).
Colony pigmentation was observed visually and recorded after 7 days growth at 30 °C on YM-SW agar. Cell morphology and motility were observed using an Olympus light microscope equipped with phase-contrast optics (magnification x400) and a transmission electron microscope. Cells were grown for 3 days at 30 °C on YM-SW agar; cell suspensions for microscopic examination were made using sterile saline. The colonies were circular, translucent, smooth-surfaced and had entire margins. The colour of the colonies ranged from cream to apricot depending on the incubation time. The cells of strain KST3-10T were coccoid and occurred in pairs or were rod-shaped, flagellated and motile (Fig. 2a). Only the rod-shaped cells showed bud formation (Fig. 2b).
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). The decomposition of hypoxanthine, tyrosine and xanthine was tested using the method described by Gordon et al. (1974). The following tests were performed with API 20NE strips (bioMérieux): nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, aesculin degradation, gelatin hydrolysis and 
-galactosidase. Catalase activity was determined with a 3 % (v/v) H2O2 solution. Oxidase activity was tested by assessing the oxidation of N,N,N',N'-tetramethyl-p-phenylenediamine. Other physiological and biochemical properties were tested with API 20NE and API ZYM strips (bioMérieux) according to the manufacturer's instructions. Strain KST3-10T utilized Tween 80 as a sole source of carbon and energy for growth, but did not use the following substrates: 
- and -cyclodextrin, dextrin, glycogen, inulin, N-acetyl-D-glucosamine, L-arabinose, D-arabitol, arbutin, L-fucose, D-gluconic acid, -D-glucose, -D-lactose, lactulose, maltotriose, D-melezitose, methyl -D-glucoside, methyl -D-glucoside, methyl -D-mannoside, D-psicose, D-raffinose, salicin, stachyose, D-trehalose, D-xylose, - and -hydroxybutyric acids, p-hydroxyphenylacetic acid, lactamide, L-lactic acid, methylpyruvate, propionic acid, pyruvic acid, succinamic acid, N-acetyl-L-glutamic acid, L-alaninamide, D- and L-alanine, L-alanyl glycine, L-glutamic acid, glycyl L-glutamic acid, L-pyroglutamic acid, L-serine, glycerol, 2'-deoxyadenosine, inosine, thymidine, uridine, adenosine 5'-monophosphate, thymidine 5'-monophosphate, uridine 5'-monophosphate, D-fructose 6-phosphate, -D-glucose-1-phosphate or DL--glycerol phosphate. Other physiological characteristics are given in Table 1 and in the species description.
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) together with other phenotypic features (e.g. cell morphology, respiratory quinone and cellular fatty acid profile) indicate that the isolate should be assigned to the genus Blastococcus. Phospholipid composition has been used as a key chemotaxonomic marker in the delineation of actinomycete genera or genus groups (Lechevalier et al., 1977). However, there are reports of phospholipid profiles varying according to species (Xu et al., 1999; Lee et al., 2001). The differentiating characteristics of the isolate and the type species of the genus Blastococcus are given in Table 1
. On the basis of the phenotypic and genotypic data, the novel organism represents a novel species of the genus Blastococcus, for which the name Blastococcus jejuensis sp. nov. is proposed.
Emended description of Blastococcus Ahrens and Moll 1970
The description is taken from Ahrens & Moll (1970), Urzì et al. (2004) and this study. The diagnostic diamino acid and sugars of peptidoglycan are meso-diaminopimelic acid and arabinose and galactose, respectively. The predominant menaquinone is MK-9(H4). MK-9 occurs in variable amounts depending on the strain. The polar lipid profiles are characterized by the common basis comprising diphosphatidylglycerol, phosphatidylinositol and phosphatidylethanolamine. The presence of phosphatidylglycerol, phosphatidylcholine and phosphatidylmethylethanolamine is variable among the species. The major cellular fatty acids consist of saturated, unsaturated and iso-branched fatty acids. The G+C contents of the DNA are in the range 72.3–74 mol%. Gram-positive, oxidase-negative, catalase-positive and aerobic. Some cells may be microaerophilic. Cells are coccoid or rod-shaped and reproduce by budding and binary or multiple fission. Vibrioid or non-motile cocci occur in pairs or tend to form aggregates, whereas rod-shaped cells occur singly and show motility by means of flagella. Bud formation varies according to the species. Most strains utilize a broad spectrum of organic compounds. Phylogenetically, the genus belongs to the family Geodermatophilaceae. The type species is Blastococcus aggregatus.
Description of Blastococcus jejuensis sp. nov.
Blastococcus jejuensis (je.ju.en'sis. N.L. masc. adj. jejuensis of Jeju, Republic of Korea, the site from which the type strain was isolated).
Aerobic, motile, non-spore-forming, oxidase-negative, catalase-positive, Gram-positive. Cells are cocci that occur in pairs or rods. Bud formation is observed for rod-shaped cells. Colonies are circular, smooth, transparent and apricot in colour. Starch and casein are hydrolysed, but elastin is not. Hypoxanthine, tyrosine and xanthine are not decomposed. In API 20NE tests, glucose fermentation and indole production from tryptophan are not observed. Activities of arginine dihydrolase, urease and -galactosidase are not present. Nitrate is not reduced to nitrite. Aesculin degradation and gelatin hydrolysis are not detected. Caprate, adipate, citrate and phenylacetate are not assimilated. In API ZYM tests, results for leucine arylamidase and -glucosidase are positive and weakly positive, respectively, whilst results for esterase lipase (C8), lipase (C14), trypsin, -chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, -galactosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are negative. The temperature range for growth is 10–37 °C, with the optimum at 30 °C. Growth occurs at pH values in the range 6.1–10.1, with the optimum at pH 7.1. Growth was observed in the presence of 0–1 % NaCl but not 2 % NaCl. The following substrates are used as sole carbon and energy sources: mannan, N-acetyl--D-mannosamine, amygdalin, D-cellobiose, D-fructose, D-galactose, D-galacturonic acid, gentiobiose, myo-inositol, D-melibiose, methyl -D-galactoside, methyl -D-galactoside, 3-methyl-D-glucoside, palatinose, L-rhamnose, sedoheptulosan, D-sorbitol, sucrose, D-tagatose, turanose, xylitol, acetic acid, 
-hydroxybutyric acid, -ketovaleric acid, D-lactic acid methyl ester, succinic acid, L-asparagine, putrescine, 2,3-butanediol, adenosine and D-glucose 6-phosphate, but not Tween 40, D-ribose, -ketoglutaric acid or methylsuccinate. The polar lipid profile contains phosphatidylcholine, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine and phosphatidylinositol. Mycolic acids are not present. The major cellular fatty acids are iso-C16 : 0, C17 : 18c and iso-C15 : 0. The predominant menaquinone is MK-9(H4). Whole-cell hydrolysates contain meso-diaminopimelic acid as the diagnostic diamino acid and arabinose and galactose as diagnostic sugars. The DNA G+C content is 72.3 mol%.
The type strain, KST3-10T (=NRRL B-24440T=KCCM 42251T), was isolated from sand sediment from Gwakji beach on Jeju Island, Republic of Korea.
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ACKNOWLEDGEMENTS |
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