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Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, South Korea
Correspondence
Tae-Kwang Oh
otk{at}kribb.re.kr
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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Published online ahead of print on 22 April 2005 as DOI 10.1099/ijs.0.63613-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain DSW-10T is AY822043.
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with the transfer of Bacillus pantothenticus to Virgibacillus pantothenticus. Subsequently, four further species, Virgibacillus proomii (Heyndrickx et al., 1999) and Virgibacillus carmonensis, Virgibacillus necropolis and Virgibacillus picturae (Heyrman et al., 2003), were described. Two Salibacillus species (Wainø et al., 1999; Arahal et al., 2000) have been reclassified in the genus as Virgibacillus marismortui and Virgibacillus salexigens (Heyrman et al., 2003). Recently, Bacillus halodenitrificans was transferred to the genus as Virgibacillus halodenitrificans (Yoon et al., 2004). The genus Virgibacillus currently comprises eight recognized species. Here we report on the taxonomic characterization of a slightly halophilic Virgibacillus-like bacterial strain, DSW-10T, which was isolated from sea water at Dokdo, an island in the East Sea, Korea.
Sea water collected at Dokdo provided the source for isolation of the bacterial strains. A single isolate, DSW-10T, was obtained by the standard dilution plating technique on marine agar 2216 (MA; Difco) at 30 °C. V. pantothenticus DSM 26T, V. proomii DSM 13055T and V. marismortui DSM 12325T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany, and cultivated according to recommended procedures of the DSMZ. Cell morphology was examined by light microscopy (Nikon E600) and transmission electron microscopy (TEM). For the latter, cells were negatively stained with 1 % (w/v) phosphotungstic acid and, after air-drying, the grids were examined by using a model CM-20 transmission electron microscope (Philips). Presence of flagella was examined by TEM using cells from exponentially growing cultures. The Gram-reaction was determined using the bioMérieux Gram Stain kit according to the manufacturer's instructions. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on MA and MA supplemented with nitrate, both of which had been prepared anaerobically. The pH range for growth was determined in marine broth 2216 (MB; Difco) supplemented with 3 % (w/v) NaCl that was adjusted to various pH values (initial pH 4·0–12·0 at intervals of 0·5 pH units). The pH (below 10·5) was adjusted prior to sterilization to various levels by the addition of HCl or Na2CO3. Growth at various NaCl concentrations was investigated in MB or trypticase soy broth (TSB; Difco). Growth in the absence of NaCl was investigated in TSB lacking NaCl. Growth at various temperatures (4–55 °C) was measured on MA supplemented with 3 % (w/v) NaCl. Oxidase and catalase activities and hydrolysis of casein, starch, urea and Tweens 20, 40, 60 and 80 were determined as described by Cowan & Steel (1965). Hydrolysis of aesculin and gelatin and nitrate reduction were determined as described by Lanyi (1987) with a modification that artificial sea water was used for preparation of media. The artificial sea water contained (per litre 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 by Bruns et al. (2001). Hydrolysis of hypoxanthine, tyrosine and xanthine was performed on MA supplemented with 3 % NaCl using the substrate concentrations given by Cowan & Steel (1965). Acid production from carbohydrates was determined as described by Leifson (1963), and utilization of various substrates as sole carbon and energy sources was tested according to the method of Baumann & Baumann (1981) using supplementation with 2 % (v/v) Hutner's mineral base (Cohen-Bazire et al., 1957), 1 % (v/v) vitamin solution (Staley, 1968) and 0·01 % (w/v) yeast extract. Enzyme activity and other physiological properties were determined using the API ZYM and API 20E systems (bioMérieux) with a modification that suspension media supplemented with 5 % (w/v) NaCl were used.
Cell mass for analyses of the cell wall, menaquinones and polar lipids and for DNA extraction was produced in MB supplemented with 3 % NaCl at 37 °C. The isomer type of diaminopimelic acid in the peptidoglycan was determined by the method described by Komagata & Suzuki (1987). Menaquinones were analysed as described by Komagata & Suzuki (1987) using reversed-phase HPLC. Polar lipids were extracted using the procedures described by Minnikin et al. (1984) and were identified by two-dimensional TLC followed by spraying with appropriate detection reagents (Komagata & Suzuki, 1987). For fatty acid methyl ester analysis, cell mass of strain DSW-10T was harvested from agar plates after cultivation for 2 days at 37 °C on MA supplemented with 3 % NaCl. The fatty acid methyl esters were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). Chromosomal DNA was isolated and purified according to the method described by Yoon et al. (1996), with the exception that RNase T1 was used in combination with RNase A. The DNA G+C content was determined by the method of Tamaoka & Komagata (1984) with a modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC. The 16S rRNA gene was amplified by PCR using two universal primers as described by Yoon et al. (1998). Sequencing of the 16S rRNA gene and phylogenetic analysis were performed as described by Yoon et al. (2003). DNA–DNA hybridization was performed fluorometrically by the method of Ezaki et al. (1989) using photobiotin-labelled DNA probes and microdilution wells. Hybridization was performed with five replications for each sample. The highest and lowest values obtained for each sample were excluded and the means of the remaining three values were quoted as DNA–DNA relatedness values.
The 16S rRNA gene sequence of strain DSW-10T determined in this study comprised 1520 nt, representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence. Comparative 16S rRNA gene sequence analyses showed that strain DSW-10T is most closely related to Virgibacillus species (Fig. 1). In the phylogenetic tree based on the neighbour-joining algorithm, strain DSW-10T joined V. pantothenticus at a bootstrap confidence level of 99·3 % (Fig. 1). Although there were differences in topologies between the phylogenetic tree based on the neighbour-joining algorithm and those based on the maximum-likelihood and maximum-parsimony algorithms, the relationships among strain DSW-10T and some Virgibacillus species were also recovered in the trees based on the latter two algorithms (Fig. 1). Strain DSW-10T exhibited 16S rRNA gene sequence similarity values of 98·7 % to V. pantothenticus IAM 11061T and 95·3–97·0 % to the type strains of the other Virgibacillus species (Fig. 1). Sequence similarities to all other species included in the phylogenetic analysis were lower than 94·3 % (Fig. 1). DNA–DNA hybridization was performed to determine the genetic relatedness between strain DSW-10T and the type strains of three Virgibacillus species that showed 16S rRNA gene sequence similarity values of >97·0 % to strain DSW-10T. Strain DSW-10T exhibited DNA–DNA relatedness levels of 17·5, 8·4 and 9·2 % to V. pantothenticus DSM 26T, V. proomii DSM 13055T and V. marismortui DSM 12325T, respectively.
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or are given in the species description below. Strain DSW-10T contained meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan. The predominant isoprenoid quinone found in strain DSW-10T was unsaturated menaquinone with seven isoprene units (MK-7), and a minor amount of MK-6 was also detected. Major components of the fatty acids (>1 %) detected in strain DSW-10T were branched fatty acids anteiso-C15 : 0 (34·4 %), iso-C15 : 0 (19·4 %), anteiso-C17 : 0 (15·4 %), iso-C16 : 0 (12·3 %), iso-C17 : 0 (7·2 %), iso-C14 : 0 (4·7 %) and iso-C13 : 0 (1·1 %) and straight-chain fatty acid C16 : 0 (2·4 %). This fatty acid profile was similar to those of Virgibacillus species, although there were differences in the proportions of some fatty acids that might have been caused by different cultivation conditions (Heyndrickx et al., 1999; Wainø et al., 1999; Heyrman et al., 2003). Major polar lipids detected in strain DSW-10T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and unidentified phospholipids. An unknown aminophospholipid that was ninhydrin-positive was not detected. The DNA G+C content of strain DSW-10T was 36·7 mol%. The results obtained from chemotaxonomic analyses, showing the properties that are shared by Virgibacillus species, were in agreement with the result of phylogenetic classification based on 16S rRNA gene sequence analysis.
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). In particular, strain DSW-10T is clearly distinguished from V. pantothenticus, its nearest phylogenetic neighbour, by differences in acid production from some substrates (Table 1). The phylogenetic distinctiveness and DNA–DNA relatedness data are sufficient to categorize strain DSW-10T as representing a species that is distinct from recognized Virgibacillus species (Wayne et al., 1987; Stackebrandt & Goebel, 1994). Therefore, on the basis of the data presented, strain DSW-10T should be classified in the genus Virgibacillus as a member of a novel species, for which the name Virgibacillus dokdonensis sp. nov. is proposed.
Description of Virgibacillus dokdonensis sp. nov.
Virgibacillus dokdonensis (dok.do.nen'sis. N.L. masc. adj. dokdonensis of Dokdo, a Korean island located at the edge of the East Sea in Korea from where the type strain was isolated).
Cells are rod-shaped, 0·6–0·8x2·5–5·0 µm, Gram-variable and motile by means of peritrichous flagella. Terminal or subterminal ellipsoidal or spherical endospores are observed in swollen sporangia. Colonies are irregular, flat, translucent, milky white in colour and 3–5 mm in diameter after 2 days of incubation at 37 °C on MA supplemented with 3 % (w/v) NaCl. Optimal growth temperature is 37 °C; growth occurs at 15 and 50 °C, but not at 10 or 55 °C. Optimal pH for growth is 7·0–8·0; growth occurs at pH 5·5, but not at pH 5·0. Optimal growth occurs in the presence of 4–5 % (w/v) NaCl; growth occurs without NaCl and in the presence of 23 % (w/v) NaCl, but not in the presence of >24 % (w/v) NaCl. Growth occurs under anaerobic conditions on MA and MA supplemented with nitrate. Oxidase-positive. Urease-negative. Starch and Tweens 20, 40, 60 and 80 are hydrolysed. Hypoxanthine, xanthines and tyrosine are not hydrolysed. Voges–Proskauer reaction is negative. Indole is not produced. Arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase are absent. When assayed with the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8), 
-chymotrypsin, naphthol-AS-BI-phosphohydrolase and -glucosidase are present, but lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, acid phosphatase, -galactosidase, 
-galactosidase, -glucuronidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are absent. Acid is produced from D-cellobiose, lactose, maltose, D-ribose, sucrose, myo-inositol and D-sorbitol. Acid is not produced from L-arabinose, D-melezitose, D-raffinose or D-xylose. The following substrates are utilized: D-cellobiose, D-mannose, maltose, acetate, citrate, pyruvate and salicin. D-Galactose, D-trehalose, benzoate, succinate, L-malate, formate and L-glutamate are not utilized. The cell-wall peptidoglycan contains meso-diaminopimelic acid. The predominant menaquinone is MK-7. The major polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and unidentified phospholipids. The major fatty acids are anteiso-C15 : 0 (34·4 %), iso-C15 : 0 (19·4 %), anteiso-C17 : 0 (15·4 %) and iso-C16 : 0 (12·3 %). The DNA G+C content is 36·7 mol%.
The type strain, DSW-10T (=KCTC 3933T=DSM 16826T), was isolated from Dokdo, an island located at the edge of the East Sea, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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