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1 Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-Ku, Tokyo 113-0032, Japan
2 Microbiological and Analytical Group, Food Research Laboratories, Mitsui Norin Co. Ltd, 223-1 Miyahara, Fujieda, Shizuoka 426-0133, Japan
3 Marine Biotechnology Co. Ltd, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan
Correspondence
Sun-Young An
an12su{at}hotmail.com
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). The genus Virgibacillus currently consists of 10 recognized species (Heyndrickx et al., 1999; Heyrman et al., 2003; Lee et al., 2006; Yoon et al., 2004, 2005), with Virgibacillus pantothenticus as the type species. The members of the genus Virgibacillus are motile, Gram-positive rods that bear oval to ellipsoidal endospores. They have DNA G+C contents ranging from 36 to 43 mol%, their cell walls contain peptidoglycan of the meso-diaminopimelic type and they possess anteiso-C15 : 0 as the major cellular fatty acid (Heyrman et al., 2003). Here, we report two novel strains, 5B73CT and 5B133E, isolated from field soil in Kakegawa, Shizuoka, Japan, in the course of an environmental investigation and characterized phenotypically, chemotaxonomically and in terms of their 16S rRNA gene sequences.
Strains 5B73CT and 5B133E were isolated by suspending soil samples in a 0.9 % NaCl solution and heating the suspension at 80 °C for 10 min. The suspension was diluted serially, spread on plate count agar (Merck) and incubated at 35 °C. Purified colonies were selected, and all cultivations and phenotypic tests were carried out in media containing 50 % Herbst's artificial seawater and incubated at 30 °C. Herbst's artificial seawater contains the following (per litre distilled water): NaCl, 30 g; KCl, 0.7 g; MgSO4.7H2O, 5.3 g; CaSO4.2H2O, 1.3 g; and MgCl2.6H2O, 10.8 g. Cell morphology and motility were examined by using phase-contrast microscopy (BX60 microscope; Olympus). Growth under anaerobic conditions was determined after 1 week incubation in an AnaeroPack (Mitsubishi Gas Chemical). Catalase was determined with 3 % H2O2, the production of bubbles representing a positive reaction. Oxidase was determined using cytochrome oxidase paper (Nissui Pharmaceutical). API 20E and API 50 CH microtest galleries (bioMérieux) were used to determine physiological and biochemical characteristics. The API tests were read after 48 h. The isolates were Gram-positive and strictly aerobic, and the cells were motile and rod-shaped. Morphological and physiological characteristics of the isolates are given in the species description. The isolates were similar to Virgibacillus species in terms of morphological and some physiological characteristics, but were distinct regarding anaerobic growth, growth temperature, nitrate reduction and H2S production (Table 1).
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, 2002), the strains were grouped within the Virgibacillus cluster. However, they were found to be distinct from previously described species of the genus Virgibacillus. 16S rRNA gene sequences were determined using an Applied Biosystems 16S rRNA gene kit, according to the instructions of the manufacturer. The 16S rRNA gene sequences of strains 5B73CT and 5B133E were compared with sequences obtained from GenBank. The sequences were aligned with the CLUSTAL W software package (Thompson et al., 1994), and evolutionary distances and Knuc values (Kimura, 1980) were generated. Alignment gaps and ambiguous bases were not taken into consideration. A phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987), and the topology of the phylogenetic tree was evaluated by using the bootstrap resampling method of Felsenstein (1985), based on 1000 replicates. Similarity values were calculated using MEGA3 (Kumar et al., 2004). Almost-complete 16S rRNA gene sequences of strains 5B73CT and 5B133E were determined and subjected to a comparative analysis. The 16S rRNA gene sequences of the two isolates shared 100 % similarity and were on the same phylogenetic branch. Strain 5B73CT showed the highest level of 16S rRNA gene sequence similarity with Virgibacillus marismortui (96.4 %), followed by Virgibacillus carmonensis (96.2 %), Virgibacillus halodenitrificans (96.2 %) and Virgibacillus proomii (95.9 %). On the other hand, the strains showed lower levels of sequence similarity (<95 %) with respect to other recognized low-G+C, Gram-positive species, including Lentibacillus salarius (94.9 %), Lentibacillus salicampi (94.0 %) and Oceanobacillus iheyensis (94.0 %). Generally, around 95 % would be a practicable border zone for genus definition (Ludwig et al., 1998). The phylogenetic tree shown in Fig. 1 indicates that strains 5B73CT and 5B133E are closely related to the genus Virgibacillus but form a separate clade within that genus.
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observed, strains with less than 97 % 16S rRNA gene sequence similarity have DNA–DNA relatedness values below 70 %. Thus, in the absence of a close relative showing significant 16S rRNA gene sequence similarity, strains 5B73CT and 5B133E can be considered as representing a novel species.
Cellular fatty acids from strains 5B73CT and 5B133E grown on trypticase soy agar (BD BBL) for 48 h at 30 °C were prepared, separated and identified with the Microbial Identification System (MIDI). The major fatty acids of strains 5B73CT and 5B133E were anteiso-C15 : 0 (35.8–40.9 %), iso-C15 : 0 (21.2–20.6 %), anteiso-C17 : 0 (16.1–19.9 %) and iso-C16 : 0 (10.3–6.5 %). This fatty acid profile is quite similar to those of species of the genus Virgibacillus (Table 2).
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. The G+C content of the total DNA was measured by HPLC according to the method described by Mesbah et al. (1989). The DNA G+C contents of 5B73CT and 5B133E were 42.6 and 42.6 mol%, which is a little higher than those for known species of the genus Virgibacillus (Table 1
). DNA–DNA hybridization was performed by using the photobiotin-labelling method of Ezaki et al. (1989) with a multi-well plate reader (CytoFluoR; PerSeptive Biosystems). The DNA–DNA hybridization values for strains 5B73CT and 5B133E and for 5B133E and 5B73CT were 95.4 and 101.5 %, respectively. The two strains should therefore be considered as representing a single species (Stackebrandt et al., 2002).
Analysis of the cell-wall peptidoglycan of strain 5B73CT (selected as the representative strain for the two isolates) was carried out by using the methods of Schleifer & Kandler (1972). Strain 5B73CT possessed the meso-diaminopimelic-type cell wall. Analysis of the respiratory quinones of strain 5B73CT was performed as described by Collins & Jones (1981): the major isoprenoid quinone was MK-7.
On the basis of phenotypic, chemotaxonomic and phylogenetic data, we conclude that strains 5B73CT and 5B133E represent a novel species of the genus Virgibacillus, for which we propose the name Virgibacillus halophilus sp. nov.
Description of Virgibacillus halophilus sp. nov.
Virgibacillus halophilus (ha.lo.phi'lus. Gr. n. hals salt; Gr. adj. philos loving; N.L. masc. adj. halophilus salt-loving).
Cells are Gram-positive, strictly aerobic, motile rods (0.5x1.75 µm). Ellipsoidal spores are formed subterminally. Colonies grown on trypticase soy agar containing 50 % Herbst's artificial seawater are circular, convex and pale yellow. The growth temperature and pH are 5–45 °C and 5.0–10.0, respectively. Growth occurs both in the absence of NaCl and in the presence of 18 % NaCl (w/v). Catalase and oxidase activities are positive. H2S and indole are not produced. Nitrate is reduced to nitrite whereas nitrite is not reduced. Acetoin is produced. Urease, gelatinase and 
-galactosidase are hydrolysed. Negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase and citrate utilization. Acid is produced from glucose, fructose, mannose, mannitol, N-acetylglucosamine, arbutin, aesculin, salicin, cellobiose, lactose, sucrose and trehalose, but not from erythritol, D-arabinose, L-xylose, adonitol, methyl -D-xyloside, sorbose, rhamnose, dulcitol, inositol, sorbitol, methyl 
-D-mannoside, methyl -D-glucoside, amygdalin, maltose, melibiose, inulin, melezitose, raffinose, starch, glycogen, xylitol, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate. Acid production from glycerol, L-arabinose, ribose, D-xylose, galactose and gentiobiose is weak. The cell wall contains peptidoglycan of the meso-diaminopimelic acid type. The major isoprenoid quinone system is MK-7. The major cellular fatty acids are anteiso-C15 : 0, iso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The genomic DNA G+C content of the type strain is 42.6 mol%.
The type strain, 5B73CT (=IAM 15308T=KCTC 13935T), was isolated from field soil in Kakegawa, Shizuoka, Japan.
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