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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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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JG-30T is AY550276.
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, b). Some new genera or species have been isolated from jeotgal (Yoon et al., 2001a, b, 2003a, b). In this study, we report on the taxonomic characterization of a halotolerant, orange/yellow-pigmented Bacillus-like bacterial strain, JG-30T, which was isolated from jeotgal.
Strain JG-30T was isolated by means of the usual dilution plating technique on marine agar 2216 (MA; Difco) at 30 °C. The cell morphology was examined by light microscopy (model E600; Nikon) and transmission electron microscopy. The flagellum type was also examined by transmission electron microscopy (model CM-20; Philips) 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 with MA that had been prepared anaerobically using nitrogen. Growth in the absence of NaCl was investigated in trypticase soy broth without NaCl. Growth at various NaCl concentrations was investigated in marine broth 2216 (MB; Difco) or trypticase soy broth. Growth at various temperatures (4–50 °C) was measured on MA. Catalase and oxidase activities and the hydrolysis of casein, gelatin, hypoxanthine, starch, Tween 80, tyrosine and xanthine were tested for as described by Cowan & Steel (1965). Hydrolysis of aesculin, Voges–Proskauer and methyl red reactions and nitrate reduction were determined as described by Lanyi (1987). H2S production was tested for as described previously (Bruns et al., 2001). Acid production from carbohydrates was determined as described by Leifson (1963). Utilization of substrates as sole carbon and energy sources was tested as described by Baumann & Baumann (1981) using supplementation with 2 % (v/v) Hutner's mineral base (Cohen-Bazire et al., 1957) and 1 % (v/v) vitamin solution (Staley, 1968). Enzyme activity was determined by using the API ZYM system (bioMérieux). Other physiological and biochemical tests were performed with the API 20E system (bioMérieux).
Cell biomass for analyses of the cell-wall peptidoglycan and isoprenoid quinones and for DNA extraction was obtained from cultivation in MB at 37 °C. The isomer type of the diamino acid of the cell-wall peptidoglycan was determined by the method of Komagata & Suzuki (1987). Isoprenoid quinones were extracted and analysed as described previously (Komagata & Suzuki, 1987) using reversed-phase HPLC. For fatty acid methyl ester analysis, a loop of cell mass was harvested from agar plates after cultivation for 2 days at 37 °C on MA. 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 previously (Yoon et al., 1996), with the exception that ribonuclease T1 was used together with ribonuclease A. The DNA G+C content was determined by the method of Tamaoka & Komagata (1984), with the modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC. The 16S rRNA gene was amplified by a PCR using 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. (2003a).
The morphological, physiological and biochemical characteristics of strain JG-30T are shown in Table 1 or are given in the species description (see below). Strain JG-30T contained meso-diaminopimelic acid as the diamino acid in the cell-wall peptidoglycan and it had an unsaturated menaquinone with seven isoprene units (MK-7) as the predominant isoprenoid quinone. The cellular fatty acid profile showed a large amount of branched fatty acids; the major components were iso-C15 : 0 (31·8 %), iso-C14 : 0 (26·7 %), iso-C16 : 0 (15·6 %) and anteiso-C15 : 0 (15·4 %). The DNA G+C content of strain JG-30T was 45 mol%. An almost-complete 16S rRNA gene sequence of strain JG-30T comprising 1493 nt (approx. 96 % of the Escherichia coli 16S rRNA sequence) was determined directly after PCR amplification. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain JG-30T fell within the radiation of the cluster comprising Bacillus species and Bacillus subtilis, the type species of the genus Bacillus (Fig. 1). Strain JG-30T exhibited levels of 16S rRNA gene sequence similarity to Bacillus species of less than 95·7 % (Fig. 1). Sequence similarities to other species included in the phylogenetic analysis were below 92·4 % (Fig. 1). The results obtained from the phenotypic and phylogenetic analyses justify a taxonomic position for strain JG-30T as a member of the genus Bacillus. A widely recognized criterion for species definition in current bacteriology (Stackebrandt & Goebel, 1994) suggests that strains with more than a 3 % difference in 16S rRNA gene sequences represent different species. The phylogenetic distinctiveness of strain JG-30T warrants separation of this strain from Bacillus species with validly published names (Stackebrandt & Goebel, 1994). Strain JG-30T can be differentiated from several phylogenetically related Bacillus species by means of some phenotypic characteristics (Table 1). Therefore, on the basis of the data presented, strain JG-30T should be placed in the genus Bacillus as a novel species, for which the name Bacillus cibi sp. nov. is proposed.
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Cells are aerobic rods that are 0·6–0·8x1·5–3·5 µm in size. Gram-variable. Motile by means of peritrichous flagella. Central or subterminal ellipsoidal endospores are observed in swollen sporangia. Colonies are smooth, glistening, circular to slightly irregular, raised, orange–yellow in colour and 3–4 mm in diameter after 2 days incubation at 37 °C on MA. Optimal growth temperature is 37 °C; growth does not occur at 4 and 50 °C. Optimal pH for growth is 6·5–7·5. Growth is observed at pH 5·5 but not at pH 5·0. Growth occurs in the presence of 0–12 % (w/v) NaCl, with an optimum at 0–1 % (w/v) NaCl. Urease-negative. Hypoxanthine, tyrosine and xanthine are not hydrolysed. H2S and indole are not produced. Voges–Proskauer and methyl-red reactions are negative. When assayed with the API 20E system, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. When assayed with the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8), naphthol-AS-BI-phosphohydrolase and 
-galactosidase are present, but lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, 
-chymotrypsin, acid phosphatase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are absent. Acid is produced from D-fructose, melibiose, D-galactose, D-cellobiose, stachyose, sucrose, maltose, D-trehalose and D-raffinose. Acid is not produced from adonitol, D-sorbitol, myo-inositol, D-ribose, L-arabinose, D-melezitose or L-rhamnose. The following substrates are utilized for growth: D-glucose, D-fructose, D-trehalose, lactose, acetate, succinate and L-malate. D-Xylose, L-arabinose, citrate, benzoate, formate and L-glutamate are not utilized. The cell-wall peptidoglycan contains meso-diaminopimelic acid. The predominant menaquinone is MK-7. The major fatty acids are iso-C15 : 0 and iso-C14 : 0. The DNA G+C content is 45 mol% (determined by HPLC).
The type strain, JG-30T (=KCTC 3880T=DSM 16189T), was isolated from jeotgal, a traditional Korean fermented seafood.
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ACKNOWLEDGEMENTS |
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