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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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through the reclassification of Sporosarcina halophila as Halobacillus halophilus and the description of two novel species, Halobacillus litoralis and Halobacillus trueperi. Subsequently, six further Halobacillus species, Halobacillus salinus (Yoon et al., 2003), Halobacillus karajensis (Amoozegar et al., 2003), Halobacillus locisalis (Yoon et al., 2004), Halobacillus aidingensis and Halobacillus dabanensis (Liu et al., 2005) and Halobacillus yeomjeoni (Yoon et al., 2005), have been described. In this study, we describe a bacterial strain, ASL-17T, which is related phylogenetically to the genus Halobacillus. However, strain ASL-17T was found to have cell-wall peptidoglycan based on meso-diaminopimelic acid instead of L-ornithine, as found in other members of the genus Halobacillus. Accordingly, the aim of the present study was to determine the exact taxonomic position of strain ASL-17T by a polyphasic characterization that included determination of the phenotypic and chemotaxonomic properties, detailed phylogenetic analysis based on 16S rRNA gene sequences and genetic analysis.
Strain ASL-17T was isolated from a sediment collected from a marine solar saltern of the Yellow Sea, Korea, by means of the dilution-plating technique at 30 °C on marine agar 2216 (MA; Difco) supplemented with 8 % (w/v) NaCl. The type strains of nine Halobacillus species were used as reference strains for DNA–DNA hybridization: H. halophilus KCTC 3685T, H. litoralis KCTC 3687T and H. trueperi KCTC 3686T were obtained from the Korean Collection for Type Cultures (KCTC), Taejon, Korea; H. karajensis DSM 14948T was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany; H. salinus HSL-3T, H. locisalis MSS-155T and H. yeomjeoni MSS-402T were obtained in our laboratory (Yoon et al., 2003, 2004, 2005); and H. aidingensis JCM 12771T and H. dabanensis JCM 12772T were obtained from the Japan Collection of Microorganisms (JCM), Saitama, Japan.
The morphological, physiological and biochemical characteristics of strain ASL-17T were investigated using routine cultivation on MA supplemented with 6 % (w/v) NaCl at 37 °C. Cell morphology was examined by light microscopy (Nikon E600) 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 supplemented with 6 % (w/v) NaCl and on MA supplemented with 6 % (w/v) NaCl and potassium nitrate, both of which had been prepared anaerobically using nitrogen. The pH range for growth was determined in marine broth 2216 (MB; Difco) supplemented with 6 % (w/v) NaCl that was adjusted to various pH values (pH 4.5–9.5 at intervals of 0.5 pH units). Growth in the absence of NaCl was investigated using trypticase soy broth, using supplementation with 0.45 % (w/v) MgCl2 . 6H2O, prepared according to the formula of the Difco medium except that NaCl was omitted. Growth at various NaCl concentrations was investigated in MB or trypticase soy broth (Difco). Growth at various temperatures (4–50 °C) was measured on MA supplemented with 6 % (w/v) NaCl. Catalase and oxidase activities and hydrolysis of casein, starch and Tweens 20, 40, 60 and 80 were determined as described by Cowan & Steel (1965). Hydrolysis of hypoxanthine, tyrosine and xanthine was tested on MA supplemented with 6 % (w/v) NaCl using the substrate concentrations described by Cowan & Steel (1965). Hydrolysis of aesculin, gelatin and urea and nitrate reduction were investigated as described previously (Lanyi, 1987) with the modification that artificial seawater supplemented with 6 % (w/v) NaCl was used for preparation of media. The artificial seawater contained (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 supplemented with 6 % (w/v) NaCl by using antibiotic discs with the following amounts: polymyxin B, 100 U; streptomycin, 50 µg; penicillin G, 20 U; chloramphenicol, 100 µg; ampicillin, 10 µg; cephalothin, 30 µg; gentamicin, 30 µg; novobiocin, 5 µg; tetracycline, 30 µg; kanamycin, 30 µg; lincomycin, 15 µg; oleandomycin, 15 µg; neomycin, 30 µg; carbenicillin, 100 µg. Acid production from carbohydrates was tested as described by Leifson (1963) using supplementation with 7 % (w/v) NaCl. Utilization of various substrates for growth was determined as described by Baumann & Baumann (1981) using supplementation with 7 % (w/v) NaCl, 2 % (v/v) Hutner's mineral salts solution (Cohen-Bazire et al., 1957) and 1 % (v/v) vitamin solution (Staley, 1968). Enzyme activities were determined by using the API ZYM system (bioMérieux).
Cell biomass for DNA extraction and for analyses of cell-wall and isoprenoid quinones was obtained from cultivation in MB supplemented with 6 % (w/v) NaCl at 37 °C. 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 to minimize contamination with RNA. The 16S rRNA gene was amplified by 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. (2003). The isomer type of the diamino acid in the cell-wall peptidoglycan was analysed using TLC according to the method described by Komagata & Suzuki (1987). Preparation of whole-cell hydrolysates and determination of the isomer type of the diamino acid was also performed at the DSMZ by using TLC and the solvent system of Rhuland et al. (1955). Isoprenoid quinones were analysed as described by Komagata & Suzuki (1987) using reversed-phase HPLC. For cellular fatty acid analysis, cell mass of strain ASL-17T was harvested from agar plates after cultivation for 3 days at 37 °C on MA supplemented with 6 % (w/v) NaCl. 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 the method of Tamaoka & Komagata (1984) with the modification that DNA was hydrolysed using nuclease P1 (Sigma) and the resultant nucleotides were analysed by reversed-phase HPLC. 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 in each sample were excluded, and the means of the remaining three values were quoted as DNA–DNA relatedness values.
Morphological, cultural, physiological and biochemical characteristics of strain ASL-17T are given in the species description and in Table 1. The almost-complete 16S rRNA gene sequence of strain ASL-17T determined in this study comprised 1522 nucleotides, representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence. Comparative 16S rRNA gene sequence analysis revealed that strain ASL-17T was most closely related phylogenetically to the genus Halobacillus. In the phylogenetic tree based on the neighbour-joining algorithm, strain ASL-17T fell within the radiation of the cluster comprising Halobacillus species, joining the type strain of H. halophilus with a bootstrap confidence value of 90.3 % (Fig. 1). The relationship between strain ASL-17T and the type strain of H. halophilus was also maintained in trees based on the maximum-likelihood and maximum-parsimony algorithms (Fig. 1). Strain ASL-17T exhibited 16S rRNA gene sequence similarity values of 97.7–98.6 % to the type strains of Halobacillus species and of less than 97.1 % to other species used in the phylogenetic analysis. Strain ASL-17T had cell-wall peptidoglycan based on meso-diaminopimelic acid as the diagnostic diamino acid, unlike previously described Halobacillus species, which have cell-wall peptidoglycan based on L-Orn–D-Asp (Spring et al., 1996; Amoozegar et al., 2003; Yoon et al., 2003, 2004, 2005; Liu et al. 2005). The predominant isoprenoid quinone detected in strain ASL-17T was menaquinone-7 (MK-7). The fatty acid profile of strain ASL-17T was composed of the following (each constituting >0.5 % of total fatty acids): branched fatty acids anteiso-C15 : 0 (50.8 %), anteiso-C17 : 0 (21.2 %), iso-C16 : 0 (12.5 %), iso-C15 : 0 (5.1 %), iso-C14 : 0 (3.5 %) and iso-C17 : 0 (2.5 %); unsaturated fatty acid C16 : 1
7c alcohol (1.6 %); straight-chain fatty acids C16 : 0 (1.2 %) and C15 : 0 (0.6 %); and summed feature 4 (iso-C17 : 1 and/or anteiso-C17 : 1, 0.6 %). This fatty acid profile was similar as those of Halobacillus species, although there are differences in the proportions of some fatty acids, probably because of differences in cultivation conditions and extraction procedures (Liu et al., 2005; Yoon et al., 2005). The DNA G+C content of strain ASL-17T was 42.1 mol%. Strain ASL-17T is clearly distinguishable from Halobacillus species by the difference in the cell-wall peptidoglycan type (Table 1). However, the phylogenetic similarities between strain ASL-17T and Halobacillus species may be too great to place strain ASL-17T in a new genus separate from the genus Halobacillus. Moreover, strain ASL-17T does not show noteworthy differences from members of the genus Halobacillus in isoprenoid quinone or fatty acid profiles. Accordingly, it appears to be appropriate that strain ASL-17T be classified in the genus Halobacillus.
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). Strain ASL-17T can be differentiated from recognized Halobacillus species by differences in several phenotypic characteristics and in cell-wall type (Table 1
). The phylogenetic and genetic distinctiveness and differential phenotypic properties are sufficient to allocate strain ASL-17T to a species that is separate from recognized Halobacillus species (Wayne et al., 1987; Table 1). Therefore, on the basis of the data presented, strain ASL-17T should be placed within a novel species of the genus Halobacillus, for which the name Halobacillus campisalis sp. nov. is proposed.
Description of Halobacillus campisalis sp. nov.
Halobacillus campisalis (cam.pi.sa'lis. L. n. campus field; L. gen. n. salis of salt; N.L. gen. n. campisalis of a field of salt).
Cells are cocci or oval (0.7–1.4x1.0–1.6 µm) on MA supplemented with 6 % (w/v) NaCl at 37 °C. Gram-positive, but Gram-variable in old cultures. Colonies are circular, slightly convex, smooth, glistening, light-yellow in colour and 1.5–2.0 mm after 3 days incubation at 37 °C on MA supplemented with 6 % (w/v) NaCl. Mg2+ ions are required for growth. Optimal growth temperature is 37 °C; growth occurs at 4 and 41 °C, but not at 42 °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 approximately 8 % (w/v) NaCl; growth occurs without NaCl and in the presence of 22 % (w/v) NaCl, but not in the presence of >23 % (w/v) NaCl. Tweens 20, 40 and 60, hypoxanthine and xanthine are not hydrolysed. H2S is not produced. In assays with the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8) and 
-galactosidase are present, but lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, 
-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are absent. D-Glucose, D-fructose, D-galactose, D-cellobiose, D-mannose, trehalose, sucrose and maltose are utilized, but L-arabinose, D-xylose, acetate, citrate, succinate, benzoate, L-malate, pyruvate, salicin, formate and L-glutamate are not. Acid is produced from D-cellobiose, lactose, D-mannose, D-melezitose, melibiose, D-raffinose and L-rhamnose, but not from L-arabinose, myo-inositol, D-ribose or D-sorbitol. Susceptible to ampicillin, carbenicillin, cephalothin, chloramphenicol, gentamicin, kanamycin, lincomycin, neomycin, novobiocin, oleandomycin, penicillin G, streptomycin and tetracycline, but not to polymyxin B. The cell-wall peptidoglycan contains meso-diaminopimelic acid as the diamino acid. The predominant menaquinone is MK-7. The major fatty acids (>10 % of total fatty acids) are anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The DNA G+C content of the type strain is 42.1 mol%.
The type strain, ASL-17T (=KCTC 13144T =CCUG 54360T), was isolated from a marine solar saltern of the Yellow Sea in Korea.
Emended description of the genus Halobacillus Spring et al. 1996
The description of the genus Halobacillus is as given by Spring et al. (1996), Yoon et al. (2003, 2004, 2005), Amoozegar et al. (2003) and Liu et al. (2005) with the following amendment. The cell-wall peptidoglycan is based on L-Orn–D-Asp or meso-diaminopimelic acid.
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ACKNOWLEDGEMENTS |
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Yoon, J.-H., Kang, K. H., Oh, T.-K. & Park, Y.-H. (2004). Halobacillus locisalis sp. nov., a halophilic bacterium isolated from a marine solar saltern of the Yellow Sea in Korea. Extremophiles 8, 23–28.[CrossRef][Medline]
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