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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 Institute Co. Ltd, 3-75-1, Heita, Kamaishi, Iwate 026-0001, Japan
Correspondence
Sun-Young An
an12su{at}hotmail.com
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ABSTRACT |
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The major fatty acids of strains 002-048T, RB589 and 002-051T are listed in a supplementary table available in IJSEM Online.
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), described as the genera Halobacillus (Spring et al., 1996), Virgibacillus (Heyndrickx et al., 1998), Gracilibacillus (Wainø et al., 1999), Oceanobacillus (Lu et al., 2001), Lentibacillus (Yoon et al., 2002), Thalassobacillus (García et al., 2005) and Pontibacillus (Lim et al., 2005b). Three isolates with similar characteristics, 002-048T, RB589 and 002-051T, were isolated from field soil in Japan. On the basis of partial 16S rRNA gene sequence analyses, the strains were found to be closely related to members of the genera Oceanobacillus, Halobacillus, Thalassobacillus, Virgibacillus and Gracilibacillus. The aim of the present study was to elucidate the taxonomic position of the isolates, using polyphasic taxonomy (physiological and chemotaxonomic analyses, determination of the fatty acid composition, the quinone system and the G+C content, and 16S rRNA gene sequence analysis). On the basis of these data, it is proposed that the three isolates represent two species within a novel genus, Terribacillus saccharophilus gen. nov., sp. nov. and Terribacillus halophilus sp. nov.
Strains 002-048T, RB589 and 002-051T were obtained using the dilution plating technique on plate-count agar (Merck). 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 activity was determined by means of bubble production from a 3 % H2O2 solution. Oxidase activity was determined by using cytochrome oxidase paper (Nissui Pharmaceutical). The API 20E and API 50CH microtest galleries (bioMérieux) were used to determine the physiological and biochemical characteristics. All suspension media were supplemented with 0.5x Herbst's artificial seawater. Herbst's artificial seawater contains the following (per l 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. The API tests were read after 48 h incubation at 30 °C. The results of the morphological, biochemical and physiological analyses are shown in Tables 1 and 2.
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), and evolutionary distances and Knuc values (Kimura, 1980) were generated. Alignment gaps and ambiguous bases were not taken into consideration when the 1387 bases of the 16S rRNA gene sequence were compared. The phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987). The topology of the phylogenetic tree was evaluated by the bootstrap resampling method of Felsenstein (1985), with 1000 replicates. The similarity values were calculated using MEGA3 (Kumar et al., 2004). Almost-complete 16S rRNA gene sequences of strains 002-048T, RB589 and 002-051T were subjected to comparative analysis. The 16S rRNA gene sequence similarities for strain 002-048T with respect to RB589 and 002-051T were 100 and 99.2 %, respectively. Strains 002-048T and 002-051T exhibited 16S rRNA gene sequence similarity values of 94.3 and 93.6 %, respectively, with respect to Oceanobacillus picturae, their closest phylogenetic relative, and the similarity values with respect to other taxa were less than 93.7 %. 16S rRNA gene sequence similarities of less than 95 % between two bacteria indicate that they belong to different genera (Ludwig et al., 1998). Moreover, the phylogenetic analysis (Fig. 1) indicated that the three isolates formed a strong cluster, with 100 % bootstrap support, representing a distinct lineage. The topologies of phylogenetic trees built using the maximum-likelihood and maximum-parsimony algorithms were similar to those of the tree constructed by neighbour-joining analysis (data not shown).
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, with a multi-well plate reader (CytoFluor; PerSeptive Biosystems). The values for DNA–DNA hybridization between strain 002-048T and the other isolates were 99.9 % (RB589) and 35.4 % (002-051T). Therefore, strains 002-048T and RB589 should be incorporated into a single species, and strain 002-051T should be considered as representing a separate species relative to strain 002-048T (Stackebrandt et al., 2002).
Genomic DNA was prepared according to the method of Marmur (1961), and the DNA G+C content was measured by HPLC according to the method described by Mesbah et al. (1989). The cellular fatty acids were identified with the Sherlock Microbial Identification System (MIDI). Bacterial strains were grown on marine agar 2216 (Difco) medium for 3 days at 25 °C. The analysis of the cell-wall peptidoglycan was carried out using the methods of Schleifer & Kandler (1972). Respiratory quinone analyses were performed according to the method indicated by Collins & Jones (1981). The chemotaxonomic characteristics determined were also congruent with the phylogenetic data. The DNA G+C contents of 002-048T, RB589 and 002-051T ranged from 44 to 46 mol%. Similar values were found in other low-G+C, Gram-positive bacteria. The major fatty acids of the novel strains were anteiso-C15 : 0 (47.6–63.5 %) and anteiso-C17 : 0 (17.9–34.8 %) (see Supplementary Table S1 in IJSEM Online). The two type strains, 002-048T and 002-051T, can be differentiated from each other by the amounts of anteiso-C15 : 0 and anteiso-C17 : 0 present (Supplementary Table S1 in IJSEM Online). The cell-wall peptidoglycan of strains 002-048T and 002-051T was based on meso-diaminopimelic acid, which corresponds with that of the phylogenetically closest neighbours (with the exception of members of the genus Halobacillus, which contain D-ornithine). The major isoprenoid quinone was MK-7, as for the other phylogenetically related genera.
The three isolates showed very low levels of 16S rRNA gene sequence similarity with respect to other taxa and formed a distinct lineage. Moreover, the isolates could be differentiated from their phylogenetically related neighbours on the basis of phenotypic characteristics (Table 1
), such as motility, acetoin production, oxidase activity and nitrate reduction. The DNA G+C contents of the isolates were in the range 44–46 mol%, which is 4 % lower than the values for members of the genera Virgibacillus (36–40 mol%), Lentibacillus (37–38 mol%) and Gracilibacillus (38–39 mol%). The diamino acid in the murein of the isolates is meso-diaminopimelic acid (as opposed to ornithine for members of the genus Halobacillus). The cells of the isolates are non-motile, whereas most of the phylogenetically related genera (except Thalassobacillus) comprise members that are motile. The isolates reduce nitrates, but the members of the genus Thalassobacillus do not.
On the basis of the phylogenetic, physiological and chemotaxonomic data, therefore, strains 002-048T, RB589 and 002-051T belong to two species within a novel genus, for which the names Terribacillus saccharophilus gen. nov., sp. nov. (strains 002-048T and RB589) and Terribacillus halophilus sp. nov. (strain 002-051T) are proposed.
Description of Terribacillus gen. nov.
Terribacillus [Ter.ri.ba.cil'lus. L. n. terra earth; L. masc. n. bacillus a small staff; N.L. masc. n. Terribacillus earth (soil) bacillus (rod)].
Cells are non-motile, Gram-positive, aerobic rods. Ellipsoidal endospores are formed subterminally within swollen sporangia. Colonies are circular and convex. Catalase-positive. H2S and indole are not produced. Nitrate is not reduced to nitrite. Gelatin is liquefied. Acetoin is produced. Urease and 
-galactosidase are absent. The DNA G+C content is 44–46 mol%. The major cellular fatty acids are anteiso-C15 : 0 and anteiso-C17 : 0. MK-7 is the major component of the quinone system. The type species of the genus is Terribacillus saccharophilus.
Description of Terribacillus saccharophilus sp. nov.
Terribacillus saccharophilus (sac.cha.ro.phi'lus. Gr. n. sakkhar -aros sugar; Gr. adj. philos loving; N.L. masc. adj. saccharophilus sugar-loving).
The characteristics are the same as those given in the description of the genus, with the following additions. Cells are 0.7–1.0x2.3–4.8 µm in size. Colonies grown on trypticase soy agar containing 0.5x Herbst's artificial seawater are circular, convex and pale yellow. Temperature range for growth is 5–45 °C; pH range for growth is 6.0–10.0. Growth occurs without NaCl and in the presence of 16 % (w/v) NaCl. Optimum NaCl concentration for growth is 1–5 % (w/v). Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. Citrate utilization is negative. Utilizes glycerol, galactose, glucose, fructose, mannose, rhamnose, mannitol, N-acetylglucosamine, amygdalin, arbutin, aesculin, salicin, cellobiose, lactose, melibiose, sucrose, trehalose, raffinose, gentiobiose and D-tagatose, but does not utilize erythritol, D-arabinose, L-arabinose, ribose, D-xylose, L-xylose, adonitol, methyl -D-xyloside, sorbose, dulcitol, inositol, sorbitol, methyl 
-D-mannoside, methyl -D-glucoside, maltose, inulin, melezitose, starch, glycogen, xylitol, D-turanose, D-lyxose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate. Acid is produced from glucose, mannitol and sucrose, but not from inositol, sorbitol, rhamnose, melibiose, amygdalin or arabinose. The cell-wall peptidoglycan contains meso-diaminopimelic acid. The predominant menaquinone system is MK-7. The major cellular fatty acids are anteiso-C15 : 0 and anteiso-C17 : 0. The DNA G+C content of the type strain is 44.0 mol%.
The type strain, 002-048T (=IAM 15309T=KCTC 13936T), was isolated from field soil from Tama City (Tokyo, Japan). The reference strain, RB589, was isolated from field soil in Saitama City (Japan).
Description of Terribacillus halophilus sp. nov.
Terribacillus halophilus (ha.lo.phi'lus. Gr. n. hals salt; Gr. adj. philos loving; N.L. masc. adj. halophilus salt-loving).
The characteristics are the same as those given in the description of the genus, with the following additions. Cells are 0.6–0.8x2.8–4.8 µm in size. Colonies grown on trypticase soy agar containing 0.5x Herbst's artificial seawater are circular, convex and pale yellow. Temperature range for growth is 5–45 °C; pH range for growth is 5.0–10.0. Growth occurs without NaCl and in the presence of 19 % (w/v) NaCl. Optimum NaCl concentration for growth is 1–5 % (w/v). Negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase and citrate utilization. Utilizes glycerol, L-arabinose, glucose, fructose, mannose, rhamnose, mannitol, N-acetylglucosamine, amygdalin, arbutin, aesculin, salicin, cellobiose, lactose, sucrose, gentiobiose and D-tagatose but does not utilize erythritol, D-arabinose, ribose, D-xylose, L-xylose, adonitol, methyl -D-xyloside, galactose, sorbose, dulcitol, inositol, sorbitol, methyl -D-mannoside, methyl -D-glucoside, maltose, melibiose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, D-turanose, D-lyxose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate. Acid is produced from mannitol, but not from glucose, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin or arabinose. The cell-wall peptidoglycan contains meso-diaminopimelic acid. The major quinone system is MK-7. The major cellular fatty acids are anteiso-C15 : 0 and anteiso-C17 : 0. The DNA G+C content of the type strain is 45.8 mol%.
The type strain, 002-051T (=IAM 15310T=KCTC 13937T), was isolated from field soil in Tama City (Tokyo, Japan).
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Collins, M. D. & Jones, D. (1981). Distribution of isoprenoid quinone structural types in bacteria and their taxonomic implications. Microbiol Rev 45, 316–354.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in micro-dilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]
García, M. T., Gallego, V., Ventosa, A. & Mellado, E. (2005). Thalassobacillus devorans gen. nov., sp. nov., a moderately halophilic, phenol-degrading, Gram-positive bacterium. Int J Syst Evol Microbiol 55, 1789–1795.
Heyndrickx, M., Lebbe, L., Kersters, K., De Vos, P., Forsyth, C. & Logan, N. A. (1998). Virgibacillus: a new genus to accommodate Bacillus pantothenticus (Proom and Knight 1950). Emended description of Virgibacillus pantothenticus. Int J Syst Bacteriol 48, 99–106.
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]
Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2004). MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.
Lee, J. S., Lim, J. M., Lee, K. C., Lee, J. C., Park, Y. H. & Kim, C. J. (2006). Virgibacillus koreensis sp. nov., a novel bacterium from a salt field, and transfer of Virgibacillus picturae comb. nov. with emended descriptions. Int J Syst Evol Microbiol 56, 251–257.
Lim, J. M., Jeon, C. O., Park, D. J., Kim, H. R., Yoon, B. J. & Kim, C. J. (2005a). Pontibacillus marinus sp. nov., a moderately halophilic bacterium from a solar saltern, and emended description of the genus Pontibacillus. Int J Syst Evol Microbiol 55, 1027–1031.
Lim, J. M., Jeon, C. O., Song, S. M. & Kim, C. J. (2005b). Pontibacillus chungwhensis gen. nov., sp. nov., a moderately halophilic Gram-positive bacterium from a solar saltern in Korea. Int J Syst Evol Microbiol 55, 165–170.
Lu, J., Nogi, Y. & Takami, H. (2001). Oceanobacillus iheyensis gen. nov., sp. nov., a deep-sea extremely halotolerant and alkaliphilic species isolated from a depth of 1050 m on the Iheya Ridge. FEMS Microbiol Lett 205, 291–297.[CrossRef][Medline]
Ludwig, W., Strunk, O., Klugbauer, S., Klugbauer, N., Weizenegger, M., Neumaier, J., Bachleitner, M. & Schleifer, K.-H. (1998). Bacterial phylogeny based on comparative sequence analysis. Electrophoresis 19, 554–568.[CrossRef][Medline]
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J Mol Biol 3, 208–218.
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36, 407–477.
Spring, S., Ludwig, W., Marquez, M. C., Ventosa, A. & Schleifer, K. H. (1996). Halobacillus gen. nov., with descriptions of Halobacillus litoralis sp. nov. and Halobacillus trueperi sp. nov., and transfer of Sporosarcina halophila to Halobacillus halophilus comb. nov. Int J Syst Bacteriol 46, 492–496.
Stackebrandt, E., Frederiksen, W., Garrity, G. M., Grimont, P. A. D., Kämpfer, P., Maiden, M. C. J., Nesme, X., Rossello-Mora, R., Swings, J. & other authors (2002). Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52, 1043–1047.[Abstract]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Wainø, M., Tindall, B. J., Schumann, P. & Ingvorsen, K. (1999). Gracilibacillus gen. nov., with description of Gracilibacillus halotolerans gen. nov., sp. nov.; transfer of Bacillus dipsosauri to Gracilibacillus dipsosauri comb. nov., and Bacillus salexigens to the genus Salibacillus gen. nov., as Salibacillus salexigens comb. nov. Int J Syst Bacteriol 49, 821–831.
Yoon, J. H., Kang, K. H. & Park, Y. H. (2002). Lentibacillus salicampi gen. nov., sp nov., a moderately halophilic bacterium isolated from a salt field in Korea. Int J Syst Evol Microbiol 52, 2043–2048.[Abstract]
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