Bacillus Persepolensis sp. nov., a Moderately Halophilic Bacterium from a Hypersaline Lake

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published in INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY on 2009, available at: https://doi.org/10.1099/ijs.0.010090-0.” 1 2 Bacillus persepolensis sp. nov., a moderately halophilic bacterium from a 3 hypersaline lake in Iran 4 5 M. A. Amoozegar1, C. Sánchez-Porro2, R. Rohban1, M. Hajighasemi1 and A. 6 Ventosa2 7 8 1Extremophiles Lab., Department of Microbiology, Faculty of Biology, College of 9 Science, University of Tehran, Tehran, Iran. 10 2Department of Microbiology and Parasitology, Faculty of Pharmacy, University of 11 Sevilla, 41012 Sevilla, Spain 12 13 14 Running title: Bacillus persepolensis sp. nov. 15 Subject category: New taxa, Other Gram-positive bacteria 16 17 Author for correspondence: Antonio Ventosa, Department of Microbiology and 18 Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain. Tel. +34 19 95 455 6765; Fax +34 95 462 81 62. E-mail: [email protected] 20 21 The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of 22 strain HS136T is FM244839. 23 24 25 26 2 A Gram-positive, moderately halophilic, endospore-forming bacterium, designated 1 strain HS136T, was isolated from the hypersaline lake Howz-Soltan in Iran. Cells 2 were motile rods, producing ellipsoidal endospores at a central-subterminal 3 position in non-swollen sporangia. Strain HS136T, a strictly aerobic bacterium, 4 grew between pH 7.0 and 10.0 (optimal growth at pH 8.0-8.5), between 25 and 5 45ºC (optimal growth at 40ºC) and at salinities of 5 to 20% (w/v) NaCl, growing 6 optimally at 10% (w/v) NaCl. On the basis of 16S rRNA gene sequence analysis, 7 strain HS136T was shown to belong to the genus Bacillus within the phylum 8 Firmicutes and showed the closest phylogenetic similarity with the species Bacillus 9 salarius BH169T (95.2%) and Bacillus qingdaonensis CM1T (94.5%). The DNA 10 G+C content of this new isolate is 37.1 mol%. The major cellular fatty acids of 11 strain HS136T were iso-C15:0, anteiso-C15:0, and anteiso-C17:0, and its polar lipid 12 pattern consisted of phosphatidylglycerol and diphosphatidylglycerol. The 13 isoprenoid quinone was MK-7. The peptidoglycan type is A1γ, with meso- 14 diaminopimelic acid as the diagnostic diamino acid. On the basis of polyphasic 15 evidence from this study, a new species of the genus Bacillus, Bacillus persepolensis 16 sp. nov. is proposed, with strain HS136T (=CCM 7595T =DSM 21632T =JCM 17 15720T =LMG 24785T) as the type strain. 18 _______________________________________________________ 19 Endospore-forming moderately halophilic Gram-positive rods are frequently isolated 20 from saline and hypersaline environments such as soils and aquatic habitats (Arahal and 21 Ventosa, 2002; Ventosa, 2006). They are characterized by their optimal growth in 22 media containing 3 to 15% (w/v) NaCl (Ventosa et al. 1998). They belong to species 23 included in the genus Bacillus, as well as Bacillus-related genera. The moderately 24 halophilic species belonging to the genus Bacillus are: B. aurantiacus (Borsodi et al., 25 2008), B. chagannorensis (Carrasco et al., 2007a), B. coahuilensis (Cerritos et al., 26 2008), B. isabeliae (Albuquerque et al., 2008), B. oshimensis (Yumoto et al., 2005), B. 27 polygoni (Aino et al., 2008), B. qingdaonensis (Wang et al., 2007), B. salarius (Lim et 28 al., 2006c), and B. taeanensis (Lim et al., 2006a). Other genera phylogenetically related 29 to the genus Bacillus that include moderately halophilic species are: Alkalibacillus (Jeon 30 et al., 2005; Usami et al., 2007), Aquisalibacillus (Márquez et al., 2008), Filobacillus 31 (Schlesner et al., 2001), Gracilibacillus (Wainø et al., 1999; Carrasco et al., 2006; Jeon 32 et al., 2008), Halalkalibacillus (Echigo et al., 2007) Halobacillus (Spring et al., 1996; 33 Amoozegar et al., 2003; Yoon et al., 2003; Liu et al., 2005; Yoon et al., 2005; Hua et 34 al., 2007; Yoon et al., 2007a; An et al., 2007b; Soto-Ramírez et al., 2008; Yoon et al., 35 2008; Romano et al., 2008), Halolactibacillus (Cao et al., 2008), Lentibacillus (Yoon et 36 al., 2002; Namwong et al., 2005; Pakdeeto et al., 2007; Lee et al., 2008), Marinibacillus 37 (Yoon et al., 2004a), Oceanobacillus (Lee et al., 2006), Piscibacillus (Tanasupawat et 38 al., 2007), Pontibacillus (Lim et al., 2005), Salimicrobium (Ventosa et al, 1990; Yoon et 39 al., 2007b), Salinibacillus (Ren and Zhou, 2005b), Salsuginibacillus (Carrasco et al., 40 2007b), Sediminibacillus (Carrasco et al., 2008), Tenuibacillus (Ren and Zhou, 2005a), 41 Thalassobacillus (García et al., 2005), or Virgibacillus (Garabito et al., 1997; Arahal et 42 al., 1999; Heyrman et al., 2003; Yoon et al., 2004b; Lee et al., 2006; An et al., 2007a; 43 Wang et al., 2008; Chen et al., 2008; Hua et al., 2008). This paper describes a novel 44 4 endospore-forming moderately halophilic Gram-positive bacterium, strain HS136T, 45 isolated from the hypersaline lake Howz-Soltan in the center of Iran. 46 47 Strain HS136T was isolated from water of the hypersaline lake Howz-Soltan, which is 48 located near Qom city in the center of Iran, with an area of about 240 and 280 square 49 kilometers in dry and wet seasons, respectively. The depth of salt layer which covers 50 almost all the surface of the playa varies between 20-46 meters and the pH of the water, 51 saline soil and salt sediments differs between 6.5 and 8.2. Major chemical composition 52 of the soil, brine, mud and salt consists of NaCl, KCl, MgSO4, MgCl2 and Na2SO4. At 53 the time of sampling, the temperature of the water was 32°C and the pH 7.6. Strain 54 HS136T was isolated during studies focused on the determination of the biodiversity of 55 the lake and constituted a single isolate of the total population. It was isolated by 56 diluting a water sample in sterile 10% (w/v) salt solution, plating on 10% HM medium 57 and incubating at 37ºC aerobically. The 10% HM medium contained (g l-1): NaCl, 81; 58 MgCl2, 7; MgSO4, 9.6; CaCl2, 0.36; KCl, 2; NaHCO3, 0.06; NaBr, 0,026; proteose- 59 peptone no. 3,5; yeast extract, 10, and glucose, 1. (Ventosa et al., 1982). The pH of this 60 medium was adjusted to pH 7.5. The strain was subsequently purified three times by 61 plating on the same medium and maintained on the same HM medium and at -80ºC on 62 this medium without agar and supplemented with 30% (v/v) glycerol. In order to 63 phenotypically characterize strain HS136T, standard phenotypic tests were performed. 64 Cell morphology was examined by light microscopy (model CX 31 2; Olympus) using 65 cells from exponentially growing cultures. Gram staining was performed by the Burke 66 method (Murray et al., 1994) and the result was confirmed by the KOH test (Baron & 67 Finegold, 1990). The presence of endospores was investigated by using the Schaeffer- 68 Fulton staining method (Murray et al., 1994). Motility was analysed by the wet-mount 69 method (Murray et al., 1994). Catalase, oxidase and urease activities, nitrate reduction, 70 5 hydrolysis of aesculin and production of indole were tested as recommended by Smibert 71 & Krieg (1994). Hydrolysis of Tween-80 was examined as described by Harrigan & 72 McCance (1976). Methyl red and Voges–Proskauer tests were done as recommended by 73 Smibert & Krieg (1994). Determination of acid production from carbohydrates, as well 74 as utilization of carbon and nitrogen sources, was performed as recommended by 75 Ventosa et al. (1982). Antibiotic susceptibility tests were performed on Mueller–Hinton 76 agar plus 10% (w/v) sea salts (Ventosa et al., 1982) seeded with a bacterial suspension 77 containing 1.5 ×106 c.f.u. ml-1 using discs (HiMedia) impregnated with various 78 antibiotics. The plates were incubated at 37°C for 48 h and the inhibition zone was 79 interpreted according the manufacturer’s manual. To determine the optimal temperature 80 and pH for growth of the strain, broth cultures were incubated at temperatures of 10–50 81 °C at intervals of 5°C and at pH 5–11 at intervals of 0.5 pH units. pH values below 6, 82 pH 6–9 and pH values above 9 were obtained using sodium acetate/acetic acid, 83 Tris/HCl and glycine/sodium hydroxide buffers, respectively. Growth at different salt 84 contents (0, 2.5, 5, 7.5, 10, 15, 20, 25 and 30%, w/v) was tested on HM medium at pH 85 8.0. Growth was monitored by turbidity at OD600 using a spectroscopic method (model 86 UV-160 A; Shimadzu). Other physiological and biochemical tests were performed as 87 described previously (Mata et al., 2002; Quesada et al., 1984; Ventosa et al., 1982). 88 89 Strain HS136T was Gram-positive, motile and strictly aerobic. Cells were rods with a 90 width of 1.0–1.5 µm and length of 2.5–3.0 µm. Ellipsoidal endospores were formed at 91 the central to subterminal position in non-swollen sporangia. When grown for 2 days at 92 37 ºC on 10% HM medium, the colonies were circular, entire, smooth, cream and with a 93 diameter of 2 mm. This isolate was moderately halophilic and slightly alkaliphilic, 94 growing in media containing 5 to 20% (w/v) NaCl and optimally in media containing 95 10% (w/v) NaCl. No growth was observed in the absence of NaCl. Strain HS136T grew 96 6 between pH 7.0 and 10.0, and optimally in media with pH 8.0-8.5. Other phenotypic 97 features are included in the species description. 98 99 Genomic DNA from strain HS136T was prepared using the method described by 100 Marmur (1961). The 16S rRNA gene was amplified by PCR with the forward primer 101 16F27 and the reverse primer 16R1488. Direct sequence determination of the PCR- 102 amplified DNA was carried out using an automated DNA sequencer model ABI 103 3130XL (Applied Biosystems). The 16S rRNA gene sequence analysis was performed 104 with the ARB software package (Ludwig et al., 2004). The 16S rRNA gene sequence 105 was aligned with the published sequences of the closely related bacteria and the 106 alignment was confirmed and checked against both primary and secondary structures of 107 the 16S rRNA molecule using the alignment tool of the ARB software package. 108 Phylogenetic trees were constructed using three different methods: maximum-likelihood 109 (Felsenstein, 1981), maximum-parsimony (Fitch, 1971) and neighbour-joining (Saitou 110 & Nei, 1987), algorithms integrated in the ARB software for phylogenetic inference. 111 The 16S rRNA gene sequences used for phylogenetic comparisons were obtained from 112 the GenBank database and their strain designations and accession numbers are shown in 113 Figure 1. 114 115 Almost-complete 16S rRNA gene sequence (1474 bp) of strain HS136T was obtained 116 and used for initial BLAST searches in GenBank and phylogenetic analysis. The 117 identification of phylogenetic neighbours and calculation of pairwise 16S rRNA gene 118 sequence similarity were achieved using the EzTaxon server (http://www.eztaxon.org/; 119 Chun et al., 2007). The phylogenetic analysis, based on the maximum-parsimony 120 algorithm, revealed that strain HS136T formed a phyletic group with Bacillus salarius 121 BH169T (95.2% similarity) and Bacillus qingdaonensis CM1T (94.5%) (Fig. 1). The 122 7 16S rRNA sequence similarity with other Bacillus species is equal or lower than 94.5%. 123 Neighbour-joining and maximum likelihood methods resulted in highly similar tree 124 topologies and only maximum-parsimony results are shown. Based on the sequence 125 divergence, it was evident that strain HS136T constituted a different taxon separated 126 from other Bacillus species. DNA-DNA hybridization between strain HS136T and the 127 nearest phylogenetic neighbours was not attempted since strains differing by more than 128 3% of the 16S rRNA gene sequence are unlikely to exhibit more than 70% relatedness 129 at the whole-genome level (Stackebrandt & Goebel, 1994; Stackebrandt et al., 2002; 130 Stackebrandt & Ebers, 2006). 131 132 The G+C content of the genomic DNA was determined from the midpoint value of the 133 thermal denaturation profile (Marmur & Doty, 1962) using the equation of Owen & Hill 134 (1979). The G+C content of the DNA of strain HS136T was 37.1 mol%. This value is 135 within the range for Bacillus but is lower than those of B. salarius and B. 136 quingdaonensis (Table 1). 137 138 Fatty acids analysis was performed using the MIDI system (Microbial Identification 139 System). Cells were cultured on the marine agar containing 10% (w/v) NaCl at 30ºC for 140 48 h. The extraction and the analysis were performed conform to the 141 recommentadations of the MIDI system. This analysis was carried out by the 142 Identification Service of the Belgian Co-ordinated Collections of Microorganisms, 143 Laboratory of Microbiology of Gent (BCCM/LMG), Gent, Belgium. The cellular fatty 144 acid profile of strain HS136T was characterized by the fatty acids iso-C15:0 (41.6%) and 145 anteiso-C15:0 (35.8%) as the major fatty acids (Table 2). The fatty acid composition is 146 similar to those found in Bacillus salarius BH169T and Bacillus qingdaonensis CM1T, 147 except that iso-C15:0 was lower ans anteiso-C17:0 higher for strain HS136Tthan for these 148 8 two Bacillus species. In order to complete the chemotaxonomic characterization of 149 strain HS136T the analysis of polar lipids, quinones and peptidoglycan of the cell wall 150 was carried out by the Identification Service of the DSMZ (Braunschweig, Germany). 151 The polar lipids detected were phosphatidylglycerol and diphosphatidylglycerol. Strain 152 HS136T contained MK-7 as the unique menaquinone and a cell wall type based on 153 meso-diaminopimelic acid. The respiratory lipoquinone, polar lipids and peptidoglycan 154 type of the cell wall of strain HS136T were typical of those found in members of the 155 genus Bacillus (Priest et al., 1988; Heyrman et al., 2004, 2005; Wieser et al., 2005; Lim 156 et al., 2006a, 2006b, 2006c). 157 158 The characteristics that differentiate strain HS136T from other related Bacillus species 159 are summarized in Table 1. The differences in some features, such as endospore 160 position and shape, motility, optimum temperature, acid production from carbohidrates 161 as well as the genomic DNA G+C content or fatty acids composition can be used to 162 distinguish this strain from the phylogenetically related taxa (Tables 1 and 2). Therefore, 163 on the basis of these polyphasic taxonomic data, we propose that the novel strain 164 represents a new species of the genus Bacillus, for which the new name B. 165 persepolensis sp. nov. is proposed. 166 167 Description of Bacillus persepolensis sp. nov. 168 Bacillus persepolensis (per. se. po. len’ sis. N.L. mac. adj. Persepolensis, related to the 169 ancient city Persepolis). 170 Cells are Gram-positive, motile, endospore-forming rods, 1.0–1.5 by 2.5–3.0 µm in size. 171 Ellipsoidal endospores are produced at a central-subterminal position in non-swollen 172 sporangia. Colonies are circular, entire, smooth, cream, with 2 mm in diameter on 10% 173 HM agar medium after 48 h of incubation at 37 ºC. Strictly aerobic. Slightly 174 15 Lee, J.S., Lim, J.M., Lee, K.C., Lee, J.C., Park, Y.H. & Kim, C.J. (2006). Virgibacillus 307 koreensis sp. nov., a novel bacterium from a salt field, and transfer of Virgibacillus picturae to 308 the genus Oceanobacillus as Oceanobacillus picturae comb. nov. with emended descriptions. 309 Int J Syst Evol Microbiol 56, 251-257. 310 Lim, J.-M., Jeon, C. O. & Kim, C.–J. (2006a). Bacillus taeanensis sp. nov., a halophilic 311 Gram-positive bacterium from a solar saltern in Korea. Int J Syst Evol Microbiol 56, 2903-2908. 312 Lim, J.-M., Jeon, C. O., Lee, J. C., Ju, Y. J., Park, D. J. & Kim, C.–J. (2006b). Bacillus 313 koreensis sp. nov., a spore-forming bacterium, isolated from the rhizosphere of willow roots in 314 Korea. Int J Syst Evol Microbiol 56, 59-63. 315 Lim, J.-M., Jeon, C. O., Lee, S.-M., Xu, L.H., Jiang, C.-L., & Kim, C.–J. (2006c). Bacillus 316 salarius sp. nov., a halophilic, spore-forming bacterium isolated from a salt lake in China. Int J 317 Syst Evol Microbiol 56, 373-377. 318 Lim, J.M., Jeon, C.O., Song, S.M. & Kim, C.J. (2005). Pontibacillus chungwhensis gen. nov., 319 sp. nov., a moderately halophilic Gram-positive bacterium from a solar saltern in Korea. Int J 320 Syst Evol Microbiol 55, 165-170. 321 Liu WY, Zeng J, Wang L, Dou YT, Yang SS. (2005). Halobacillus dabanensis sp. nov. and 322 Halobacillus aidingensis sp. nov., isolated from salt lakes in Xinjiang, China. 323 Int J Syst Evol Microbiol 55, 1991-1996. 324 Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar, Buchner, A., 325 Lai, T., Steppi, S., Jobb, G. & 22 other authors (2004). ARB: a software environment for 326 sequence data. Nucleic Acids Res 32, 1363–1371. 327 Mata, J. A., Martinez-Canovas, J., Quesada, E. & Bejar, V. (2002). A detailed phenotypic 328 characterization of the type strains of Halomonas species. Syst Appl Microbiol 25, 360–375. 329 16 Márquez, M.C., Carrasco, I.J., Xue, Y., Ma, Y., Cowan, D.A., Jones, B.E., Grant, W.D. & 330 Ventosa, A. (2008). Aquisalibacillus elongatus gen. nov., sp. nov., a moderately halophilic 331 bacterium of the family Bacillaceae isolated from a saline lake. Int J Syst Evol Microbiol 58, 332 1922-1926. 333 Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from micro-organisms. 334 J Molec Biol 3, 208-218. 335 Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid 336 from its thermal denaturation temperature. J Mol Biol 5, 109-118. 337 Murray, R. G. E., Doetsch, R. N. & Robinow, C. F. (1994). Determinative and cytological 338 light microscopy. In Methods for General and Molecular Bacteriology, pp. 21–41. Edited by P. 339 Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for 340 Microbiology. 341 Namwong, S., Tanasupawat, S., Smitinont, T., Visessanguan, W., Kudo, T. & Itoh, T., 342 (2005). Isolation of Lentibacillus salicampi strains and Lentibacillus juripiscarius sp. nov. from 343 fish sauce in Thailand. Int J Syst Evol Microbiol 55, 315-320. 344 Owen, R. J. & Hill, L. R. (1979). The estimation of base compositions, base pairing and genome 345 size of bacterial deoxyribonucleic acids. In Identification Methods for Microbiologists, 2nd edn, pp. 346 217-296. Edited by F. A. Skinner & D. W. Lovelock. London: Academic Press. 347 Pakdeeto, A., Tanasupawat, S., Thawai, C., Moonmangmee, S., Kudo, T. & Itoh, T. (2007). 348 Lentibacillus kapialis sp. nov., from fermented shrimp paste in Thailand. 349 Int J Syst Evol Microbiol 57, 364-369. 350 Priest, F. G., Goodfellow, M. & Todd, C. (1988). A numerical classification of the genus 351 Bacillus. J Gen Microbiol 134, 1847-1882. 352 17 Quesada, E., Ventosa, A., Ruiz-Berraquero, F. & Ramos-Cormenzana, A. (1984). Deleya 353 halophila, a new species of moderately halophilic bacteria. Int J Syst Bacteriol 34, 287–292. 354 Ren, P.G. & Zhou, P.J. (2005a). Tenuibacillus multivorans gen. nov., sp. nov., a moderately 355 halophilic bacterium isolated from saline soil in Xin-Jiang, China. Int J Syst Evol Microbiol 55, 356 95-99. 357 Ren, P.G. & Zhou, P.J. (2005b). Salinibacillus aidingensis gen. nov., sp. nov. and 358 Salinibacillus kushneri sp. nov., moderately halophilic bacteria isolated from a neutral saline 359 lake in Xin-Jiang, China. Int J Syst Evol Microbiol 55, 949-953. 360 Romano, I., Finore, I., Nicolaus, G., Huertas, F.J., Lama, L., Nicolaus, B. & Poli, A. (2008). 361 Halobacillus alkaliphilus sp. nov., a halophilic bacterium isolated from a salt lake in Fuente de 362 Piedra, southern Spain. Int J Syst Evol Microbiol 58, 886-890. 363 Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing 364 phylogenetic trees. Mol Biol Evol 4, 406–425. 365 Schlesner, H., Lawson, P. A., Collins, M. D., Weiss, N., Wehmeyer, U., Völker, H. & 366 Thomm, M. (2001). Filobacillus milensis gen. nov., sp. nov., a new halophilic spore-forming 367 bacterium with Orn-D-Glu-type peptidoglycan. Int J Syst Evol Microbiol 51, 425-431. 368 Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization In Methods for General 369 and Molecular Bacteriology, pp. 607–654. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood 370 & N. R. Krieg. Washington, DC: American Society for Microbiology. 371 Soto-Ramírez, N., Sánchez-Porro, C., Rosas-Padilla, S., Almodóvar, K., Jiménez, G., 372 Machado-Rodríguez, M., Zapata, M., Ventosa, A. & Montalvo-Rodríguez, R. (2008). 373 Halobacillus mangrovi sp. nov., a moderately halophilic bacterium isolated from the black 374 mangrove Avicennia germinans. Int J Syst Evol Microbiol 58, 125-130. 375 18 Spring, S., Ludwig, W., Marquez, M. C., Ventosa, A. & Schleifer, K.-H. (1996). 376 Halobacillus gen. nov., with descriptions of Halobacillus litoralis sp. nov. and Halobacillus 377 trueperi sp. nov., and transfer of Sporosarcina halophila to Halobacillus halophilus comb. nov. 378 Int J Syst Bacteriol 46, 492-496. 379 Stackebrandt, E. & Ebers, J. (2006). Taxonomic parameters revisited: tarnished gold 380 standards. Microbiol Today 33, 152-155. 381 Stackebrandt, E., Fredericksen, W., Garrity, G.M., Grimont, P.A.D., Kampfer, P., 382 Maiden, M.C.J., Nesme, X., Rossello-Mora, R., Swings, J. & 4 other authors (2002). Report 383 of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst 384 Evol Microbiol 52, 1043-1047. 385 Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA 386 reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. 387 Int J Syst Bacteriol 44, 846-849. 388 Tanasupawat, S., Namwong, S., Kudo, T. & Itoh, T. (2007). Piscibacillus salipiscarius gen. 389 nov., sp. nov., a moderately halophilic bacterium from fermented fish (pla-ra) in Thailand. Int J 390 Syst Evol Microbiol 57, 1413-1417. 391 Usami, R., Echigo, A., Fukushima, T., Mizuki, T., Yoshida, Y. & Kamekura, M. (2007). 392 Alkalibacillus silvisoli sp. nov., an alkaliphilic moderate halophile isolated from non-saline 393 forest soil in Japan. Int J Syst Evol Microbiol 57, 770-774. 394 Ventosa, A. (2006). Unusual micro-organisms from unusual habitats: hypersaline 395 environments. In Prokaryoticdiversity: Mechanisms and significance, pp. 223-253. Edited by 396 N.A. Logan, H.M. Lappin-Scott & P.C.F. Oyston. Cambridge: Cambridge University Press. 397 Ventosa, A., García, M.T., Kamekura, M., Onishi, H. & Ruiz-Berraquero, F. (1990). 398 Bacillus halophilus sp. nov., a moderately halophilic Bacillus species. Syst Appl Microbiol 12, 399 162-166. 400 19 Ventosa, A., Quesada, E., Rodriguez-Valera, F., Ruiz-Berraquero, F. & Ramos- 401 Cormenzana, A. (1982). Numerical taxonomy of moderately halophilic Gram-negative rods. J 402 Gen Microbiol 128, 1959–1968. 403 Ventosa, A., Nieto, J. J. & Oren, A. (1998). Biology of moderately halophilic aerobic bacteria. 404 Microbiol Mol Biol Rev 62, 504–544. 405 Wainø, M., Tindall, B.J., Schumann, P. & Ingvorsen, K. (1999). Gracilibacillus gen. nov., 406 with description of Gracilibacillus halotolerans gen. nov., sp. nov.; transfer of Bacillus 407 dipsosauri to Gracilibacillus dipsosauri comb. nov., and Bacillus salexigens to the genus 408 Salibacillus gen. nov., as Salibacillus salexigens comb. nov. Int J Syst Evol Microbiol 49, 821- 409 831. 410 Wang, C.Y., Chang, C.C., Ng, C.C., Chen, T.W. & Shyu, Y.T. (2008). Virgibacillus 411 chiguensis sp. nov., a novel halophilic bacterium isolated from Chigu, a previously commercial 412 saltern located in southern Taiwan. Int J Syst Evol Microbiol 58, 341-345. 413 Wang, Q.F., Li, W., Liu, Y.L., Cao, H.H., Li, Z. & Guo G.Q. (2007). Bacillus qingdaonensis 414 sp. nov., a moderately haloalkaliphilic bacterium isolated from a crude sea-salt sample collected 415 near Qingdao in eastern China. Int J Syst Evol Microbiol 57, 1143-1147. 416 Wieser, M., Worliczek, H., Kämpfer, P. & Busse, H.-J. (2005). Bacillus herbersteinensis sp. 417 nov. Int J Syst Evol Microbiol 55, 2119-2123. 418 Yoon, J.H., Kang, S.J., Jung, Y.T. & Oh,T.K. (2007a). Halobacillus campisalis sp. nov., 419 containing meso-diaminopimelic acid in the cell-wall peptidoglycan, and emended description 420 of the genus Halobacillus. Int J Syst Evol Microbiol 57, 2021-2025. 421 Yoon J.H., Kang S.J., Lee C.H., Oh H.W. & Oh T.K. (2005). Halobacillus yeomjeoni sp. 422 nov., isolated from a marine solar saltern in Korea. Int J Syst Evol Microbiol 55, 2413-2417. 423 Yoon, J.H., Kang, S.J. & Oh, T.K. (2007b). Reclassification of Marinococcus albus Hao et al. 424 1985 as Salimicrobium album gen. nov., comb. nov. and Bacillus halophilus Ventosa et al. 1990 425 20 as Salimicrobium halophilum comb. nov., and description of Salimicrobium luteum sp. nov. Int 426 J Syst Evol Microbiol 57, 2406-2411. 427 Yoon, J.H., Kang, S.J. & Oh, T.K. (2008). Halobacillus seohaensis sp. nov., isolated from a 428 marine solar saltern in Korea. Int J Syst Evol Microbiol 58, 622-627. 429 Yoon, J.H., Kang, K.H. & Park, Y.H. (2002). Lentibacillus salicampi gen. nov., sp. nov., a 430 moderately halophilic bacterium isolated from a salt field in Korea. Int J Syst Evol Microbiol 431 52, 2043-2048. 432 Yoon, J.H., Kang, K.H. & Park, Y.H. (2003). Halobacillus salinus sp. nov., isolated from a 433 salt lake on the coast of the East Sea in Korea. Int J Syst Evol Microbiol 53, 687-693. 434 Yoon, J.H., Kim, I.G., Schumann, P., Oh, T.K., Park, Y.H. (2004a). Marinibacillus 435 campisalis sp. nov., a moderate halophile isolated from a marine solar saltern in Korea, with 436 emended description of the genus Marinibacillus. Int J Syst Evol Microbiol 54, 1317-1321. 437 Yoon, J.H., Oh, T.K. & Park, Y.H. (2004b). Transfer of Bacillus halodenitrificans Denariaz et 438 al. 1989 to the genus Virgibacillus as Virgibacillus halodenitrificans comb. nov. Int J Syst Evol 439 Microbiol 54, 2163-2167. 440 Yumoto, I., Hirota, K., Goto, T., Nodasaka, Y. & Nakajima, K. (2005). Bacillus oshimensis 441 sp. nov., a moderately halophilic, non-motile alkaliphile. Int J Syst Evol Microbiol 55, 907-911. 442 443 Table 1. Characteristics that distinguish strain HS136T from other related species of the genus Bacillus. Strains: 1, Strain HS136T; 2, Bacillus salarius BH169T (Lim et al, 2006c); 3, Bacillus qingdaonensis CM1T(Wang et al., 2007). Symbols: +, positive; - negative; ND, not determined. Characteristic 1 2 3 Cell size 1.0-1.5 x 2.5-3.0 0.5-0.7 x 1.3- 1.9 0.3-0.4 x 1.8- 3.2 Pigmentation Cream Cream-white to yellow Cream Endospore formation + + - Endospore position/shape Centralsubterminal/ellipsoidal Spherical/ Terminal - Oxidase + + - Motility + - - NaCl concentration for growth (%, w/v) 5-20 3-20 2.5-20 Optimum NaCl concentration (%, w/v) 10 10-12 12 Temperature range (ºC) 25-45 15-40 25-45 Optimum temperature ºC) 40 30 37 pH range 7.0-10.0 6.8-9.5 6.5-10.5 22 Optimum pH 8.0-8.5 8.0 9.0 Acid production from: D-fructose - + + D-mannose - + + Maltose - + ND D-melibiose - + ND D-xylose - + + Nitrate reduction - - + Arginine dehydrolase - ND + Hydrolisis of: Casein + - ND Aesculin - + ND Urease activity - - + DNA G+C content (mol%) 37.1 43 48 23 Table 2. Cellular fatty acid composition (%) of strain HS136T and the closely related Bacillus species, Bacillus salarius BH169T (Lim et al., 2006c) and Bacillus qingdaonensis CM1T (Wang et al., 2007). Fatty acid composition Strain HS136T Bacillus salarius BH169T Bacillus qingdaonensis CM1T Iso-C14:0 0.5 - 1.6 C14:0 1.3 - 1.3 C15:0 - - 1.2 Iso-C15:0 41.6 8.9 5.9 Anteiso-C15:0 35.8 53.1 34.8 Iso-C16:0 1.3 6.6 14.0 C16:1 11c 1.5 - - C16:0 5.8 - 7.7 Iso-C17:0 3.7 - 4.4 Anteiso-C17:0 8.4 18.7 21.4 C18:0 - - 2.1 24 Legend to figure Fig. 1. Maximum-parsimony tree, based on the 16S rRNA gene sequence comparison, showing the relationship of strain HS136T with related species. The accession numbers of the sequences used in this study are shown in parentheses after the strain designation. Brevibacillus brevis JCM 2503T was used as an outgroup. The scale bar represents 0.01 substitutions per nucleotide position.