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Bacillus Okuhidensis Sp. Nov., Isolated from the Okuhida Spa Area of Japan

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Bacillus Okuhidensis Sp. Nov., Isolated from the Okuhida Spa Area of Japan International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1205–1209 DOI: 10.1099/ijs.0.01962-0 Bacillus okuhidensis sp. nov., isolated from the NOTE Okuhida spa area of Japan 1,2 Department of Urology1 Zhiyu Li,1 Yoshiaki Kawamura,2 Osamu Shida,3 Shuzo Yamagata,4 and Department of 1 2 Microbiology2 , Gifu Takashi Deguchi and Takayuki Ezaki University School of Medicine, Tsukasa-Machi 40, Gifu 500-8705, Japan Author for correspondence: Zhiyu Li. Tel: j81 58 267 2240. Fax: j81 58 267 0156. e-mail: lizhiyu70!hotmail.com 3 Research Laboratory, Higeta Shoyu Co. Ltd, Chiba 288, Japan Two Gram-positive, endospore-forming, alkaliphilic bacteria were isolated 4 Department of from water samples obtained from the Okuhida hot spa area of Japan. The Biotechnology, Faculty of Agriculture, Gifu unknown bacteria were characterized using phenotypic and molecular University, Gifu 501-1193, taxonomic methods. On the basis of phylogenetic evidence and phenotypic Japan distinctiveness, a new species, Bacillus okuhidensis sp. nov., is proposed. The type strain of Bacillus okuhidensis is GTC 854T (l JCM 10945T l DSM 13666T). Keywords: Bacillus okuhidensis, 16S rRNA, alkaliphilic, DNA–DNA hybridization Alkaliphilic micro-organisms, species of the genus data show that the isolated strains represent a new Bacillus in particular, are of considerable biotechnolo- species of Bacillus, for which we propose the name gical importance because of their ability to produce Bacillus okuhidensis. some extracellular alkaline enzymes such as protease (Horikoshi, 1971), pectinase (Horikoshi, 1972) and Two hot-water samples were collected from the Oku- amylases (Boyer & Ingle, 1972) that are resistant to hida hot spa area in Japan. The temperatures and pH high-pH or high-temperature conditions (Hamamoto values of the sources of the two isolates were 59 mC\pH & Horikoshi, 1992; Nielsen et al., 1994). These unusual 6n4 and 51 mC\pH 8n1. Strains were deposited with the Gifu Type Culture collection (GTC) and designated as enzymes can be used in industrial and other processes T (Nielsen et al., 1995; Yamagata, 1993). GTC 854 and GTC 855. The primary culture medium was heart infusion broth (Difco). To determine the Since Vedder (1934) first isolated the aerobic, endo- optimal pH for growth, isolates were cultured at 45 mC spore-forming, obligate alkaliphilic micro-organism by shaking in an incubator (Eyela Shaker III; Tokyo Bacillus alcalophilus, many strains of obligate or Rikakikai) at speed 5. The pH of the media was facultative alkaliphiles have been isolated for indus- adjusted with 1 M solutions of K$PO% and HCl. trial applications and physiological studies (Horikoshi, Growth was monitored by determining the turbidity at 1991; Nielsen et al., 1995; Yumoto et al., 1998). 650 nm with a spectrophotometer (Ultraspec 3000; Gordon & Hyde (1982) grouped these alkaliphilic Pharmacia) according to the method described by strains into the Bacillus firmus and Bacillus lentus Yamagata (1993). Analysis of the effect of NaCl on the groups and the B. firmus–B. lentus complex. These growth of cells was carried out in heart infusion broth, strains and other isolates were redefined by Fritze et al. adjusted to pH 10n5, and the culture was shaken at (1990). On the basis of the phenotypic characteristics 45 mC as described above. Both isolates showed growth correlated with distinct mol% GjC contents, they at the lowest temperature, 30 mC, and showed optimal concluded that these alkaliphilic Bacillus strains growth at temperatures between 45 and 50 mC in heart should be classified as various species according to the infusion medium with the pH adjusted to 10n5. Strain T low DNA–DNA similarity values between strains in GTC 854 grew at up to 60 mC, but the maximum the same group. In the present investigation, two temperature for growth of strain GTC 855 was 55 mC. alkaliphilic bacteria isolated from water samples were Both strains grew well at pH values ranging from 6n0to subjected to a polyphasic study designed to establish 11n0at45mC. Optimal growth was observed at pH their taxonomic positions. Genotypic and phenotypic 10n5. They also grew in the presence of up to 10% NaCl (w\v) at pH 10n5 and 45 mC (Table 1). ................................................................................................................................................. Published online ahead of print on 28 January 2002 as DOI 10.1099/ Organisms were observed through a phase-contrast ijs.0.01962-0. light microscope (Optiphot XF-NT; Nikon) to de- The DDBJ/GenBank accession numbers for the 16S rRNA gene sequences of termine the Gram-staining and endospore-forming GTC 854T and GTC 855 are AB047684 and AB047685, respectively. characteristics. Detailed morphological studies were 01962 # 2002 IUMS Printed in Great Britain 1205 Z. Li and others Table 1. Phenotypic characteristics of B. okuhidensis and other alkaliphilic Bacillus species ................................................................................................................................................................................................................................................................................................................. Species: 1, B. okuhidensis;2,B. halodurans;3,B. alcalophilus;4,B. pseudalcalophilus;5,B. pseudofirmus;6,B. cohnii;7,B. clausii;8,B. horikoshii;9,B. clarkii; 10, B. agaradhaerens; 11, B. gibsonii; 12, B. halmapalus. j, Positive; k, negative; , variable; , no data. All species grow in 5% NaCl and hydrolysed gelatin and starch. Data for species 2–12 are from Nielsen et al. (1995) and Yumoto et al. (1998). Characteristic 1 23456789101112 Growth at: 20 mC kjjjj jjjjjj 40 mC jjjjjjjjjjkj 45 mC jjkkjjjkjjkk 50 mC jjkkkkjkkkkk 55 mC jjkkkkkkkkkk 60 mC * kkkkkkkkkkk pH pH 6 jkkkkkkkkkkk pH 7 j kk jjkkjj pH 9 jjjjjjkkjjkk pH 10 j j kkkjjkk pH 11 jkkk kkkjjkk Growth in NaCl at: 8% jjjjj jjjjjk 10% jjkjjk kjj k 12% kjkkjkkkjj k 13% k kkjkkkjjkk Reduction of nitrate j kkjjkjj k Deamination of phenylalanine kkkkjkkkkkkk Utilization of: -Arabinose j jj jk jjk -Xylose jj j j j Salicin jjjj jk jj Glycogen jjjj jj jkj Inositol jj k k kkk Melibiose jjjkk jk jjk Gentiobiose j jj jk Sorbitol jkkkk jk kk Turanose j jj j jjj Galactose jjjj k j k Mannose kj k j jjj Rhamnose jjj k jk k -Tagatose k k jk kk Raffinose k jkk jk jjk Lactose jjjjk k jk Melezitose kj kk jk jk N-Acetylglucosamine jjjkj jj jkj -Mannitol j Glycerol j Arabitol j k k kk Xylitol j kk jk kkk Hydrolysis of: Casein jjjjj jjjkjj Hippurate k kkkjkjjkkj Tween 20 k kk kkkkkk Tween 40 kjj j k jj k Tween 60 kjj jjk jj k * The result is strain dependent, and only strain GTC 854T is positive. 1206 International Journal of Systematic and Evolutionary Microbiology 52 Bacillus okuhidensis, a novel alkaliphile Cellular fatty acid composition was analysed as de- scribed by Komagata & Suzuki (1987). We analysed strains GTC 854T, GTC 855 and 10 type strains of related Bacillus species (including Bacillus halodurans DSM 497T, B. alcalophilus JCM 5262T, Bacillus pseudoalcaliphilus DSM 8725T, Bacillus pseudofirmus DSM 8715T, Bacillus clausii DSM 8716T, Bacillus gibsonii DSM 8722T, Bacillus clarkii DSM 8720T, Bacillus halmapalus DSM 8723T, Bacillus horikosii DSM 8719T and Bacillus agaradhaerens DSM 8721T). The cellular fatty acids of strains GTC 854T and GTC 855 consisted of C"%:! (0n6 and 1n2%), C"':! (3n7 and 4n3%), iso-C"%:! (1n6 and 1n9%), iso-C"&:! (43n2 and 44n2%), iso-C"':! (3n7 and 3n0%), iso-C"(:! (7n4 and 5n2%), anteiso-C"&:! (25n3 and 26n2%), anteiso-C"(:! (9n3 and 5n9%), respectively. The branched saturated fatty acids iso-C"&:! and anteiso-C"&:! represented the major cellular fatty acids in all strains tested. It was difficult to distinguish the novel isolates from B. alcalophilus or B. pseudoalcaliphilus by analysis of fatty acid composition. However, the two isolated strains could be distinguished from B. halodurans on the basis of significant differences in the iso-C"&:! fatty acid content (B. halodurans,29n4%). To determine the phylogenetic positions of the novel bacteria, the 16S rRNA genes (corresponding to positions 8–1485 in the Escherichia coli gene) were PCR-amplified and sequenced as described previously (Ezaki et al., 1994). A comparison with the sequences ................................................................................................................................................. of all established Bacillus species and related low- Fig. 1. Morphological characteristics of B. okuhidensis GTC 854T. (top) Phase-contrast micrograph showing endospores. GjC-content genera indicated that both strains clus- (bottom) Transmission electron micrograph showing flagella. tered in the genus Bacillus, and 16S rDNA sequence Bars, 5 µm. similarities with the genus Bacillus were above 90% (data not shown). The highest 16S rDNA sequence similarity was with B. halodurans (99n3%). They also carried out with a transmission electron microscope exhibited higher levels of similarity with B. alcalophilus and B. pseudalcalophilus (96n4 and 95n2%). The se- (JEM-100SX; JEOL) (Yamagata, 1993). In the expon- T ential growth phase, the cells of both isolates were quence similarity between strains GTC 854 and GTC Gram-positive rods 0n5–1n0 µm in diameter and 5–7 855 was 99n7%. A phylogenetic tree based on an µm in length; however, they stained Gram-negative in almost complete 16S rDNA sequence revealed the the stationary phase of growth. These organisms relationships
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