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Bacillus Horti Sp. Nov., a New Gram-Negative a I Ka I I P H I I Ic Baci

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Bacillus Horti Sp. Nov., a New Gram-Negative a I Ka I I P H I I Ic Baci International Journal of Systematic Bacteriology (1998), 48, 565-571 Printed in Great Britain Bacillus horti sp. nov., a new Gram-negative a Ika I i p h i I ic baciI I us lsao Yumoto,’ Koji Yamazaki,2 Tomoo Sa~abe,~Kazuaki Nakan~,~ Kosei Kawasaki,’ Yoshio Ezura3 and Haruo Shinano2 Author for correspondence: Isao Yumoto. Tel: + 81 11 857 8925. Fax: +81 11 857 8900. e-mail : yumoto@ hniri.go.jp 1 Bioscience and Chemistry Novel Gram-negative alkaliphilic strains were isolated from soil obtained from Division, Hokkaido Atsuma, Hokkaido, Japan. The isolates were strictly aerobic rods that produced National Industrial Research Institute, 2-17-2- subterminally located ellipsoidal spores. Chemotaxonomic characteristics of 1 Tsukisamu-Higashi, the isolates included the presence of meso-diaminopimelic acid in the cell wall Toyohira-ku, Sapporo and a DNA G+C content of 40.2-409 mol%. The major isoprenoid quinone was 062-8517, Japan menaquinone-7 and the cellular fatty acid profile consisted of a significant 213 Department of Marine amount of 15-C branched-chain acids, iso-C,,:, and anteiso-C,,:,. The growth B ioresources Chemistry*, and Department of rate was higher at pH 8-10 than at pH 7. Comparative sequence analysis of 165 Fisheries Oceanography & rDNA of 14 alkaliphilic Bacillus strains indicates that the isolated strain has an Marine Science3, Faculty equidistant relationship to three already defined rRNA groups of alkaliphilic of Fisheries, Hokkaido University, Hakodate Bacillus species. Based on the morphological and physiological characteristics, 041-0821, Japan as well as phylogenetic position as determined by 16s rDNA analysis and DNA-DNA relatedness data, it is concluded that these isolates should be designated as a new species, for which the name Bacillus horti is proposed. The type strain is K13l(= JCM 99433. Keywords : Bacillus horti sp. nov., alkaliphilic, Gram-negative bacillus chemotaxonomic analysis, 16s rDNA analysis INTRODUCTION properties correlated with a distinct DNA G+C content. However, considering the low DNA-DNA Since Vedder (1934) isolated the obligate alkaliphile, relatedness values between strains in the same group, Bacillus alcalophilus, many strains of obligate and they concluded that these alkaliphilic Bacillus strains facultative alkaliphiles have been isolated for indus- could be assigned to numerous species. During the trial applications and physiological studies (Horikoshi, investigation by Fritze et al. (1990), a new species, 1991 ;Krulwich, 1995). Most of the isolates are Gram- Bacillus cohnii, was proposed which was distinct positive, spore-forming, motile rods. Catalase- and because it had oval spores and no diaminopimelic acid oxidase-positive aerobes were classified under the (DAP) in the cell wall (Spanka & Fritze, 1993). The genus Bacillus. However, most of these bacteria were phenotypic and genotypic heterogeneity of alkaliphilic not identified up to the species level. Gordon & Hyde Bacillus strains (Fritze et al., 1990) was reflected in (1982) grouped alkaliphilic bacilli with Bacillus phylogenetic studies by 16s rDNA sequence analysis Jirmus, Bacillus lentus groups 1-111, and the B. (Nielsen et al., 1994). In addition to the two existing firmus-B. lentus complex according to the results of species, B. alcalophilus and B. cohnii, nine new species physiological and biochemical characterization. A were proposed by Nielsen et al. (1995) on the basis of reclassification of the strains that were first classified numerical analysis. In the present study, two new by Gordon & Hyde (1982), and of other additional strains of Gram-negative alkaliphilic bacilli were isolates, was reported by Fritze et al. (1990). Their isolated. The characteristics of the isolates are reported reclassification was based on certain phenotypic and differences between the new isolates and pre- viously reported alkaliphilic Bacillus species are dis- ........................................................................................................... ....... ...................... ........ cussed. This study focuses on phenotypic character- Abbreviation: DAP, diaminopirnelic acid. ization, phylogenetic analysis and DNA-DNA re- The GenBanWEMBUDDBJ accession number for the 165 rRNA sequence latedness data of the isolates with respect to strains of reported in this paper is D87035. other alkaliphilic Bacillus species. 00582 0 1998 IUMS 565 I. Yumoto and others METHODS Analysis of cellular fatty acids. Whole-cell fatty acids were extracted with n-hexane from 100 mg lyophilized cells, Bacterial strains. Seventy-nine alkaliphilic bacteria were which were cultivated in PYA medium until the late isolated from seven sampling station sites in Hokkaido, exponential phase of growth, esterified by acid methanolysis Japan using PYA (peptone/yeast extract/alkaline) agar and analysed using a gas-liquid chromatograph equipped medium consisting of 8 g peptone (Kyokuto), 3 g yeast with a flame ionization detector (model GC-7A; Shimadzu) extract (Merck), 15 g agar, 1 g K,HPO,, 3.5 mg EDTA, and a 50 m BPX70 column (SGE) at an oven temperature of 3 mg ZnSO,. 7H,O, 10 mg FeSO,. 7H,O, 2 mg MnSO,. 170 "C. Identification of the fatty acid components was nH,O, 1 mg CuSO, . 5H,O, 2 mg Co(NO,), .6H,O, and 1 mg accomplished by comparison with fatty acid methyl ester H,BO, in 1 1 NaHCO,/Na,CO, buffer (100 mM ; pH 10) in standards purchased from Supelco and GL Science using a deionized water. Based on preliminary taxonomic charac- gas chromatograph-mass spectrometer (model INCOS 50 ; terization of the isolates, only strains K13T and K38 were Finnigan mat) connected to a gas-liquid chromatograph identified as Gram-negative Bacillus species. These strains (model 3400 ; Varian). were isolated from garden soil samples obtained from Astuma, Hokkaido, Japan. In addition to the isolates, B. Cell wall analysis. Identification of meso-DAP in the cell wall alcalophilus JCM 5262T (= DSM 485T), B. cohnii DSM was performed by TLC (DC-Alufoline Cellulose; Merck), as 6307T, Bacillus clarkii DSM 8720T and Bacillus agarad- described by Yamada & Komagata (1970). haerens DSM 8721Twere used as reference strains. DNA base composition and DNA-DNA hybridization. Bac- Phenotypic characterization. For phenotypic characteri- terial DNA was prepared according to the method of zation, PYA medium (pH 10) was used as the basal medium, Marmur (1961). The DNA obtained was digested with and the cultures were incubated at 30 "C for 2 weeks unless nuclease P1 (Yamasa Shoyu) and the resulting nucleotides otherwise stated. Hydrolysis of casein, gelatin, starch, DNA, were separated by HPLC (Tamaoka & Komagata, 1984). hippurate and Tween 20,40,60 and 80, oxidase and catalase The HPLC system was as described above. An equimolar reactions, reduction of NO, to NO,, indole production, mixture of 4-deoxyribonucleotides (Yamasa Shoyu) was ONPG hydrolysis, H,S production, urea hydrolysis, and used as the standard. Levels of DNA relatedness were deamination of phenylalanine were performed as described determined fluorometrically by the method of Ezaki et al. in Cowan & Steel's manual (Barrow & Feltham, 1993). Acid (1989) using photobiotin-labelled DNA probes and micro- production from carbohydrates was determined by the plates. method of Hugh & Leifson (1953), using thymol blue instead of bromothymol blue at pH 10. Growth at pH 7 was tested 16s rDNA sequencing. 16s rDNA fragments from strain in PYA broth containing 100 mM NaH,PO,/Na,HPO, K13T were analysed using PCR-direct sequencing, as de- buffer (pH 7) on a reciprocal shaker (140 strokes per min) at scribed by Shima et al. (1994). Sequences were compiled 30 "C for 1 week incubation. The requirement and tolerance from overlapping sequence data using the DNASIS computer for Na+ was determined using a medium containing 2g program (Hitachi Software Engineering). Multiple align- glucose, 1 g peptone (Difco), 0.1 g yeast extract (Difco) and ments of the sequences were performed, nucleotide sub- 0-200 g NaCl in 1 1 KHCO,/K,CO, buffer (50 mM; pH 10) stitution rates (&,, value) were calculated, and a neighbour- in deionized water. joining phylogenetic tree (Kimura, 1980; Satiou & Nei, 1987) was constructed using the CLUSTAL w program Growth experiment. The growth experiment was performed (Thompson et al., 1994). The similarity values of the using a 500 ml flask containing 250 ml PYA broth with a pH sequences were calculated using the GENETYX program of 7-10 with reciprocal shaking (140 strokes per min) at (Software Development). 30 "C. Growth was estimated by monitoring OD,,,. PYA media containing 100 mM Na,HPO,/NaH,PO, and 100 mM NaHCO,/Na,CO, buffer, at pH 7-8 and pH 9-10, RESULTS AND DISCUSSION respectively, were used. Electron microscopy. Cells were suspended in physiological Strains K13T (type strain) and K38, which were saline. A small drop of the suspension was introduced on a isolated from a soil sample, were Gram-negative, carbon-coated copper grid and negatively stained with 1 % aerobic rods (Table 1) whose cells were 06-04 by phosphowolframic acid for examination. Immediately after 1-5-6.0 pm long and produced subterminally located staining, cells were observed with a transmission electron ellipsoidal spores (Fig. 1a). During Gram-staining of microscope (model H-800; Hitachi). the isolates, the crystal violet-iodine dye was easily Analysis of isoprenoid quinones. Isoprenoid quinones were extracted from the cells with ethanol, even when freshly extracted by treating 500 mg lyophilized cells grown in PYA prepared samples (within 12
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