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 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 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 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 h of culture) were used. broth with 150 ml chloroform :methanol [2 : 1 (v/v)] for 2 h, However, the extraction rate of the dye with ethanol using a reciprocal shaker (120 strokes per min) at room was not as fast as that in Gram-negative bacteria. This temperature. The extracted solution was concentrated using indicates that the peptidoglycan layers of the isolates a rotary vacuum evaporator and transferred by redissolving are thicker than those of Gram-negative bacteria but in acetone. The resulting solution was evaporated, separated thinner than those of ordinary Gram-positive bacteria. by TLC using n-hexane : dimethyl ether [85 : 15 (v/v)] as the However, further studies will be necessary to clarify solvent, and recovered from the TLC plate with acetone. Isoprenoid quinones thus obtained were analysed by HPLC. this point. The cells were peritrichously flagellated The HPLC system equipped with a Novapak C18 column (Fig. lb) and strains K13T and K38 exhibited positive (Waters) consisted of a CCPM-I1 (Tosoh) solvent delivery oxidase and catalase reactions (Table 1). Growth of pump and a UV-8020 spectrophotometer/detector (Tosoh). strains K13Tand K38 was studied at pH 7-10. Growth Methanol: 2-propanol [l : 1 (v/v)] was used as the mobile was more rapid at pH 8-10 than at neutral pH. The phase (flow rate of 0.15 ml min-l). growth curve of strain K13T is shown in Fig. 2.

566 International Journal of Systematic Bacteriology 48 Gram-negative alkaliphilic bacillus

Table 1. Phenotypic characteristics of the two strains of B. horti ...... , ...... , ...... All tests except the growth temperature range test were performed at 30 "C. + , Positive; -, negative ; w, weakly positive.

Characteristic Strain K13T/K38

Colour of colonies Whitelwhite Form Rods/rods Motility +I+ Flagella Peritrichous/peritrichous Spore formation +I+ Gram stain -1- Catalase +I+ Oxidase +I+ Indole production -1- ONPG hydrolysis +I+ Urea hydrolysis (at pH 7) -1- H,S production -1- H,S production (at pH 7) +I+ Growth at ("C): 5 -1- 10 -1- 15 +I+ 40 +I+ 45 -1- 50 -1- 55 -1- Acid produced from : D-Glucose +I+ D-Xylose +I+ D-Ribose +I+ D-Fructose +I+ D-Mannose WI - Lactose WI- Melibiose -1- Raffinose -1- Sorbitol -1- D-Arabinose -1- L-Rhamnose -1- rnyo-Inositol -1- rneso-Ery t hri to1 -I- Sucrose -1- Hydrolysis of: Casein +I+ Gelatin +I+ Starch +I+ DNA +I+ Hippurate +/- Tweens 20,40,60 and 80 -1- Growth in : 0% NaCl -I+ 3, 5, 7,9, 10 and 11 % NaCl +I+ 12, 13, 14, 15, 16, 17, 18 and 20% NaCl -1- Deamination of phenylalanine -1- Reduction of NO, to NO, +I+

The DNA G+C contents of strains K13T and K38 cell walls. The major menaquinone detected in strain were 40-9 and 40.2 mol%, respectively (Table 3). K13T was menaquinone-7. Whole-cell fatty acids of Strains K13T and K38 contained meso-DAP in their strain K13T consisted of the straight saturated fatty lnternational Journal of Systematic Bacteriology 48 567 I. Yumoto and others

Fig. 1. (a) Light microphotograph of B. horti K13T. Arrows indicate spores. Bar, 10pm. (b) Electron micrograph of negatively stained cells of B. horti K13T showing peritrichous flagellation. Bar, 1 pm.

anteiso-C,,: ,(4.4 YO),iso-C,, : , (1.4 YO),and anteiso- 2-0 Cl.,:o plus an unknown fatty acid (7.9%). The fatty acid composition of strain K38 was almost identical in quality and quantity to that of strain K13T. The fatty acid compositions of B. alcalophilus JCM 5262T, an 1.5 obligate alkaliphile which cannot grow at pH 7, and B. cohnii DSM 6307T, a facultative alkaliphile which can grow at pH 7, were also analysed. Some of the strains 0 of B. cohnii are obligate alkaliphiles (Spanka & Fritze, 0" 1993); however, the strain used, DSM 6307T, grew at 0 1.0 pH 7. The fatty acid compositions of the two strains, B. alcalophilus and B. cohnii, were similar to those in previous reports (Clejan et al., 1986; Spanka & Fritze, 1993), although they had lower unsaturated fatty acid contents (data not shown). The fatty acid composition of the isolates is more similar to that of B. alcalophilus than to that of B. cohnii. This indicates that the isolates are more similar to obligate alkaliphiles than to 6 12 18 24 facultative alkaliphiles in terms of fatty acid com- Time (h) position. Branched 15-C fatty acids, iso-C,,: ol and anteiso-C,,: ,, were the predominant fatty acids in the alkaliphiles examined. In B. alcalophilus and B. cohnii, Fig. 2. Growth of B. horti K13T at pH 7-10. PYA medium anteiso-C,, : ,outweighed iso-C,, :,, while the reverse (described in the text) containing 100 mM NaHC03/Na2C0, was found for the isolates. buffer was used at pH9 (0) and 10 (0)and l00mM NaH2P04/Na2HP04buffer was used instead of NaHC03/Na2C03 buffer at pH 7 (A) and 8 (A). The 16s rRNA gene DNA of strain K13T was sequenced to determine its phylogenetic position. The almost complete 16s rDNA sequence of strain K1 3T, consisting of 1512 nt, was compared with the sequences of 14 other alkaliphilic Bacillus and 13 ~~~~~C1,~,(1~2%),~,,~,(l~l%),C1,:,(1.9%),and the neutrophilic species belonging to genus Bacillus, as monounsaturated C,,: , (5-3YO). Branched saturated well as other related taxa. fatty acids included anteiso-C,, :, (2.4 %), iso-C,, : , (38.4 %), anteiso-C,,: , (30.2 %), iso-C,, : , (4.4 YO), The phylogenetic tree constructed using the neighbour-

568 International Journal of Systematic Bacteriology 48 Gram-negative alkaliphilic bacillus

Fig. 3. Phylogenetic tree derived from 165 rDNA sequence data of B. horti K13T, members of other alkaliphilic Bacillus strains Alicyclobacillus acidocaldarius and other species belonging to the genus Paenibacillus macerans Bacillus and some related organisms. Pa eniba cillus polymyxa Bar, 0.1 K,,,. The GenBank nucleotide sequence accession numbers for the Aneurinibacillus aneurinolyticus I organisms used are as follows: B. alcalophilus DSM 48ST, X76436; B. cohnii DSM 6307T, X76437; Bacillus sp. DSM 8714, X76438; B. pseudofirmus DSM 871 ST, X76439; B. clausii DSM 871ST, X76440; Bacillus sp. DSM 8717, X76441; B. halodurans DSM 871 8, X76442; B. clarkii DSM 8720T, X76444; B. agaradhaerens DSM 8721T, X76445; B. gibsonii DSM 8722T, X76446; B. halmapalus DSM 8723T, X76447; L B. pseudalcaliphilus DSM 872ST, X76449; Bacillus cereus NCDO 1 77 1, X55060; Bacillus coagulans NCDO 1761, X60614; Paenibacillus macerans NCDO 1764, X60624; Bacillus megaterium DSM 32, X60629; Paenibacillus L polymyxa NCDO 1774, X60632; Bacillus subtilis NCDO 1769, X60646; Aneurinibacillus aneurinolyticus ATCC 12856, D78455; Brevibacillus brevis JCM 2503T, D78457; - Bacillus ha Imapalus Bacillus psychrophilus ATCC 23304, X60634; Amphibacillus xylanus JCM 7361T, 226923; - Bacillus cereus Sporolactobacillus inulinus NRlC 1134, Bacillus subtilis D16284; Bacillus thermoalcalophilus DSM Bacillus coagulans 6866, 226931 ; and Alicyclobacillus - Sporolacto bacillus in ulinus acidocaldarius DSM 446, X60742. The nucleotide sequence data of B. horikoshii 0.0 1 DSM 871gT are obtained as a result of - personal communication with Dr F. A. Rainey.

joining method (Fig. 3) and 16s rDNA similarity that to members of rRNA group 6 was 89.4-91.4%; values (data not shown) show that strain K13T is and that to the remaining two species was 90.4-9 1.4 %. phylogenetically related to members of the . These values indicate that strain K13T has an equi- The similarity values (89-1-91.4 %) of strain K13T to distant relationship to the three groups. Therefore, alkaliphilic Bacillus strains were generally greater than strain K1 3T belongs to an independent group. those (85.0-89.8 YO)of the neutrophilic Bacillus strains. The levels of DNA-DNA relatedness were estimated Although strain K13T was grown at pH 7, the simi- using the isolated strains and representative alkali- larity values (89.9-9 1.4 YO)to the obligate alkaliphiles philic bacilli from the three groups: group 1 (B. (B. alcalophilus, B. agaradhaerens, B. clarkii and cohnii), group 6 (B. alcalophilus) and the remaining Bacillus pseudalcaliphilus) were higher than those two species (B.agaradhaerens and B. clarkii) (Table 2). (89-1-89-9%) of facultative alkaliphiles (Bacillus sp. A DNA relatedness value of 93 %, indicates that DSM 8714 and DSM 8717, Bacillus clausii, Bacillus strains K13T and K38 are of the same species. The horikoshii, Bacillus gibsonii and Bacillus halmapalus). levels of DNA relatedness between strain K1 3T and B. Nielsen et al. (1994) classified 14 alkaliphilic Bacillus cohnii, B. alcalophilus, B. agaradhaerens and B. clarkii strains into three groups on the basis of 16s rDNA sequence analysis: those belonging to rRNA group 1 were less than 6 %. of Bacillus (Ash et al., 1991) (B. halmapalus, B. Bacillus strains K13T and K38 can be differentiated horikoshii and B. cohnii); those belonging to the newly from other existing alkaliphilic Bacillus species in terms designated rRNA group 6 (Bacillus pseudofirmus, B. of phenotypic characteristics, even species with similar gibsonii, Bacillus halodurans, B. pseudalcaliphilus, B. DNA G+C contents, as follows: B. pseudojirmus alcalophilus, B. clausii, Bacillus sp. DSM 8714 and deaminates phenylalanine, hydrolyses Tween 40 and Bacillus sp. DSM 8717); and the two strains (B. Tween 60, grows at 10 "C and is negative for nitrate agaradhaerens and B. clarkii) that showed an equi- reduction; B. horikoshii is negative for nitrate reduc- distant relationship to the first two groups. Based on tion and growth in 10% NaCl and grows at 10 "C; phylogenetic analysis, strain K13T clustered in the last B. agaradhaerens hydrolyses Tween 40 and Tween 60, group. The 16s rRNA sequence similarity of strain and grows at 10 "C and is negative for growth at pH 7 K13Tto alkaliphilesin rRNA group 1was 89489.9YO ; and hydrolysis of casein, gelatin and starch; and B.

International Journal of Systematic Bacteriology 48 569 I. Yumoto and others

Table 2. DNA base compositions and levels of relatedness between strain K13T DNA and DNAs of strain K38 and other alkaliphilic Bacillus strains

Strain G + C content Hybridization (mol %)* with DNA from strain K13T (%)

Bacillus horti K13T 40.9 100 Bacillus horti K38 40.2 93 Bacillus cohnii DSM 6307T 35.8 5 Bacillus clarkii DSM 8720T 41.8 4 Bacillus agaradhaerens DSM 872 1 39.1 3 Bacillus alcalophilus JCM 5262T 37.3 2 * Determined by HPLC.

Table 3. Characteristics differentiating B. horti from other alkaliphilic Bacillus species ...... , ...... , ...... + , Positive; v, variable among strains; -, negative. Characteristics of other alkaliphilic Bacillus species were cited from Nielsen et al. (1995) and Spank & Fritze (1993). 1, B. horti; 2, B. alcalophilus; 3, B. cohnii; 4, B. pseudojirmus; 5, B. clausii; 6, B. halodurans; 7, B. horikoshii; 8, B. clarkii; 9, B. agaradhaerens; 10, B. gibsonii; 11, B. halmapalus; 12, B. pseudalcaliphilus.

Characteristic 1 2 3 4 5 6 I 8 9 10 11 12

- ~ Hydrolysis of Casein + V + + + + Gelatin + + + + + + Starch + + + + + + Tween 20 V V - V - - Tween 40 + V + + V V Tween 60 + + + + V V Deamination of - - + - - - phenylalanine Reduction of nitrate V + - V - - Growth at: 10 "C + V + - + + 40 "C + + + + + + 50 "C - - - + - - PH 7 - V V V + - Growth in: 5% NaCl + + + + + + 10% NaCl - - + + - + DNA G + C (mol %) 36.2-38.4 339-35.0 39+40+3 42.143.9 41.142.0 382-39.0

gibsonii is negative for hydrolysis of starch and growth Growth occurs at pH 7, with optimum growth at at 40 "C (Table 3). pH 8-10. There is growth at 10 YONaCl and no growth at 15% NaCl. Nitrate is reduced to nitrite. Acid, but On the basis of the above results, the name Bacillus no gas, is produced from D-glucose, D-xylose, D-ribose horti sp. nov. is proposed and the type strain is and D-fructose when grown at pH 10. No acid is designated K13T. A description of the new species is produced from melibiose, raffinose, sorbitol, D- given below. arabinose, L-rhamnose, myo-inositol, meso-erythritol, D-galactose and sucrose. Hydrolysis of casein, gelatin, starch and DNA, but not Tween 20,40,60 or 80, was Description of Bacillus horti sp. nov. observed. Phenylalanine is not deaminated. The main Bacillus horti (hor'ti. L. masc. n. hortus garden, horti quinone is menaquinone-7. iso-C,, :,, (38-42 %) and from the garden). anteiso-C,,: (27-30 %) represent the main fatty acids produced during growth in an alkaline medium Cells are rod-shaped (06-0.8 by 1.5-6.0 pm) and (PH 10). The DNA G + C content is 40-2-40.9 mol %, produce subterminally located ellipsoidal spores. Cells as determined by HPLC. Strains K13T and K38 were from freshly prepared cultures are Gram-negative and isolated from a soil sample obtained from Atsuma, motile by means of peritrichous flagella. Colonies are Hokkaido, Japan. The type strain is K13T (= JCM white. Catalase and oxidase reactions are positive. 9943T).

570 International Journal of Systematic Bacteriology 48 Gram-negative alkaliphilic bacillus

ACKNOWLEDGEMENTS Krulwich, T. A. (1995). Alkaliphiles : ‘basic’ molecular problems of pH tolerance and bioenergetics. Mol Microbioll5,403410. The authors would like to thank Dr Y. Nodasaka (Hokkaido University) for his help with electron microscopy and Dr M. Marmur, 1. (1961). A procedure for the isolation of de- Morita (Hokkaido National Industrial Research Institute) oxyribonucleic acid from micro-organisms. J Mol Biol 3, for his help with fatty acid analysis. 208-2 18. Nielsen, P., Rainey, F. A., Outtrup, H., Priest, F. G. & Fritze, D. REFERENCES (1994). Comparative 16s rDNA sequence analysis of some Ash, C., Farrow, J. A. E., Wallbanks, 5. & Collins, M. D. (1991). alkaliphilic bacilli and the establishment of a sixth rRNA group Phylogenetic heterogeneity of the genus Bacillus revealed within the genus Bacillus. FEMS Microbiol Lett 117, 61-66. by comparative analysis of small-subunit-ribosomal RNA Nielsen, P., Fritze, D. & Priest, F. G. (1995). Phenetic diversity of sequences. Lett Appl Microbioll3, 202-206. alkaliphilic Bacillus strains : proposal for nine new species. Barrow, G. L. & Feltham, R. K. A. (1993). Cowan and Steel‘s Microbiology 141, 1745-1 76 1. Manual for the IdentiJication of Medical Bacteria, 3rd edn. Satiou, N. & Nei, M. (1987). The neighbor-joiningmethod: a new Cambridge: Cambridge University Press. method for reconstructing phylogenetic trees. Mol Biol EvoE4, Clejan, S., Krulwich, T. A., Mondrus, K. R. & Seto-Young, D. 406-425. (1986). Membrane lipid composition of obligately and faculta- Shima, S., Yanagi, M. & Saiki, H. (1994). The phylogenetic tively alkalophilic strains of Bacillus spp. J Bacteriol 168, position of Hydrogenobacter acidophilus based on 16s rRNA 334-340. sequence analysis. FEMS Microbiol Lett 119, 119-122. Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric Spanka, R. & Fritze, D. (1993). Bacillus cohnii sp. nov., a new deoxyribonucleic acid-deoxyribonucleic acid hybridization in obligately alkaliphilic, oval-spore-formingBacillus species with microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine ornithine and aspartic acid instead of diaminopimelic acid in genetic relatedness among bacterial strains. Int J Syst Bacteriol the cell wall. Int J Syst Bacteriol40, 92-97. 39,224-229. Tamaoka, J. & Komagata, K. (1984). Determination of DNA base Fritze, D., Flossdorf, 1. & Claus, D. (1990). of composition by reversed-phase high-performance liquid chro- alkaliphilic Bacillus strains. Int J Syst BacterioE40, 92-97. matography. FEMS Microbiol Lett 25, 125-128. Gordon, R. E. & Hyde, 1. L. (1982). The Bacillus~rmus-Bacillus Thompson, J. D., Higgins, D. G. & Gibson, T. 1. (1994). CLUSTAL lentus complex and pH 7.0 variants of some alkalophilic strains. w : improving the sensitivity of progressive multiple sequence J Gen Microbioll28, 1109-1 116. weighing, position-specific gap penalties and weight matrix Horikoshi, K. (1 991). Microorganisms in Alkaline Environments. choice. Nucleic Acids Res 76, 43504354. Weinheim : VCH. Vedder, A. (1934). Bacillus alcalophilus n. sp. ; benevens enkele Hugh, R. & Leifson, E. (1953). The taxonomic significance of ervaringen met sterk alcalische voedingbodems. Antonie fermentative versus oxidative metabolism of carbohydrates by Leeuwenhoek J Microbiol Serol 1, 143-147. various Gram-negative bacteria. J Bacteriol66, 24-26. Yamada, K. & Komagata, K. (1970). Taxonomic studies on Kimura, M. (1980). A simple method for estimating evolutionary coryneform bacteria. 11. Principal amino acids in the cell wall rates of base substitution through comparative studies of and their taxonomic significance. J Gen Appl Microbiol 16, nucleotide sequences. J Mol Evoll6, 11 1-120. 103-1 13.

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