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Int J Syst Evol Microbiol 53 (2003), 1081-1084; DOI  10.1099/ijs.0.02409-0
© 2003 International Union of Microbiological Societies

Alicyclobacillus sendaiensis sp. nov., a novel acidophilic, slightly thermophilic species isolated from soil in Sendai, Japan

Naoki Tsuruoka1, Yuri Isono1, Osamu Shida2, Hisashi Hemmi1, Toru Nakayama1 and Tokuzo Nishino1

1 Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 07, Sendai, Miyagi 980-8579, Japan
2 Research Laboratory, Higeta Shoyu Co. Ltd, Choshi, Chiba 288, Japan

Correspondence
Tokuzo Nishino
nishino{at}mail.cc.tohoku.ac.jp


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An acidophilic, slightly thermophilic bacterium, designated strain NTAP-1T, that produces a thermostable extracellular acid collagenase activity with potential industrial applications was isolated from soil of Aoba-yama Park, Sendai, Japan. The temperature range for growth was 40–65 °C, with an optimum at 55 °C, and the pH range for growth was 2·5–6·5, with an optimum at pH 5·5. Analysis of the 16S rDNA sequence of strain NTAP-1T showed that it is most closely related to strains of the genus Alicyclobacillus. Consistently, the major constituents of the cell-membrane lipid of strain NTAP-1T were {omega}-alicyclic fatty acids. However, DNA–DNA reassociation studies showed only low similarities (less than 33 %) to any type strain of Alicyclobacillus. On the basis of the phenotypic and genotypic properties, a novel species is proposed, Alicyclobacillus sendaiensis sp. nov., represented by strain NTAP-1T (=JCM 11817T =ATCC BAA-609T).

Published online ahead of print on 6 December 2002 as DOI 10.1099/ijs.0.02409-0.

The DDBJ accession number for the 16S rDNA sequence of strain NTAP-1T is AB084128.


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Three thermoacidophilic species of the genus Bacillus, Bacillus acidocaldarius, Bacillus acidoterrestris and Bacillus cycloheptanicus, were reclassified into the genus Alicyclobacillus on the basis of the distinctive phylogenetic position of the organisms in relation to those of other Bacillus species, as well as their unique chemotaxonomic properties, in particular the occurrence of {omega}-alicyclic fatty acids as major fatty acid constituents of their cell-membrane lipids (Wisotzkey et al., 1992Go). Recently, novel species of the genus Alicyclobacillus have also been proposed: Alicyclobacillus hesperidum (Albuquerque et al., 2000Go) and Alicyclobacillus herbarius (Goto et al., 2002Go).

During the course of our screening programme for thermostable ‘acid collagenase’ (which has potential applications in biotechnology), we isolated, from soil at Aoba-yama Park, Sendai, Miyagi, Japan, an acidophilic bacterium that produces an extracellular thermostable collagenase with an optimum pH for catalytic activity at 3·9 (Nakayama et al., 2000Go). This bacterium, strain NTAP-1T, was an aerobic, endospore-forming, rod-shaped bacterium that stained Gram-negative and was tentatively assigned as a strain of the genus Bacillus. In this study, we have carried out extensive physiological, chemotaxonomic and phylogenetic analyses to show that strain NTAP-1T represents a novel species of the genus Alicyclobacillus, which we have named Alicyclobacillus sendaiensis sp. nov.

Strain NTAP-1T was isolated from soil using an agar medium (pH 4·8) containing 1·5 % gelatin, 0·01 % yeast extract, 0·85 % NaCl, 0·5 % KH2PO4 and 0·001 % MgSO4.7H2O at 60–70 °C (Nakayama et al., 2000Go). The type strains Alicyclobacillus acidocaldarius DSM 446T, Alicyclobacillus acidoterrestris DSM 3922T, Alicyclobacillus cycloheptanicus DSM 4006T and A. hesperidum DSM 12489T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. Cells of strain NTAP-1T were stored at -80 °C in broth cultures supplemented with 15 % (w/v) glycerol.

Bacterial growth was monitored for up to 7 days after inoculation by measuring the turbidity (600 nm) of cultures in 5 ml liquid Bacillus acidocaldarius medium (BAM; Deinhard et al., 1987Go) at specified pH values (see below) in 25 ml metal-capped test tubes incubated in a water-bath incubator. For the measurement of turbidity, an uninoculated control was used as a blank. The growth temperature range of the organisms was examined at pH 4·0 between 23 and 75 °C in 5 °C steps. To determine the pH range for growth, the organisms were grown at 55 °C as described above except that the pH of the medium was adjusted to different values with 1·0 M H2SO4; all pH measurements were performed at room temperature. The effects of NaCl (0, 2, 3, 4, 5, 7 and 9 %, w/v) on growth were examined at pH 5·5 and 55 °C. Anaerobic growth was tested using incubation at 55 °C in 10 ml rubber-sealed screw-cap tubes containing liquid BAM (pH 5·5, 9 ml) covered with liquid paraffin.

Cell morphology was determined using light microscopy during the exponential growth phase in BAM (pH 5·5). Sporulation was observed by using phase-contrast microscopy with cells grown to stationary phase. Gram staining was performed using exponentially growing cells according to Hucker's modification (Cowan & Steel, 1965Go), with reagents produced by Nacalai Tesque. Flagellation was examined using Leifson's method (Cowan & Steel, 1965Go). The following physiological tests were carried out as described previously (Albuquerque et al., 2000Go): catalase and oxidase reactions, the Voges–Proskauer reaction, the oxidation/fermentation test and tests for H2S production, nitrate reduction, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase, {beta}-galactosidase, gelatinase and urease. Acid production from carbon sources (see Table 2Go) was examined using API 50 CH test strips (bioMérieux) and BAM (pH 4·0) without glucose. Cells were resuspended in BAM without glucose at a cell density corresponding to tube no. 2 of the McFarland series of standard opacities (Cowan & Steel, 1965Go). Cell suspensions (200 µl) were added to API 50 CH test-strip wells as recommended by the manufacturer and incubated at 55 °C. Acidification was observed every day for 7 days by measuring the pH of cultures, using a compact pH meter (model B-212; Horiba). Medium showing a pH drop of more than 0·5 pH units, relative to a control, was regarded as positive with respect to acid production. Cultures for analysis of cellular fatty acids, isoprenoid quinones and cell-wall diamino acids were grown in 2 l Erlenmeyer flasks containing 1 l BAM at pH 5·5 and 55 °C in a reciprocal shaker until the exponential phase of growth. Analyses for cellular fatty acids, isoprenoid quinones and cell-wall diamino acids were carried out as described by Komagata & Suzuki (1987)Go.


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  		Table 2. Phenotypic characteristics that differentiate species of the genus Alicyclobacillus

Strains: 1, NTAP-1T; 2, A. acidocaldarius DSM 446T; 3, A. acidoterrestris DSM 3922T; 4, A. hesperidum DSM 12489T; 5, A. cycloheptanicus DSM 4006T (data in columns 1–5 from this study); 6, A. herbarius DSM 13609T (data from Goto et al., 2002Go). +, Positive; W, weakly positive; -, negative; NR, not reported. All strains were positive for acid production from D-ribose, D-xylose, D-glucose, D-fructose, D-mannose and mannitol. All were negative for acid production from L-xylose, adonitol, methyl {beta}-xyloside, L-sorbose, dulcitol, N-acetyl-D-glucosamine, inulin, D-lyxose, D-tagatose, L-fucose, L-arabitol, gluconate and 2-ketogluconate.

 
Isolation and purification of chromosomal DNA and estimation of DNA base composition by HPLC were performed according to Tamaoka & Komagata (1984)Go. DNA reassociation values were determined as described by Ezaki et al. (1989)Go. Briefly, probes for DNA hybridization were prepared from cells of A. acidocaldarius DSM 446T, A. acidoterrestris DSM 3922T, A. cycloheptanicus DSM 4006T and A. hesperidum DSM 12489T. Probe DNAs were biotinylated with photobiotin and hybridized with single-stranded unlabelled chromosomal DNA fragments of strain NTAP-1T. Hybridized DNA fragments were visualized using alkaline phosphatase colour-detection methods (Ezaki et al., 1989Go). 16S rDNA was amplified by a PCR (Weisburg et al., 1991Go) with the universal bacterial primers 10F (5'-AGTTTGSTCCTGGCTC-3') and 1500R (5'-GGCTACCTTGTTACGA-3'). PCR products were purified with MicroSpin columns S-400 HR (Amersham Biosciences) and sequenced using a CEQ2000XL DNA Analysis system (Beckman Coulter). Previously published 16S rDNA sequences were obtained from the GenBank/EMBL/DDBJ databases (see Fig. 2Go). Multiple alignment of sequences, calculation of nucleotide substitution rates (Knuc values; Kimura, 1980Go), construction of a neighbour-joining phylogenetic tree (Saitou & Nei, 1987Go) and bootstrap analysis with 1000 replicates for the evaluation of phylogenetic tree topology (Felsenstein, 1985Go) were carried out using CLUSTAL W version 1.81.



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  		Fig. 2. Phylogenetic relationships of Alicyclobacillus species and some aerobic, rod-shaped, endospore-forming bacteria, based on 16 rRNA gene sequences. GenBank/EMBL/DDBJ accession numbers are shown in parentheses. The branching pattern was generated by the neighbour-joining method. Numbers indicate bootstrap percentages greater than 90 %. Bar, 0·01 nucleotide substitution per site.

 
Cells of strain NTAP-1T are rod-shaped, 2–3 µm long and 0·8 µm in diameter. Cells stained Gram-negative and were non-motile. Ellipsoid endospores were produced (Fig. 1Go). Colonies of strain NTAP-1T formed on BAM were circular, convex, white and semi-transparent, 1 mm in diameter after 30 h growth at 55 °C.



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  		Fig. 1. Phase-contrast micrograph of sporulating cells of Alicyclobacillus sendaiensis sp. nov. Cells were cultured on liquid BAM for 3 days at 55 °C.

 
A 1438-nt stretch of the 16S rDNA sequence, representing 93 % of the Escherichia coli 16S rDNA sequence, was determined and compared with available 16S rDNA sequences to construct a phylogenetic tree (Fig. 2Go). The results clearly showed that strain NTAP-1T falls within the radiation of the genus Alicyclobacillus. Similarity values between the 16S rDNA sequence of strain NTAP-1T and those of A. acidoterrestris DSM 3923T, A. acidocaldarius DSM 11297T, A. hesperidum DSM 12766T, A. cycloheptanicus DSM 4006T, A. herbarius DSM 13609T and Bacillus tusciae DSM 2912T were respectively 98·2, 97·9, 95·1, 93·5, 92·4 and 89·2 %. The G+C content of strain NTAP-1T was determined, by HPLC, to be 62·3 mol%, which is higher than the values determined in this laboratory for A. acidoterrestris DSM 3922T (51·5 mol%), A. cycloheptanicus DSM 4006T (57·9 mol%) and A. acidocaldarius DSM 446T (60·8 mol%). DNA–DNA reassociation studies of strain NTAP-1T were performed using type strains whose 16S rDNA sequences showed more than 95 % similarity to that of strain NTAP-1T. Strain NTAP-1T showed 33, 26 and 5 % DNA–DNA reassociation, respectively, with A. acidocaldarius DSM 446T, A. hesperidum DSM 12489T and A. acidoterrestris DSM 3922T. These values are significantly lower than the threshold value (70 %) recommended for the delineation of a novel species. Thus, strain NTAP-1T can be distinguished from representatives of all described species of Alicyclobacillus.

The fatty acid composition of strain NTAP-1T is shown in Table 1Go. {omega}-Alicyclic fatty acids were the major cellular fatty acids, as is the case for strains of the genus Alicyclobacillus. Examination of the respiratory lipoquinone content of strain NTAP-1T showed that menaquinones were the only respiratory lipoquinones detected, menaquinone-7 being the predominant type. Strain NTAP-1T contained meso-diaminopimelic acid as the wall diamino acid.


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  		Table 1. Cellular fatty acid composition of strain NTAP-1T and Alicyclobacillus strains

Strains: 1, NTAP-1T; 2, A. acidocaldarius ATCC 27009T; 3, A. acidoterrestris DSM 3923T; 4, A. cycloheptanicus DAM 4006T; 5, A. herbarius DSM 13609T (data in columns 2–5 from Goto et al., 2002Go); 6, A. hesperidum DSM 12489T (data from Albuquerque et al., 2000Go). Values are percentages of total fatty acids. TR, Trace.

 
Strain NTAP-1T was able to grow in BAM at pH 2·5–6·5 (at 55 °C) or at 40–65 °C (at pH 4·0). The optimum pH and temperature values for growth were pH 5·5 and 55 °C. Anaerobic growth did not occur. The organism grew in the presence of 4 % (w/v) NaCl; however, no growth was observed at NaCl concentrations of 5 % or higher. NTAP-1T was positive for the Voges–Proskauer reaction, nitrate reduction and gelatinase activity, but negative for the oxidation/fermentation test and tests for H2S production, catalase, oxidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase, {beta}-galactosidase and urease. Acid production from various carbon sources is summarized in Table 2Go. This acid-production pattern also distinguishes strain NTAP-1T from known species of Alicyclobacillus.

Description of Alicyclobacillus sendaiensis sp. nov.
Alicyclobacillus sendaiensis (sen.dai.en'sis. N.L. masc. adj. sendaiensis of Sendai, a city in Miyagi Prefecture, Japan, where the type strain was isolated).

Rod-shaped, endospore-forming, strictly aerobic organism. Cells stain Gram-negative. Round spores lie terminally in swollen sporangia. The rods measure 2–3x0·8 µm. Cell wall contains meso-diaminopimelic acid. The predominant isoprenoid quinone is menaquinone-7. Major fatty acid components are {omega}-alicyclic acids (of the {omega}-cyclohexyl type). The temperature range for growth is 40–65 °C (optimum 55 °C). The pH range for growth is 2·5–6·5 (optimum pH 5·5). Grows in the presence of 4 % (w/v) NaCl in BAM. Positive in the Voges–Proskauer reaction and the nitrate-reduction test, but negative in the oxidation/fermentation test and for H2S production, catalase, oxidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase, {beta}-galactosidase and urease. The pattern of acid production is shown in Table 2Go. The G+C content of the DNA of the type strain is 62·3 mol%.

The type strain, strain NTAP-1T (=JCM 11817T =ATCC BAA-609T), was isolated from soil of Aoba-yama Park, Sendai, Japan.


   ACKNOWLEDGEMENTS
 
We thank Professor Milton S. da Costa, Departamento de Bioquímica, Universidade de Coimbra, Portugal, for his invaluable comments and help with improving this paper. This work was supported in part by the industrial technology research grant program in 2001 from the New Energy and Industrial Technology Development Organization (NEDO) of Japan (to H. H.).


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Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of Medical Bacteria. Cambridge: Cambridge University Press.

Deinhard, G., Saar, J., Krischke, W. & Poralla, K. (1987). Bacillus cycloheptanicus sp. nov., a new thermoacidophile containing {omega}-cycloheptane fatty acids. Syst Appl Microbiol 10, 68–73.

Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution 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.[Abstract/Free Full Text]

Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]

Goto, K., Matsubara, H., Mochida, K., Matsumura, T., Hara, Y., Niwa, M. & Yamasato, K. (2002). Alicyclobacillus herbarius sp. nov., a novel bacterium containing {omega}-cycloheptane fatty acids, isolated from herbal tea. Int J Syst Evol Microbiol 52, 109–113.[Abstract]

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]

Komagata, K. & Suzuki, K. (1987). Lipid and cell-wall analysis in bacterial systematics. Methods Microbiol 19, 161–207.

Nakayama, T., Tsuruoka, N., Akai, M. & Nishino, T. (2000). Thermostable collagenolytic activity of a novel thermophilic isolate, Bacillus sp. strain NTAP-1. J Biosci Bioeng 89, 612–614.

Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]

Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.

Weisburg, W. G., Barns, S. M., Pelletier, D. A. & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173, 697–703.[Abstract/Free Full Text]

Wisotzkey, J. D., Jurtshuk, P., Jr, Fox, G. E., Deinhard, G. & Poralla, K. (1992). Comparative sequence analyses on the 16S rRNA (rDNA) of Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus and proposal for creation of a new genus, Alicyclobacillus gen. nov. Int J Syst Bacteriol 42, 263–269.[Abstract/Free Full Text]




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