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1 Institute of Biological Resources and Function, Hokkaido Centre, National Institute of Advanced Industrial Science and Technology, Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan
2 Department of Bioscience and Technology, School of Engineering, Hokkaido Tokai University, Minaminosawa, Minami-ku, Sapporo 005-8601, Japan
3 Laboratory of Electron Microscopy, School of Dentistry, Hokkaido University, Kita-ku, Sapporo 060-8586, Japan
4 Laboratory of Environmental Molecular Biology, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan
Correspondence
Isao Yumoto
i.yumoto{at}aist.go.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA sequence reported in this paper is AB110008.
A more complete version of the phylogenetic tree presented in Fig. 1
is available as supplementary material in IJSEM Online.
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MAIN TEXT |
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; Hiraishi et al., 1999; Nübel et al., 2002). Research on hot-spring biomats represents a rather appropriate case study for determining microbial ecosystems in environments with extreme temperatures. It has been reported that the isolation of micro-organisms from such biomats is difficult (Hiraishi et al., 1999). Therefore, several non-culture methods have been applied to the analysis of microbial flora in hot-spring biomats (Yamamoto et al., 1998; Hiraishi et al., 1999). Nevertheless, there have been several attempts at isolating micro-organisms from hot-spring biomats: in the course of such studies, a number of micro-organisms have been isolated and identified (Pierson et al., 1985; Hanada et al., 1995, 2002). In 1978, drilling opened up the Voinovskie Hot Springs in Kamchatka, Russia. Prior to this, no hot springs had been found in this area. Natural springs are located 300–400 m upstream of the Mutnovskaya River on both banks. In Voinovskie Hot Springs, hair-like microbial vegetation, called ‘Veronica's hair’, was observed. We began to investigate the bacterial flora of this vegetation. By using light microscopic observation of a sample obtained from the hot spring, we observed that the relatively larger cells appeared dominant.
In this study, a relatively large, moderately thermophilic micro-organism among our isolates was isolated from a biomat sample obtained from the Voinovskie Hot Springs. Phenotypic characterization, phylogenetic analysis based on 16S rRNA gene sequences and DNA–DNA hybridization with close relatives showed that the isolate merited classification as a novel Anoxybacillus species.
Water samples collected from Voinovskie Hot Springs in Kamchatka, Russia, were plated on TH agar medium (pH 7·5) consisting of the following (per litre distilled water): 5 g peptone (Kyokuto), 3 g yeast extract (Kyokuto), 5 g NaCl, 15 g agar, 3·5 mg EDTA, 3 mg ZnSO4.7H2O, 10 mg FeSO4.7H2O, 2 mg MnSO4.nH2O, 1 mg CuSO4.5H2O, 2 mg Co(NO3)2.6H2O and 1 mg H3BO3. The cultures were then incubated aerobically at 70 °C for 24 h. This isolate was picked and subcultured five times for purification and maintained on TH agar medium at 70 °C. In addition to the isolate, Anoxybacillus flavithermus NBRC 15317T (=IFO 15317T) and Anoxybacillus pushchinoensis DSM 12423T were used as reference strains for DNA–DNA hybridization experiments. These micro-organisms were cultivated in TH broth in a rotary shaker at 105 r.p.m. and 50 °C.
For phenotypic characterization, TH medium was used as the basal medium. The isolate was incubated at 50 °C for 1 week and all the experiments were performed three times. Acid production from carbohydrates was determined by the method of Hugh & Leifson (1953). Growth experiments at pH 7–10 were performed using TH medium containing 100 mM NaH2PO4/Na2HPO4 buffer (pH 7–8) or 100 mM NaHCO3/Na2CO3 buffer (pH 9–10). Anaerobic growth was tested on a TH plate by substituting air with argon gas. Other physiological and biochemical characteristics were examined according to Kawasaki et al. (2002) and as described in Barrow & Feltham (1993).
For the morphological observation of negatively stained cells under a transmission electron microscope and of platinum- and palladium-coated cells under a scanning electron microscope, cells were grown on a TH agar slant for 1 day. The transmission electron microscope and scanning electron microscope preparations and observations were performed as described previously (Yumoto et al., 2000).
Analyses of whole-cell fatty acids and isoprenoid quinones were performed as described previously (Yumoto et al., 2001, 2002).
Bacterial DNA was prepared according to Marmur (1961). DNA base composition was determined according to Tamaoka & Komagata (1984). The level of DNA–DNA hybridization was determined fluorometrically according to Ezaki et al. (1989), using photobiotin-labelled DNA probes and black microplates.
16S rRNA gene sequence analysis was performed as reported previously (Yumoto et al., 2002). Multiple alignments of the sequence were performed and the nucleotide substitution rate (Knuc) was calculated. A phylogenetic tree was constructed by the neighbour-joining method (Kimura, 1980; Saitou & Nei, 1987) using the CLUSTAL W program (Thompson et al., 1994). Sequence similarity was calculated using the GENETYX computer program (Software Development).
The morphological, physiological and biochemical characteristics of the isolate are given in the species description. The isolate was revealed to be Gram-positive and moderately thermophilic (optimum temperature 54 °C). Electron microscopic observation showed that cells were non-flagellated, straight rods (0·4–0·6x1·5–5·0 µm). The cell surface was smooth when observed using scanning electron microscopy. No pronounced structures were observed under transmission electron microscopy of thin sections.
GLC analysis of the fatty acids of strain TH13T revealed that the major components were isoC14 : 0 (1·3 %), C14 : 0 (1·3 %), isoC15 : 0 (54·7 %), anteisoC15 : 0 (8·0 %), isoC16 : 0 (7·1 %), C16 : 0 (1·9 %), isoC17 : 0 (3·9 %), anteisoC17 : 1 (7·1 %) and C17 : 1 (2·6 %).
The 16S rRNA gene sequence of strain TH13T was analysed to determine its phylogenetic position. The sequence of 1506 bases of the 16S rRNA gene of strain TH13T was compared with those of two other Anoxybacillus spp. and other related taxa. The phylogenetic tree constructed using the neighbour-joining method (Fig. 1) and the 16S rRNA gene sequence similarity showed that strain TH13T is a member of the genus Anoxybacillus. The highest similarity values were observed with A. flavithermus DSM 2641T (95·7 %), Anoxybacillus gonensis NCIMB 13933T (94·8 %) and A. pushchinoensis DSM 12423T (94·5 %). These results demonstrate that strain TH13T is apparently distinct from other Anoxybacillus spp.
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According to the results of the 16S rRNA gene sequence analysis, strain TH13T is closely related to A. flavithermus DSM 2641T and A. pushchinoensis DSM 12423T. Therefore, DNA–DNA hybridization between strain TH13T and closely related strains, given above, was estimated. DNA–DNA relatedness data indicated that the isolate is distinct from A. flavithermus NBRC 15317T (20·6 % similarity) and A. pushchinoensis DSM 12423T (30·2 % similarity).
Strain TH13T differed from other relatively closely related species in terms of the following phenotypic characteristics: catalase production, hydrolysis of casein and starch, reduction of 
to 
, pH range for growth, and utilization of substrates (Table 1).
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Description of Anoxybacillus voinovskiensis sp. nov.
Anoxybacillus voinovskiensis (vo.in.ov.ski.en'sis. N.L. adj. voinovskiensis from Voinovskie, referring to the Voinovskie Hot Springs, the place of isolation).
Cells are Gram-positive, non-flagellated, straight rods (0·4–0·6x1·5–5·0 µm). It is facultatively aerobic. Colonies are circular and pale cream in colour. Catalase and oxidase reactions are positive. Negative for H2S production and hydrolysis of casein, gelatin, starch, DNA, Tween 20 and Tween 80. Hydrolyses Tween 40 and Tween 60. Growth occurs at pH 7–8, but not at pH 9–10. Growth occurs at 30–64 °C with the optimum temperature at 54 °C. Grows in the absence of NaCl, but not at concentrations higher than 3 % (w/v). Nitrate is reduced to nitrite. Acid is produced from D-glucose, D-xylose, D-arabinose, D-fructose, maltose, D-mannose, sucrose, sorbitol and cellobiose in aerobic conditions. No acid is produced from D-galactose, raffinose, melibiose, inositol, mannitol, trehalose, L-rhamnose or lactose in aerobic conditions. The DNA G+C content is 43·9 mol%, as determined by HPLC. Strain TH13T was isolated from a water sample collected from a hot spring in Kamchatka.
The type strain is TH13T (=NCIMB 13956T=JCM 12111T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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