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1 Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan
2 Department of Bioscience and Technology, School of Engineering, Hokkaido Tokai University, Minamisawa, Minami-ku, Sapporo 005-8601, Japan
3 Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo 060-8589, Japan
4 Laboratory of Electron Microscopy, Graduate School of Dentistry, Hokkaido University, Kita-ku, Sapporo 060-8586, Japan
5 Department of Biological Science, Faculty of Science, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
Correspondence
Isao Yumoto
i.yumoto{at}aist.go.jp
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ABSTRACT |
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Supplementary figures showing maximum-parsimony and minimum-evolution phylogenetic trees are available with the online version of this paper.
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; Krulwich et al., 2007), ion transport systems (Krulwich et al., 2007) and cell wall components (Aono et al., 1999). Most of these studies were performed using a limited number of strains representative of alkaliphiles. However, studies of the cytochrome content (Yumoto et al., 1997), lipid composition (Clejan et al., 1986) and cell wall composition (Aono & Horikoshi, 1983) of various alkaliphilic Bacillus strains have indicated that the environmental adaptation mechanisms of alkaliphilic Bacillus strains differ. Therefore, it is considered that such Bacillus strains have diverse physiological alkaline adaptation mechanisms because they exhibit taxonomic diversity and have a wide ecological distribution.
Nearly 30 alkaliphilic Bacillus species have been identified to date (Nielsen et al., 1995; Nogi et al., 2005; Yumoto, 2007). These species are not scattered phylogenetically, but are grouped into three main clusters on the basis of their 16S rRNA gene sequence. Alkaliphiles exist not only in specific environments such as alkaline soda lakes (Duckworth et al., 1996), but also in ordinary soils and faeces (Horikoshi, 1991). Some alkaliphiles can thrive in different alkaline environments, whereas some alkaliphiles can thrive only in specific environments. Furthermore, some alkaliphiles can thrive in diverse environments regardless of ambient pH. Such species may differ in their physiological functions for adapting to the ambient environment, even among strains of the same species. For example, although alkaliphilic Bacillus species isolated from the gut of higher termites exhibit similar taxonomic characteristics to a Bacillus strain isolated from soil, some of them show distinct sensitivity to NaCl compared to the corresponding soil isolate (Thongaram et al., 2003).
Since the original isolation of strain YN-1T from an indigo ball, the NADH dehydrogenase (Xu et al., 1991; Kitazume et al., 2006), cyanide-insensitive non-proteinaceous respiratory material (Higashibata et al., 1998), flagella rotatory system (Hirota & Imae, 1983) and polyamines (Hamasaki et al., 1993) of this strain have been studied. On the basis of these data, it is important to study the phylogenetic position of this alkaliphilic isolate. In consideration of the relationship between the taxonomic characteristics and physiological function of alkaliphiles, their characterization and identification are crucial. In the present study phenotypic and chemotaxonomic characteristics have been used together with phylogenetic analysis based on the 16S rRNA gene sequence of strain YN-1T to show that the new isolate merits classification as a novel species of Bacillus.
Strain YN-1T was originally isolated and identified as a Bacillus species by Ota et al. (1975). In the present study, PYA (peptone/yeast extract/alkaline) agar plates (pH 10) containing 8 g peptone (Kyokuto), 3 g yeast extract (Merck) and 15 g agar in 1 l NaHCO3/Na2CO3 buffer (100 mM in deionized water; pH 10) (Yumoto et al., 1998) was used as the basal medium for the strain. Cells for chemotaxonomic analyses were cultured with reciprocal shaking (120 r.p.m.) at 27 °C for 18 h. In addition to strain YN-1T, Bacillus clarkii DSM 8720T, Bacillus agaradhaerens DSM 8721T and Bacillus cellulosilyticus JCM 9156T were used as reference strains for DNA–DNA hybridization. These were cultured using PYA broth containing 100 mM NaHCO3/Na2CO3 buffer (pH 10).
For phenotypic characterization, PYA medium was also used as the basal medium. The culture was incubated at 27 °C for 2 weeks and the experiment was performed three times. Acid production from carbohydrate was determined by the method of Hugh & Leifson (1953) using thymol blue (0.008 %, w/v) instead of bromthymol blue at pH 10. Growth experiments at pH 7–10 were performed using PYA medium containing 100 mM NaH2PO4/Na2HPO4 buffer (pH 7–8), 100 mM NaHCO3/Na2CO3 buffer (pH 9–10), Na2HPO4/NaOH buffer (pH 11) or KCl/NaOH buffer (pH 12). Hydrolysis of cellulose and xylan was examined according to the method of Teather & Wood (1982) using medium containing 1 % substrate in the basal medium. Other physiological and biochemical characteristics were examined according to the methods of Yumoto et al. (1998) and Barrow & Feltham (1993).
To observe negatively stained cells by TEM, strain YN-1T cells were grown on a PYA agar slant. The procedures for TEM preparation and observation were as described previously (Yumoto et al., 2001). The morphological, physiological and biochemical characteristics of the isolate are given in the species description. The isolate was Gram-positive and produced ellipsoidal spores centrally positioned within a sporangium which was not swollen. TEM showed that the cells were non-flagellate rods (0.4–0.5x1.0–3.5 µm).
Whole-cell fatty acids and isoprenoid quinones were analysed as described previously (Yumoto et al., 1998, 2001). The results of GLC of the fatty acids of strain YN-1T grown at pH 10 are shown in Table 1. The fatty acids were mostly of the branched type. Differences were observed between YN-1T and its closest neighbour, B. clarkii DSM 8720T. The major isoprenoid quinone was MK-7.
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. The DNA base composition was determined by the method of Tamaoka & Komagata (1984). The DNA G+C content of strain YN-1T was 42.9 mol%, which is similar to that of B. clarkii DSM 8720T (41.6 mol%)
The 16S rRNA gene was amplified by PCR using primers 9F (5'-GAGTTTGATCCTGGCTCAG-3') and 1541R (5'-AAGGAGGTGATCCAGCC-3'). The approximately 1.5 kb PCR product was sequenced directly by the dideoxynucleotide chain-termination method using a DNA sequencer (PRISM 3100; Applied Biosystems). Multiple alignments of the sequences were performed using the CLUSTAL W program (Thompson et al., 1994). A phylogenetic tree was constructed by the neighbour-joining (Kimura, 1980; Saitou & Nei, 1987), maximum-parsimony and minimum-evolution methods in MEGA4 (Tamura et al., 2007). The confidence values for the branches of the phylogenetic tree were determined using bootstrap analysis (Felsenstein, 1985) based on 1000 resamplings. The similarity between sequences was calculated using the GENETYX computer program (Software Development). The 16S rRNA gene sequence (1558 bp) of strain YN-1T was obtained. Similarity with previously reported strains was determined, and a phylogenetic tree was constructed (Fig. 1 and supplementary Figs S1 and S2, available with the online version of this paper). These results suggest that strain YN-1T is a member of group 7 (alkaliphiles) of the genus Bacillus (Nielsen et al., 1994). The strain showed the highest similarity with B. clarkii DSM 8720T (99.5 %) among the strains identified as species. It also showed high sequence similarities with other phylogenetic neighbours, namely B. agaradhaerens DSM 8721T (96.1 %) and B. cellulosilyticus JCM 9156T (95.6 %).
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using photobiotin-labelled DNA probes and black microplates. The hybridization temperature was 38.1 °C. According to the sequence similarities and phylogenetic analysis based on the 16S rRNA gene sequence, strain YN-1T is most closely related to B. clarkii DSM 8720T among the strains identified as species. Therefore, DNA–DNA hybridizations between strain YN-1T and B. clarkii DSM 8720T, B. agaradhaerens DSM 8721T and B. cellulosilyticus JCM 9156T were performed. DNA–DNA hybridization data indicated that the isolate is distinct from B. clarkii DSM 8720T (7 %), B. agaradhaerens DSM 8721T (8 %) and B. cellulosilyticus JCM 9156T (10 %). Therefore, it is proposed that strain YN-1T represents a novel species.
Strain YN-1T can also be differentiated from B. clarkii DSM 8720T and other phylogenetic neighbours on the basis of several phenotypic and chemotaxonomic characteristics (Table 2).
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reported that strain YN-1T possesses caa3-type cytochrome c oxidase, the oxidase reaction was negative in our study. This may be due to a greater dependence on cyanide-insensitive non-proteinaceous material than on cytochrome c oxidase for oxygen metabolism in this strain. Although most alkaliphilic Bacillus strains are aerobes, Nogi et al. (2005) reported that several alkaliphilic Bacillus species are oxidase-negative. This suggests that several alkaliphilic Bacillus species have an alternative system, such as one based on cyanide-insensitive non-proteinaceous material. Second, although Hirota & Imae (1983) reported on Na+-driven flagellar motors in strain YN-1T, we found that the strain is non-motile and has no flagella. This may be explained by considering that extensive reisolation had been performed before the study by Higashibata et al. (1998), and we have studied the reisolated strain. Strain YN-1T exhibited notable growth at pH 12. To the best of our knowledge, this is the first report of an alkaliphilic Bacillus species that can grow at pH 12. This is understandable because the strain was isolated from indigo fermentation fluid and the initial stage of indigo fermentation is carried out at a high pH (approx. pH 12).
On the basis of the above results, it is suggested that the isolate is a novel species for which the name Bacillus polygoni sp. nov. is proposed.
Description of Bacillus polygoni sp. nov.
Bacillus polygoni (po.ly'gon.i. N.L. gen. n. polygoni of Polygonum tinctorium, referring to polygonum, the fermentation product from which the type strain was isolated).
The strain is a non-motile, Gram-positive, aerobic and straight rod (0.4–0.5x1.0–3.5 µm) and produces centrally located ellipsoidal spores that do not swell the sporangium. Colonies are circular and white. The isolate grows at 3–14 % NaCl. The growth temperature range is 5–47 °C with an optimum growth temperature range of 29–31 °C at pH 10. The maximum generation time was 45 min at 35–39 °C. pH range for growth is pH 8–12 with an optimum of pH 9. Positive for catalase, ONPG and nitrate reduction; negative for oxidase, indole production, deamination of phenylalanine and H2S production. Positive for hydrolysis of casein and Tweens 20, 40 and 60. Negative for hydrolysis of gelatin, starch, xylan, cellulose, MUG (4-methylumbelliferone glucuronide), aesculin, DNA, Tween 80, pullulan and hippurate. Acid, but no gas, is produced from D-xylose, D-arabinose, D-glucose, D-fructose, maltose, D-mannose, raffinose, glycerol, D-mannitol, sucrose and myo-inositol when grown at pH 10. No acid is produced from D-galactose, lactose, melibiose, sorbitol, rhamnose, salicin, xylitol and meso-erythritol. The major isoprenoid quinone is MK-7. The major cellular fatty acids consist of anteiso-C15 : 0, anteiso-C17 : 0 and anteiso-C17 : 1. The DNA G+C content is 42.9 mol%.
The type strain, YN-1T (=JCM 14604T=NCIMB 14282T), was originally isolated from indigo balls from Ibaraki, Japan.
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