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Institute of Cell Biology, School of Life Sciences, Lanzhou University, Lanzhou 730000, People's Republic of China
Correspondence
Guang-Qin Guo
gqguo{at}lzu.edu.cn
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ABSTRACT |
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Phylogenetic trees based on maximum-parsimony and minimum-evolution analyses of the 16S rRNA gene sequences of strain CM1T and related species are available as supplementary figures in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Arahal et al., 1999; Lim et al., 2006; Nielsen et al., 1994; Ventosa et al., 1989; Yoon et al., 2004). Because of their potential usefulness in the production of compatible solutes or hydrolytic enzymes (Margesin & Schinner, 2001), alkaline enzymes (Horikoshi, 1971), cyclomaltodextrin glucanotransferase (CGTase) (Yamamoto et al., 1972) and cellulase (Horikoshi et al., 1971), this group of bacteria has attracted a great deal of attention recently. While screening halophilic bacteria from a crude sea-salt sample collected near Qingdao in eastern China, we isolated an aerobic, Gram-positive, moderately halophilic bacterium, designated as strain CM1T. On the basis of its phenotypic characteristics and genotypic data, this strain should be assigned to the genus Bacillus as a novel species.
CM medium (Seghal & Gibbons, 1960) at pH 7.0 and containing 12 % (w/v) NaCl was used for bacterial enrichment and growth. The sample was first inoculated into the liquid medium and cultured on a shaker (120 r.p.m.) at 37 °C in the dark until turbid, then streaked onto solid medium to obtain single colonies. The streaking was repeated several times to obtain pure single colonies. The purified strain was cultured and maintained in liquid or solid ATCC 213 medium (Garrity et al., 2004) at pH 7.0, with 12 % (w/v) NaCl, in the dark.
Cell morphology was determined by using phase-contrast microscopy. Gram staining was performed according to Dussault (1955) and electron microscopy was performed with negative staining according to Bouchotroch et al. (2001).
Bacterial growth was determined by measuring absorbance at 660 nm at certain intervals during liquid culture. The effects of different concentrations of NaCl on the growth of strain CM1T were tested in liquid ATCC 213 medium by measuring the absorbance at 660 nm after 7 days incubation.
The susceptibility of strain CM1T to different antibiotics, including ampicillin, tetracycline, hygromycin, kanamycin, streptomycin, rifampicin, bacitracin, penicillin, chloramphenicol, neomycin and erythromycin (Sigma) was tested on solid ATCC 213 medium after 7 days incubation at 37 °C. Analysis of the whole-cell fatty acid pattern was performed according to previously described methods (Miller, 1982) using the Sherlock system (MIDI). Isoprenoid quinones were analysed by HPLC with a reversed-phase column according to the method of Komagata & Suzuki (1987).
Physiological and biochemical tests were performed as described by Buchanan & Gibbons (1974) and Dong & Cai (2001). Anaerobic growth of strain CM1T with nitrate as the electron acceptor was tested according to the method of Mancinelli & Hochstein (1986). Growth with DMSO and by the fermentation of L-arginine as alternative electron acceptors was tested as described by Oren et al. (1997) and Oren & Trüper (1990) using closed tubes fully filled with the growth medium and then kept in the dark for 3 weeks before comparison of the growth with that found on medium lacking the test compounds. The DNA G+C content was determined by the thermal denaturation method (Sly et al., 1986) using a Beckman UV-800 spectrophotometer equipped with a TB-85 thermostat. We used the optical renaturation method (De Ley et al., 1970; Huß et al., 1983; Jahnke, 1992) to perform DNA–DNA hybridization experiments.
PCR amplification of the 16S rRNA gene was performed according to Reddy et al. (2003). The PCR product was ligated to the T-vector and transformed into Escherichia coli JM109 for purification and sequencing.
The sequences used for comparison with the 16S rRNA gene sequence from strain CM1T were obtained from GenBank by using the BLAST program. The sequence from strain CM1T was aligned with closely related 16S rRNA gene sequences using CLUSTAL_X software (version 1.83; Thompson et al., 1997). The phylogenetic tree was constructed by using neighbour-joining (Saitou & Nei, 1987), maximum-parsimony and minimum-evolution methods in MEGA, version 3.1 (Kumar et al., 2001, 2004). The confidence values for the branches of the phylogenetic tree were determined using bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings.
Cells of strain CM1T were Gram-positive rods, 0.3–0.4 µm wide and 1.8–3.2 µm long (Fig. 1). The colonies were cream–white to yellow in colour, wet and smooth-surfaced with clear edges on ATCC 213 medium containing 12 % (w/v) NaCl. Strain CM1T grew as single cells or clusters (Fig. 1). The novel strain did not produce spores, even in the presence of 0.1 g MnCl2 ml–1 and under other conditions used for stimulating spore formation (Hageman et al., 1984).
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Growth of strain CM1T could be inhibited by the antibiotics rifampicin, penicillin, hygromycin, ampicillin and bacitracin. No inhibition was observed when the strain was grown in the presence of kanamycin, erythromycin, streptomycin, tetracycline, chloramphenicol or neomycin.
The major fatty acids of strain CM5T were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0; the fatty acid profile is shown in Table 1. The major isoprenoid quinones were MK-7(H2) (95.15 %) and MK-6(H2) (4.85 %).
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. Strain CM1T had a DNA G+C content of 48 mol% and had a DNA–DNA reassociation value of 35.4 % with Bacillus salarius BH169T.
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and Supplementary Figs S1 and S2 available in IJSEM Online).
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). In conclusion, strain CM1T represents a novel species of the genus Bacillus, for which the name Bacillus qingdaonensis sp. nov. is proposed.
Description of Bacillus qingdaonensis sp. nov.
Bacillus qingdaonensis (qing.da.o.nen'sis. N.L. masc. adj. qingdaonensis pertaining to Qingdao, the name of the place from which the type strain was isolated).
Cells are rod-shaped, 0.3–0.4x1.8–3.2 µm and Gram-positive. Colonies are cream–white to yellow, smooth and circular. Aerobic, non-spore-forming and non-motile (non-flagellated). Grows at 25–45 °C and at 2.5–20 % (w/v) NaCl, with optima around 37 °C and 12 % (w/v) NaCl. The pH range for growth is 6.5–10.5 in 12 % (w/v) NaCl (optimum, pH 9.0). Oxidase-negative and catalase-positive. Negative for hydrolysis of soluble starch, gelatin and Tweens 20 and 80 and for production of H2S and indole. Positive for hydrolysis of urea and arginine and for nitrate reduction. Utilizes, and produces acid, from sucrose, D-glucose, D-fructose, D-galactose, cellobiose, D-mannose, D-mannitol, D-galactose, lactose, D-xylose and dextran as sole carbon sources. Voges–Proskauer and methyl red reactions are negative. The major fatty acids are anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The major isoprenoid quinones are MK-7(H2) and MK-6(H2). The DNA G+C content of the type strain is 48 mol%.
The type strain, CM1T (=CGMCC 1.6134T=JCM 14087T), was isolated from a crude sea-salt sample collected near Qingdao in eastern China.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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