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1 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China
2 Graduate School of the Chinese Academy of Sciences, Beijing 100049, China
Correspondence
Yanfen Xue
xueyf{at}im.ac.cn
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 8-1T is AY321433.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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), are distributed globally from oceanic to diverse terrestrial saline environments, including salterns, saline soils, salted foods and soda lakes. Numerous MHB are assigned taxonomically to the genera Halobacillus (Spring et al., 1996), Virgibacillus (Heyndrickx et al., 1998), Gracilibacillus (Wainø et al., 1999), Filobacillus (Schlesner et al., 2001), Jeotgalibacillus (Yoon et al., 2001), Lentibacillus (Yoon et al., 2002), Tenuibacillus (Ren & Zhou, 2005a) and Salinibacillus (Ren & Zhou, 2005b), many of which were originally affiliated to the genus Bacillus.
In recent years, several Gram-positive MHB belonging to the family Bacillaceae have been isolated from the salt lakes in Xin-Jiang, China (Jeon et al., 2005; Ren & Zhou, 2005a, b). Here we present phenotypic and genotypic data from a moderately halophilic strain, designated 8-1T, and show that this represents a novel species of the genus Lentibacillus.
The sampling site (Ai-Ding Lake), sample collection and treatment and the isolation of MHB were as described by Ren & Zhou (2005a). Strain 8-1T was grown routinely on HM medium (Ventosa et al., 1982) with the addition of 10 % (w/v) NaCl. Cell morphology was observed by using an AOM Optical 1-20 light microscope and a transmission electron microscope (Hitachi S-570) as described by Zhu et al. (2003). Gram staining was performed as described by Gerhardt et al. (1981). Motility was determined in wet mounts by using phase-contrast microscopy (AOM Optical 1-20) and by culture in soft-agar medium. The presence of flagella was demonstrated by staining (Kodaka et al., 1982) and transmission electron microscopy. On HM medium with 10 % (w/v) NaCl, strain 8-1T formed creamy, circular colonies that were slightly convex at the centre. Cells of strain 8-1T were rod-shaped, measuring approximately 0.5x1.5–2.5 µm. Cells were non-motile and no flagella were observed. In contrast, Jeon et al. (2005) reported that cells of Lentibacillus salarius KCTC 3911T were motile by means of two flagella on the sides of the cells.
General physiological and biochemical tests were performed as described by Smibert & Krieg (1981). The NaCl, temperature and pH ranges for growth of the new isolate were determined as described by Ren & Zhou (2005a). Utilization of carbon and energy sources was investigated by using basal medium to which appropriate substrates (0.5 %, w/v) had been added (Xin et al., 2001). Twenty-six different saccharides, polysaccharides and sugar alcohols were each used as sole carbon sources. Hydrolysis of starch, casein, gelatin and Tweens 20, 40, 60 and 80 was determined on a modified HM plate (Ventosa et al., 1982) by replacing the saccharide in the medium with the appropriate substrate. Susceptibility to antibiotics was determined on agar-medium plates by using absorbent paper discs impregnated with antibiotics. Strain 8-1T showed moderate halophily, growing in the presence of 5–30 % (w/v) NaCl. It grew optimally on media with 8–12 % (w/v) NaCl. Strain 8-1T grew in the pH range 6.0–9.0 (optimum pH 7.0–7.5) and a temperature range of 22–45 °C (optimum 30 °C). Other biochemical properties are detailed in Table 1 and in the species description below.
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), determination of the cellular fatty acid profile (MIDI Sherlock microbial identification system; Microbial ID) and peptidoglycan composition (Schleifer & Kandler, 1972) and analysis of polar lipids (Minnikin et al., 1984) and isoprenoid quinones (Collins et al., 1977; Groth et al., 1996) was obtained by cultivating strain 8-1T at 30 °C and pH 7.2 on HM medium with 10 % (w/v) NaCl. The genomic DNA G+C content was determined by the thermal denaturation method according to Marmur & Doty (1962). The major isoprenoid quinone of strain 8-1T was MK-7. Strain 8-1T contained meso-diaminopimelic acid (meso-DAP) as a diagnostic diamino acid in the cell-wall peptidoglycan; by contrast, Jeon et al. (2005) reported that L. salarius KCTC 3911T did not contain any diamino acids in its cell-wall peptidoglycan. The major polar lipids of strain 8-1T were phosphatidylglycerol, diphosphatidylglycerol and two unidentified glycolipids. Cellular fatty acid analysis of strain 8-1T revealed that the main components were anteiso-C15 : 0 (42.41 %), iso-C15 : 0 (20.73 %), anteiso-C17 : 0 (17.17 %), iso-C16 : 0 (7.73 %), iso-C17 : 0 (5.3 %), iso-C14 : 0 (3.1 %) and C16 : 0 (2.16 %), contrasting with several closely related species of the genus Lentibacillus. The genomic DNA G+C content of strain 8-1T was 43.4 mol%, a value similar to those reported for L. salarius KCTC 3911T (43 mol%) and Lentibacillus salicampi KCCM 41560T (44 mol%). Characteristics that are useful in the differentiation of strain 8-1T from several closely related taxa are shown in Table 1
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The 16S rRNA gene of strain 8-1T was amplified by PCR with universal primers as described by Duckworth et al. (1996). The almost-complete nucleotide sequence (1466 bp) was determined by direct sequencing and compared with available 16S rRNA gene sequences in the GenBank database by using the BLAST program. Multiple alignment with closely related members was performed by using the CLUSTAL W program (Thompson et al., 1994). Ambiguous and unalignable bases were manually omitted and the phylogenetic tree was then constructed by using the neighbour-joining, minimum-evolution and maximum-parsimony methods in the MEGA program (version 3.1; Kumar et al., 2004). The stability of relationships was assessed by bootstrap analysis (1000 replications). The phylogenetic tree (Fig. 1) based on the neighbour-joining method indicated that strain 8-1T formed a coherent cluster with recognized species of the genus Lentibacillus. Strain 8-1T was related most closely to L. salarius KCTC 3911T (98.0 % 16S rRNA gene sequence similarity) and showed levels of similarity of 94.5–95.9 % to other recognized species of the genus Lentibacillus. Similar tree topologies were obtained with the maximum-likelihood and maximum-parsimony algorithms (data not shown). DNA–DNA hybridization was performed by the spectrophotometric renaturation rate method (Huß et al., 1983; De Ley et al., 1970) to determine the level of relatedness between strain 8-1T and L. salarius KCTC 3911T, its closest relative as determined from 16S rRNA gene sequence analysis. The level of DNA–DNA relatedness between strain 8-1T and L. salarius KCTC 3911T was 40 % (mean of three independent experiments, which did not differ by more than 6 %). In combination with 16S rRNA gene sequence data, this indicates that strain 8-1T is not genotypically related to any recognized species of the genus Lentibacillus (Stackebrandt & Goebel, 1994).
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Description of Lentibacillus halodurans sp. nov.
Lentibacillus halodurans (ha.lo.du'rans. Gr. n. hals salt; L. pres. part. durans enduring; N.L. part. adj. halodurans salt-enduring).
Cells are Gram-positive, rod-shaped, 0.5x1.5–2.5 µm, non-motile and strictly aerobic. Spherical or oval endospores form at the cell-terminal position in swollen sporangia. Colonies are white, slightly convex and 4–5 mm in diameter with regular margins after 2 days cultivation on HM medium with 10 % (w/v) NaCl. Oxidase- and catalase-positive but urease-negative. Growth occurs at a temperature range of 22–45 °C (optimum 30 °C) and NaCl concentration of between 5 and 30 % (w/v) (optimum 8–12 %). No growth occurs without NaCl. The pH range for growth is 6.0–9.0 (optimum pH 7.0–7.5). Nitrate is not reduced to nitrite. D-Glucose, D-mannose, D-fructose, cellobiose, L-sorbose, inulin, D-raffinose, D-galactose, salicin, lactose, sucrose, aesculin, maltose, mannitol, melibiose, D-sorbose, trehalose, dulcitol, glycerol, inositol, erythritol, melezitose, arabinose, rhamnose and xylose are used as carbon sources. Acid is produced from D-mannose, D-fructose and D-glucose. Gelatin and Tween 40 are hydrolysed, but casein, starch, aesculin, and Tweens 20 and 80 are not. Cells are resistant to norfloxacin (10 µg), neomycin (30 µg), bacitracin (0.04 U), tetracycline (30 µg), streptomycin (10 µg), chloramphenicol (30 µg), kanamycin (30 µg) and penicillin G (10 U), but susceptible to novobiocin (30 µg), rifampicin (5 µg), erythromycin (15 µg), ciprofloxacin (5 µg), vancomycin (30 µg) and ampicillin (10 µg). The predominant menaquinone is MK-7. The diagnostic diamino acid in the cell-wall peptidoglycan is meso-DAP, and major polar lipids are phosphatidylglycerol, diphosphatidylglycerol and two unidentified glycolipids. The major fatty acids are anteiso-C15 : 0, iso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The genomic DNA G+C content is 43.4 mol% (Tm).
The type strain, 8-1T (=CGMCC 1.3702T=DSM 18342T), was isolated from a sediment sample of a neutral salt lake in Xin-Jiang, China.
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ACKNOWLEDGEMENTS |
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