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1 Environmental Biotechnology National Core Research Center, Division of Environmental Biotechnology, Gyeongsang National University, 660-701, Korea
2 Korea Research Institute of Bioscience and Biotechnology, 52 Oeundong, Yusong, Daejeon 305-333, Republic of Korea
3 Yunnan Institute of Microbiology, Yunnan University, Kunming, Yunnan, 650091, P. R. China
Correspondence
Chang-Jin Kim
changjin{at}kribb.re.kr
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain BH139T is AY667493.
A transmission electron micrograph showing the general morphology and flagella of strain BH139T is available as supplementary material in IJSEM Online.
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). They are attracting interest because they have great biotechnological potential for the production of compatible solutes or hydrolytic enzymes (Margesin & Schinner, 2001). Taxonomically, this group of bacteria includes both Gram-positive and Gram-negative micro-organisms. Aerobic, spore-forming, moderately halophilic, Gram-positive rods are also taxonomically very diverse and were originally assigned to the genus Bacillus (Ventosa et al., 1998; Nielsen et al., 1994). However, molecular and chemical analyses have shown that they form several phylogenetically distinct lineages such as the genera Halobacillus (Spring et al., 1996), Gracilibacillus (Wainø et al., 1999), Virgibacillus (Heyndrickx et al., 1998), Filobacillus (Schlesner et al., 2001) and Pontibacillus (Lim et al., 2005) within the group classically defined as the genus Bacillus (Ash et al., 1991; Stackebrandt & Liesack, 1993; Nielsen et al., 1994).
It has been reported that some members of the genera Filobacillus and Halobacillus show Gram-negative or Gram-variable reactions despite belonging phylogenetically to a Gram-positive group (Schlesner et al., 2001; Yoon et al., 2003). Yoon et al. (2002) indicated that Lentibacillus salicampi isolated from the Yellow sea in Korea also produced a Gram-variable reaction. In the course of screening of halophilic bacteria, an aerobic, Gram-positive, moderately halophilic bacterium, strain BH139T, was isolated from Xinjiang Province, China. In this study, this isolate is described as a novel species within the genus Lentibacillus, on the basis of phylogenetic and phenotypic characteristics.
Strain BH139T was isolated from soil sediment of a salt lake in Xinjiang Province, China, using marine agar 2216 (MA; Difco), to which 15 % (w/v) NaCl had been added (final NaCl concentration: 16·94 %, w/v), at 28 °C for 3 days. NaCl requirements and tolerance were determined in trypticase soy broth (per litre: 17·0 g casein, 3·0 g soy-bean meal and 2·5 g glucose, 5·0 g sodium chloride and 2·5 g dipotassium phosphate) supplemented with modified artificial sea water (per litre: 0–30 %, w/v, NaCl; 5·94 g MgSO4.7H2O; 4·53 g MgCl2.6H2O; 0·64 g KCl; 1·3 g CaCl2). To investigate its morphological and physiological characteristics, strain BH139T was cultivated on MA with 12 % (w/v) NaCl at 30 °C, except where indicated otherwise. Anaerobic growth was determined by incubation in an anaerobic chamber for 5 days at 30 °C on MA supplemented with 10 % (w/v) NaCl. Optimum growth was tested at different temperatures (4–55 °C) on MA containing 12 % (w/v) NaCl and at different pH values (5·0–10·0) in trypticase soy broth supplemented with artificial sea water containing 12 % (w/v) NaCl.
Cell morphology was studied using light microscopy and transmission electron microscopy. Motility was observed on agar-coated wet mounts by using a light microscope (Nikon E600) after 12 and 36 h cultivation. The flagellum type was examined by transmission electron microscopy using cells from the exponential growth phase. Cells were mounted on Formvar-coated copper grids (Electron Microscopy Science) and negatively stained with 2 % (w/v) uranyl acetate for 15 s, then subjected to transmission electron microscopy (JEM-1010; JEOL) operated at 80 kV. Endospores were stained using Schaeffer–Fulton stain (Smibert & Krieg, 1981).
Gram staining was performed using the bioMérieux Gram Stain kit according to the manufacturer's instructions. Catalase activity was determined from the production of oxygen bubbles in a 3 % (v/v) aqueous hydrogen peroxide solution. Oxidase activity was tested for by the oxidation of 1 % (w/v) tetramethyl-p-phenylenediamine (Merck). Hydrolysis of aesculin, casein, starch, Tween 80, urea, hypoxanthine, tyrosine and xanthine was determined on MA according to methods described previously (Cowan & Steel, 1965; Lanyi, 1987; Smibert & Krieg, 1994). Nitrate reduction was performed according to the method of Lanyi (1987). Acid production from carbohydrates was determined as described by Leifson (1963); all suspension media were supplemented with artificial sea water containing 12 % (w/v) NaCl.
For quantitative analysis of whole-cell fatty acids, strain BH139T was cultivated on MA and on MA with the addition of 10 % (w/v) NaCl, at 30 °C for 5 and 2 days, respectively. The preparation of fatty acid methyl esters and their analysis were performed according to the instructions of the Microbial Identification System (Microbial ID; MIDI). Analysis of the peptidoglycan and the polar lipids of the strain was carried out using the methods described by Komagata & Suzuki (1987). Isoprenoid quinones were analysed as described by Komagata & Suzuki (1987), using HPLC with a reversed-phase column (GROM-SIL 100 ODS-2FE, GROM). The DNA G+C content of strain BH139T was determined by reversed-phase HPLC using the method of Tamaoka & Komagata (1984).
Sequencing and assembly of 16S rRNA genes were carried out as described previously (Bakermans & Madsen, 2002). The resultant sequences of 16S rRNA genes were compared with available 16S rRNA sequences from GenBank, using the BLAST program (http://www.ncbi.nlm.nih.gov/blast/), and aligned with closely related members by using CLUSTAL W software (Thompson et al., 1994). Sequence similarity values were computed using Similarity Matrix version 1.1 (Ribosomal Database Project II; http://35.8.164.52/html/; Cole et al., 2003). Gaps at the 5' and 3' ends of the alignment were omitted from further analyses. The phylogenetic trees were constructed using three different algorithms – neighbour-joining, maximum-likelihood and maximum-parsimony – available within PHYLIP software, version 3.6 (Felsenstein, 2002). Distance matrices were calculated according to the algorithm of the Kimura two-parameter model (Kimura, 1980) for the neighbour-joining method. To evaluate the stability of the phylogenetic tree, a bootstrap analysis (1000 replications) was performed with the SEQBOOT, DNADIST, NEIGHBOR and CONSENSE programs in the PHYLIP package.
On MA medium with 12 % (w/v) NaCl, strain BH139T formed smooth, creamy, circular colonies and the colony morphologies were consistent at various salinities in the range 1–20 % (w/v) NaCl. Strain BH139T showed moderate halophily, growing in media containing 1–20 % (w/v) NaCl; optimum growth occurred on media with 12–14 % (w/v) NaCl. Growth was observed at temperatures between 15 and 45 °C; the optimum growth temperature was 30–35 °C. Strain BH139T grew in the pH range 6·0–8·5 in 12 % (w/v) NaCl-containing trypticase soy broth; optimal growth was observed at pH 7·0–7·5. Cells of strain BH139T from early and late growth phases revealed obvious Gram-positive reactions; however, it was reported that L. salicampi KCCM 41560T showed a Gram-variable reaction (Yoon et al., 2002). Cells of the isolate were slender and strictly rod-shaped, being 0·2–0·3 µm wide and 1·5–3·0 µm long after 2 days incubation at 30 °C on MA with 12 % (w/v) NaCl. The isolate produced a spherical terminal endospore within a swollen sporangium. Cell motility was facilitated by two flagella on the sides of the cells (see the Supplementary Figure in IJSEM Online). In contrast, Namwong et al. (2005) reported that Lentibacillus juripiscarius JCM 12147T was non-motile and lacked flagella. Anaerobic growth was not observed after 5 days at 30 °C on MA with 12 % (w/v) NaCl under anaerobic conditions.
The major isoprenoid quinone of strain BH139T was MK-7. The fatty acid profile of the strain was characterized by the presence of branched and saturated fatty acids such as iso-C16 : 0, anteiso-C15 : 0, iso-C15 : 0, iso-C14 : 0 and anteiso-C17 : 0 (the major fatty acids). Although this fatty acid profile was shared by L. salicampi KCCM 41560T (currently the closest relative, on the basis of 16S rRNA gene sequence), the fatty acid compositions of the two micro-organisms were somewhat different (Table 1). Analysis of cell-wall peptidoglycan showed that strain BH139T did not contain any amino acids. Attempts to analyse the peptidoglycan of the isolate failed, even after several re-examinations and analysis by the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany). However, it was reported that the phylogenetically closest strain, L. salicampi KCCM 41560T, possesses meso-diaminopimelic acid as the diagnostic diamino acid (Yoon et al., 2002). The major polar lipids of strain BH139T were diphosphatidylglycerol and phosphatidylglycerol, which were the same as those of L. salicampi KCCM 41560T. The genomic DNA G+C content of strain BH139T was about 43 mol%. In Table 2, typical phenotypes of strain BH139T are summarized and compared with those of the type strains of closely related taxa.
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). The topologies of phylogenetic trees obtained using the maximum-likelihood and maximum-parsimony algorithms were similar to that of the tree constructed using neighbour-joining analysis (data not shown). The strain shared 16S rRNA gene sequence similarity values of 96·9 and 95·5 %, respectively, with the closely related strains L. salicampi KCCM 41560T and L. juripiscarius JCM 12147T; these levels of 16S rRNA gene sequence similarity are sufficient to justify defining a novel species (Rosselló-Mora & Amann, 2001; Stackebrandt et al., 2002).
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Description of Lentibacillus salarius sp. nov.
Lentibacillus salarius (sa.la'ri.us. L. masc. adj. salarius of, or belonging to, salt, because of the isolation of this micro-organism from saline sediment).
Cells are approximately 0·2–0·3 µm wide and 1·5–3·0 µm long and are strictly aerobic motile rods with flagella. Colonies are cream-coloured, low convex, smooth and circular on MA supplemented with 10 % (w/v) NaCl. Oxidase-negative and catalase-positive. Growth occurs at 15–50 °C (optimum: 30–35 °C), pH 6·0–8·5 (optimum: pH 7·0–7·5) and 1–20 % (w/v) NaCl (optimum: 12–14 %). No growth occurs without NaCl or in the presence of more than 20 % (w/v) NaCl. Nitrate is reduced to nitrite. Aesculin is hydrolysed. Hydrolysis of urea, L-tyrosine, hypoxanthine, casein, starch, Tween 80 and xanthine is not observed. Acids are produced from D-xylose, D-ribose, glycerol, D-glucose, maltose, D-trehalose, L-arabinose, 
-D-lactose, D-mannitol, D-fructose and D-mannose, but not from rhamnose, adonitol, raffinose, arbutin, D-salicin or D-melibiose. The major polar lipids are diphosphatidylglycerol and phosphatidylglycerol. The major fatty acids are iso-C16 : 0, anteiso-C15 : 0, iso-C15 : 0, iso-C14 : 0 and anteiso-C17 : 0. The DNA G+C content is about 43 mol% (HPLC).
The type strain is BH139T (=KCTC 3911T=DSM 16459T), isolated from saline soil of Xinjiang Province, China.
Emended description of the genus Lentibacillus Yoon et al. 2002
Lentibacillus (Len.ti.ba.cil'lus. L. adj. lentus slow; L. dim. n. bacillus small rod; N.L. masc. n. Lentibacillus slowly growing bacillus).
Cells are rods. Gram-variable. Spherical or oval endospores lie terminally in swollen sporangia. Motile or non-motile. Colonies are cream-coloured, smooth and circular to slightly irregular. Catalase-positive and oxidase-variable. Urease-negative. The cell-wall peptidoglycan contains meso-diaminopimelic acid. The predominant menaquinone is MK-7. The major polar lipids are diphosphatidylglycerol and phosphatidylglycerol. The major fatty acids are anteiso-C15 : 0 and iso-C16 : 0. The G+C content of the DNA is in the range 42–44 mol%.
The type species is Lentibacillus salicampi.
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ACKNOWLEDGEMENTS |
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