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1 Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
2 Department of Applied Biology, Faculty of Science, King Mongkut's Institute of Technology Ladkrabang, Bangkok 10520, Thailand
3 Thailand Institute of Scientific and Technological Research, Pathumthani 12120, Thailand
4 Japan Collection of Microorganisms, RIKEN BioResource Center, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
Correspondence
Somboon Tanasupawat
Somboon.T{at}chula.ac.th
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ABSTRACT |
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A scanning electron micrograph of endospore-containing cells of strain PN7-6T and a table showing strain designations, isolation sources, DNA G+C contents and levels of DNA–DNA relatedness for strains PN5-2 and PN7-6T and related Lentibacillus species are available as supplementary material in IJSEM Online.
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), Bacillus (Ventosa et al., 1989), Amphibacillus (Niimura et al., 1990; Zhilina et al., 2001), Halobacillus (Spring et al., 1996), Tetragenococcus (Satomi et al., 1997), Virgibacillus (Heyndrickx et al., 1998), Gracilibacillus (Wainø et al., 1999), Filobacillus (Schlesner et al., 2001), Oceanobacillus (Lu et al., 2001), Lentibacillus (Yoon et al., 2002), Paraliobacillus, Halolactibacillus (Ishikawa et al., 2002, 2005), Cerasibacillus (Nakamura et al., 2004), Pontibacillus (Lim et al., 2005a), Tenuibacillus (Ren & Zhou, 2005a), Salinibacillus (Ren & Zhou, 2005b), Alkalibacillus (Jeon et al., 2005b) and Thalassobacillus (García et al., 2005). Several novel species of the genus Lentibacillus have been described recently, Lentibacillus salicampi, Lentibacillus juripiscarius, Lentibacillus salarius, Lentibacillus lacisalsi and Lentibacillus halophilus (Yoon et al., 2002; Namwong et al., 2005; Jeon et al., 2005a; Lim et al., 2005b; Tanasupawat et al., 2006). A fermented shrimp paste (‘ka-pi’), produced in Thailand, contains a high concentration of NaCl which permits the growth of various halophilic micro-organisms (Phithakpol et al., 1995; Tanasupawat & Komagata, 2001). In this study, a red-pigmented novel species isolated from ka-pi was identified as belonging to the genus Lentibacillus on the basis of phenotypic and chemotaxonomic characterization and 16S rRNA gene sequence analysis.
The halophilic bacteria investigated in this work were isolated from samples of fermented shrimp paste collected from a market in Nakhonsrithammarat Province in southern Thailand. The spread-plate technique was used on agar plates of JCM medium no. 168; incubation was performed at 37 °C for 7 days. Liquid cultures were cultivated in Erlenmeyer flasks containing the same medium and incubated on a rotary shaker. All media contained 20 % (w/v) NaCl, except when NaCl tolerance was being investigated. Cell shape, size and arrangement and colony size were examined using cells grown on JCM medium no. 168 agar at 37 °C for 5 days. The Hucker–Conn modification was used for Gram staining (Hucker & Conn, 1923). Spore formation was examined using Gram-stained specimens and colonies subjected to a temperature of 80 °C for 30 min. Critical-point-dried cells were observed under a scanning electron microscope. The presence of flagella was determined as described by Forbes (1981) and observed by using transmission electron microscopy. The physiological and biochemical characteristics were determined as described by Thornley (1960), Leifson (1963) and Barrow & Feltham (1993), using medium supplemented with 15 % (w/v) NaCl. Growth under anaerobic conditions on agar plates with or without nitrate (1 %, w/v) was investigated using a GasPak (BBL) anaerobic jar. Growth at various temperatures (10–50 °C), pH values (5.0, 6.0, 7.0, 7.5, 8.0 and 9.0) and NaCl concentrations (0–30 %, w/v) was assessed. At the lower NaCl concentrations (0.0–2.0 %, w/v), MgSO4.7H2O was omitted from the test medium, while KCl and C6H5Na3O7.2H2O (trisodium citrate dehydrate) were added. Growth was monitored by measuring culture turbidity at 660 nm (Namwong et al., 2005).
The presence of meso-diaminopimelic acid in the cell wall peptidoglycan and menaquinone profiles were analysed as described previously (Komagata & Suzuki, 1987). Polar lipid profiles were determined according to the methods of Minnikin et al. (1984) and Albert et al. (2005). Quantitative analysis of the cellular fatty acids was performed as described previously (Sasser, 1990; Kämpfer & Kroppenstedt, 1996). DNA was isolated from cells grown in JCM medium no. 168 broth, supplemented with 15 % (w/v) NaCl and purified according to the method of Saito & Miura (1963). The DNA G+C content was determined by using the method of Tamaoka & Komagata (1984), with reversed-phase HPLC. DNA–DNA hybridization was conducted in microdilution-well plates, as reported by Ezaki et al. (1989), and was detected by using the colorimetric method described by Tanasupawat et al. (2000). The 16S rRNA gene of the novel isolate was amplified, purified and sequenced according to the methods of Seearunruangchai et al. (2004). The sequence determined (1516 bases) was aligned with selected sequences (obtained from GenBank) by using CLUSTAL W, version 1.81 (Thompson et al., 1994). The alignment was manually edited to remove gaps and ambiguous nucleotides prior to the construction of the phylogenetic tree. The phylogenetic tree was constructed by using the neighbour-joining method (Saitou & Nei, 1987) in MEGA, version 2.1 (Kumar et al., 2001). The confidence values for the branches of the phylogenetic tree were determined using bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings.
Two strains of moderately halophilic rods were characterized on the basis of their phenotypic and chemotaxonomic characteristics; the results are listed in the species description and in Tables 1 and 2. In the case of strain PN7-6T, endospores were observed after high-temperature treatment (80 °C) of a culture for 30 min (see Supplementary Fig. S1 available in IJSEM Online). Flagella were not observed. The cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. The predominant isoprenoid quinone found was MK-7. Polar lipid analysis revealed the presence of phosphatidylglycerol, diphosphatidylglycerol and two unidentified glycolipids, whereas L. salicampi JCM 11462T contained phosphatidylglycerol, diphosphatidylglycerol and one unidentified glycolipid (result not shown).
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). On the basis of these characteristics and the data from 16S rRNA gene sequencing, strain PN7-6T was included in a monophyletic cluster consisting of the Lentibacillus species, as shown in Fig. 1 (Yoon et al., 2002; Jeon et al., 2005a; Lim et al., 2005b; Namwong et al., 2005). The values for 16S rRNA gene sequence similarity between strain PN7-6T and L. salarius KCTC 3911T, L. salicampi JCM 11462T, L. juripiscarius JCM 12147T, L. lacisalsi KCTC 3915T and L. halophilus JCM 12149T were 96.5, 96.3, 96.3, 95.6 and 94.7 %, respectively. In addition, strain PN7-6T showed 94.5–95.1 % 16S rRNA gene sequence similarity to members of the genus Virgibacillus and 90.8–94.2 % sequence similarity to the other related bacteria (Fig. 1). The DNA–DNA hybridization study revealed that strains PN5-2 and PN7-6T were closely related, exhibiting 98–118 % similarity, but they showed only low levels of DNA–DNA relatedness to L. salicampi JCM 11462T (2.3–16.5 %) and L. juripiscarius JCM 12147T (5.9–16.2 %) (see Supplementary Table S1 available in IJSEM Online), indicating that the two novel strains are unrelated to these Lentibacillus species (Wayne et al., 1987). The DNA G+C contents of strains PN5-2 and PN7-6T were in the range 41.2–41.6 mol%. The two novel strains could be differentiated from L. salarius KCTC 3911T and related species on the basis of pigmentation, motility, maximum growth temperature, NaCl tolerance, oxidase activity, hydrolysis of aesculin, casein and Tween 80, acid production from maltose, D-trehalose and D-xylose and DNA G+C content, as shown in Table 1. Thus, strains PN5-2 and PN7-6T represent a novel species within the genus Lentibacillus, for which we propose the name Lentibacillus kapialis sp. nov.
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Cells are Gram-positive, slender-rod-shaped, strictly aerobic, red-pigmented, non-motile and approximately 0.2–0.4x0.8–2.5 µm in size. Spherical endospores are formed terminally in swollen sporangia. Flagella are not observed. Colonies are low convex, smooth and circular (0.2–1.3 mm in diameter). Growth occurs at temperatures between 15 and 45 °C (optimum at 37 °C), at pH 5–9 (optimum at pH 7.0) and with 5–30 % NaCl (optimum at 15 % NaCl). Anaerobic growth is not observed in the presence of 1 % (w/v) nitrate. Positive in tests for catalase, oxidase, and urease activity and nitrate reduction. Hydrolyses arginine and gelatin, but not aesculin, casein, Tween 80, tyrosine, starch, xanthine or hypoxanthine. Produces acid from D-fructose, D-galactose, D-glucose, glycerol, D-mannitol, D-mannose, D-ribose, sorbitol and sucrose, but not from amygdalin, L-arabinose, cellobiose, aesculin, gluconate, myo-inositol, inulin, lactose, maltose, melibiose, melezitose, methyl 
-D-glucoside, raffinose, L-rhamnose, salicin, D-trehalose or D-xylose. Contains meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan. MK-7 is the major menaquinone. The fatty acid profile includes anteiso-C15 : 0 (38.3 %), iso-C16 : 0 (23.4 %) and iso-C14 : 0 (14.8 %). Phosphatidylglycerol, diphosphatidylglycerol and two unidentified glycolipids are predominant in the polar lipid profile. The DNA G+C content of the type strain is 41.6 mol%.
The type strain, PN7-6T (=JCM 12580T=PCU 259T=TISTR 1551T), was isolated from fermented shrimp paste (‘ka-pi’) produced in Thailand.
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ACKNOWLEDGEMENTS |
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