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1 Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, 254 Phayathai Road, Wangmai, Pathumwan, Bangkok 10330, Thailand
2 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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19.4 %). Therefore, the strains represent a novel species of the genus Lentibacillus, for which the name Lentibacillus halophilus sp. nov. is proposed. The type strain is PS11-2T (=JCM 12149T=TISTR 1549T=PCU 240T).
A scanning electron micrograph of sporulating cells of strain PS11-2T, a thin-layer chromatogram of polar lipids and details of the cellular fatty acids and levels of DNA–DNA relatedness of the novel strains and related taxa are available as supplementary material in IJSEM Online.
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; Spring et al., 1996; Heydrickx et al., 1998; Arahal et al., 2000; Schlesner et al., 2001; Lu et al., 2001; Heyrman et al., 2003; Yoon et al., 2002, 2004; Lim et al., 2005a). The genus Lentibacillus, which forms a phylogenetically coherent group, currently comprises four species: Lentibacillus salicampi, L. juripiscarius, L. salarius and L. lacisalsi (Yoon et al., 2002; Namwong et al., 2005; Jeon et al., 2005; Lim et al., 2005b). In this paper, we report the isolation and identification of some novel extremely halophilic strains representing a novel Lentibacillus species.
Fish-sauce samples were collected from fish-sauce factories in Thailand during the early, middle and late stages of the fermentation process. Halophilic bacteria were isolated from the samples by using the spread plate technique on agar plates of JCM medium no. 168 (containing, l–1, 200 g NaCl, 5 g Casamino acids, 5 g yeast extract, 1 g glutamic acid, 2 g KCl, 3 g trisodium citrate, 20 g MgSO4.7H2O, 36 mg FeCl2.4H2O, 0.36 mg MnCl2.4H2O and 20 g agar; pH 7.2] with incubation at 37 °C for 7 days. Liquid cultures were cultivated in Erlenmeyer flasks containing the same medium without agar and were incubated on a rotary shaker. All media contained 20 % (w/v) NaCl, except those used to investigate NaCl tolerance. Cell shape, cell size and cell arrangement were examined 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 on Gram-stained specimens. Critical-point-dried cells were observed under a scanning electron microscope. Flagella were examined as described by Forbes (1981) and observed by transmission electron microscopy. Catalase activity, oxidase activity and the hydrolysis of aesculin were investigated as described by Barrow & Feltham (1993), while urease activity and the hydrolysis of gelatin, casein, starch, Tween 80, tyrosine, phenylalanine, xanthine and hypoxanthine were tested as described by Namwong et al. (2005). Arginine hydrolysis was investigated by using the medium reported by Thornley (1960). Acid production from carbohydrate was determined in the medium described by Leifson (1963), supplemented with 20 % NaCl. Growth under anaerobic conditions on agar plates with or without KNO3 (1 %, w/v) was performed in a Gaspak (BBL) anaerobic jar. Growth at various temperatures (10–50 °C), pH values (5, 6, 7, 7.5, 8 and 9) and NaCl concentrations (0–30 %, w/v) was tested by using JCM medium no. 168 as a basal medium (Namwong et al., 2005).
The diaminopimelic acid in the peptidoglycan and the menaquinone composition were determined as described previously (Komagata & Suzuki, 1987). Polar lipids were investigated according to the methods of Minnikin et al. (1984) and Albert et al. (2005). A loop of cell mass was used for the extraction and quantitative analysis of the cellular fatty acids by means of the Microbial Identification System (MIDI) (Sasser, 1990; Kämpfer & Kroppenstedt, 1996).
DNA was isolated from cells grown in JCM medium no. 168 broth and purified according to the method of Saito & Miura (1963). The DNA G+C content was determined by the method of Tamaoka & Komagata (1984), using 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 isolate was amplified, purified and sequenced as described previously (Seearunruangchai et al., 2004). The sequence determined (1410 bases) was aligned with selected sequences (obtained from the GenBank/EMBL/DDBJ database) 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 of branches of the phylogenetic tree were determined using bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings.
Fifteen aerobic, extremely halophilic bacteria were isolated from the fish-sauce samples, and were characterized according to their morphological, cultural, physiological and biochemical properties. The results are listed in the species description and Table 1. All 15 strains contained meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan. The menaquinones, cellular fatty acids and polar lipids of four of these strains, namely PS11-2T, CB0-1, DS26-3 and DB9-1, were analysed. The four strains contained the following: MK-7 as a major menaquinone; anteiso-C15 : 0 (58.0–62.7 %), anteiso-C17 : 0 (24.6–33.8 %), C16 : 0 (1.7–3.0 %), iso-C15 : 0 (2.7–7.8 %) and iso-C16 : 0 (1.8–2.3 %) as the cellular fatty acids (see Supplementary Table S1 available in IJSEM Online); and phosphatidylglycerol, diphosphatidylglycerol and two unidentified glycolipids as major polar lipids (see Supplementary Fig. S1 available in IJSEM Online).
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). The same topology was obtained by applying the maximum-parsimony method (result not shown). The 16S rRNA gene sequence similarity values between PS11-2T and L. juripiscarius JCM 12147T, L. salarius KCTC 3911T, L. lacisalsi KCTC 3915T and L. salicampi JCM 11462T were 97.3, 95.5, 95.4 and 95.3 %, respectively. Furthermore, strain PS11-2T showed 93.3–94.2 % 16S rRNA gene sequence similarity to members of the genus Virgibacillus. Hybridization studies revealed high levels of DNA–DNA relatedness between PS11-2T and PB7-3 and to the other isolates (>70 %), but only low levels with respect to L. salicampi JCM 11462T (4.9–19.4 %) and L. juripiscarius JCM 12147T (17.0–17.1 %), as shown in Supplementary Table S2 (available in IJSEM Online).
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; Namwong et al., 2005; Jeon et al., 2005; Lim et al., 2005b). The fatty acid profiles of the four strains examined were qualitatively similar to those of other Lentibacillus species, although the levels of iso-C14 : 0 and iso-C16 : 0 were significantly lower than those of the other Lentibacillus species reported (0.7 % iso-C14 : 0 and 1.8–2.3 % iso-C16 : 0 for the isolates; 5.7–13.9 % iso-C14 : 0 and 16.3–26.5 % iso-C16 : 0 for Lentibacillus species). When the cellular fatty acid profile of L. juripiscarius JCM 12147T grown on JCM medium no. 168 (containing 20 % NaCl) was compared with the profile from cells grown on the Lentibacillus medium (JCM medium no. 377, containing 10 % NaCl), the levels of iso-C14 : 0 and iso-C16 : 0 were significantly lower (2.1 and 6.3 %, respectively: see Supplementary Table S1 in IJSEM Online). Thus, the levels of iso-C14 : 0 and iso-C16 : 0 were affected by NaCl concentration during growth. The morphological, cultural, physiological and biochemical characteristics that differentiate the isolates from Lentibacillus species are shown in Table 1
. It is noteworthy that all of the isolates are extreme halophiles requiring at least 12 % NaCl for growth. In addition, the isolates do not reduce nitrate and do not produce acid from sugars, unlike Lentibacillus species. The 16S rRNA gene sequence of PS11-2T shows a relatively high level of similarity to that of the type strain of L. juripiscarius (97.3 %). However, the low levels of DNA–DNA relatedness (19.4 %) demonstrate that the novel strains do not belong to the species L. juripiscarius. On the other hand, inclusion of the 15 strains in the same species is supported by the fact that they share the same phenotypic properties and demonstrate high levels of DNA–DNA relatedness to each other (>70 %) (Wayne et al., 1987). Therefore, these 15 isolates represent a novel species in the genus Lentibacillus, for which we propose the name Lentibacillus halophilus sp. nov. The type strain is PS11-2T (=JCM 12149T=TISTR 1549T=PCU 240T).
Description of Lentibacillus halophilus sp. nov.
Lentibacillus halophilus (ha.lo'phi.lus. Gr. n. hals, halos salt; Gr. adj. philos loving; N.L. masc. adj. halophilus salt-loving).
Cells are Gram-positive, aerobic, motile rods and are mostly 0.4–0.6 µm wide and 1.0–3.0 µm long. Longer cells (up to 6 µm) or short filaments are observed. Spherical endospores are formed terminally in swollen sporangia (see Supplementary Fig. S2 available in IJSEM Online). Colonies are white to cream, low-convex or raised, smooth and circular (0.1–0.8 mm in diameter). Catalase- and oxidase-positive. Urease-negative. Growth occurs between 15 °C (weakly) and 42 °C (optimum, 30–37 °C) but not at 10, 45 or 50 °C. Growth is observed between pH 6 and 8 (optimum, pH 7.0–7.5) but not at pH 5 or 9. Extremely halophilic, growing in the presence of 12–30 % (w/v) NaCl but not at or below 10 % (w/v) NaCl (optimum, 20–26 % NaCl, w/v). Anaerobic growth is not observed in the presence of 1 % KNO3 (w/v) or in other media. Aesculin, arginine, casein, gelatin, Tween 80, tyrosine, starch, phenylalanine, xanthine and hypoxanthine are not hydrolysed. Acid is not produced from D-glucose, glycerol, D-ribose, D-xylose, L-arabinose, cellobiose, D-fructose, D-galactose, lactose, maltose, D-mannitol, D-mannose, D-melibiose, D-melezitose, myo-inositol, raffinose, L-rhamnose, salicin, sorbitol, sucrose or D-trehalose. Contains meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan. MK-7 is the major menaquinone. The fatty acid profile consists of anteiso-C15 : 0 (58.0–62.7 %), anteiso-C17 : 0 (24.6–33.8 %), iso-C15 : 0 (2.4–7.8 %), C16 : 0 (1.7–3.0 %), iso-C16 : 0 (1.8–2.3 %), iso-C17 : 0 (0.5–1.4 %), C14 : 0 (0–0.9 %), iso-C14 : 0 (0–0.7 %), C15 : 0 (0–0.6 %) and C12 : 0 (0–0.5 %). Phosphatidylglycerol, diphosphatidylglycerol and two unidentified glycolipids are predominant in the polar lipid profile. The DNA G+C content is 42.1–43.1 mol% (type strain, 42.4 mol%).
The type strain, PS11-2T (=JCM 12149T=TISTR 1549T=PCU 240T), was isolated from a fish-sauce fermentation in Thailand.
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ACKNOWLEDGEMENTS |
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