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1 Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Republic of Korea
2 Department of BioEnvironmental Chemistry, College of Agriculture and Life Sciences, Chungnam National University, Taejon, Republic of Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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ABSTRACT |
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A table detailing the cellular fatty acid compositions of strain AHS-1T and related Lentibacillus species is available as supplementary material with the online version of this paper.
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MAIN TEXT |
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with the description of a single species, Lentibacillus salicampi. Subsequently, six further species of the genus, Lentibacillus salarius (Jeon et al., 2005), L. lacisalsi (Lim et al., 2005), L. juripiscarius (Namwong et al., 2005), L. halophilus (Tanasupawat et al., 2006), L. kapialis (Pakdeeto et al., 2007) and L. halodurans (Yuan et al., 2007), have been described. Here we report on the taxonomic characterization of a novel bacterial strain, AHS-1T, which was isolated from a marine solar saltern in Korea.
Sediment samples collected from a marine solar saltern of the Yellow Sea, Byunsan, Korea, were used as the source for the isolation of bacterial strains. The sediment samples (each 0.5 mg) were inoculated in 100 ml modified S-G (MSG) medium (Sehgal & Gibbons, 1960) containing (per litre of distilled water): 100 g NaCl, 20 g MgSO4 . 7H2O, 2 g KCl, 3 g trisodium citrate, 10 g yeast extract and 7.5 g Casamino acids (pH 7.2). This medium was incubated at 30 °C on a horizontal shaker at 150 r.p.m. After incubation for 30 days, 0.1 ml of the suspension was transferred into 100 ml fresh medium and the medium was reincubated under the same conditions. Strain AHS-1T was isolated from the enrichment culture by means of dilution plating on MSG medium solidified with 1.5 % (w/v) agar. L. salarius KCTC 3911T, which was used as a reference strain for DNA–DNA hybridization experiments, was obtained from the Korean Collection for Type Cultures (KCTC), Taejon, Korea. The morphological, cultural, physiological and biochemical characteristics of strain AHS-1T were investigated by using routine cultivation at 37 °C on MSG medium (pH 7.2). Cell morphology was examined by light microscopy (Nikon E600) and transmission electron microscopy. Flagellation was determined by using a Philips CM-20 transmission electron microscope with cells from exponentially growing cultures: for this purpose, cells were negatively stained with 1 % (w/v) phosphotungstic acid and the grids were examined after being air-dried. The Gram reaction was determined by using the bioMérieux Gram stain kit according to the manufacturer's instructions. Growth under anaerobic conditions was determined after incubation in a Forma anaerobic chamber on MSG agar and on MSG agar supplemented with potassium nitrate (0.1 %, w/v), both of which had been prepared anaerobically under nitrogen. The pH range for growth was determined in MSG broth that was adjusted to various pH values (pH 4.5–10.5 at intervals of 0.5 pH units). Growth at various NaCl concentrations (0–30 % at intervals of 1 %) was investigated by using liquid media prepared according to the formula of the MSG medium except that NaCl was excluded. Growth at various temperatures (4–50 °C) was measured on MSG agar. Catalase and oxidase activities were determined as described by Cowan & Steel (1965). Hydrolysis of casein, starch, hypoxanthine, tyrosine and xanthine was tested on MSG agar by using the substrate concentrations described by Cowan & Steel (1965). Hydrolysis of aesculin, gelatin, Tween 80 and urea and reduction of nitrate were investigated as described by Lanyi (1987) with the modification that artificial seawater (Bruns et al., 2001) supplemented with 8 % (w/v) NaCl was used for preparation of media. Acid production from carbohydrates was determined as reported by Leifson (1963) but using supplementation with 8 % (w/v) NaCl.
Cell biomass for DNA extraction and for the analyses of cell-wall, isoprenoid quinones and polar lipids was obtained from cultivation in MSG broth at 37 °C. Chromosomal DNA was isolated and purified according to the method described by Yoon et al. (1996), with the exception that RNase T1 was used in combination with RNase A to minimize RNA contamination. The 16S rRNA gene was amplified by PCR by using two universal primers as given by Yoon et al. (1998). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed as described by Yoon et al. (2002). The isomer type of the diamino acid in the cell-wall peptidoglycan was analysed via TLC according to the method described by Komagata & Suzuki (1987). Isoprenoid quinones were analysed according to Komagata & Suzuki (1987) by using reversed-phase HPLC. Polar lipids were extracted according to the procedures of Minnikin et al. (1984) and were identified by two-dimensional TLC followed by spraying with appropriate detection reagents (Minnikin et al., 1984; Komagata & Suzuki, 1987). For cellular fatty acid analysis, cell mass of strain AHS-1T was harvested from agar plates after cultivation for 2 days at 37 °C on marine agar 2216 (Difco) supplemented with 10 % (w/v) NaCl. Fatty acids were extracted and fatty acid methyl esters were prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). The DNA G+C content was determined according to the method of Tamaoka & Komagata (1984) with the modification that DNA was hydrolysed by using nuclease P1 (Sigma) and the resultant nucleotides were analysed by reversed-phase HPLC. DNA–DNA hybridization experiments were performed fluorometrically according to the method of Ezaki et al. (1989). Hybridization was performed with five replications for each sample. The highest and lowest values obtained in each sample were excluded, and the means of the remaining three values were quoted as DNA–DNA relatedness values.
Morphological, cultural, physiological and biochemical characteristics of strain AHS-1T are detailed in Table 1 or are given in the species description below. Growth occurred at 15–45 °C (optimum, 37–40 °C), at pH 6.0–9.5 (optimum, pH 6.5–7.0) and in the presence of 3–24 % (w/v) NaCl (optimum, 10–12 % NaCl). After incubation for 4 days on MSG agar at 37 °C, colonies were circular, slightly convex, smooth, entire, cream-yellow in colour and 0.5–1.2 mm in diameter.
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). Strain AHS-1T showed highest 16S rRNA gene sequence similarity to the type strain of L. salarius (97.0 %), and showed values of 95.0–96.7 % to the type strains of other recognized Lentibacillus species. The mean level of DNA–DNA relatedness between strain AHS-1T and L. salarius KCTC 3911T was 8.5 %, when their DNAs were used individually as labelled DNA probes for cross-hybridization, indicating that these taxa represent different genomic species.
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; Jeon et al., 2005; Tanasupawat et al., 2006; Yuan et al., 2007). The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The predominant isoprenoid quinone detected in strain AHS-1T was menaquinone-7 (MK-7) and the major polar lipids were diphosphatidylglycerol, phospatidylglycerol and two unidentified glycolipids. Strain AHS-1T contained large amounts of branched- and straight-chain fatty acids; the major components (>10 % of the total fatty acids) were anteiso-C15 : 0 (54.0 %) and anteiso-C17 : 0 (27.0 %) (see Supplementary Table S1 in IJSEM Online). The fatty acid profile of strain AHS-1T was similar to those of recognized Lentibacillus species, although there were some differences in the proportions of some components, perhaps due to differences in the cultivation, extraction and analysis conditions used (see Supplementary Table S1 in IJSEM Online). The DNA G+C content of strain AHS-1T was 49.0 mol%, a value higher than those of other Lentibacillus species. Strain AHS-1T was distinguishable from recognized Lentibacillus species by differences in several phenotypic characteristics (Table 1
). Accordingly, phylogenetic and genetic distinctiveness, together with differential phenotypic properties, was sufficient to allocate strain AHS-1T in a species that is separate from recognized Lentibacillus species (Wayne et al., 1987; Stackebrandt & Goebel, 1994). On the basis of the data presented, strain AHS-1T represents a novel species of the genus Lentibacillus, for which the name Lentibacillus salinarum sp. nov. is proposed.
Description of Lentibacillus salinarum sp. nov.
Lentibacillus salinarum (sa.lin.a'rum. L. gen. pl. n. salinarum of salt-works).
Cells are Gram-positive, facultatively anaerobic, rod-shaped and 0.7–1.2x2.0–4.0 µm in size. Motile by means of peritrichous flagella. Colonies on MSG agar are circular, slightly convex, smooth, entire, cream-yellow in colour and 0.5–1.2 mm in diameter after incubation for 4 days at 37 °C. Optimal growth temperature is 37–40 °C; growth occurs at 15 and 45 °C, but not at 10 or 46 °C. Optimal pH for growth is 6.5–7.0; growth occurs at pH 6.0, but not at pH 5.5. Optimal growth occurs in the presence of approximately 10–12 % (w/v) NaCl; growth occurs in the presence of 24 % (w/v) NaCl, but not in the absence of NaCl or in the presence of >25 % (w/v) NaCl. Growth occurs under anaerobic conditions on MSG agar and on MSG agar supplemented with potassium nitrate. Catalase- and oxidase-positive. Urease-negative. Hypoxanthine, xanthine and tyrosine are not hydrolysed. Positive for nitrate reduction. The cell-wall peptidoglycan contains meso-diaminopimelic acid as the diamino acid. The predominant menaquinone is MK-7. Major fatty acids (>10 % of the total) are anteiso-C15 : 0 and anteiso-C17 : 0. Phosphatidylglycerol, diphosphatidylglycerol and two unidentified glycolipids are present as major polar lipids. The DNA G+C content of the type strain is 49.0 mol%.
The type strain, AHS-1T (=KCTC 13162T=CCUG 54822T), was isolated from a marine solar saltern of the Yellow Sea, Byunsan, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of Medical Bacteria. London: Cambridge University Press.
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Yuan, S., Ren, P., Liu, J., Xue, Y., Ma, Y. & Zhou, P. (2007). Lentibacillus halodurans sp. nov., a moderately halophilic bacterium isolated from a salt lake in Xin-Jiang, China. Int J Syst Evol Microbiol 57, 485–488.
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