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Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Republic of Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain MSS-402T is AY881246.
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; at present, it comprises six species with validly published names, Halobacillus halophilus, Halobacillus litoralis and Halobacillus trueperi (Spring et al., 1996), Halobacillus salinus (Yoon et al., 2003), Halobacillus karajensis (Amoozegar et al., 2003) and Halobacillus locisalis (Yoon et al., 2004).
The genus Halobacillus is differentiated from the genus Bacillus and other related genera as having a cell-wall peptidoglycan type based on L-orn–D-Asp (Spring et al., 1996; Shida et al., 1997; Yoon et al., 2001, 2002; Lim et al., 2005). Recently, in the course of screening for novel micro-organisms present in marine solar salterns of the west coast of the Korean peninsula, a Halobacillus-like bacterial strain, MSS-402T, was isolated and characterized taxonomically. In this study, we focused on strain MSS-402T because it was found to have an interesting morphological property, i.e. some cells are crooked, long, filamentous rods. The aim of the present work was to investigate the exact taxonomic status of strain MSS-402T by using polyphasic taxonomic characterization.
Water collected from a marine solar saltern at Seosan, Korea, provided the source for isolation of the bacterial strains. A standard dilution plating technique was used to isolate strain MSS-402T at 30 °C on marine agar 2216 (MA; Difco) supplemented with 8 % (w/v) NaCl. H. halophilus KCTC 3685T, H. litoralis KCTC 3687T and H. trueperi KCTC 3686T were obtained from the Korean Collection for Type Cultures, Taejon, Korea. H. karajensis DSM 14948T was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany. H. salinus HSL-3T and H. locisalis MSS-155T were obtained from the studies of Yoon et al. (2003, 2004). To investigate its morphological, physiological and biochemical characteristics, strain MSS-402T was routinely cultivated on MA supplemented with 3 % (w/v) NaCl at 30 °C. The cell morphology was examined by light microscopy (E600; Nikon) and transmission electron microscopy. For the latter, the cells were negatively stained with 1 % (w/v) phosphotungstic acid and, after air-drying, the grids were examined by using a model CM-20 transmission electron microscope (Philips). The presence of flagella was examined by transmission electron microscopy using cells from exponentially growing cultures. The Gram-reaction was determined using the bioMérieux Gram Stain kit according to the manufacturer's instructions. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on MA supplemented with 3 % (w/v) NaCl and on MA supplemented with 3 % (w/v) NaCl and nitrate, both of which had been prepared anaerobically using nitrogen. The optimal pH for growth was determined in trypticase soy broth (Difco), supplemented with 3 % (w/v) NaCl and 4·53 g MgCl2.6H2O (l distilled water)–1, that was adjusted to various pH values (pH 4·0 initially, rising to pH 10·5 at intervals of 0·5 pH units). The pH was adjusted prior to sterilization to various levels by the addition of HCl or Na2CO3. Growth in the absence of NaCl was investigated in trypticase soy broth without NaCl, supplemented with 4·53 g MgCl2.6H2O (l distilled water)–1. The requirement for ions for growth was investigated in trypticase soy broth supplemented with 3 % (w/v) NaCl using MgCl2.6H2O, MgSO4.7H2O, KCl or CaCl2 at the concentrations indicated by Bruns et al. (2001). Growth at various NaCl concentrations was investigated in marine broth 2216 (Difco) (more than 2 %, w/v, NaCl at intervals of 1 %) and in trypticase soy broth supplemented with 4·53 g MgCl2.6H2O (l distilled water)–1 (less than 2 %, w/v, NaCl at intervals of 0·5 %). Growth at various temperatures (4–50 °C) was measured on MA supplemented with 3 % (w/v) NaCl. Oxidase and catalase activities and hydrolysis of casein and starch were determined as described by Cowan & Steel (1965). Hydrolysis of Tweens 20, 40, 60 and 80 was determined as described by Cowan & Steel (1965), with the modification that artificial sea water was used instead of distilled water. Hydrolysis of aesculin, gelatin and urea and nitrate reduction were determined as described by Lanyi (1987), with the modification that artificial sea water was used for preparation of media. The artificial sea water contained the following (l distilled water)–1: 23·6 g NaCl, 0·64 g KCl, 4·53 g MgCl2.6H2O, 5·94 g MgSO4.7H2O and 1·3 g CaCl2.2H2O (Bruns et al., 2001). H2S production was tested as described by Bruns et al. (2001). Hydrolysis of hypoxanthine, tyrosine and xanthine was performed on MA supplemented with 3 % (w/v) NaCl, using the substrate concentrations indicated by Cowan & Steel (1965). Acid production from carbohydrates was determined as described by Leifson (1963). Enzyme activities and other physiological properties were determined using the API ZYM and API 20E systems (bioMérieux); the cell suspension used to inoculate the systems was prepared by using artificial sea water (Bruns et al., 2001).
Cell mass for analyses of the cell wall and menaquinones and for DNA extraction was produced in marine broth 2216 supplemented with 3 % (w/v) NaCl at 37 °C. Preparation of cell-wall peptidoglycan was carried out using the method described by Schleifer & Kandler (1972), and the peptidoglycan structure was determined using an automated amino acid analyser (model L-8500A; Hitachi). Menaquinones were analysed as described by Komagata & Suzuki (1987), using reverse-phase HPLC. Chromosomal DNA was isolated and purified according to the method described previously (Yoon et al., 1996), with the exception that RNase T1 was used in combination with RNase A. For fatty acid methyl ester analysis, cell mass of strain MSS-402T was harvested from agar plates after cultivation for 2 days at 37 °C on MA supplemented with 3 % (w/v) NaCl. The fatty acid methyl esters were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). The DNA G+C content was determined by the method of Tamaoka & Komagata (1984), with the modification that the DNA was hydrolysed and the resultant nucleotides were analysed by reverse-phase HPLC. The 16S rRNA gene was amplified by using a PCR with two universal primers, as described previously (Yoon et al., 1998). Sequencing of the 16S rRNA gene and phylogenetic analysis were performed as described by Yoon et al. (2003). DNA–DNA hybridization was performed fluorometrically by the method of Ezaki et al. (1989), using photobiotin-labelled DNA probes and microdilution wells. Hybridization was performed with five replications for each sample. The highest and lowest values obtained for each sample were excluded, and the means of the remaining three values were quoted as the DNA–DNA binding values.
The morphological, cultural, physiological and biochemical characteristics of strain MSS-402T are shown in Table 1 or are given in the species description below. Cells of strain MSS-402T were Gram-variable rods or long filamentous rods, measuring 0·4–0·6x2·0–100 µm after incubation on MA supplemented with 3 % (w/v) NaCl at 37 °C; a few cells greater than 100 µm in length were also observed (Fig. 1). This cellular morphological property was not found in other Halobacillus species (Spring et al., 1996; Amoozegar et al., 2003; Yoon et al., 2003, 2004). Strain MSS-402T required Na+ and Mg2+ ions for growth, but did not require K+ or Ca2+. The 16S rRNA gene sequence of strain MSS-402T determined in this study comprised 1522 nt, representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence. Comparative 16S rRNA gene sequence analyses showed that strain MSS-402T is phylogenetically most closely affiliated to the genus Halobacillus (Fig. 2). In the phylogenetic tree based on the neighbour-joining algorithm, strain MSS-402T fell within the radiation of the cluster comprising Halobacillus species (Fig. 2). The same tree topology was found in trees generated with the maximum-likelihood and maximum-parsimony algorithms (data not shown). The 16S rRNA gene sequence of strain MSS-402T had similarity levels of 98·0–99·2 % with respect to sequences of the type strains of recognized Halobacillus species (Fig. 2). Sequence similarities with respect to all other species included in the phylogenetic analysis were lower than 94·8 % (Fig. 2).
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, based on L-orn–D-Asp, as described by Schleifer & Kandler (1972). This cell-wall peptidoglycan type is a key marker that differentiates strain MSS-402T and the genus Halobacillus from other aerobic or facultatively anaerobic, endospore-forming, rod-shaped genera (Spring et al., 1996; Shida et al., 1997; Wainø et al., 1999; Yoon et al., 2001). The predominant isoprenoid quinone found in strain MSS-402T was unsaturated menaquinone with seven isoprene units (MK-7). The fatty acids detected in strain MSS-402T were anteiso-C15 : 0 (40·4 %), anteiso-C17 : 0 (23·0 %), iso-C16 : 0 (19·3 %), iso-C15 : 0 (8·8 %), iso-C17 : 0 (3·5 %), iso-C14 : 0 (3·4 %) and C16 : 0 (1·6 %). This fatty acid profile was similar to those of the type strains of Halobacillus species, although there were differences in the proportions of some fatty acids (Yoon et al., 2003, 2004). The DNA G+C content of strain MSS-402T was 42·9 mol%.
There were some differences between strain MSS-402T and recognized Halobacillus species in terms of phenotypic characteristics (Table 1
). DNA–DNA binding data were sufficient to categorize strain MSS-402T as representing a species that is distinct from recognized Halobacillus species (Wayne et al., 1987). The mean DNA–DNA relatedness levels between strain MSS-402T and the type strains of six recognized Halobacillus species were in the range 13–26 %. Therefore, on the basis of the data presented, strain MSS-402T should be placed in the genus Halobacillus as a member of a novel species, for which the name Halobacillus yeomjeoni sp. nov. is proposed.
Description of Halobacillus yeomjeoni sp. nov.
Halobacillus yeomjeoni (yeom.jeo'ni. N.L. gen. n. yeomjeoni of yeomjeon, the Korean name for a marine solar saltern).
Cells are rods or long filamentous rods, measuring 0·4–0·6x2·0–100 µm, on MA supplemented with 3 % (w/v) NaCl at 37 °C; a few cells in old cultures are greater than 100 µm in length (Fig. 1). Gram-positive, but Gram-variable in old cultures. Central or subterminal ellipsoidal endospores are observed in swollen sporangia. Colonies are circular, slightly raised, glistening, light yellow in colour and 1–2 mm in size after 2 days incubation at 30–37 °C on MA supplemented with 3 % (w/v) NaCl. Na+ and Mg2+ ions are required for growth. The optimal growth temperature is 37 °C; growth occurs at 15 and 48 °C, but not at 10 or 49 °C. The optimal pH for growth is 7·0–8·0; growth occurs at pH 6·0, but not at pH 5·5. Optimal growth occurs in the presence of 3–5 % (w/v) NaCl; growth occurs in the presence of 0·5 % (w/v) and 21 % (w/v) NaCl, but not without NaCl or in the presence of >22 % (w/v) NaCl. Tweens 20, 40 and 60 are hydrolysed. Hypoxanthine and xanthine are not hydrolysed. The Voges–Proskauer test is negative. Indole and H2S are not produced. Arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase are absent. In assays with the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8), naphthol-AS-BI-phosphohydrolase and -galactosidase are present, but lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, 
-chymotrypsin, acid phosphatase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are absent. Acid is produced from D-cellobiose, D-ribose and D-mannose. Acid is not produced from L-arabinose, lactose, D-melezitose, melibiose, D-raffinose, L-rhamnose, myo-inositol or D-sorbitol. The cell wall contains peptidoglycan based on L-orn–D-Asp. The predominant menaquinone is MK-7. The major fatty acids are anteiso-C15 : 0 (40·4 %), anteiso-C17 : 0 (23·0 %) and iso-C16 : 0 (19·3 %). The DNA G+C content is 42·9 mol%.
The type strain, MSS-402T (=KCTC 3957T=DSM 17110T), was isolated from a marine solar saltern of the Yellow Sea in Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Spring, S., Ludwig, W., Marquez, M. C., Ventosa, A. & Schleifer, K.-H. (1996). Halobacillus gen. nov., with description of Halobacillus litoralis sp. nov. and Halobacillus trueperi sp. nov., and transfer of Sporosarcina halophila to Halobacillus halophilus comb. nov. Int J Syst Bacteriol 46, 492–496.
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Wainø, M., Tindall, B. J., Schumann, P. & Ingvorsen, K. (1999). Gracilibacillus gen. nov., with description of Gracilibacillus halotolerans gen. nov., sp. nov.; transfer of Bacillus dipsosauri to Gracilibacillus dipsosauri comb. nov., and Bacillus salexigens to the genus Salibacillus gen. nov., as Salibacillus salexigens comb. nov. Int J Syst Bacteriol 49, 821–831.
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Yoon, J.-H., Weiss, N., Lee, K.-C., Lee, I.-S., Kang, K. H. & Park, Y.-H. (2001). Jeotgalibacillus alimentarius gen. nov., sp. nov., a novel bacterium isolated from jeotgal with L-lysine in the cell wall, and reclassification of Bacillus marinus Rüger 1983 as Marinibacillus marinus gen. nov., comb. nov. Int J Syst Evol Microbiol 51, 2087–2093.[Abstract]
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