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Int J Syst Evol Microbiol 55 (2005), 1945-1948; DOI  10.1099/ijs.0.63332-0
© 2005 International Union of Microbiological Societies

Bacillus litoralis sp. nov., isolated from a tidal flat of the Yellow Sea in Korea

Jung-Hoon Yoon and Tae-Kwang Oh

Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, South Korea

Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr


   ABSTRACT
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A Gram-variable, motile, endospore-forming, slightly halophilic bacterial strain, designated SW-211T, was isolated from a tidal flat of the Yellow Sea in Korea, and was characterized taxonomically by using a polyphasic approach. The organism grew optimally at 37 °C and in the presence of 2–3 % NaCl. Comparative 16S rRNA gene sequence analysis showed that strain SW-211T forms a distinct phylogenetic lineage within the radiation of the cluster comprising Bacillus species. Strain SW-211T had cell-wall peptidoglycan based on meso-diaminopimelic acid. The predominant menaquinone was MK-7 and the major fatty acids were anteiso-C15 : 0 (34·8 %), iso-C15 : 0 (15·6 %), iso-C16 : 0 (12·5 %) and iso-C14 : 0 (10·0 %). The DNA G+C content was 35·2 mol%. Strain SW-211T exhibited levels of 16S rRNA gene sequence similarity of <96·2 % with respect to the type strains of recognized Bacillus species. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain SW-211T (=KCTC 3898T=DSM 16303T) was classified in the genus Bacillus as the type strain of a novel species, for which the name Bacillus litoralis sp. nov. is proposed.

Published online ahead of print on 13 May 2005 as DOI 10.1099/ijs.0.63332-0.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain SW-211T is AY608605.


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Tidal sediments have been utilized as excellent sources for isolating novel and useful micro-organisms (Yi et al., 2003Go; Yoon et al., 2003aGo). Some new genera or species have recently been isolated from tidal sediments in Korea (Yi et al., 2003Go; Yoon et al., 2003aGo, bGo, 2004aGo, bGo). In the course of screening novel micro-organisms from a tidal flat near Daepo beach in the Yellow Sea in Korea, many moderately halophilic or halotolerant bacteria have been isolated and characterized taxonomically. Of these isolates, a rod-shaped, endospore-forming bacterial strain, SW-211T, is described here. Its exact taxonomic position was determined by using a polyphasic approach that included phenotypic properties and phylogenetic analysis based on 16S rRNA gene sequences.

Strain SW-211T was isolated by a standard dilution plating technique on marine agar 2216 (MA; Difco) at 30 °C. Cell morphology was examined by light microscopy (Nikon E600) and transmission electron microscopy (TEM). Flagellum type was examined by TEM using cells from exponentially growing cultures. 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 an anaerobic chamber on MA and on MA supplemented with nitrate, both of which had been prepared anaerobically using nitrogen. The pH range for growth was determined in marine broth 2216 (MB; Difco) that was adjusted to various pH values (initial pH 4·5–9·5 at intervals of 0·5 pH units). The pH was adjusted prior to sterilization by the addition of HCl or Na2CO3. Growth at various NaCl concentrations was investigated in MB or trypticase soy broth (Difco). Growth in the absence of NaCl was investigated in trypticase soy broth lacking NaCl. Growth at various temperatures (4–50 °C) was measured on MA. Catalase and oxidase activities and hydrolysis of casein, gelatin, hypoxanthine, starch, Tween 80, tyrosine and xanthine were determined as described by Cowan & Steel (1965)Go. Hydrolysis of aesculin and nitrate reduction were determined as described by Lanyi (1987)Go. H2S production was tested as described by Bruns et al. (2001)Go. Acid production from carbohydrates was determined according to the method of Leifson (1963)Go. Utilization of substrates as sole carbon and energy sources was tested as described by Baumann & Baumann (1981)Go, with the substrates supplemented with 2 % (v/v) Hutner's mineral base (Cohen-Bazire et al., 1957Go) and 1 % (v/v) vitamin solution (Staley, 1968Go). Enzyme activity was determined by using the API ZYM system (bioMérieux). Other physiological and biochemical tests were performed with the API 20E system (bioMérieux). Morphological, cultural, physiological and biochemical characteristics of strain SW-211T are shown in Table 1Go or are given in the species description.


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  		Table 1. Phenotypic characteristics of Bacillus litoralis sp. nov. and some related Bacillus species

Species: 1, B. litoralis sp. nov.; 2, B. fastidiosus (data from Claus & Berkeley, 1986Go); 3, Bacillus cibi (Yoon et al., 2005Go); 4, Bacillus indicus (Suresh et al., 2004Go); 5, Bacillus horikoshii (Nielsen et al., 1995Go; Li et al., 2002Go; Logan et al., 2002Go); 6, Bacillus halmapalus (Nielsen et al., 1995Go; Li et al., 2002Go; Logan et al., 2002Go); 7, Bacillus cohnii (Spanka & Fritze, 1993Go; Suresh et al., 2004Go); 8, Bacillus cereus (Claus & Berkeley, 1986Go; Priest et al., 1988Go); 9, Bacillus flexus (Claus & Berkeley, 1986Go; Suresh et al., 2004Go); 10, Bacillus megaterium (Claus & Berkeley, 1986Go; Reva et al., 2002Go; Täubel et al., 2003Go); 11, Bacillus simplex (Priest et al., 1988Go; De Clerck et al., 2004Go). +, Positive; –, negative; ND, not determined; NG, no growth; V, variable. Data in parentheses are for the type strain. Cells of all species are rod-shaped and catalase-positive.

 
Cell biomass for DNA extraction and for cell-wall and isoprenoid quinone analyses was obtained from cultivation in MB at 37 °C. Chromosomal DNA was isolated and purified according to the method described by Yoon et al. (1996)Go, with the modification that RNase T1 was used in combination with RNase A to minimize contamination with RNA. The 16S rRNA gene was amplified by PCR using two universal primers as described by Yoon et al. (1998)Go. Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed as described by Yoon et al. (2003a)Go. The isomer type of diamino acid of the cell-wall peptidoglycan was determined by the method of Komagata & Suzuki (1987)Go. Isoprenoid quinones were extracted and analysed as described by Komagata & Suzuki (1987)Go using reversed-phase HPLC. For fatty acid methyl ester analysis, a loop of cell mass was harvested from MA plates after cultivation for 2 days at 37 °C. The fatty acid methyl esters were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990Go). The DNA G+C content was determined by the method of Tamaoka & Komagata (1984)Go with a modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC.

An almost complete 16S rRNA gene sequence of strain SW-211T comprising 1505 nt (approximately 96 % of the Escherichia coli 16S rRNA gene sequence) was analysed. Comparative 16S rRNA gene sequence analyses showed that strain SW-211T is phylogenetically affiliated to Bacillus species. The phylogenetic tree based on the neighbour-joining algorithm showed that strain SW-211T fell within the radiation of the cluster comprising Bacillus species and joined Bacillus fastidiosus with a bootstrap resampling value of 98·0 % (Fig. 1Go). Strain SW-211T exhibited 16S rRNA gene sequence similarity levels of 96·2 % to the type strain of B. fastidiosus and lower than 95·3 % with respect to the type strains of other recognized Bacillus species. Sequence similarities to all other species included in the phylogenetic analysis were below 91·4 % (Fig. 1Go). Strain SW-211T contained meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan. The predominant isoprenoid quinone found in strain SW-211T was unsaturated menaquinone with seven isoprene units (MK-7). Major components (>5 %) of the fatty acids detected in strain SW-211T were branched fatty acids anteiso-C15 : 0 (34·8 %), iso-C15 : 0 (15·6 %), iso-C16 : 0 (12·5 %), iso-C14 : 0 (10·0 %) and anteiso-C17 : 0 (6·4 %) and straight-chain fatty acid C16 : 0 (5·5 %). The DNA G+C content of strain SW-211T was 35·2 mol%.



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  		Fig. 1. Neighbour-joining tree showing the phylogenetic positions of SW-211T, some Bacillus species and representatives of other related taxa based on 16S rRNA gene sequence analysis. Bootstrap values (expressed as percentages of 1000 replications) greater than 50 % are shown at branch points. Bar, 0·01 substitutions per nucleotide position.

 
The results obtained from the phenotypic and phylogenetic characterizations indicated that strain SW-211T belongs to the genus Bacillus. The phylogenetic distinctiveness (16S rRNA gene sequence similarity of <97 %) confirms that strain SW-211T represents a species distinct from recognized Bacillus species (Stackebrandt & Goebel, 1994Go). There were some phenotypic differences between strain SW-211T and several phylogenetically related Bacillus species (Table 1Go). Therefore, on the basis of the data presented, strain SW-211T should be classified in the genus Bacillus as a member of a novel species, for which the name Bacillus litoralis sp. nov. is proposed.


   Description of Bacillus litoralis sp. nov.
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 Description of Bacillus...
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Bacillus litoralis (li.to.ra'lis. L. masc. adj. litoralis of the shore).

Cells are aerobic rods, 0·8–1·0x2·5–4·5 µm in size. Gram-variable. Motile by means of peritrichous flagella. Central, subterminal or terminal ellipsoidal endospores are observed in swollen sporangia. Colonies grown on MA are circular to slightly irregular, smooth, slightly raised, yellowish white in colour and 2–3 mm in diameter after 2 days of incubation at 37 °C; colony colour becomes dark from the centre as cultures age. Optimal growth temperature is 37 °C; growth occurs at 4 and 45 °C but not above 46 °C. Optimal pH for growth is 7·5. Growth is observed at pH 5·5 but not at pH 5·0. Optimal growth occurs in the presence of 2–3 % (w/v) NaCl; growth does not occur without NaCl or in the presence of >11 % (w/v) NaCl. Anaerobic growth does not occur on MA or on MA supplemented with nitrate. Catalase-positive. Urease-negative. Hypoxanthine, tyrosine and xanthine are not hydrolysed. H2S and indole are not produced. Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. When assayed with the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8), acid phosphatase and naphthol-AS-BI-phosphohydrolase are present, but lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, {alpha}-chymotrypsin, {alpha}-galactosidase, {beta}-galactosidase, {beta}-glucuronidase, {alpha}-glucosidase, {beta}-glucosidase, N-acetyl-{beta}-glucosaminidase, {alpha}-mannosidase and {alpha}-fucosidase are absent. Acid is produced from L-arabinose, D-cellobiose, D-fructose, D-galactose, D-melezitose, melibiose, D-raffinose, L-rhamnose, D-ribose, sucrose and D-trehalose. Acid is not produced from adonitol, D-sorbitol, myo-inositol or maltose. The following substrates are utilized for growth: L-arabinose, D-fructose, D-glucose, D-trehalose, D-xylose, lactose, acetate and L-malate. Benzoate, citrate, formate, succinate or L-glutamate are not utilized. The cell-wall peptidoglycan contains meso-diaminopimelic acid. The predominant menaquinone is MK-7. The major fatty acids are anteiso-C15 : 0 (34·8 %), iso-C15 : 0 (15·6 %), iso-C16 : 0 (12·5 %) and iso-C14 : 0 (10·0 %). The DNA G+C content is 35·2 mol% (determined by HPLC).

The type strain, SW-211T (=KCTC 3898T=DSM 16303T), was isolated from a tidal flat near Daepo beach of the Yellow Sea in Korea.


   ACKNOWLEDGEMENTS
 
This work was supported by the 21C Frontier programme of Microbial Genomics and Applications (grant MG02-0401-001-1-0-0) from the Ministry of Science and Technology (MOST) of the Republic of Korea.


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