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1 Korea Research Institute of Bioscience and Biotechnology, 52 Oeundong, Yusong, Daejeon 305-333, Republic of Korea
2 Environmental Biotechnology National Core Research Center, PMBBRC, Division of Environmental Biotechnology, Gyeongsang National University, Jinju 660-701, Korea
Correspondence
Chang-Jin Kim
changjin{at}kribb.re.kr
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ABSTRACT |
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peptidoglycan with meso-diaminopimelic acid as the diagnostic diamino acid. The major cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0, iso-C14 : 0 and iso-C16 : 0. The major cellular phospholipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and unknown phospholipids (PL1, PL2). The genomic DNA G+C content was 36 mol%. Comparative 16S rRNA gene sequence analyses showed that strain BR030T formed a distinct phyletic line within the genus Bacillus and was most closely related to Bacillus flexus DSM 1320T, with 16S rRNA gene sequence similarity of 96·8 %. Sequence similarities to other type strains were lower than 96·2 %. On the basis of physiological and molecular properties, the isolate represents a novel species within the genus Bacillus, for which the name Bacillus koreensis sp. nov. is proposed. The type strain is BR030T (=KCTC 3914T=DSM 16467T).
Published online ahead of print on 2 September 2005 as DOI 10.1099/ijs.0.63701-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Bacillus koreensis sp. nov. BR030T is AY667496.
A transmission electron micrograph showing the spore morphology of Bacillus koreensis sp. nov. and tables detailing fatty acid profiles and the results of DNA hybridization studies are available as supplementary material in IJSEM Online.
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; Somers et al., 2004). Due to their potential for increasing agricultural yields and controlling plant diseases, bacteria in the rhizosphere and rhizoplane of grasses and crop plants have attracted a great deal of attention (Berge et al., 2002; Penrose & Glick, 2003; Sessitsch et al., 2004). In the course of an investigation of rhizosphere micro-organisms, a novel aerobic Gram-positive bacterium, designated strain BR030T, was isolated from the rhizosphere of willow roots (Salix koreensis Andersson) in Korea. On the basis of phylogenetic and phenotypic characteristics, strain BR030T was classified as a novel species of the genus Bacillus. Strain BR030T was isolated from the roots of a willow tree, more than 3 m tall, growing on hillside silt soil of the Daejeon area in Korea. We sampled root branches from the willow tree and removed soil debris from the roots by tapping. The root branches were washed and the resulting solution was serially diluted using 0·9 % (w/v) saline. The diluted solution was spread on nutrient agar (NA; Difco) and incubated for 3 days at 30 °C. The isolated strain was routinely grown aerobically on Luria–Bertani (LB; Difco) agar for 2 days at 35 °C, except where indicated otherwise. Requirement for and tolerance of NaCl were determined in LB broth with different amounts of NaCl after 2 days incubation at 35 °C. Anaerobic growth was determined by incubation in an anaerobic chamber at 35 °C for 5 days on LB agar. Optimum growth conditions were established by incubation at temperatures between 4 and 55 °C on LB agar and at pH values between 5·0 and 10·0 in LB broth.
Cell morphology was studied using light microscopy and transmission electron microscopy (TEM). Motility was observed at 12 and 36 h on agar-coated wet mounts with a light microscope (E600; Nikon). The agar-coated wet mounts were prepared by placing 10 µl culture under a cover slip on a glass slide that had been previously coated with a film of 0·5 % (w/v) agarose (Cambrex) in distilled water and then air-dried. The flagellum type was examined by TEM using cells from the exponential growth phase as described by Lee et al. (2005). For investigation of the spore shape of the isolate, specimens were prepared according to Jeon et al. (2003) and then subjected to TEM (JEM-1010; JEOL). Endospores were stained according to the method of Schaeffer-Fulton (Smibert & Krieg, 1981).
Gram staining was determined using the bioMérieux Gram Stain kit according to the manufacturer's instructions. Catalase activity was determined by the production of oxygen bubbles in 3 % (v/v) aqueous hydrogen peroxide solution. Oxidase activity was tested by oxidation of 1 % (w/v) tetramethyl-p-phenylenediamine (Merck). Hydrolysis of aesculin, casein, starch, Tween 80, urea, hypoxanthine, tyrosine, gelatin and xanthine was determined on LB agar according to methods described previously (Cowan & Steel, 1965; Lanyi, 1987; Smibert & Krieg, 1994). Nitrate reduction tests were performed as described by Lanyi (1987). Acid production from carbohydrates was tested according to Leifson (1963).
For quantitative analyses of whole-cell fatty acids, strain BR030T was cultivated on LB agar at 35 °C for 2 days. Preparation and analysis of fatty acid methyl esters were conducted according to the instructions of the MIDI microbial identification system (Microbial ID). Preparation of cell walls from strain BR030T and analysis of peptidoglycan were carried out using methods described by Schleifer (1985), with the modification that TLC was performed on cellulose sheets rather than by paper chromatography. Isoprenoid quinones and polar lipids were analysed according to Komagata & Suzuki (1987). The DNA G+C content of strain BR030T was determined by reverse-phase HPLC using the method of Tamaoka & Komagata (1984). DNA–DNA hybridization was performed to evaluate the genomic DNA–DNA relatedness between strain BR030T and Bacillus flexus DSM 1320T, obtained from the DSMZ. Both strains were grown aerobically on NA for 2 days at 35 °C and chromosomal DNA was isolated and purified according to Yoon et al. (1996). Randomly primed DNA labelling with digoxigenin (DIG)-dUTP and detection of hybrids by enzyme immunoassay on nylon membranes were performed using a DIG High Prime DNA labelling and detection starter kit II (Roche Applied Science) according to the manufacturer's instructions and Lim et al., 2005.
Sequencing and assembly of 16S rRNA genes were carried out as described by Bakermans & Madsen (2002). The 16S rRNA gene sequence of strain BR030T was compared with available 16S rRNA gene sequences from GenBank using the BLAST program (http://www.ncbi.nlm.nih.gov/blast/) to determine the approximate phylogenetic affiliation and was aligned with closely related members using CLUSTAL W software (Thompson et al., 1994). Unmatched regions of the 5'- and 3'-ends from the alignment, which were caused by different lengths of the 16S rRNA gene sequence data, were deleted. The resulting 16S rRNA gene sequence of strain BR030T that was used for phylogenetic analysis comprised 1348 nucleotides. Phylogenetic trees were constructed by three different methods, neighbour-joining, maximum-likelihood and maximum-parsimony, using algorithms available in the PHYLIP software, version 3.6 (Felsenstein, 2002). Using the FASTA3 program in EBI, 16S rRNA gene sequence comparisons for similarity calculations were made between strain BR030T and other members of the Bacillaceae. A bootstrap analysis was performed according to the Kimura two-parameter model (Kimura, 1980) of the neighbour-joining method in the PHYLIP package (Jeon et al., 2005).
Strain BR030T grew in LB broth containing 0–9 % (w/v) NaCl; optimum growth occurred on media with 0–3 % (w/v) NaCl. Colonies of the strain were yellow–cream, smooth, convex and circular/slightly irregular on LB agar medium. Strain BR030T grew at a wide range of pH values, 4·5–9·0 in LB broth; optimal growth was observed at pH 7·0. Growth was observed at temperatures between 15 and 48 °C with an optimum growth temperature of 35–40 °C. Cells of the isolate were motile short rods (0·8–1·6 µm wide and 1·6–2·8 µm long) with peritrichous flagella (data not shown). The isolate produced an oval-type central endospore (see Supplementary Fig. S1 in IJSEM Online). Growth was not observed under anaerobic conditions.
Strain BR030T showed obviously Gram-positive, catalase-positive and oxidase-negative reactions. The strain did not reduce nitrate to nitrite. Analysis of cell-wall peptidoglycan showed that strain BR030T possessed A1 type peptidoglycan with meso-diaminopimelic acid (m-DAP) as the diagnostic diamino acid, in common with the great majority of the genus Bacillus (Priest et al., 1988). The predominant isoprenoid quinone of strain BR030T was menaquinone-7 (MK-7) and the genomic DNA G+C content was 36 mol%. The strain contained phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE) and unknown phospholipids (PL1, PL2) as the major polar lipids. The cellular fatty acid profile of strain BR030T was characterized by saturated branched fatty acids such as anteiso-C15 : 0 (27·25 %), iso-C15 : 0 (25·87 %), iso-C14 : 0 (16·76 %) and iso-C16 : 0 (14·16 %) as the major fatty acids. The fatty acid profile of strain BR030T was similar to the profiles of its close relatives, B. flexus DSM 1320T and Bacillus megaterium DSM 32T, but the fatty acid compositions are somewhat different (see Supplementary Table S1 in IJSEM Online). The major fatty acid profiles, major lipoquinones and DNA G+C content of strain BR030T were typical of those of members of the genus Bacillus (Priest et al., 1988; Nielsen et al., 1995; Heyrman et al., 2004, 2005). Typical phenotypic and chemotaxonomic properties of strain BR030T are compared with those of phylogenetically related species in Table 1.
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(Stackebrandt & Liesack, 1993; Nielsen et al., 1994; Schlesner et al., 2001) (Fig. 1). The topologies of phylogenetic trees built using the maximum-likelihood and maximum-parsimony algorithms also supported the conclusion that BR030T belongs to the genus Bacillus (data not shown). Comparative 16S rRNA gene sequence analyses showed that strain BR030T was most closely related to B. flexus DSM 1320T, with a sequence similarity of 96·8 %. Sequence similarities to other members used in the phylogenetic analysis were lower than 96·2 %. DNA hybridization was carried out to evaluate the genomic DNA–DNA relatedness between strain BR030T and the most closely related type strain, B. flexus DSM 1320T. A DNA–DNA relatedness value of 57 % was obtained (see Supplementary Table S2 in IJSEM Online). On the basis of its distinctive physiological, morphological and other key traits, it is proposed that strain BR030T represents a novel species within the genus Bacillus, for which we propose the name Bacillus koreensis sp. nov.
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Colonies are yellow–cream, smooth, convex and circular/slightly irregular. Cells are approximately 0·8–1·6 µm wide and 1·6–2·8 µm long. Strictly aerobic, spore-forming motile rods. Gram-positive and KOH test-negative. Catalase-positive and oxidase-negative. Nitrate is not reduced to nitrite. Growth occurs at 15–48 °C (optimum 35–40 °C), pH 4·5–9·0 (optimum pH 7·0) and 0–9 % (w/v) NaCl (optimum 0–3 %). Aesculin, casein, starch and urea are hydrolysed. Hydrolysis of Tween 80, L-tyrosine, hypoxanthine and xanthine is not observed. Acids are produced from D-glucose, D-fructose, D-ribose, 
-D-lactose, maltose, D-trehalose, D-raffinose, glycerol, sucrose and D-melibiose, but not from D-mannitol, D-xylose, L-arabinose, L-rhamnose, adonitol or D-mannose. The cell wall contains A1 type peptidoglycan with m-DAP as the diagnostic diamino acid. Predominant polar lipids are PG, DPG, PE and unknown PL1 and PL2. Major isoprenoid quinone is MK-7. DNA G+C content is 36 mol% (HPLC). Predominant cellular fatty acids are anteiso-C15 : 0, iso-C15 : 0, iso-C14 : 0 and iso-C16 : 0.
The type strain, BR030T (=KCTC 3914T=DSM 16467T), was isolated from the rhizosphere of willow roots in Korea.
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ACKNOWLEDGEMENTS |
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