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1 Korean Agricultural Culture Collection (KACC), Microbial Genetics Division, National Institute of Agricultural Biotechnology, Rural Development Administration, Suwon 441-707, Republic of Korea
2 Applied Microbiology Division, National Institute of Agricultural Science and Technology, Rural Development Administration, Suwon 441-707, Republic of Korea
Correspondence
Byung-Yong Kim
kimby{at}rda.go.kr
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ABSTRACT |
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Phase-contrast micrographs of the novel strains and tables showing their phenotypic characteristics and fatty acid compositions and levels of DNA–DNA hybridization between the novel isolates are available as supplementary material with the online version of this paper.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Samples were spread on tryptic soy agar (TSA; Difco) at pH 7.0 and incubated at 30 °C for 3 days. Light-yellow colonies were produced on TSA and nutrient agar (Difco). Gram staining was performed with a Gram-stain kit (Difco) according to the manufacturer's protocol. A KOH test and an L-alanine aminopeptidase assay were also performed (Gregersen, 1978
). The cell morphology was observed by using transmission electron microscopy (model 912AB; LEO) and phase-contrast microscopy (AXIO; Zeiss) after cultivation on TSA for 2 days.
Various physiological tests were carried out according to Gordon et al. (1973) and Claus & Berkeley (1986). The Voges–Proskauer test was performed along with tests for catalase activity, temperature ranges for growth (from 5 to 65 °C, using 5 °C increments), resistance to lysozyme (0.001 %), growth in the presence of NaCl (at 0, 3, 5, 7,10 and 15 %), pH range for growth (pH 4–9 in increments of 1.0 pH unit), acid production from carbohydrates (D-glucose, L-arabinose, D-xylose and D-mannitol), gas formation from glucose, utilization of citrate and propionate, production of indole and dihydroxyacetone, deamination of phenylalanine, decomposition of casein and tyrosine and liquefaction of gelatin. The oxidase test and tests for the hydrolysis of aesculin, gelatin and starch were conducted according to Smibert & Krieg (1994). Motility tests were performed on motility medium, which was composed of 0.1 % yeast extract, 0.01 % K2HPO4 and 0.2 % agar. Anaerobic growth was tested in serum bottles by adding thioglycolate (1 g l–1) to tryptic soy broth medium and replacing the upper airspace with nitrogen gas. To test for the reduction of nitrate and nitrite, each isolate was inoculated into three serum bottles (25 ml) containing tryptic soy broth medium, while nitrate and nitrite were added as KNO3 and NaNO2 at concentrations of 10 mM. The reduction of nitrate and nitrite was monitored using an ion chromatograph (model 790 personal IC; Metrohm) equipped with a conductivity detector and an anion-exchange column (Metrosep Anion Supp 4; Metrohm). The urease test was performed according to Kim et al. (2006).
The 16S rRNA gene of each isolate was amplified by means of a PCR with two universal primers, as described previously (Kwon et al., 2003). The sequences of amplified 16S rRNA genes were analysed using a DNA sequencer (ABI3100; Applied Biosystems). The 16S rRNA gene sequences were aligned using CLUSTAL W software (Thompson et al., 1994). Nucleotide similarity values were calculated from the alignment. An evolutionary distance matrix was constructed using a two-parameter model (Kimura, 1980), and phylogenetic trees for the datasets were inferred with the neighbour-joining method (Saitou & Nei, 1987) using MEGA, version 3.0 (Kumar et al., 2004). The stability of relationships was assessed by performing bootstrap analyses of the neighbour-joining data (based on 1000 resamplings).
DNA–DNA hybridization was carried out using the filter hybridization method described by Seldin & Dubnau (1985). Probe labelling was conducted by using the non-radioactive DIG-High prime system (Roche), and hybridized DNA was visualized using a DIG luminescent detection kit (Roche). DNA–DNA relatedness was quantified by using a densitometer (Bio-Rad). Chemotaxonomic characteristics of these strains in this study were determined using cells grown at 30 °C on TSA for 24 h. Isoprenoid quinones were analysed by HPLC (Shimadzu) as described previously (Groth et al., 1996). DNA G+C contents (mol%) were determined by performing HPLC analysis of deoxyribonucleosides, as described by Mesbah et al. (1989). Analysis of the fatty acid methyl esters was performed by GLC according to the instructions for the Microbial Identification System (MIDI).
The five strains, 4T19T, 4T12, 5M45, 5M53 and 5T52, showed similar morphologies. The cells were rod-shaped, occurring singly or in filamentous chains. Single cells were 0.5–0.7 µm in diameter and 2.0–3.0 µm in length. Spores were ellipsoidal or oval, occurring subterminally or terminally in swollen sporangia (see Supplementary Fig. S1 in IJSEM Online).
The isolates were motile on the medium used in this study. The growth temperature range for the isolates was 15–40 °C and the pH range for growth was pH 6.0–8.0 (optimum, pH 7.0), these ranges being different from those for Bacillus litoralis and Bacillus herbersteinensis. The isolates differed from each other with regard to growth ranges for NaCl (%): strain 4T19T grew in the presence of NaCl (w/v) at concentrations up to 5 %, 4T12 could grow with NaCl concentrations up to only 3 % and the other isolates did not grow in the presence of 3 % NaCl. All isolates were positive for catalase, urease, nitrate reduction, acid production from D-glucose, L-arabinose, D-xylose and D-mannitol and hydrolysis of aesculin, gelatin and starch. All isolates were negative for oxidase, phenylalanine deamination, the formation of indole and dihydroxyacetone, in the Voges–Proskauer test and for the utilization of citrate and propionate. Detailed results of the physiological characterizations are given in the species description, and features that serve to distinguish between the isolates and related Bacillus species are listed in Table 1 and in Supplementary Table S1 (available in IJSEM Online).
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). All of the isolates, which fell within the radiation of the genus Bacillus including Bacillus subtilis, formed a compact cluster with 100 % bootstrap support (Fig. 1). The 16S rRNA gene sequence similarity between strain 4T19T and the other isolates was approximately 99.5–99.7 %. However, strain 4T19T exhibited relatively low levels of 16S rRNA gene sequence similarity with respect to Bacillus species. The closest relatives of strain 4T19T were the type strains of B. herbersteinensis (96.7 %), B. litoralis (96.2 %) and Bacillus cohnii (96.0 %).
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) (Supplementary Table S2). Meanwhile, the low levels of 16S rRNA gene sequence similarity (<97 %) between 4T19T and the type strains of Bacillus species with validly published names confirm that this strain represents a species that is distinct from recognized Bacillus species (Stackebrandt & Goebel, 1994).
Fatty acid analysis revealed some differences between the isolates and related Bacillus species (Table 1
). The major fatty acid components of the isolates were anteiso-C15 : 0, C16 : 0, anteiso-C17 : 0, iso-C16 : 0 and iso-C14 : 0 (Supplementary Table S3). The isolates contained an unsaturated menaquinone with seven isoprene units (MK-7) as the predominant isoprenoid quinone. The G+C contents of the isolates were in the range 37.7–40.9 mol%, which is higher than the values for B. litoralis, B. herbersteinensis, B. cohnii and Bacillus fastidiosus.
On the basis of the polyphasic data, the five isolates represent a novel species of the genus Bacillus, for which the name Bacillus niabensis sp. nov. is proposed.
Description of Bacillus niabensis sp. nov.
Bacillus niabensis (niab.en'sis. N.L. masc. adj. niabensis arbitrary name formed from NIAB, the acronym for the National Institute of Agricultural Biotechnology, Korea, where taxonomic studies on this species were performed).
Cells are Gram-positive, motile, spore-forming rods, 0.5–0.7x2.0–3.0 µm, motile by means of single polar flagella. Cells form ellipsoidal or oval spores that occur subterminally or terminally in swollen sporangia. Colonies are yellowish white, 2–3 mm in diameter and circular with clear margins after incubation on TSA for 2 days. Growth occurs at temperatures ranging from 15 to 50 °C, the optimum being 30 °C. Growth occurs at pH 6.0–8.0, the optimum being pH 7.0. Capable of growing anaerobically. NaCl growth range is variable, being in the range 0–5 % (w/v). Positive for catalase, urease, acid production from D-glucose, L-arabinose, D-xylose and D-mannitol and hydrolysis of aesculin, gelatin and starch. Negative for oxidase, formation of indole and dihydroxyacetone, phenylalanine deamination, utilization of citrate and propionate, degradation of tyrosine, growth with lysozyme and in the Voges–Proskauer test. Nitrate is reduced under anaerobic conditions. The major cellular fatty acids are anteiso-C15 : 0, C16 : 0, anteiso-C17 : 0 and iso-C16 : 0. The major quinone is MK-7. The DNA G+C contents are 37.7–40.9 mol%, as determined by HPLC.
The type strain, 4T19T (=KACC 11279T =DSM 17723T), was isolated from cotton-waste composts in Suwon, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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