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1 Korean Agricultural Culture Collection (KACC), Genetic Resources Division, National Institute of Agricultural Biotechnology, Rural Development Administration (RDA), Suwon 441-707, Republic of Koreaand
2 Applied Microbiology Division, National Institute of Agricultural Science and Technology, Rural Development Administration (RDA), Suwon 441-707, Republic of Korea
3 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
Correspondence
Soon-Wo Kwon
swkwon{at}rda.go.kr
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ABSTRACT |
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9c (25.5 %), 15 : 0 iso (18.7 %) and 17 : 0 iso (14.6 %), and the major hydroxy fatty acids were 11 : 0 iso 3-OH (5.0 %), 13 : 0 iso 3-OH (3.4 %) and 17 : 0 iso 3-OH (1.0 %). The major isoprenoid quinone was Q-8. The G+C content of the DNA of the type strain was 63.0 mol%. On the basis of the data from this study, strain R2A16-10T represents a novel species of the genus Dyella, for which the name Dyella yeojuensis sp. nov. is proposed. The type strain is R2A16-10T (=KACC 11405T=DSM 17673T).
Differentiating properties among strain R2A16-10T, members of the genus Dyella and Frateuria aurantia are shown in Supplementary Table S1 available in IJSEM Online.
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. At present, the genus Dyella includes two species, Dyella japonica and Dyella koreensis (An et al., 2005). This genus is closely related to the genera Fulvimonas, Frateuria and Rhodanobacter on the basis of 16S rRNA gene sequence analysis. D. japonica can be differentiated from Frateuria aurantia on the basis of fatty acid profiles and some phenotypic features such as growth at pH 4.5, acid production from carbohydrates and enzyme reactions.
Strain R2A16-10T was cultivated on R2A medium (Difco) at pH 7.0 and 28 °C, and maintained on R2A medium. Frateuria aurantia DSM 6220T, D. japonica DSM 16301T and D. koreensis BB4T were used as reference strains. Standard physiological and biochemical tests were performed at 28 °C. Gram staining was performed by using a Difco Gram-stain kit. Catalase activity was tested with a 3 % (v/v) H2O2 solution. Oxidase activity, degradation of agar (1.5 %, w/v), hydrolysis of aesculin, starch, casein, gelatin and DNA and indole production were tested according to the methods of Smibert & Krieg (1994). Hydrolysis of tyrosine (0.5 %, w/v) was tested on R2A agar medium. Hydrolysis of carboxymethylcellulose and Whatman powder CF11 was tested by overlaying R2A agar with a thin layer of 0.1 % each component in tap-water agar. The urease test was performed using the method described by MacFaddin (2000). Growth on AE broth [1.5 % (w/v) glucose, 0.2 % (w/v) yeast extract, 0.3 % (w/v) peptone, 6.5 % (v/v) acetic acid and 2 % (v/v) ethanol] (Entani et al., 1985) was checked at 28 °C on a rotary shaker for 30 days. The pH range for growth was determined in R2A broth adjusted with citrate-phosphate buffer or Tris/HCl buffer (Breznak & Costilow, 1994) to pH 4.0–10.0, using increments of 0.5 pH units. Growth at 1, 2, 3, 5 and 7 % NaCl (w/v) was investigated in R2A broth. Growth at various temperatures (5–50 °C) was measured on R2A medium. Physiological and biochemical properties were further determined with API ZYM, API 20NE and API 50 CH kits (bioMérieux). Tests involving commercial systems were generally performed according to the manufacturer's instructions. The API ZYM test strip was read after 4 h incubation at 30 °C, and API test strips were examined after 48 h at 28 °C. In the case of the API 50 CH test strips, Frateuria aurantia DSM 6220T produced reaction results after 48 h at 28 °C, whereas the reactions of type strains of the genus Dyella were delayed, being observed after 7 days at 28 °C.
Cells grown on R2A agar plates for 48 h were used for the analysis of the cellular fatty acid composition. Fatty acid methyl esters were extracted and prepared by using the standard protocol of the Microbial Identification System (MIDI; Microbial ID). Isoprenoid quinones were analysed by HPLC as described previously (Groth et al., 1996). The G+C content (mol%) was determined by HPLC analysis of deoxyribonucleosides as described by Mesbah et al. (1989), using a reverse-phased column (Supelcosil LC-18-S; Supelco).
The 16S rRNA gene sequence was determined by PCR amplification (Kwon et al., 2003) and direct sequencing (Hiraishi, 1992). A phylogenetic analysis was carried out using 16S rRNA gene sequences of 1498 nucleotide bases, from positions 37 to 1510 (Escherichia coli numbering system). The neighbour-joining and maximum-parsimony methods were carried out using MEGA, version 2.1 (Kumar et al., 2001), and the maximum-likelihood method (DNAML) was carried out using PHYLIP, version 3.5 (Felsenstein, 1993). The resulting trees and topology were evaluated by bootstrap analysis (Felsenstein, 1985) based on 1000 resamplings.
After 2 days growth on R2A, the colonies of strain R2A16-10T were circular, yellow in colour and convex with clear margins. The strain grew well on R2A, tryptic soy agar (Difco) and nutrient agar (Difco) but grew weakly on MacConkey agar (Difco). Strain R2A16-10T was a Gram-negative rod, 0.3–0.4x1.5–3.5 µm in size. In API 20NE tests, the micro-organism assimilated D-glucose, D-mannose, N-acetylglucosamine and D-maltose and showed positive reactions for aesculin and gelatin hydrolysis and 
-galactosidase activity. In API 50 CH tests, there were positive reactions only for D-galactose, D-glucose, D-fructose, D-mannose and aesculin. In API ZYM tests, the micro-organism showed positive reactions for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, 
-galactosidase, -galactosidase, -glucosidase, -glucosidase and N-acetyl--glucosaminidase activities (see Supplementary Table S1 available in IJSEM Online). Differentiating properties among strain R2A16-10T, members of the genus Dyella and Frateuria aurantia are shown in Table 1 and Supplementary Table S1.
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9c (21.5 %), 16 : 0 iso (21.3 %), 15 : 0 iso (14.5 %) and 17 : 0 iso (7.9 %) as the major constituents. The major hydroxy fatty acids were 11 : 0 iso 3-OH (4.2 %), 13 : 0 iso 3-OH (2.4 %) and 12 : 0 iso 3-OH (1.0 %). The fatty acid profiles of Dyella species and isolate R2A16-10T could be clearly differentiated from that of Frateuria aurantia (Table 2). The DNA G+C content of strain R2A16-10T was 63.0 mol%.
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). Within the cluster, strains of Frateuria aurantia formed another compact cluster distinct from that for members of the genus Dyella. On the basis of the data from polyphasic studies including analyses of biochemical properties, fatty acid composition and the 16S rRNA gene sequence, strain R2A16-10T represents a novel species within the genus Dyella, for which the name Dyella yeojuensis sp. nov. is proposed.
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Cells are Gram-negative, aerobic, motile, catalase-positive and oxidase-positive. Colonies on R2A medium are yellow. Colonies after 2 days growth on R2A medium are circular, yellow in colour and convex with clear margins. Optimal growth occurs at 28 °C. Growth occurs at temperatures in the range 5–37 °C, at pH values in the range 4.5–8.5 and at 0–5 % (w/v) NaCl. Does not grow on AE medium. Hydrolyses aesculin, casein, DNA, gelatin and tyrosine. Does not hydrolyse starch, urea, CM-cellulose or Whatman powder CF11. The major isoprenoid quinone is Q-8. The predominant fatty acids are 17 : 1 iso 9c, 16 : 0 iso, 15 : 0 iso and 17 : 0 iso. The major hydroxy fatty acids are 11 : 0 iso 3-OH, 13 : 0 iso 3-OH and 12 : 0 iso 3-OH. The DNA G+C content of the type strain is 63.0 mol%.
The type strain, R2A16-10T (=KACC 11405T=DSM 17673T), was isolated from greenhouse soil in Yeoju, Korea.
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REFERENCES |
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Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
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Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for General and Molecular Bacteriology, pp. 607–654. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology.
Swings, J., De Ley, J. & Gillis, M. (1984). Genus III. Frateuria Swings, Kersters, De Vos, Gossele and De Ley, 1980, 547VP. In Bergey's Manual of Systematic Bacteriology, vol. 1, pp. 210–213. Edited by N. R. Krieg & J. G. Holt. Baltimore: Williams & Wilkins.
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