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Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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, b, 2006a, b). One of these strains, DS-56T, which is phylogenetically closely related to the genus Devosia, is the subject of this study. The genus Devosia was created by the reclassification of Pseudomonas riboflavina as Devosia riboflavina (Nakagawa et al., 1996). Phylogenetic analyses based on 16S rRNA gene sequences have shown that the genus Devosia falls within the Alphaproteobacteria (Nakagawa et al., 1996; Lee et al., 2005). At the time of writing, the genus Devosia comprises four species with validly published names: Devosia riboflavina (Nakagawa et al., 1996), Devosia neptuniae (Rivas et al., 2003), Devosia limi (Vanparys et al., 2005) and Devosia soli (Yoo et al., 2006). The aim of the present study was to determine the exact taxonomic position of strain DS-56T by means of a polyphasic characterization that included phenotypic and chemotaxonomic determinations and a detailed phylogenetic analysis based on 16S rRNA gene sequences.
Strain DS-56T was isolated from a soil sample collected from Dokdo (37° 14' 12'' N 131° 52' 07'' E), Korea, by means of the standard dilution plating technique performed at 25 °C on 10x diluted nutrient agar (Difco). The type strains of D. riboflavina, D. limi, D. neptuniae and D. soli were used as reference strains for fatty acid analyses; D. riboflavina DSM 7230T and D. limi DSM 17137T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany), D. neptuniae LMG 21357T was obtained from the Laboratorium voor Microbiologie Universiteit Gent (Ghent, Belgium) and D. soli KACC 11509T was obtained from the Korean Agricultural Culture Collection (Suwon, Korea). The morphological, physiological and biochemical characteristics of strain DS-56T were investigated using routine cultivation on trypticase soy agar (TSA; Difco) at 25 °C. Cell morphology was examined by using light microscopy (E600; Nikon) and transmission electron microscopy. The presence of flagella was determined by transmission electron microscopy using cells from exponentially growing cultures. For transmission electron microscopic observations, the cells were negatively stained with 1 % (w/v) phosphotungstic acid and the grids were examined after air-drying with a Philips CM-20 transmission electron microscope. The Gram reaction was determined using the bioMérieux Gram-stain kit according to the manufacturer's instructions. Growth at various temperatures (4–40 °C) was measured on TSA. Growth in the absence of NaCl and at various NaCl concentrations (0.5 and 1.0–5.0 %, w/v, in increments of 1.0 %) was investigated using trypticase soy broth prepared according to the formula of the Difco medium except that no NaCl was used. The pH range for growth was determined in nutrient broth (Difco) adjusted to various pH values (pH 4.5–10.5, in increments of 0.5 pH units), prior to sterilization, by the addition of HCl or Na2CO3. Growth under anaerobic conditions was determined after incubation of the novel strain in an anaerobic chamber on TSA and on TSA supplemented with nitrate, both of which had been prepared anaerobically using nitrogen. Catalase and oxidase activities and hydrolysis of casein, gelatin, hypoxanthine, starch, Tweens 20, 40, 60 and 80, tyrosine, urea and xanthine were determined as described by Cowan & Steel (1965). Hydrolysis of aesculin and reduction of nitrate were studied as described previously (Lanyi, 1987). Susceptibility to antibiotics was tested on TSA plates, using antibiotic discs containing the following amounts (µg unless otherwise indicated): polymyxin B, 100 U; streptomycin, 50; penicillin G, 20 U; chloramphenicol, 100; ampicillin, 10; cephalothin, 30; gentamicin, 30; novobiocin, 5; tetracycline, 30; kanamycin, 30; lincomycin, 15; oleandomycin, 15; neomycin, 30; carbenicillin, 100. The assimilation of various substrates and the activities of various enzymes were tested by using the API 20E, API 20NE and API ZYM systems (bioMérieux).
Cell biomass for DNA extraction and for isoprenoid quinone analysis was obtained from cultures grown in trypticase soy broth (Difco) at 25 °C. Chromosomal DNA was isolated and purified according to the method described by Yoon et al. (1996), with the exception that RNase T1 was used in combination with RNase A to minimize contamination with RNA. The 16S rRNA gene was amplified by using a PCR with two universal primers, as described previously (Yoon et al., 1998). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed as described by Yoon et al. (2003). Isoprenoid quinones were extracted according to the method of Komagata & Suzuki (1987) and analysed using reversed-phase HPLC and a YMC ODS-A (250x4.6 mm) column. For fatty acid methyl ester analysis, cell mass of strain DS-56T was harvested from TSA plates after incubation for 7 days at 25 °C, and cell mass of D. riboflavina DSM 7230T, D. limi DSM 17137T, D. neptuniae LMG 21357T and D. soli KACC 11509T was harvested from TSA plates after incubation for 3 days at 28 °C and from TSA plates after incubation for 7 days at 25 °C. 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 resulting nucleotides were analysed by reversed-phase HPLC.
Cells of strain DS-56T were Gram-negative, non-spore-forming and oval- or rod-shaped. Strain DS-56T showed optimal growth at pH 6.5–7.5 and at 25 °C. The growth of strain DS-56T was slower than that of the type strains of the four recognized Devosia species. In tests with the API 20NE system, strain DS-56T did not assimilate any substrates (after incubation for 10 days). Morphological, cultural, physiological and biochemical characteristics of strain DS-56T are given in the species description (see below) or are shown in Table 1. The almost-complete 16S rRNA gene sequence of strain DS-56T determined in this study comprised 1437 nt, representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence. In the phylogenetic tree constructed using the neighbour-joining algorithm, strain DS-56T joined ‘Devosia ginsengisoli’ Gsoil 326 at a bootstrap resampling value of 100 %, and this cluster joined the clade comprising recognized Devosia species at a bootstrap confidence value of 100 % (Fig. 1). Strain DS-56T exhibited 16S rRNA gene sequence similarity values of 96.4, 95.8, 95.7, 95.4 and 99.1 % with respect to D. riboflavina DSM 7230T, D. neptuniae J1T, D. limi LMG 22951T, D. soli GH2-10T and ‘D. ginsengisoli’ Gsoil 326, respectively. The sequence similarities with respect to other species included in the phylogenetic analysis were below 92.4 % (Fig. 1). The fatty acid profile of strain DS-56T comprised major amounts of unsaturated, branched, straight-chain and 11-methyl C18 : 1
7c; the major fatty acids (>10 % of total fatty acids) were 11-methyl C18 : 17c (29.6 %), C18 : 17c (18.5 %), iso-C17 : 0 (14.5 %) and C16 : 0 (13.6 %) (Table 2). The predominant isoprenoid quinone detected in strain DS-56T was Q-11, at a peak area ratio of approximately 85 %; a minor amount of Q-10 (<10 % approx.) was present. The DNA G+C content of strain DS-56T was 66.2 mol%.
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). D. riboflavina and D. soli have been reported to have Q-10 as the predominant ubiquinone (Nakagawa et al., 1996; Yoo et al., 2006). It was found in this study that the type strains of D. riboflavina, D. neptuniae and D. limi have Q-10 as the predominant ubiquinone. Strain DS-56T can be differentiated from recognized Devosia species on the basis of the fatty acid profiles, e.g. by the presence of branched fatty acids, by the absence of hydroxy fatty acids and by the presence or absence of some other fatty acids (Table 2). Strain DS-56T also shows slight differences from recognized Devosia species in terms of DNA G+C content (Table 1). However, the many phylogenetic similarities between strain DS-56T and Devosia species preclude the placement of strain DS-56T in a novel genus. In particular, the closest phylogenetic neighbour, ‘D. ginsengisoli’ Gsoil 326, has been shown to contain Q-10 as the predominant menaquinone (W.-T. Im and S.-T. Lee, personal communication). Accordingly, it appears to be appropriate for strain DS-56T to be classified within the genus Devosia. The phylogenetic distinctiveness of DS-56T, together with the differential phenotypic properties, is sufficient to allocate the strain to a species that is distinct from recognized Devosia species (Stackebrandt & Goebel, 1994). On the basis of the data presented, strain DS-56T represents a novel species of the genus Devosia, for which the name Devosia insulae sp. nov. is proposed.
Description of Devosia insulae sp. nov.
Devosia insulae (in.su'lae. L. fem. gen. n. insulae of an island, from where the type strain was isolated).
Cells are Gram-negative, non-spore-forming and oval- or rod-shaped (0.3–0.5x0.5–2.0 µm). Motile by means of a single flagellum. Colonies on TSA are circular, convex, smooth, glistening, ivory in colour and 0.3–0.5 mm in diameter after 7 days incubation at 25 °C. Optimal temperature for growth is 25 °C. Growth occurs at 10 and 31 °C, but not at 4 or 32 °C. Optimal pH for growth is between 6.5 and 7.5. Growth does not occur in the absence of NaCl or in the presence of >2.0 % (w/v) NaCl; optimal growth occurs in the presence of 0.5 % (w/v) NaCl. Anaerobic growth does not occur on TSA or on TSA supplemented with nitrate. Hypoxanthine, xanthine and Tweens 20, 40, 60 and 80 are not hydrolysed. H2S is not produced. Lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase, valine arylamidase and trypsin are absent. Susceptible to penicillin G, cephalothin, novobiocin and carbenicillin, but not to lincomycin, neomycin, oleandomycin or streptomycin. The predominant ubiquinone is Q-11. The major fatty acids (>10 % of total fatty acids) are 11-methyl C18 : 17c, C18 : 17c, iso-C17 : 0 and C16 : 0. The DNA G+C content is 66.2 mol% (determined by HPLC). Other phenotypic characteristics are given in Table 1.
The type strain, DS-56T (=KCTC 12821T=DSM 17955T), was isolated from soil from Dokdo, Korea.
Emended description of the genus Devosia Nakagawa et al. 1996
The description of the genus Devosia is as given by Nakagawa et al. (1996) and emended by Rivas et al. (2003), Vanparys et al. (2005) and Yoo et al. (2006), with the following further amendments. The predominant ubiquinone is Q-10 or Q-11. The fatty acid profile consists of unsaturated and straight-chain fatty acids, 11-methyl C18 : 17c and hydroxy or branched fatty acids. The DNA G+C contents are in the range 59.5–66.2 mol%.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Komagata, K. & Suzuki, K. (1987). Lipid and cell wall analysis in bacterial systematics. Methods Microbiol 19, 161–203.
Lanyi, B. (1987). Classical and rapid identification methods for medically important bacteria. Methods Microbiol 19, 1–67.
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Nakagawa, Y., Sakane, T. & Yokota, A. (1996). Transfer of "Pseudomonas riboflavina" (Foster 1944), a gram-negative, motile rod with long-chain 3-hydroxy fatty acids, to Devosia riboflavina gen. nov., sp. nov., nom. rev. Int J Syst Bacteriol 46, 16–22.
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Yoo, S.-H., Weon, H. Y., Kim, B. Y., Hong, S.-B., Kwon, S.-W., Cho, Y.-H., Go, S.-J. & Stackebrandt, E. (2006). Devosia soli sp. nov., isolated from greenhouse soil in Korea. Int J Syst Evol Microbiol 56, 2689–2692.
Yoon, J.-H., Kim, H., Kim, S.-B., Kim, H.-J., Kim, W. Y., Lee, S. T., Goodfellow, M. & Park, Y.-H. (1996). Identification of Saccharomonospora strains by the use of genomic DNA fragments and rRNA gene probes. Int J Syst Bacteriol 46, 502–505.
Yoon, J.-H., Lee, S. T. & Park, Y.-H. (1998). Inter- and intraspecific phylogenetic analysis of the genus Nocardioides and related taxa based on 16S rRNA gene sequences. Int J Syst Bacteriol 48, 187–194.
Yoon, J.-H., Kang, K. H. & Park, Y.-H. (2003). Psychrobacter jeotgali sp. nov., isolated from jeotgal, a traditional Korean fermented seafood. Int J Syst Evol Microbiol 53, 449–454.
Yoon, J.-H., Kang, S.-J., Lee, S.-Y., Lee, M.-H. & Oh, T.-K. (2005a). Virgibacillus dokdonensis sp. nov., isolated from a Korean island, Dokdo, located at the edge of the East Sea in Korea. Int J Syst Evol Microbiol 55, 1833–1837.
Yoon, J.-H., Kang, S.-J., Lee, C.-H. & Oh, T.-K. (2005b). Dokdonia donghaensis gen. nov., sp. nov., isolated from sea water. Int J Syst Evol Microbiol 55, 2323–2328.
Yoon, J.-H., Kang, S.-J. & Oh, T.-K. (2006a). Dokdonella koreensis gen. nov., sp. nov., isolated from soil. Int J Syst Evol Microbiol 56, 145–150.
Yoon, J.-H., Kang, S.-J. & Oh, T.-K. (2006b). Variovorax dokdonensis sp. nov., isolated from soil. Int J Syst Evol Microbiol 56, 811–814.
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