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1 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea
2 NeoPharm Co., Ltd, BVC-307, KRIBB, 52 Oeun-dong, Yuseong-gu, Daejeon 305-333, Republic of Korea
3 Department of Oriental Medicinal Material and Processing College of Life Science, Kyung Hee University, 1 Seocheon, Kihung Yongin, Kyunggi 449-701, Republic of Korea
Correspondence
Wan-Taek Im
wandra{at}kaist.ac.kr
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ABSTRACT |
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7c, iso-C15 : 0 and C16 : 0) supported the affiliation of strain Gsoil 043T to the genus Dyadobacter. The results of physiological and biochemical tests enabled strain Gsoil 043T to be differentiated genotypically and phenotypically from the three Dyadobacter species with validly published names. The novel isolate therefore represents a novel species for which the name Dyadobacter ginsengisoli sp. nov. is proposed, with the type strain Gsoil 043T (=KCTC 12589T=LMG 23409T).
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MAIN TEXT |
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). One of these isolates, strain Gsoil 043T, was found to be a member of the genus Dyadobacter (family ‘Flexibacteraceae’) and was subjected to a taxonomic investigation.
The genus Dyadobacter was described by Chelius & Triplett (2000) to accommodate Gram-negative, rod-shaped cells that are straight to curved, occur in pairs in young cultures and form chains of coccoid cells in old cultures and produce a flexirubin-like pigment (Reddy & Garcia-Pichel, 2005). At present, the genus comprises three species: Dyadobacter fermentans (Chelius & Triplett, 2000), Dyadobacter crusticola (Reddy & Garcia-Pichel, 2005) and Dyadobacter hamtensis (Chaturvedi et al., 2005).
For the present study, we conducted phylogenetic (16S rRNA gene), phenotypic, genotypic and chemotaxonomic analyses to determine the precise taxonomic position of strain Gsoil 043T. On the basis of the results obtained, we propose that strain Gsoil 043T should be placed in the genus Dyadobacter as the type strain of novel species.
Strain Gsoil 043T was originally isolated from soil from a ginseng field in Pocheon province, South Korea. The soil sample was thoroughly suspended with 50 mM phosphate buffer (pH 7.0) and the suspension was spread on one fifth-strength modified R2A (0.25 g tryptone, 0.25 g peptone, 0.25 g yeast extract, 0.125 g malt extract, 0.125 g beef extract, 0.25 g Casamino acids, 0.25 g soytone, 0.5 g glucose, 0.3 g soluble starch, 0.2 g xylan, 0.3 g sodium pyruvate, 0.3 g K2HPO4, 0.05 g MgSO4, 0.05 g CaCl2 and 15 g agar 1 l–1) agar plates after serial dilution with 50 mM phosphate buffer (pH 7.0). The plates were incubated at 30 °C for 1 month. Single colonies on the plates were purified by transferring them onto new plates and these were incubated again on modified R2A or half-strength modified R2A. Purified colonies were tentatively identified by partial sequences of the 16S rRNA gene (Im et al., 2005). Strain Gsoil 043T was one of the predominant isolates on modified R2A agar plates in aerobic conditions. Strain Gsoil 043T was routinely cultured on R2A agar at 30 °C and maintained as a glycerol suspension (20 %, w/v) at –70 °C.
Gram-reaction testing was performed by the non-staining method as described by Buck (1982). Cell morphology was observed at x 1000 magnification with a light microscope (Nikon) using cells grown for 3 days at 30 °C on R2A agar. Catalase activity was determined by bubble production in 3 % (v/v) H2O2 and oxidase activity was determined using 1 % (w/v) tetramethyl p-phenylenediamine. For single carbon source assimilation studies, a defined liquid medium containing basal salt media was used composed of (g l–1); 1.8 g K2HPO4, 1.08 g KH2PO4, 0.5 g NaNO3, 0.5 g NH4Cl, 0.1 g KCl, 0.1g MgSO4 and 0.05 g CaCl2. A vitamin solution (Widdel & Bak, 1992), trace element solution SL-10 (Widdel et al., 1983) and selenite/tungstate solution (Tschech & Pfennig, 1984) were added to this medium and the pH was adjusted to 6.8. This liquid medium was aliquoted into 96-well trays and filter-sterilized carbon sources were added to each well (individually at 0.1 % w/v). Growth was visually examined on 96-well plates incubated at 30 °C for up to 7 days. A negative control well, containing no carbon sources, was included. A positive control culture was grown in a well containing R2A broth.
Fermentative and oxidative acid production from carbohydrates was tested by growth in O–F basal medium with bromothymol blue (Atlas, 1993) supplemented with 1 % carbohydrate [soft agar stabs with (fermentative) and without (oxidative) sterile mineral oil overlay]. The O–F medium tubes were incubated at 30 °C for 5 days. Some physiological characteristics were determined with API 20E galleries according to the manufacturer's instructions (bioMérieux). Anaerobic growth was tested in serum bottles by adding 1 g thioglycolate l–1 to R2A broth and substituting the upper air layer with nitrogen gas. The anaerobic nitrate reduction test to determine the final electron acceptor was performed in serum bottles by adding 1 g thioglycolate l–1 and 10 mM KNO3 to R2A broth under nitrogen gas. Aerobic nitrate reduction was later confirmed by inoculation into 12 ml R2A broth supplemented with 10 mM KNO3 in three 25 ml serum bottles. The reduction of nitrate was monitored by an ion chromatograph (model 790 personal IC; Metrohm) equipped with a conductivity detector and an anion exchange column (Metrosep Anion Supp 4; Metrohm). Tests for the degradation of DNA (DNase agar Scharlau by flooding plates with 1 M HCl), casein, chitin, starch (Atlas, 1993), lipid (Kouker & Jaeger, 1987), xylan and cellulose (Ten et al., 2004) were performed and evaluated after 5 days. Growth at different temperatures (4, 15, 25, 30, 37 and 42 °C) and various pH values (pH 5.0–10.0 at intervals of 0.5 pH units) was assessed after 5 days incubation. Salt tolerance was tested on R2A agar supplemented with 1–10 % (w/v) NaCl after 5 days incubation. Growth on nutrient agar, trypticase soy agar (TSA) and MacConkey agar at 30 °C was also evaluated. Pigment was extracted according to a previously described method (Weeks, 1981) and a spectrum was obtained using a UV-visible spectrophotometer (Beckman). Additionally, the presence of flexirubin-type pigments was tested spectrophotometrically as described previously (Güde, 1980) using 20 % KOH.
Extraction of genomic DNA was performed with a commercial genomic DNA extraction kit (Core Biosystem) and PCR-mediated amplification of the 16S rRNA gene and sequencing of the purified PCR product were carried out according to Kim et al. (2005). Full sequences of the 16S rRNA gene were compiled using SeqMan software (DNASTAR). The 16S rRNA gene sequences of related taxa were obtained from GenBank. Multiple alignments were performed with CLUSTAL_X (Thompson et al., 1997). Gaps were edited using BIOEDIT (Hall, 1999). Evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1983). Phylogenetic trees were constructed using the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) methods using the MEGA3 program (Kumar et al., 2004) with bootstrap values based on 1000 replications (Felsenstein, 1985).
For the measurement of the G+C content of chromosomal DNA, genomic DNA of the novel strain was extracted and purified as described by Moore & Dowhan (1995) and was enzymically degraded into nucleosides. DNA G+C content was determined as described by Mesbah et al. (1989) using a reverse-phase HPLC. Isoprenoid quinones were extracted with chloroform/methanol (2 : 1, v/v), evaporated under vacuum conditions and reextracted in n-hexane/water (1 : 1, v/v). The crude n-hexane–quinone solution was purified using Sep-Pak Vac cartridges silica (Waters) and subsequently analysed by HPLC as previously described (Hiraishi et al., 1996). Cellular fatty acid profiles were determined for strains grown on TSA (Difco) for 2 days. Cellular fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI). The fatty acids analysed by GC (6890; Hewlett Packard) were identified by the Microbial Identification software package (Sasser, 1990).
Strain Gsoil 043T was aerobic, Gram-negative, non-motile and rod-shaped. Colonies grown on R2A agar plates for 5 days were smooth, circular, transparent, yellowish and 3–7 mm in diameter. The novel strain grew well on nutrient agar and TSA. On R2A agar medium, strain Gsoil 043T was able to grow at 4–30 °C, but not 37 °C. A flexirubin-like yellow-coloured pigment was produced. The pigment exhibited peaks at 412 and 448 nm when extracted in ethanol. The addition of alkali (20 % KOH) changed the colour of the pigment to orange and also broadened the peak, thus confirming that it is a flexirubin-type pigment (Weeks, 1981). Other physiological characteristics of strain Gsoil 043T are summarized in the species description and comparisons of selective characteristics with closely related type strains are shown in Table 1.
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). Strain Gsoil 043T and the three species of the genus Dyadobacter mentioned above formed a monophyletic clade with a high bootstrap value (100 %) which was supported by both tree-making methods employed in this study.
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).
The G+C content of the genomic DNA of strain Gsoil 043T was 48 mol%. MK-7 was the predominant menaquinone. The fatty acid profile of strain Gsoil 043T (Table 2) comprised mainly C16 : 17c (44.6 %), iso-C15 : 0 (20.3 %) and C16 : 0 (11.0 %). Significant differences in fatty acid profiles were found between strain Gsoil 043T and the other species of the genus Dyadobacter. D. hamtensis and D. fermentans contained significantly higher amounts of iso-C17 : 0 3-OH, whereas D. crusticola and strain Gsoil 043T contained significantly higher amounts of C16 : 17c. However, strain Gsoil 043T contained significantly lower amounts of C16 : 15c. The species of the genus Dyadobacter could be differentiated from one another by the fatty acid profiles.
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Description of Dyadobacter ginsengisoli sp. nov.
Dyadobacter ginsengisoli (gin.sen.gi.so'li. N.L. n. ginsengum ginseng; L. n. solum soil; N.L. gen. n. ginsengisoli of the soil of a ginseng field, the source of the type strain).
Cells are Gram-negative, aerobic, non-motile and rod-shaped, 0.6–0.8 µm in diameter and 3.0–6.0 µm in length after 2 days culture on R2A agar. Colonies grown on R2A agar for 2 days are smooth, circular, transparent and yellowish. Grows well at 4–30 °C and at pH 5.5–8.5, but does not grow at 37 °C. Growth occurs in the absence of NaCl and in the presence of 1.0 % (w/v) NaCl, but not with 2 % (w/v) NaCl. Nitrate is reduced in aerobic conditions. Anaerobic growth does not occur. Does not degrade DNA, chitin or xylan. Substrate utilization, enzyme production, acid production and other physiological characteristics are given in Table 1
. In addition, is able to utilize L-rhamnose, D-lyxose, N-acetylglucosamine, salicin, D-lactose, D-maltose, D-melibiose and inulin as sole carbon sources. Does not utilize D-fucose, ethanol, L-xylose, pyruvic acid, formic acid, propionate, 3-hydroxybutyrate, valerate, caprate, maleic acid, phenylacetate, benzoic acid, 3-hydroxybenzoate, 4-hydroxybenzoate, malate, succinic acid, glutaric acid, itaconate, adipate, suberate, oxalic acid, gluconate, xylitol, amygdalin, methanol or glycogen as sole carbon sources. Resistant to (µg per disc) tetracycline (100), ampicillin (100), kanamycin (100), rifampicin (100), chloramphenicol (100), carbenicillin (100), hygromycin (100) and streptomycin (50), but sensitive to streptomycin (100). The predominant menaquinone is MK-7. Major cellular fatty acids are C16 : 17c, iso-C15 : 0, and C16 : 0. The DNA G+C content of the type strain is 48 mol% (determined by HPLC).
The type strain, Gsoil 043T (=KCTC 12589T=LMG 23409T), was isolated from soil of a ginseng field in Pocheon province, South Korea.
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ACKNOWLEDGEMENTS |
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