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1 Department of Biology, College of Natural Sciences, Sunchon National University, Suncheon 540-742, Republic of Korea
2 Department of Dental Hygiene, Gwangyang Health College, Gwangyang 545-703, Republic of Korea
3 Saint Garlo Medical Center, Suncheon 540-719, Republic of Korea
Correspondence
Chi Nam Seong
scnu{at}scnu.ac.kr
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7c (34.8 %), iso-C15 : 0 (24.2 %) and C16 : 0 (9.4 %). The DNA G+C content was 44 mol%. A phylogenetic tree based on 16S rRNA gene sequences showed that strain WPCB159T forms a lineage within the genus Dyadobacter (family ‘Flexibacteraceae’) and is closely related to Dyadobacter hamtensis HHS 11T (97.8 % sequence similarity) and to other members of the genus Dyadobacter (95.2–96.8 % sequence similarity). The phenotypic characteristics and DNA–DNA hybridization relatedness data indicate that strain WPCB159T should be distinguished from D. hamtensis HHS 11T. On the basis of the evidence presented in this study, strain WPCB159T represents a novel species of the genus Dyadobacter, for which the name Dyadobacter koreensis sp. nov. is proposed. The type strain is WPCB159T (=KCTC 12537T=NBRC 101116T).
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; Reddy & Garcia-Pichel, 2005; Chaturvedi et al., 2005; Liu et al., 2006). In the course of our study on wetland microbial diversity, a rod-shaped bacterial strain, designated WPCB159T, was isolated from freshwater samples and was the subject of a polyphasic taxonomic investigation. On the basis of the data from this investigation, strain WPCB159T represents a novel species within the genus Dyadobacter.
Strain WPCB159T was isolated from a freshwater sample collected in July 2000 from the wetland of Woopo (35° 33' N 128° 25' E) located in the Republic of Korea; the standard dilution plating technique was used. Isolation was achieved using PYGV (Staley, 1968) at 25 °C for 7 days. The isolate was routinely cultured on R2A and maintained as a glycerol suspension (20 %, w/v) at –80 °C.
Preparation of bacterial DNA and PCR amplification and sequencing of the 16S rRNA gene were carried out as described previously (Chun & Goodfellow, 1995). The resulting sequence of strain WPCB159T was aligned manually against sequences obtained from the GenBank database. Phylogenetic trees were inferred from the regions available for all sequences (positions 33–1380; Escherichia coli numbering system) using the Fitch–Margoliash (Fitch, 1971; Fitch & Margoliash, 1967) and neighbour-joining (Saitou & Nei, 1987) methods. Evolutionary distance matrices were generated according to Jukes & Cantor (1969). The resulting neighbour-joining tree topology was evaluated by means of bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings. Alignment and phylogenetic analyses were carried out using the jPHYDIT program and PAUP 4.0 (Swofford, 1998) as described previously (Chun et al., 2000).
A preliminary sequence comparison with 16S rRNA gene sequences held in GenBank indicated that our isolate was closely related to the genus Dyadobacter. The newly determined sequence was then aligned manually against representatives of the genus Dyadobacter. Strain WPCB159T showed the highest level of 16S rRNA gene sequence similarity with respect to D. hamtensis HHS 11T (97.8 %), followed by D. ginsengisoli Gsoil 043T (96.0 %), D. fermentans NS114T (95.4 %) and D. crusticola CP183-8T (95.2 %). To elucidate the phylogenetic relationship between the novel isolate and species of the genus Dyadobacter, phylogenetic trees were constructed by using two different tree-making algorithms. The neighbour-joining tree (Fig. 1) showed that strain WPCB159T formed a monophyletic clade with D. hamtensis HHS 11T, with 98 % bootstrap support.
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), respectively. Pigment was extracted according to a previously described method (Weeks, 1981). The absorbance spectrum was obtained using a UV-visible spectrophotometer (Shimadzu). Additionally, the presence of flexirubin-type pigments was tested spectrophotometrically, as described previously (Güde, 1980), using 20 % KOH. Other physiological and biochemical tests were performed using API 20E, API 20NE and API 50 CH (bioMérieux). Enzyme activities were tested using the API ZYM kit (bioMérieux) according to the manufacturer's instructions. Antibiotic resistance was determined by using the disc diffusion method. The results were interpreted according to the guidelines set by the NCCLS.
Strain WPCB159T was found to be aerobic, Gram-negative, non-motile and rod-shaped. Colonies grown on TSA plates for 5 days at 25 °C were circular, low-convex with entire margins, smooth, translucent, light yellow in colour and approximately 3.0 mm in diameter. The novel strain grew well on R2A, PCA and TSA. On TSA medium, strain WPCB159T 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 427, 453 and 475 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).
Detailed results of the physiological and biochemical analyses are given in Table 1 and in the species description. It is evident from Table 1 that there are several phenotypic characteristics that clearly separate strain WPCB159T from phylogenetically related species (D. ginsengisoli, D. hamtensis, D. crusticola and D. fermentans).
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. The cellular fatty acid profile of strain WPCB159T is given in Table 2. The DNA G+C content of strain WPCB159T was 44 mol%.
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) using DIG High Prime DNA Labelling and Detection Starter kit II (Roche). When DNAs were used individually as labelled DNA probes for reciprocal hybridization experiments (conducted in duplicate), the DNA–DNA relatedness value between strain WPCB159T and D. hamtensis HHS 11T was found to be 19.4 %. It is clear from the 16S rRNA gene sequence and DNA–DNA hybridization data that strain WPCB159T represents a novel species within the genus Dyadobacter (Wayne et al., 1987).
WPCB159T occupies a distinctive phyletic line within the radiation of the genus Dyadobacter, indicating that this strain can be assigned to a novel species within that genus. In addition, a number of physiological and chemotaxonomic characteristics clearly distinguish the isolate from other phylogenetically related species (Tables 1 and 2
). Therefore strain WPCB159T represents a novel species within the genus Dyadobacter, for which the name Dyadobacter koreensis sp. nov. is proposed.
Description of Dyadobacter koreensis sp. nov.
Dyadobacter koreensis (ko.re.en'sis. N.L. masc. adj. koreensis pertaining to Korea, where the type strain was isolated).
Cells are rod-shaped, Gram-negative, non-motile and aerobic. Grows best on media such as R2A, PCA and TSA, but weakly on nutrient agar. Colonies on TSA agar are circular, low-convex with entire margins, smooth, translucent, light yellow in colour and approximately 3.0 mm in diameter after incubation at 5 days at 25 °C (pH 7). Growth occurs at pH 5–11 (optimum pH 7) and at 4–30 °C (optimum 25 °C). Oxidase- and catalase-positive. Does not reduce nitrate to nitrite. Negative for glucose fermentation. Does not produce gelatinase, arginine dihydrolase, urease, lysine decarboxylase, ornithine decarboxylase or tryptophan deaminase. Does not produce H2S or indole. Produces alkaline phosphatase and 
-galactosidase, but not lipase (C14). Utilizes the following substrates as sole carbon and energy sources: glucose, galactose, fructose, mannose, melibiose, D-arabinose, L-arabinose, D-xylose, aesculin and salicin. Does not utilize the following substrates: glycerol, ribose, adonitol, sorbose, dulcitol, inositol, mannitol, sorbitol and inulin. Cells are sensitive to the following antibiotics (µg per disc, unless otherwise indicated): amikacin (30), ampicillin (10), ampicillin/sulbactam (10/10), cefotaxime (30), gentamicin (10), imipenem (10), novobiocin (30), piperacillin (100), teicoplanin (30), vancomycin (30) and isepamicin (30), but resistant to aztreonam (30), amoxycillin/clavulanic acid (20/10), bacitracin (10 U), cefepime (30), cefmetazole (30), ceftazidime (30), ciprofloxacin (5), lincomycin (2), moxalactam (30), oxacillin (1), piperacillin/tazobactam (100/10), tetracycline/clavulanic acid (75/10) and tobramycin (10). Other physiological and biochemical characteristics are given in Table 1. The major fatty acids are C16 : 17c (34.8 %), iso-C15 : 0 (24.2 %) and C16 : 15c (11.0 %); the complete fatty acid composition is given in Table 2. The DNA G+C content is 44 mol%.
The type strain, WPCB159T (=KCTC 12537T=NBRC 101116T), was isolated from samples of fresh water from Woopo wetland, Republic of Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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