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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
Tae-Kwang Oh
otk{at}kribb.re.kr
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ABSTRACT |
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9c, iso-C17 : 0 and iso-C15 : 0 as the major fatty acids. Major polar lipids detected in the two strains were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an amino-group-containing lipid that was ninhydrin-positive. Their DNA G+C contents were 71·0 mol%. Strains DS-123T and DS-140 exhibited no difference in their 16S rRNA gene sequences and possessed a mean DNA–DNA relatedness level of 92 %. Phylogenetic trees based on 16S rRNA gene sequences showed that strains DS-123T and DS-140 formed a distinct evolutionary lineage within the Gammaproteobacteria. The 16S rRNA gene sequences of strains DS-123T and DS-140 exhibited similarity values of less than 94·1 % to members of the Gammaproteobacteria. Strains DS-123T and DS-140 were distinguished from phylogenetically related genera by differences in some phenotypic properties. On the basis of phenotypic, phylogenetic and genetic data, it is proposed that strains DS-123T (=KCTC 12396T=DSM 17203T) and DS-140 be classified in a novel genus and species, Dokdonella koreensis gen. nov., sp. nov.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains DS-123T and DS-140 are AY987368 and AY987369, respectively.
A transmission electron micrograph of strain DS-123T is available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Soil samples collected in Dokdo (37° 14' 12'' N 131° 52' 07'' E) provided the source for isolation of bacterial strains. Strains DS-123T and DS-140 were isolated by the standard dilution plating technique on twofold diluted nutrient agar (Difco) at 30 °C. Frateuria aurantia LMG 1558T and Fulvimonas soli LMG 19981T, which were used as reference strains, were obtained from the BCCM/LMG (the Belgian Co-ordinated Collections of Microorganisms/Laboratorium voor Microbiologie, Universiteit Gent), Gent, Belgium. To investigate their morphological and physiological characteristics, strains DS-123T and DS-140 were routinely cultivated at 30 °C on trypticase soy agar (TSA; Difco) under aerobic conditions. Cell morphology was examined by light microscopy (Nikon E600) and transmission electron microscopy. Presence of flagella was determined by transmission electron microscopy using cells from exponentially growing cultures. Gram-reaction was determined by using the bioMérieux Gram Stain kit according to the manufacturer's instructions. The pH range for growth was determined in nutrient broth (Difco) that was adjusted to various pH values (pH 4·5–10·5 at intervals of 0·5 pH units). The pH was adjusted prior to sterilization to various levels by the addition of HCl or Na2CO3. Growth in the absence of NaCl was investigated in trypticase soy broth prepared according to the formula of the Difco medium except that no NaCl was used. Growth at various NaCl concentrations (0·5, 1·0, 2·0, 3·0, 4·0 and 5·0 %, w/v) was investigated in trypticase soy broth (TSB; Difco). Growth at various temperatures (4–45 °C) was measured on TSA. Growth under anaerobic conditions was determined after incubation 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, starch, urea, hypoxanthine, tyrosine, xanthine and Tweens 20, 40, 60 and 80 were determined as described by Cowan & Steel (1965)
. Hydrolysis of aesculin and nitrate reduction were determined as described previously (Lanyi, 1987). Utilization of various substrates as sole carbon and energy sources was determined as described by Shirling & Gottlieb (1966). Enzyme activity was determined by using the API ZYM system (bioMérieux). Susceptibility to antibiotics was tested on TSA plates using antibiotic discs containing the following concentrations: polymyxin B, 100 U; streptomycin, 50 µg; penicillin G, 20 U; chloramphenicol, 100 µg; ampicillin, 10 µg; cephalothin, 30 µg; gentamicin, 30 µg; novobiocin, 5 µg; tetracycline, 30 µg; kanamycin, 30 µg; lincomycin, 15 µg; oleandomycin, 15 µg; neomycin, 30 µg; carbenicillin, 100 µg. Other physiological and biochemical tests were performed with the API 20E system (bioMérieux).
Cell biomass of strains DS-123T and DS-140 for DNA extraction and for respiratory lipoquinone and polar lipid analyses was obtained from cultivation in TSB at 30 °C. Cell mass of Frateuria aurantia LMG 1558T and Fulvimonas soli LMG 19981T for respiratory lipoquinone analysis was obtained by cultivation in TSB at 30 °C. Chromosomal DNA was isolated and purified according to the method described previously (Yoon et al., 1996), with the exception that ribonuclease T1 was treated in combination with ribonuclease A to minimize contamination with RNA. The 16S rRNA gene was amplified by PCR using 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). Respiratory lipoquinones 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. Polar lipids were extracted according to the procedures described by Minnikin et al. (1984) and identified by two-dimensional TLC followed by spraying with appropriate detection reagents (Minnikin et al., 1984; Komagata & Suzuki, 1987). For fatty acid methyl ester analysis, cell mass of strains DS-123T and DS-140, Frateuria aurantia LMG 1558T and Fulvimonas soli LMG 19981T was harvested from agar plates after incubation for 6 days at 30 °C on TSA. 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 a modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC. DNA–DNA hybridization was performed fluorometrically by the method of Ezaki et al. (1989) using photobiotin-labelled DNA probes and microdilution wells. Hybridization was performed with five replications for each sample. The highest and lowest values obtained in each sample were excluded, and the means of the remaining three values were quoted as DNA–DNA relatedness values.
Morphological, cultural, physiological and biochemical characteristics of strains DS-123T and DS-140 are given in the genus and species descriptions (see later) or are shown in Table 1, together with those of the four phylogenetically related genera Frateuria, Rhodanobacter, Fulvimonas and Dyella. Strains DS-123T and DS-140 were motile by means of polar peritrichous flagella (supplementary figure in IJSEM Online). Almost complete 16S rRNA gene sequences of strains DS-123T and DS-140, comprising 1494 nucleotides (approx. 96 % of the Escherichia coli 16S rRNA sequence), were determined in this study. The 16S rRNA gene sequences of strains DS-123T and DS-140 were identical. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, the two strains joined the genus Aquimonas by a bootstrap resampling value of 62·3 %, and this cluster joined the clade comprising the genera Dyella, Frateuria, Fulvimonas and Rhodanobacter by a bootstrap resampling value of 98·7 % (Fig. 1). The relationship between this cluster and the clade comprising the genus Xanthomonas and some other genera was supported by a bootstrap resampling value of 100 % (Fig. 1). Strains DS-123T and DS-140 exhibited 16S rRNA gene sequence similarity levels of 94·1 % to Aquimonas voraii and of less than 92·2 % to other species used in the phylogenetic analysis. Strains DS-123T and DS-140 exhibited a mean level of DNA–DNA relatedness of 92 %, when their DNA was used individually as labelled DNA probes, indicating that the two strains are members of the same genomic species (Wayne et al., 1987).
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9c, iso-C17 : 0 and iso-C15 : 0 (Table 2). Major polar lipids detected in strains DS-123T and DS-140 were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an amino-group-containing lipid that was ninhydrin-positive. The DNA G+C contents of strains DS-123T and DS-140 were 71·0 mol%.
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, Im et al. (2004), Xie & Yokota (2005) and An et al. (2005) and those generated in this study. However, strains DS-123T and DS-140 were differentiated from phylogenetically related genera by differences in some phenotypic characteristics, including DNA G+C contents, fatty acid profiles and others (Tables 1 and 2
). The DNA G+C contents of strains DS-123T and DS-140 were higher than those of the genera Frateuria, Rhodanobacter and Dyella (Table 1). The fatty acid profiles, particularly proportions of the major fatty acid(s), distinguish strains DS-123T and DS-140 from phylogenetically related genera Frateuria, Fulvimonas, Dyella and Aquimonas and Rhodanobacter fulvus (Table 2). In particular, the genus Frateuria was clearly differentiated from the genera Fulvimonas and Dyella as well as from strains DS-123T and DS-140 by the proportion of iso-C15 : 0 and the presence of hydroxy (2- and 3-) C12 : 0 and cyclo C17 : 0 (Table 2). Unfortunately, the ubiquinone type and fatty acid profile of the type species of the genus Rhodanobacter, Rhodanobacter lindaniclasticus, were not reported previously and could not be analysed in this study. It was confirmed by the original author and LMG that R. lindaniclasticus is not extant because the culture they held was contaminated and the original author's backup culture was lost. Therefore, on the basis of the phenotypic, phylogenetic and genetic data, strains DS-123T and DS-140 should be classified in a novel genus and species, for which the name Dokdonella koreensis gen. nov., sp. nov. is proposed.
Description of Dokdonella gen. nov.
Dokdonella (Dok.do.nel'la. N.L. fem. dim. n. Dokdonella of Dokdo, an island located on the East Sea in Korea, from where the organisms were isolated).
Cells are Gram-negative, non-spore-forming and rod-shaped. Strictly aerobic. Motile by means of polar peritrichous flagella. Catalase- and oxidase-positive. Urease-negative. The predominant ubiquinone is Q-8. The major fatty acids are iso-C17 : 19c, iso-C17 : 0 and iso-C15 : 0. Major polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an amino-group-containing lipid that is ninhydrin-positive. The DNA G+C content is 71·0 mol%. The type species is Dokdonella koreensis.
Description of Dokdonella koreensis sp. nov.
Dokdonella koreensis (ko.re.en'sis. N.L. fem. adj. koreensis pertaining to Korea, where Dokdo is located).
Exhibits the following properties in addition to those given in the genus description. Cells are Gram-negative, strictly aerobic rods, 0·3–0·5x1·0–2·0 µm. Colonies on TSA are circular, convex, glistening, smooth, moderate yellow in colour and 1·0–2·0 mm in diameter after incubation for 6 days at 30 °C. Growth occurs at 10 and 39 °C with an optimum temperature of 30 °C; growth does not occur at 4 and 40 °C. Optimal growth occurs without NaCl; growth does not occur in the presence of >3 % (w/v) NaCl. Optimal pH for growth is 6·5; good growth occurs at pH 6·5–7·5. Growth is observed at pH 5·0 and 9·0, but not at pH 4·5 and 9·5. Urease-negative. Tyrosine and Tweens 20, 40, 60 and 80 are hydrolysed. Hypoxanthine and xanthine are not hydrolysed. Indole is not produced. Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. In assays with the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8), acid phosphatase and naphthol-AS-BI-phosphohydrolase are present, but lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, 
-chymotrypsin, -galactosidase, 
-glucuronidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are absent. Susceptible to polymyxin B, chloramphenicol, gentamicin and tetracycline, but not to streptomycin, penicillin G, ampicillin, cephalothin, novobiocin, kanamycin, lincomycin, oleandomycin, neomycin or carbenicillin. Pyruvate is utilized for growth, but D-glucose, D-fructose, D-galactose, D-cellobiose, D-trehalose, D-xylose, succinate, benzoate, formate, L-glutamate and salicin are not utilized. The predominant ubiquinone is Q-8. The major fatty acids are iso-C17 : 19c, iso-C17 : 0 and iso-C15 : 0. Major polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an amino-group-containing lipid that is ninhydrin-positive. The DNA G+C content is 71·0 mol%. Other phenotypic characteristics are given in Table 1.
The type strain, DS-123T (=KCTC 12396T=DSM 17203T), was isolated from soil. The reference strain is DS-140.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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