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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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9c as the major fatty acids. The DNA G+C content was 38.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain DS-20T belonged to the genus Flavobacterium. Levels of 16S rRNA gene sequence similarity between strain DS-20T and the type strains of recognized Flavobacterium species were below 94.9 %. Strain DS-20T differed from phylogenetically related Flavobacterium species in several phenotypic characteristics. On the basis of its phenotypic and phylogenetic distinctiveness, strain DS-20T was classified in the genus Flavobacterium as representing a novel species, for which the name Flavobacterium terrigena sp. nov. is proposed. The type strain is DS-20T (=KCTC 12761T=DSM 17934T).
A table giving Biolog assimilation data for strain DS-20T is available as supplementary material in IJSEM Online.
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. Recently, descriptions of many novel Flavobacterium species, including Flavobacterium saliperosum (Wang et al., 2006), Flavobacterium soli (Yoon et al., 2006), Flavobacterium weaverense and Flavobacterium segetis (Yi & Chun, 2006), Flavobacterium daejeonense and Flavobacterium suncheonense (Kim et al., 2006), Flavobacterium croceum (Park et al., 2006), Flavobacterium indicum (Saha & Chakrabarti, 2006), Flavobacterium glaciei (Zhang et al., 2006), and Flavobacterium aquidurense and Flavobacterium hercynium (Cousin et al., 2007), have increased considerably the number of species belonging to the genus. Flavobacterium ferrugineum was reclassified recently in a new genus, Terrimonas, as Terrimonas ferruginea (Xie & Yokota, 2006). Accordingly, at the time of writing, the genus Flavobacterium comprises 45 recognized species. Here we report on the taxonomic characterization of a Flavobacterium-like bacterial strain, DS-20T.
Strain DS-20T was isolated from a soil sample collected on the island of Dokdo (37° 14' 12'' N 131° 52' 07'' E), Korea, using the standard dilution plating technique at 25 °C on nutrient agar (NA; Difco). The morphological, physiological and biochemical characteristics of strain DS-20T were investigated by using routine cultivation on NA at 25 °C. Cell morphology was examined by light microscopy (Nikon E600) and transmission electron microscopy with cells from exponentially growing cultures. 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. Gliding motility was determined as described by Bowman (2000). The Gram reaction was determined by using the bioMérieux Gram stain kit according to the manufacturer's instructions. Growth at various temperatures (4–40 °C) was measured on NA. The pH range for growth was determined in nutrient broth (NB; Difco) that, prior to sterilization, was adjusted to various pH values (pH 4.5–10.5 at intervals of 0.5 pH units) by the addition of HCl or Na2CO3. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on NA and on NA supplemented with nitrate, both of which had been prepared anaerobically under a nitrogen atmosphere. 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). DNase activity was examined by using DNase test agar with methyl green (Difco). Hydrolysis of aesculin and reduction of nitrate were studied as described by Lanyi (1987). Hydrolysis of carboxymethylcellulose (CM-cellulose) was tested as described by Bowman (2000) using NA as the basal medium. The presence of flexirubin-type pigments was investigated as described by Reichenbach (1992). Congo red adsorption was determined according to the method of Bernardet et al. (2002). Assimilation of various substrates was determined by using the Biolog GN2 MicroPlate assay as recommended by the manufacturer. Sensitivity to antibiotics was tested on NA plates by using antibiotic discs containing the following: 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) or carbenicillin (100 µg). Enzyme activities and other physiological and biochemical properties were tested by using the API 20E and API ZYM systems (bioMérieux) according to the manufacturer's instructions.
Cell biomass for DNA extraction and for isoprenoid quinone analysis was obtained from cultivation in NB 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 PCR by 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). 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 analysis, cell mass of strain DS-20T was harvested from NA plates after incubation for 3 days at 25 °C. The fatty acids were extracted and fatty acid methyl esters were prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). The DNA G+C content was determined according to the method of Tamaoka & Komagata (1984) with a modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC.
Morphological, cultural, physiological and biochemical characteristics of strain DS-20T are given in the species description or are shown in Table 1. The almost complete 16S rRNA gene sequence of strain DS-20T determined in this study comprised 1470 nt, representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain DS-20T falls within the radiation of the cluster comprising Flavobacterium species (Fig. 1). In the neighbour-joining phylogenetic tree, strain DS-20T joined the clade comprising F. saliperosum, F. suncheonense, Flavobacterium columnare, F. indicum and F. croceum with a bootstrap resampling value of 83.8 %; this tree topology was also found in trees generated with the maximum-likelihood and maximum-parsimony algorithms (Fig. 1). Strain DS-20T exhibited 16S rRNA gene sequence similarity values of 92.3–94.9 % to the type strains of recognized Flavobacterium species.
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, 2002; Kim et al., 2006; Yi et al., 2005; Yi & Chun, 2006; Yoon et al., 2006; Wang et al., 2006). The fatty acid profile of strain DS-20T comprised (>1 % of the total fatty acids): branched fatty acids iso-C15 : 0 (30.1 %), iso-C17 : 19c (11.7 %), iso-C15 : 1 (9.0 %), iso-C16 : 0 (5.2 %), iso-C16 : 1 (2.2 %); hydroxy fatty acids iso-C17 : 0 3-OH (16.0 %), iso-C15 : 0 3-OH (6.6 %) and iso-C16 : 0 3-OH (3.6 %); straight-chain fatty acid C15 : 0 (4.3 %); summed feature 3 (C16 : 17c and/or iso-C15 : 0 2-OH, 2.4 %); and unsaturated fatty acid C15 : 16c (1.3 %). This fatty acid profile was similar to those of other Flavobacterium species, although there were differences in the proportions of some components, probably because of differences in cultivation conditions and extraction procedures (Aslam et al., 2005; Bernardet et al., 1996, 2002; Wang et al., 2006). The DNA G+C content of strain DS-20T was 38.2 mol%. Strain DS-20T differed from phylogenetically related Flavobacterium species in several phenotypic characteristics, as shown in Table 1
. The phylogenetic distinctiveness, together with differential phenotypic properties, was sufficient to classify strain DS-20T as representing a species that is distinct from recognized Flavobacterium species (Stackebrandt & Goebel, 1994). Therefore, on the basis of the data presented, strain DS-20T should be classified in the genus Flavobacterium as the type strain of a novel species, for which the name Flavobacterium terrigena sp. nov. is proposed.
Description of Flavobacterium terrigena sp. nov.
Flavobacterium terrigena (ter.ri.ge'na. L. masc. or fem. n. terrigena child of the earth, referring to the isolation of the type strain from soil).
Cells are Gram-negative, aerobic short rods or rods (0.3–0.6 µmx0.7–2.5 µm). No flagella are detected. Non-motile. Colonies on NA are circular, flat to raised, smooth, glistening, dark yellow in colour and 1.0–2.0 mm in diameter after 3 days incubation at 25 °C. Optimal temperature for growth is 25 °C; growth occurs at 10 and 30 °C, but not at 4 or 31 °C. Optimal pH for growth is 6.5–7.0; growth occurs at pH 6.0 and 8.0, but not at pH 5.5 or 8.5. No anaerobic growth occurs on NA or on NA supplemented with nitrate. Tweens 20, 40, 60 and 80 are hydrolysed, but hypoxanthine, xanthine and urea are not. Negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase. In assays with the API ZYM system, positive for alkaline phosphatase, leucine arylamidase and valine arylamidase, but negative for lipase (C14), cystine arylamidase, trypsin, 
-chymotrypsin, -galactosidase, 
-glucuronidase, -glucosidase, -glucosidase, -mannosidase and -fucosidase. Sensitive to polymyxin B, streptomycin, penicillin G, chloramphenicol, ampicillin, cephalothin, gentamicin, novobiocin, tetracycline, carbenicillin, kanamycin, lincomycin and neomycin, but not to oleandomycin. Pyruvic acid methyl ester, -ketovaleric acid, succinic acid, L-glutamic acid, glycyl L-glutamic acid, L-ornithine, L-proline and L-threonine are assimilated, and -cyclodextrin, -ketobutyric acid, -ketoglutaric acid and L-serine are weakly assimilated. The predominant menaquinone is MK-6. Major fatty acids (>10 % of the total fatty acids) are iso-C15 : 0, iso-C17 : 0 3-OH and iso-C17 : 19c. The DNA G+C content of the type strain is 38.2 mol% (determined by HPLC). Other phenotypic characteristics are given in Table 1 or in Supplementary Table S1 available in IJSEM Online.
The type strain, DS-20T (=KCTC 12761T=DSM 17934T), was isolated from soil.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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