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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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ABSTRACT |
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7c and C16 : 0 as the major fatty acids. The DNA G+C content was 68.7 mol%. Strain DS-18T exhibited levels of 16S rRNA gene sequence similarity of 96.3–98.7 % to the type strains of Brevundimonas species and Mycoplana bullata. Mean DNA–DNA relatedness values between strain DS-18T and the type strains of phylogenetically related Brevundimonas species and M. bullata were in the range 15–32 %. Strain DS-18T differed from Brevundimonas species and M. bullata in several phenotypic characteristics. On the basis of phenotypic, phylogenetic and genetic data, strain DS-18T represents a novel species of the genus Brevundimonas, for which the name Brevundimonas lenta sp. nov. is proposed. The type strain is DS-18T (=KCTC 12871T =JCM 14602T).
Differential phenotypic characteristics of Brevundimonas lenta sp. nov. and related species are available as supplementary material with the online version of this paper.
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MAIN TEXT |
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, currently comprises 12 species with validly published names: Brevundimonas diminuta and B. vesicularis (Segers et al., 1994), B. alba, B. aurantiaca, B. bacteroides, B. intermedia, B. subvibrioides and B. variabilis (Abraham et al., 1999), B. nasdae (Li et al., 2004), B. mediterranea (Fritz et al., 2005), B. kwangchunensis (Yoon et al., 2006a) and B. terrae (Yoon et al., 2006b). Here, the taxonomic characterization is reported of a Brevundimonas-like bacterial strain, DS-18T, which was isolated from a soil sample from Dokdo in Korea.
Strain DS-18T was isolated by the standard dilution plating technique at 25 °C on 10x diluted nutrient agar (NA; Difco). The type strains of eight Brevundimonas species and Mycoplana bullata were used as reference strains for DNA–DNA hybridization: B. alba LMG 18360T, B. aurantiaca LMG 18359T, B. diminuta LMG 2089T, B. intermedia LMG 18361T, B. subvibrioides LMG 14903T and B. variabilis LMG 18362T were obtained from the Laboratorium voor Microbiologie Universiteit Gent (LMG), Gent, Belgium; B. kwangchunensis KSL-102T and B. terrae KSL-145T were obtained from the studies of Yoon et al. (2006a, b); and M. bullata DSM 7126T was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. The morphological, physiological and biochemical characteristics of strain DS-18T were investigated using routine cultivation on NA at 25 °C. Cell morphology was examined by light microscopy (Nikon E600) and transmission electron microscopy. Flagellation was determined by using a Philips CM-20 transmission electron microscope with cells from exponentially growing cultures: for this purpose, cells were negatively stained with 1 % (w/v) phosphotungstic acid and the grids were examined after being air-dried. 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 NA. Growth in the absence of NaCl was investigated in trypticase soy broth prepared according to the formula of the Difco medium except that NaCl was excluded. The pH range for growth was determined in nutrient broth (NB; Difco) that was adjusted to various pH values (initial pH 4.5–10.5 at intervals of 0.5 pH units) prior to sterilization 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 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 nitrate reduction were studied as described previously (Lanyi, 1987). 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 using antibiotic discs containing: 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; and carbenicillin, 100 µg. Other physiological and biochemical properties were investigated using the API 20E and API ZYM systems (bioMérieux).
Cell biomass for DNA extraction and isoprenoid quinone analysis was obtained from cultures grown 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 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-18T was harvested from NA plates after incubation for 7 days at 25 °C. 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 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 are quoted as DNA–DNA relatedness values.
Morphological, cultural, physiological and biochemical characteristics of strain DS-18T are given in the species description, Table 1 and Supplementary Table S1 (available in IJSEM Online). The almost-complete 16S rRNA gene sequence of strain DS-18T determined in this study comprised 1418 nt, representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence. 16S rRNA gene sequence analyses showed that strain DS-18T was most closely affiliated with members of the genus Brevundimonas. In the phylogenetic tree based on the neighbour-joining algorithm, strain DS-18T fell within the cluster comprising Brevundimonas species and M. bullata (Fig. 1). Strain DS-18T exhibited 16S rRNA gene sequence similarity values of 96.3–98.7 % to the type strains of Brevundimonas species and M. bullata, and less than 95.1 % to other species included in the phylogenetic analysis (Fig. 1).
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; Abraham et al., 1999; Li et al., 2004; Fritz et al., 2005; Yoon et al., 2006a, b). Strain DS-18T had a cellular fatty acid profile that contained large amounts of unsaturated, straight-chain and 11-methyl fatty acids; the major fatty acids (>10 % total fatty acids) were C18 : 17c and C16 : 0 (Table 2). This fatty acid profile was similar to those of Brevundimonas species (Table 2). The DNA G+C content of strain DS-18T was 68.7 mol%.
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and Supplementary Table S1). The phylogenetic distinctiveness, together with genetic data and differential phenotypic properties, were sufficient to allocate strain DS-18T to a species that is separate from recognized Brevundimonas species and M. bullata (Wayne et al., 1987; Stackebrandt & Goebel, 1994). Therefore, on the basis of the data presented, strain DS-18T should be placed in the genus Brevundimonas as a representative of a novel species, for which the name Brevundimonas lenta sp. nov. is proposed.
Description of Brevundimonas lenta sp. nov.
Brevundimonas lenta (len'ta. L. fem. adj. lenta sticky, referring to sticky colonies).
Cells are Gram-negative rods (0.3–0.5x0.6–3.5 µm). Motile by means of a single polar flagellum. Colonies on NA are circular, convex, glistening, sticky, greyish-yellow in colour and 0.8–1.0 mm in diameter after 7 days incubation at 25 °C. Optimal temperature for growth is 25 °C. Growth occurs at 4 and 34 °C, but not at 35 °C. Optimal pH for growth is between 6.5 and 7.0; growth occurs at pH 6.0 and 9.5, but not at pH 5.5 or 10.0. Growth occurs in the presence of 0–1 % (w/v) NaCl; optimal growth occurs without NaCl. Anaerobic growth does not occur on NA or NA supplemented with nitrate. Tweens 20, 40 and 60 are hydrolysed, but casein, hypoxanthine, xanthine and tyrosine are not. H2S and indole are not produced. Lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. In assays with the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, trypsin, acid phosphatase and naphthol-AS-BI-phosphohydrolase are present, but lipase (C14), valine arylamidase, cystine arylamidase, 
-chymotrypsin, -galactosidase, 
-glucuronidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are absent. Susceptible to polymyxin B, chloramphenicol, cephalothin, gentamicin, novobiocin, tetracycline, kanamycin and neomycin, but not to ampicillin, carbenicillin, lincomycin or oleandomycin. The predominant ubiquinone is Q-10. The major fatty acids (>10 % of total fatty acids) are C18 : 17c and C16 : 0. Other phenotypic characteristics are given in Table 1 and Supplementary Table S1.
The type strain is DS-18T (=KCTC 12871T =JCM 14602T), isolated from soil from Dokdo, Korea. The DNA G+C content of the type strain is 68.7 mol% (determined by HPLC).
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ACKNOWLEDGEMENTS |
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Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of Medical Bacteria. London: Cambridge University Press.
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Yoon, J.-H., Kang, S.-J., Oh, H. W., Lee, J.-S. & Oh, T.-K. (2006a). Brevundimonas kwangchunensis sp. nov., isolated from an alkaline soil in Korea. Int J Syst Evol Microbiol 56, 613–617.
Yoon, J.-H., Kang, S.-J., Lee, J.-S. & Oh, T.-K. (2006b). Brevundimonas terrae sp. nov., isolated from an alkaline soil in Korea. Int J Syst Evol Microbiol 56, 2915–2919.
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-hydroxybutyric acid, p-hydroxyphenylacetic acid, itaconic acid, malonic acid, quinic acid, D-saccharic acid, sebacic acid, succinamic acid, glucuronamide, L-ornithine, L-phenylalanine, L-pyroglutamic acid, D-serine, DL-carnitine, 