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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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7c and C16 : 0 as the major fatty acids. The DNA G+C content was 59.7 mol%. Strain SW-277T could be distinguished from members of the phylogenetically related genera Salipiger and Roseivivax by phenotypic differences and low 16S rRNA gene similarity values. On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic data, strain SW-277T (=KCTC 12735T=JCM 13604T) should be classified as a representative of a novel species in a new genus, Donghicola eburneus gen. nov., sp. nov.
A light micrograph of Donghicola eburneus gen. nov., sp. nov. strain SW-277T is available as supplementary data in IJSEM Online.
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, 2004a, b, c). This study is focused on one of these isolates, SW-277T. Comparative 16S rRNA gene sequence analysis indicated that strain SW-277T forms an independent branch within the Alphaproteobacteria. Accordingly, the aim of the present work was to determine the exact taxonomic position of strain SW-277T by a polyphasic characterization that included phenotypic and chemotaxonomic properties and detailed phylogenetic analysis based on 16S rRNA gene sequences.
Strain SW-277T was isolated by the standard dilution plating technique at 25 °C on marine agar 2216 (MA; Difco). The morphological, physiological and biochemical characteristics of strain SW-277T were investigated using routine cultivation on MA at 37 °C. Cell morphology and the presence of flagella were examined by light microscopy (Nikon E600; see Supplementary Fig. S1 in IJSEM Online) and transmission electron microscopy. For transmission electron microscopic observation, 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. Growth under anaerobic conditions was determined after incubation in a Forma anaerobic chamber on MA and MA supplemented with nitrate, both of which had been prepared anaerobically using nitrogen. Growth in the absence of NaCl was investigated using 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 % and 1.0–15.0 %, w/v, at intervals of 1.0 %) was investigated in marine broth 2216 (MB; Difco) or trypticase soy broth (Difco). Growth at various temperatures (4–45 °C) was measured on MA. Catalase and oxidase activities and hydrolysis of casein, starch and Tweens 20, 40, 60 and 80 were determined as described by Cowan & Steel (1965) using MA as base medium. Hydrolysis of hypoxanthine, tyrosine and xanthine was tested on MA using the substrate concentrations described by Cowan & Steel (1965). Hydrolysis of aesculin, gelatin and urea, and nitrate reduction were investigated as described previously (Lanyi, 1987) with a modification that artificial seawater was used for preparation of media. The artificial seawater contained (per l distilled water) 23.6 g NaCl, 0.64 g KCl, 4.53 g MgCl2.6H2O, 5.94 g MgSO4.7H2O and 1.3 g CaCl2.2H2O (Bruns et al., 2001). H2S production was tested as described previously (Bruns et al., 2001). For in vivo pigment-absorption spectrum analysis, two strains were cultivated aerobically in the dark at 37 °C in MB and liquid Erythromicrobium/Roseococcus medium (Yurkov et al., 1994; DSMZ medium no. 767) with the modification that D-glucose was used instead of acetate. The cultures were washed twice by centrifugation using a MOPS buffer (MOPS/NaOH, 0.01 M; KCl, 0.1 M; MgCl2, 0.001 M; pH 7.5) and disrupted by sonication with a Branson Sonifier 450. After removal of cell debris by centrifugation, the absorption spectrum of the supernatant was examined on a Beckman Coulter DU800 spectrophotometer. Susceptibility to antibiotics was detected on MA plates using antibiotic discs with 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; carbenicillin, 100 µg; kanamycin, 30 µg; lincomycin, 15 µg; neomycin, 30 µg; and oleandomycin, 15 µg. Acid production from carbohydrates was determined as described by Leifson (1963) in open tubes. Utilization of substrates as sole carbon and energy sources was tested according to the method of Baumann & Baumann (1981) using supplementation with 2 % (v/v) Hutner's mineral base (Cohen-Bazire et al., 1957) and 1 % (v/v) vitamin solution (Staley, 1968). Other physiological and biochemical tests were performed with the API 20E and API ZYM systems (bioMérieux) according to the manufacturer's instructions.
Cell biomass for respiratory lipoquinone analysis and DNA extraction was obtained from cells that were cultivated for 2 days in MB at 37 °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 of 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. (2003a). Respiratory lipoquinones were analysed as described by Komagata & Suzuki (1987) using reversed-phase HPLC. For cellular fatty acid analysis, cell mass of strain SW-277T was harvested from MA plates after cultivation for 3 days at 37 °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 by the method of Tamaoka & Komagata (1984) with the modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC.
Morphological, cultural, physiological and biochemical characteristics of strain SW-277T are given in the genus and species descriptions (see later) or are shown in Table 1. The almost complete 16S rRNA gene sequence of strain SW-277T determined in this study comprised 1416 nt, representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence. In the phylogenetic tree based on the neighbour-joining algorithm, strain SW-277T joined the clade comprising two Roseivivax species and Salipiger mucosus of the Alphaproteobacteria with a bootstrap resampling value of 72.5 % (Fig. 1). This tree topology was found in trees generated with the maximum-likelihood and maximum-parsimony algorithms. Strain SW-277T exhibited 16S rRNA gene sequence similarity values of 94.6 % to the type strain of S. mucosus and 93.8 and 94.7 % to the type strains of Roseivivax halodurans and Roseivivax halotolerans, respectively. The fatty acid profile of strain SW-277T comprised (>0.5 % of total fatty acids): unsaturated fatty acids C18 : 17c (61.6 %) and C18 : 19c (0.8 %); straight-chain fatty acids C16 : 0 (13.6 %), C18 : 0 (9.2 %), C14 : 0 (1.4 %) and C17 : 0 (1.3 %); 11-methyl fatty acid C18 : 17c (5.2 %); hydroxy fatty acid C12 : 1 3-OH (4.9 %); and C16 : 17c and/or iso-C15 : 0 2-OH (0.9 %). The predominant respiratory lipoquinone detected in strain SW-277T was ubiquinone-10 (Q-10) at a peak area ratio of approximately 94 %. The DNA G+C content of strain SW-277T was 59.7 mol%.
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). The predominant ubiquinone and fatty acid profile of strain SW-277T were similar to those of members of the phylogenetically related genera Salipiger and Roseivivax (Suzuki et al., 1999; Martínez-Cánovas et al., 2004). Members of many genera of the Alphaproteobacteria cannot be differentiated clearly by chemotaxonomic characteristics including major ubiquinone and fatty acid types (Nishimura et al., 1994; Martínez-Cánovas et al., 2004; Martens et al., 2006). Strain SW-277T could be differentiated from members of the genera Salipiger and Roseivivax by differences in some phenotypic properties as listed in Table 1. Low 16S rRNA gene sequence similarity values between strain SW-277T and all other members of the Alphaproteobacteria, together with differential phenotypic properties, suggest that strain SW-277T represents a novel species in a new genus in the Alphaproteobacteria, for which the name Donghicola eburneus gen. nov., sp. nov. is proposed.
Description of Donghicola gen. nov.
Donghicola [Dong.hi'co.la. N.L. n. Donghae the Korean name of the East Sea in Korea; L. suff. -cola (from L. n. incola) a dweller, inhabitant; N.L. masc. n. Donghicola a dweller of the East Sea in Korea].
Cells are Gram-negative, aerobic, non-spore-forming cocci or rods. The predominant ubiquinone is Q-10. The major fatty acids are C18 : 17c and C16 : 0. The type species is Donghicola eburneus.
Description of Donghicola eburneus sp. nov.
Donghicola eburneus (e.bur.ne'us. L. masc. adj. eburneus white as ivory).
Cells are Gram-negative, slightly halophilic, non-spore-forming cocci or rods (0.6–0.8x0.6–2.0 µm) existing singly, in pairs, in tetrads or long chains. Colonies on MA are circular to slightly irregular, slightly convex, smooth, ivory in colour and 2.5–3.5 mm in diameter after 3 days incubation at 37 °C. Growth occurs at 10 and 42 °C, but not at 4 or 43 °C. Optimal pH for growth is between 7.0 and 8.0; growth occurs at pH 5.0, but not at pH 4.5. Optimal growth occurs in the presence of 2 % (w/v) NaCl. Growth occurs in the presence of 11 % (w/v) NaCl, but not in the absence of NaCl or in the presence of more than 12 % (w/v) NaCl. Anaerobic growth does not occur on MA or on MA supplemented with nitrate. Hypoxanthine and Tweens 20, 40 and 60 are hydrolysed, but aesculin, casein, L-tyrosine and xanthine are not. H2S is not produced. Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. L-Malate and pyruvate are utilized as carbon and energy sources, but maltose, sucrose, D-trehalose, D-xylose, benzoate, formate and salicin are not. Acid is produced from melibiose, but not from D-melezitose, D-raffinose, L-rhamnose, D-trehalose or myo-inositol. Susceptible to ampicillin, cephalothin, gentamicin, novobiocin, carbenicillin, kanamycin, neomycin and oleandomycin, but not to lincomycin. The major fatty acids (>10 % of total fatty acids) are C18 : 17c (61.6 %) and C16 : 0 (13.6 %). Other phenotypic characteristics are given in Table 1.
The type strain is SW-277T (=KCTC 12735T=JCM 13604T), isolated from a seawater sample of Jungdongjin, the East Sea, Korea. The DNA G+C content of strain SW-277T is 59.7 mol% (determined by HPLC).
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ACKNOWLEDGEMENTS |
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