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Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, 52 Oeundong, Yusong, Daejeon 305-333, Korea
Correspondence
Jung-Sook Lee
jslee{at}kribb.re.kr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JS53-2T is AY962574.
Photomicrographs of strain JS53-2T, an extended phylogenetic tree and a table of whole-cell fatty acid profiles are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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. It was recognized as a very heterogeneous group. However, extensive systematic studies have resolved these relationships and emended the description of the genus Microbacterium. Its description was first emended in 1983 (Collins et al., 1983) and then emended again in 1998 (Takeuchi & Hatano, 1998a). The genera Microbacterium and Aureobacterium are phylogenetically intermixed, although they are separate taxa on the basis of (primarily) cell-wall differences. The presence of the L-diamino acid L-lysine in the cell wall is characteristic of microbacterium and the presence of the D-diamino acid D-ornithine in the cell wall is characteristic of Aureobacterium. The other chemotaxonomic and phenotypic characteristics of the members of the two genera have shown very similar profiles. On the basis of these data, Takeuchi & Hatano (1998a) emended the descriptions of the genera Microbacterium and Aureobacterium, uniting them in a redefined genus Microbacterium. Members of the genus Microbacterium are found in various environments, including soil, water, plants, milk products and human clinical samples. During the course of our studies on microbial diversity in the sea water of Korea, we found yellow-pigmented bacteria in sea water from the South Sea of Korea (the Korea Strait); experiments were performed to determine the morphological, biochemical and phylogenetic characteristics of those isolates. On the basis of the results, we propose that these isolates be assigned to a novel species.
Strains JS53-2T and JS53-5 were isolated (on tryptic soy agar) from sea water from the South Sea of Korea. The isolates and reference strains Microbacterium terregens KCTC 19034T (=IFO 12961T), Microbacterium lacticum KCTC 9230T (=DSM 20427T), Microbacterium aurum KCTC 19091T (=IFO 15204T) and Microbacterium schleiferi KCTC 19095T (=IFO 15075T) were cultured on tryptic soy agar for 48 h at 30 °C.
The cell morphology was examined by using light microscopy. Motility was determined with an optical microscope, using the hanging drop technique (Skerman, 1967). Anaerobic growth was recorded in an anaerobic chamber (CO2/H2/N2, 7 : 7 : 86; Forma Scientific) on tryptic soy agar for up to 1 week. Growth in the presence of various concentrations of added NaCl (0·5, 1, 2, 5, 10, 15 and 20 %, w/v) was tested with nutrient broth (Difco) as the basal medium. Growth at different temperatures was observed in trypticase soy broth at 10, 15, 20, 25, 30, 37, 42 and 50 °C: 10 ml trypticase soy broth was adjusted to pH values ranging from 4·0 to 11·0 (100 mM citric acid/200 mM Na2HPO4 at pH 4·0–5·0, 100 mM Na2HPO4/NaH2PO4 buffer at pH 6·0–8·0, 100 mM NaHCO3/Na2CO3 buffer at pH 9·0–10·0 and 50 mM Na2HPO4/100 mM NaOH buffer at pH 11·0), using a method based on that of Yumoto et al. (1998). Growth was estimated by monitoring the OD600. Physiological and biochemical characteristics were determined using API 20E, API 20NE and API 50 CHB kits (bioMérieux). All API tests were performed in accordance with the manufacturer's instructions. Catalase activity was determined by means of bubble production from 3 % (v/v) H2O2, while oxidase activity was determined using 1 % (w/v) tetramethyl-p-phenylenediamine.
The isolates were aerobic, non-motile, rod-shaped and Gram-positive. In old cultures, the rods became shorter or spherical (see Supplementary Fig. S1 available in IJSEM Online). Colonies were circular, convex with entire margins, moist, shiny and light yellow in colour. The isolates grew neither in an anaerobic chamber at 37 °C nor with NaCl at concentrations above 10 %. Optimum growth was observed without added NaCl. The isolates grew at 20–37 °C and at pH 6·0–8·0. The optimum growth temperature and pH were 30 °C and pH 7. The morphological, physiological and chemotaxonomic characteristics of the isolates are different from those of phylogenetically closely related Microbacterium species (Table 1).
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; Yang et al., 1993). The isoprenoid quinones were extracted with chloroform/methanol (2 : 1, v/v) and purified by using TLC on Merck Kieselgel 60 F254 plates (20x20 cm, 0·5 mm thickness) using petroleum ether/diethyl ether (9 : 1, v/v) as the solvent. The identities of the quinones were determined by using reversed-phase HPLC analysis, as described by Shin et al. (1996). The whole-cell sugars were determined by using the method of Komagata & Suzuki (1987). Analysis of the diamino acid in peptidoglycan hydrolysates was carried out as described by Bousfield et al. (1985) on a cellulose TLC plate.
The whole-cell fatty acid profiles for the strains are shown in Supplementary Table S1 (available in IJSEM Online). The major fatty acids in the isolates and in related Microbacterium species were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The cell-wall sugars and the major menaquinones of the isolates were found to be galactose and xylose and MK-11 and MK-12, respectively. Differential characteristics of the isolates and reference strains are shown in Table 1
. The diamino acid in cell-wall hydrolysates of the isolates and reference strains M. lacticum KCTC 9230T and M. aurum KCTC 19091T was lysine, while that of reference strains M. terregens KCTC 19034T and M. schleiferi KCTC 19095T was ornithine.
DNA was extracted and purified by using a modified version of the method described by Marmur (1961). The G+C content of the DNA was determined using the HPLC method described by Tamaoka & Komagata (1984). The DNA G+C contents of the isolates and reference strains M. lacticum KCTC 9230T, M. terregens KCTC 19034T, M. aurum KCTC 19091T and M. schleiferi KCTC 19095T were 68, 71, 68, 69 and 68 mol%, respectively.
Two universal primers (9F and 1492R) described by Stackebrandt & Liesack (1993) were used for PCR amplification of the 16S rRNA gene, and the amplified PCR product was purified using a QIAquick PCR purification kit (Qiagen). The purified 16S rRNA gene was then sequenced using an ABI Prism BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems), with an automatic DNA sequencer (model 377; Applied Biosystems). Nearly complete 16S rRNA gene sequences (1435 bp) were determined for isolates JS53-2T and JS53-5, and aligned with the 16S rRNA gene sequences of representatives of the genus Microbacterium and related taxa, using CLUSTAL W software (Thompson et al., 1994). A phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987) based on distance-matrix data. Evolutionary distances were calculated using the model of Jukes & Cantor (1969). The PHYLIP software package (Felsenstein, 1993) was used for all analyses. The topology of the phylogenetic tree was evaluated using a bootstrap analysis (Felsenstein, 1985) of the neighbour-joining method, based on 1000 replications. The 16S rRNA gene sequences of the strains were compared with those of closely related reference strains. A phylogenetic tree indicated that isolates JS53-2T and JS53-5 belong to the genus Microbacterium (Fig. 1; an extended version of this tree is available as Supplementary Fig. S2 in IJSEM Online).
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DNA–DNA hybridization was carried out by means of fluorometric hybridization in microdilution wells, using biotinylated DNA (Ezaki et al., 1989). As shown in Table 2, the DNA–DNA reassociation values between isolates JS53-2T and JS53-5 and reference strains M. lacticum KCTC 9230T, M. terregens KCTC 19034T, M. aurum KCTC 19091T and M. schleiferi KCTC 19095T were less than 23 %. The phylogenetic definition of a species generally involves ‘strains with approximately 70 % or greater DNA–DNA relatedness' (Wayne et al., 1987). According to currently available data, organisms with a sequence similarity of less than 97·0 % will not reassociate to more than 60 %, no matter which hybridization method is applied (Stackebrandt & Goebel, 1994; Rossello-Mora & Amann, 2001; Stackebrandt et al., 2002). Therefore, the phylogenetic and DNA–DNA hybridization results from this work demonstrate that the isolates are phylogenetically closer to members of the genus Microbacterium and are not related to any previously described Microbacterium species at the species level.
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Description of Microbacterium koreense sp. nov.
Microbacterium koreense (ko.re.en'se. N.L. neut. adj. koreense pertaining to Korea, where the type strain was isolated).
Aerobic, non-motile, Gram-positive organism. In young cultures, cells are rods about 0·7 µm in width by 3·0–4·0 µm in length. In old cultures, rods become shorter or spherical. Colonies are circular, convex with entire margins, moist, shiny and light yellow in colour. Does not grow in an anaerobic chamber at 37 °C. The temperature range for growth is 20–37 °C, with optimal growth at 30 °C. The pH range for growth is 6·0–8·0, with optimal growth at pH 7·0. No growth occurs at an NaCl concentration of more than 10 %; optimal growth occurs without added NaCl. Strains give positive results for aesculin hydrolysis, for catalase and for acid production from glucose, fructose, mannose, rhamnose, mannitol, N-acetylglucosamine, cellobiose, maltose, sucrose and starch. Weakly positive for acid production from D-xylose, galactoside, trehalose, glycogen and D-turanose. Negative for oxidase, nitrate reduction, indole production, glucose acidification, arginine hydrolysis, urease, gelatin hydrolysis, 
-galactosidase and acid production from glycerol, erythritol, D-arabinose, L-arabinose, ribose, L-xylose, adonitol, methyl -D-xyloside, sorbose, dulcitol, inositol, sorbitol, methyl 
-D-mannoside, methyl -D-glucoside, amygdalin, arbutin, salicin, lactose, melibiose, inulin, melezitose, raffinose, xylitol, gentiobiose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate and 5-ketogluconate. The G+C content of the DNA is 68 mol%, the major isoprenoid quinones are MK-11 and MK-12, and the major cellular fatty acids are anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The cell-wall sugars are galactose and xylose. The diamino acid in the cell-wall hydrolysates is lysine.
The type strain is JS53-2T (=KCTC 19074T=CIP 108696T=CCUG 50754T), isolated from sea water in the South Sea of Korea (the Korea Strait).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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