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1 Korea Research Institute of Bioscience and Biotechnology (KRIBB), Laboratory of Microbial Function, PO Box 115, Yusong, Taejon, Korea
2 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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ABSTRACT |
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) as Isoptericola variabilis (Stackebrandt et al., 2004). Currently, the genus Isoptericola comprises three species with validly published names: Isoptericola variabilis (Stackebrandt et al., 2004), Isoptericola hypogeus (Groth et al., 2005) and Isoptericola halotolerans (Zhang et al., 2005). In this study, we report on the taxonomic characterization of an Isoptericola-like bacterial strain, DS-3T, which was isolated from a soil sample from Dokdo in Korea.
Strain DS-3T was isolated using the standard dilution plating technique at 25 °C on nutrient agar (Difco). The type strains of three Isoptericola species were used as reference strains: I. variabilis DSM 10177T and I. hypogeus DSM 16849T were obtained from the DSMZ (Braunschweig, Germany) and I. halotolerans KCTC 19046T was obtained from the Korean Collection for Type Cultures (Taejon, Korea). The morphological, physiological and biochemical characteristics of strain DS-3T were investigated using routine cultivation on trypticase soy agar (TSA; Difco) at 30 °C. Cell morphology was examined by light microscopy (Nikon E600) and transmission electron microscopy. The presence of flagella was determined by transmission electron microscopy using cells from exponentially growing cultures. For transmission electron microscopic observation, cells were negatively stained with 1 % (w/v) phosphotungstic acid and the grids were air-dried and examined with a Philips CM-20 transmission electron microscope. The Gram reaction was determined using the bioMérieux Gram stain kit according to the manufacturer's instructions. Growth at various temperatures (4–45 °C) was measured on TSA. To investigate tolerance of NaCl, trypticase soy broth was prepared according to the formula of the Difco medium and NaCl concentrations were varied (0, 0.5 and 1.0–10.0 %, w/v, at intervals of 1.0 %). The pH range for growth was determined in nutrient broth (Difco) adjusted to various pH values (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 TSA and on TSA supplemented with nitrate. 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 was studied as described previously (Lanyi, 1987). Utilization of substrates as sole carbon and energy sources was tested according to the method of Kämpfer et al. (1991). 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 properties and enzyme activities were tested using API 20E and API ZYM systems (bioMérieux).
Cell biomass for DNA extraction and for analyses of cell-wall components, isoprenoid quinones and polar lipids was obtained from cultures grown by shaking at 150 r.p.m. in trypticase soy broth (Difco) at 30 °C. Chromosomal DNA was isolated and purified according to the method described previously (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). 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 reverse-phase HPLC. The presence or absence of diaminopimelic acid in the peptidoglycan was determined by the method described by Komagata & Suzuki (1987). Preparation of cell walls and determination of peptidoglycan structure were carried out using modified methods of Schleifer & Kandler (1972) and MacKenzie (1987). Whole-cell sugars were determined as described by Komagata & Suzuki (1987). Isoprenoid quinones were extracted according to the method of Komagata & Suzuki (1987) and analysed using reverse-phase HPLC on 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 strain DS-3T was harvested from TSA plates after incubation for 3 days at 30 °C. The fatty acid methyl esters were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). 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.
The morphological, cultural, physiological and biochemical characteristics of strain DS-3T are given in the species description (see below) or are shown in Table 1. The almost complete 16S rRNA gene sequence of strain DS-3T determined in this study comprised 1474 nt, representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence. Comparative 16S rRNA gene sequence analysis showed that strain DS-3T was most closely related to members of the genus Isoptericola. In a phylogenetic tree based on the neighbour-joining algorithm, strain DS-3T joined the clade comprising Isoptericola species at a bootstrap confidence value of 85.0 % (Fig. 1). Strain DS-3T exhibited 16S rRNA gene sequence similarity values of 98.0, 98.0 and 98.4 % to the type strains of I. halotolerans, I. hypogeus and I. variabilis, respectively, and of less than 97.6 % to other species included in the phylogenetic analysis.
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, based on L-lys–D-Asp, as described by Schleifer & Kandler (1972). The peptidoglycan structure of strain DS-3T was the same as that of I. variabilis and I. halotolerans, but different from that of I. hypogeus, which has the peptidoglycan type based on L-lys–D-Glu (Stackebrandt et al., 2004; Groth et al., 2005; Zhang et al., 2005). Strain DS-3T contained MK-9(H4), at peak area ratio of approximately 85 %, as the predominant menaquinone; smaller amounts (approx. 6–9 %) of MK-9(H2) and MK-8(H4) were also present. This menaquinone profile was similar to those of I. variabilis and I. halotolerans, but different from that of I. hypogeus, which has major amounts of MK-9(H2) and MK-9 and a minor amount of MK-9(H4) (Stackebrandt et al., 2004; Groth et al., 2005; Zhang et al., 2005). Major polar lipids detected in strain DS-3T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and two unidentified glycolipids. This polar lipid profile was similar to those of Isoptericola species, although it differed from that of I. hypogeus in that phosphatidylinositol mannoside was absent (Stackebrandt et al., 2004; Groth et al., 2005; Zhang et al., 2005). The fatty acid profile of strain DS-3T comprised (>0.5 % of total fatty acids) branched fatty acids anteiso-C15 : 0 (58.6 %), iso-C15 : 0 (11.5 %), iso-C14 : 0 (4.2 %), anteiso-C17 : 0 (4.1 %), iso-C16 : 0 (3.0 %), iso-C13 : 0 (1.1 %) and iso-C17 : 0 (0.5 %); and straight-chain fatty acids C16 : 0 (9.0 %), C14 : 0 (3.3 %), C15 : 0 (2.6 %) and C17 : 0 (0.6 %). This fatty acid profile was similar to those of Isoptericola species in that anteiso-C15 : 0 was the major fatty acid (Stackebrandt et al., 2004; Groth et al., 2005; Zhang et al., 2005). The DNA G+C content of strain DS-3T was 74.1 mol%. Based on the results of the phylogenetic and chemotaxonomic analyses, it seems appropriate that strain DS-3T be considered as a member of the genus Isoptericola, although there are some differences in chemotaxonomic properties between strain DS-3T and I. hypogeus (Stackebrandt et al., 2004; Groth et al., 2005; Zhang et al., 2005).
DNA–DNA relatedness data demonstrated that strain DS-3T represents a genomic species that is different from recognized Isoptericola species (Wayne et al., 1987). Mean DNA–DNA relatedness values between strain DS-3T and the type strains of the three recognized Isoptericola species were in the range of 11–23 %. Strain DS-3T could also be distinguished from the three recognized Isoptericola species by noteworthy differences in several phenotypic properties as shown in Table 1
. Therefore, on the basis of the data presented, strain DS-3T should be placed in the genus Isoptericola within a novel species, for which the name Isoptericola dokdonensis sp. nov. is proposed.
Description of Isoptericola dokdonensis sp. nov.
Isoptericola dokdonensis (dok.do.nen'sis. N.L. masc. adj. dokdonensis of Dokdo, Korea, where the organism was first isolated).
Cells are Gram-positive, non-motile rods or cocci (0.8–1.1x0.8–4.5 µm). Primary mycelium is formed. Colonies on TSA are circular, convex, smooth, yellow in colour and 1.0–2.0 mm in diameter after 3 days of incubation at 30 °C. Optimal temperature for growth is 30 °C. Growth occurs at 10 and 40 °C, but not at 4 or 41 °C. Optimal pH for growth is between 7.0 and 8.0; growth occurs at pH 6.5, but not at pH 6.0. Growth occurs in the presence of 0–9 % (w/v) NaCl. Anaerobic growth occurs on TSA and on TSA supplemented with nitrate. Tweens 20, 40 and 60 are not hydrolysed. H2S and indole are not produced. Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. Pyruvate is utilized as a carbon source, but benzoate, citrate, L-malate, succinate and formate are not. The cell-wall peptidoglycan type is L-lys–D-Asp. The whole-cell sugars are galactose, glucose, rhamnose and ribose. The predominant menaquinone is MK-9(H4). The major fatty acids (>10 % of total fatty acids) are anteiso-C15 : 0 and iso-C15 : 0. The major polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and two unidentified glycolipids. The DNA G+C content is 74.1 mol% (determined by HPLC). Other phenotypic characteristics are given in Table 1.
The type strain, DS-3T (=KCTC 19128T=CIP 108921T), was isolated from soil in Dokdo, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of Medical Bacteria. London: Cambridge University Press.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
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Stackebrandt, E., Schumann, P. & Cui, X.-L. (2004). Reclassification of Cellulosimicrobium variabile Bakalidou et al. 2002 as Isoptericola variabilis gen. nov., comb. nov. Int J Syst Evol Microbiol 54, 685–688.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reverse-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
Yoon, J.-H., Kim, H., Kim, S.-B., Kim, H.-J., Kim, W. Y., Lee, S. T., Goodfellow, M. & Park, Y.-H. (1996). Identification of Saccharomonospora strains by the use of genomic DNA fragments and rRNA gene probes. Int J Syst Bacteriol 46, 502–505.
Yoon, J.-H., Lee, S. T. & Park, Y.-H. (1998). Inter- and intraspecific phylogenetic analysis of the genus Nocardioides and related taxa based on 16S rDNA sequences. Int J Syst Bacteriol 48, 187–194.
Yoon, J.-H., Kang, K. H. & Park, Y.-H. (2003). Psychrobacter jeotgali sp. nov., isolated from jeotgal, a traditional Korean fermented seafood. Int J Syst Evol Microbiol 53, 449–454.
Zhang, Y.-Q., Schumann, P., Li, W.-J., Chen, G.-Z., Tian, X.-P., Stackebrandt, E., Xu, L.-H. & Jiang, C.-L. (2005). Isoptericola halotolerans sp. nov., a novel actinobacterium isolated from saline soil from Qinghai Province, north-west China. Int J Syst Evol Microbiol 55, 1867–1870.
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