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1 Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
2 School of Biological Sciences, Seoul National University, Kwanak-gu, Seoul 151-742, Korea
3 The Center for Traditional Microorganism Resources, Keimyung University, Shindang-Dong, Dalseo-gu, Daegu, Korea
4 Department of Crop Science, Chungnam National University, Yusong, Taejon, Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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8c and C18 : 19c or C17 : 0. The DNA G+C content of the two strains was 73 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strains formed a coherent cluster with a clade comprising two Janibacter species, Janibacter limosus and Janibacter terrae. Strains CM2104T and CM2110 exhibited a 16S rRNA gene sequence similarity value of 99·7 % and a mean DNA–DNA relatedness level of 89 %. Strains CM2104T and CM2110 showed 16S rRNA gene sequence similarity levels of 97·8–98·4 % to the type strains of J. limosus and J. terrae. DNA–DNA relatedness between strains CM2104T and CM2110 and the type strains of these two Janibacter species was 7–11 %. On the basis of the phenotypic and phylogenetic data and genomic distinctiveness, strains CM2104T and CM2110 should be placed within the genus Janibacter as members of a novel species, for which the name Janibacter melonis sp. nov. is proposed. The type strain is CM2104T (=KCTC 9987T=DSM 16063T=JCM 12321T).
Published online ahead of print on 17 September 2004 as DOI 10.1099/ijs.0.63167-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains CM2104T and CM2110 are AY522568 and AY522569, respectively.
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with a single species, Janibacter limosus. Subsequently, two further species, Janibacter terrae (Yoon et al., 2000) and Janibacter brevis (Imamura et al., 2000), have been described. However, J. brevis has recently been recognized as a later heterotypic synonym of J. terrae (Lang et al., 2003). All Janibacter species have been isolated from environmentally polluted samples. In this study, we describe two strains, CM2104T and CM2110, which were isolated from the inner part of abnormally spoiled oriental melon (Cucumis melo) in Korea. These isolates were considered to be Janibacter-like strains based on 16S rRNA gene sequence comparison. Accordingly, the aim of the present study was to determine the exact taxonomic positions of strains CM2104T and CM2110 with a combination of phenotypic typing, detailed phylogenetic analysis based on 16S rRNA gene sequence analysis and genomic relatedness.
Abnormally spoiled oriental melon collected from a cultivation field was used as the source for isolation of endophytic bacteria. Strains CM2104T and CM2110 were isolated by a standard dilution plating technique on nutrient agar (NA; Difco) at 30 °C. The strains were cultivated on rich (R) medium (Martin et al., 1997) or brain heart infusion (BHI) medium at 30 °C to investigate their morphological and physiological characteristics. Cell mass for analyses of the cell wall, menaquinones and polar lipids and for DNA extraction was obtained after cultivation for 2 days in liquid R medium at 30 °C. Reference strains included J. limosus DSM 11140T and J. terrae KCCM 80001T. For fatty acid methyl ester (FAME) analysis, cell mass of strains CM2104T and CM2110 was obtained from agar plates after cultivation for 7 days at 30 °C on solid R medium and trypticase soy agar (TSA; Difco). Cell morphology was examined by light microscopy (Nikon E600) and transmission electron microscopy (TEM). The presence of flagella was examined by TEM using cells from exponentially growing cultures. Growth at various temperatures and various NaCl concentrations was measured in liquid R and BHI media. Growth under anaerobic conditions was determined in an anaerobic chamber with anaerobically prepared R agar. Oxidase activity was determined by using 1 % (w/v) p-aminodimethylaniline oxalate. Catalase activity was determined by bubble production in a 3 % (v/v) hydrogen peroxide solution. Hydrolysis of aesculin, H2S production, methyl-red and Voges–Proskauer reactions, and nitrate reduction were determined as described by Lanyi (1987). Hydrolysis of casein, gelatin, hypoxanthine, starch, Tween 80, tyrosine, urea and xanthine was determined as described by Cowan & Steel (1965). Utilization of various substrates as sole carbon and energy sources was determined as described by Gordon & Mihm (1957). Acid production from carbohydrates was determined according to Hugh & Leifson (1953). Other physiological tests were performed with the API 20E system (bioMérieux).
The isomer type of the diamino acid in the cell-wall peptidoglycan was analysed using TLC according to the method described by Komagata & Suzuki (1987). Menaquinones were analysed as described by Komagata & Suzuki (1987), using reversed-phase HPLC. For quantitative analysis of the cellular fatty acid composition, a loop of cell mass was harvested and FAMEs were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). Polar lipids were extracted using the procedures described by Minnikin et al. (1984) and identified by two-dimensional TLC followed by spraying with appropriate detection reagents (Komagata & Suzuki, 1987). Chromosomal DNA was isolated and purified according to the method described previously (Yoon et al., 1996), with the exception that ribonuclease T1 was used together with ribonuclease A. The DNA G+C content was determined by the method of Tamaoka & Komagata (1984). DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC.
16S rRNA genes were amplified by PCR using two universal primers as described previously (Yoon et al., 1998). Sequencing of the amplified 16S rRNA gene was performed as described by Yoon et al. (2003). Alignment of sequences was carried out with CLUSTAL W software (Thompson et al., 1994). Gaps at the 5' and 3' ends of the alignment were omitted from further analysis. Phylogenetic trees were inferred using three tree-making algorithms, the neighbour-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Kluge & Farris, 1969) methods in the PHYLIP package (Felsenstein, 1993). Evolutionary distance matrices for the neighbour-joining method were calculated with the algorithm of Jukes & Cantor (1969) with the DNADIST program. The stability of relationships was assessed by a bootstrap analysis based on 1000 resamplings of the neighbour-joining dataset using the programs SEQBOOT, DNADIST, NEIGHBOR and CONSENSE of the PHYLIP package. 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. Of the values obtained, the highest and lowest values for each sample were excluded; DNA–DNA relatedness values are the mean of the remaining three values.
Morphological, cultural, physiological and biochemical characteristics of strains CM2104T and CM2110 are given in the species description (see below) and in Table 1, together with those of J. limosus and J. terrae. Strains CM2104T and CM2110 contained meso-diaminopimelic acid as the diagnostic diamino acid in the cell wall. The predominant isoprenoid quinone was tetrahydrogenated menaquinone with eight isoprene units [MK-8(H4)]. The major fatty acids of strains CM2104T and CM2110 were iso-C16 : 0, C17 : 18c and C18 : 19c or C17 : 0 (Table 2). There were differences in the proportions of some fatty acids when the two strains were grown on solid R medium and TSA (Table 2). These cellular fatty acid profiles were similar to those of J. limosus DSM 11140T and J. terrae KCCM 80001T, although there were differences in the proportions of some fatty acids. The cellular polar lipids found in the two strains were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylinositol. The DNA G+C content of strains CM2104T and CM2110 was 73 mol%.
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). The two sequences contained 28 of 29 signature nucleotides defined for the genus Janibacter as described by Maszenan et al. (2000); the two strains had the matching base pair G–C at positions 140–223 instead of G–T or A–T as given by Maszenan et al. (2000). In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strains CM2104T and CM2110 formed a coherent cluster, which was supported by a bootstrap resampling value of 100 %, and joined the phylogenetic clade comprising J. limosus and J. terrae (Fig. 1). This tree topology was also found in the tree generated with the maximum-parsimony algorithm (data not shown). Strains CM2104T and CM2110 exhibited 16S rRNA gene sequence similarity levels of 97·8–98·4 % to J. limosus DSM 11140T and J. terrae KCCM 80001T, 96·9–98·0 % to Knoellia sinensis DSM 12331T and Knoellia subterranea DSM 12332T and less than 97·0 % to other species (Fig. 1). The mean level of DNA–DNA relatedness between strains CM2104T and CM2110 was 89 %, when each of their DNAs was separately used as labelled DNA probe. Strains CM2104T and CM2110 exhibited levels of DNA–DNA relatedness of 7–10 % and 7–11 % to J. limosus DSM 11140T and J. terrae KCCM 80001T, respectively.
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). Chemotaxonomic data obtained from the strains were also most similar to those of the genus Janibacter (Stackebrandt & Schumann, 2000; Yoon et al., 2000; Groth et al., 2002). The meso-diaminopimelic acid type clearly distinguished strains CM2104T and CM2110 from members of the genus Ornithinicoccus, which contain L-ornithine, and the other genera of the Intrasporangiaceae, namely Intrasporangium, Terrabacter and Terracoccus, which contain LL-diaminopimelic acid (Groth et al., 1999; Stackebrandt & Schumann, 2000). Differences in major fatty acid contents also distinguished strains CM2104T and CM2110 from members of the genera Ornithinicoccus, Intrasporangium, Terrabacter and Terracoccus, which contain iso-C15 : 0 as the major fatty acid, and the genus Knoellia, which contain unsaturated fatty acids (particularly C17 : 1 and C18 : 1) as minor components (Groth et al., 1999, 2002; Maszenan et al., 2000; Yoon et al., 2000) (Table 2). In comparison with Tetrasphaera, strains CM2104T and CM2110 differed in the amounts of anteiso-branched fatty acids, which were minor components in strains CM2104T and CM2110 and comprised more than 32 % in the genus Tetrasphaera (Maszenan et al., 2000; Hanada et al., 2002). Accordingly, both phylogenetic and chemotaxonomic data indicate that strains CM2104T and CM2110 are members of the genus Janibacter.
Strains CM2104T and CM2110 showed almost identical phenotypic characteristics, except growth in liquid BHI medium and hydrolysis of casein (Table 1). The two strains were also phylogenetically similar, showing four nucleotide differences (0·3 % difference) in their 16S rRNA gene sequences (Fig. 1). DNA–DNA relatedness indicates that strains CM2104T and CM2110 are members of the same genomic species (Wayne et al., 1987). Strains CM2104T and CM2110 are differentiated from other Janibacter species by several physiological characteristics, such as temperature for growth, NaCl tolerance and hydrolysis of certain substrates (Table 1). DNA–DNA relatedness values between strains CM2104T and CM2110 and the type strains of two Janibacter species were far below the threshold value (70 %) suggested for species delineation in current bacterial systematics (Wayne et al., 1987). Genomic distinctiveness, together with differential phenotypic properties and 16S rRNA gene sequence similarity data, justify a taxonomic discrimination of strains CM2104T and CM2110 from the two accepted Janibacter species. Therefore, on the basis of the data presented, strains CM2104T and CM2110 should be placed in the genus Janibacter as members of a novel species, for which the name Janibacter melonis sp. nov. is proposed.
Description of Janibacter melonis sp. nov.
Janibacter melonis [me.lo'nis. L. gen. n. melonis of melon (Cucumis melo), referring to the fruit from which the organisms were isolated].
Cells are aerobic, non-spore-forming, non-motile cocci, 0·8–1·0 µm in diameter. Gram-positive. Non-acid-fast. Colonies are smooth, circular, convex, glistening, cream-coloured and 1·5–3·0 mm in diameter after 7 days incubation on solid R medium; those of the type strain wrinkle as cultures age. Neither substrate nor primary mycelium is formed. Optimal temperature for growth is 30 °C. Grows occurs at 10 °C but not at 4 °C. Grows occurs at 40 °C in liquid BHI medium but not in liquid R medium. Optimal pH for growth is around 7·0; growth occurs at pH 5·5 but not at pH 5·0. Tweens 20, 40 and 60 and tyrosine are hydrolysed. Hypoxanthine and xanthine are not hydrolysed. Acid is not produced from adonitol, myo-inositol, D-melezitose, melibiose or sucrose. The cell-wall peptidoglycan contains meso-diaminopimelic acid as the diagnostic diamino acid. The predominant menaquinone is MK-8(H4). The major fatty acids are iso-C16 : 0, C17 : 18c and C18 : 19c or C17 : 0. The cellular polar lipids are phosphatidylglycerol, diphosphatidylglycerol and phosphatidylinositol. The DNA G+C content is 73 mol% (determined by HPLC). Other characteristics are given in Table 1.
The type strain, CM2104T (=KCTC 9987T=DSM 16063T=JCM 12321T), was isolated from the inner part of abnormally spoiled oriental melon in Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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