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1 Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Sector 39-A, Chandigarh, 160 036, India
2 Department of Microbiology, Guru Nanak Dev University (GNDU), Amritsar, 143 005, India
Correspondence
S. Mayilraj
mayil{at}imtech.res.in
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Kitasatospora sampliensis VT-36T is AY260167.
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; Zhang et al., 1997) was composed of 19 species with validly published names, including the recently described Kitasatospora putterlickiae, K. arboriphila, K. gansuensis, K. nipponensis, K. paranensis, K. terrestris (Groth et al., 2003, 2004) and K. viridis (Liu et al., 2005). Strain VT-36T was isolated from a sugar-cane field soil sample collected at Sampli village, Punjab state, India, by using Streptomyces agar (1·0 % malt extract, 0·4 % yeast extract, 0·4 % glucose, 0·2 % calcium carbonate and 1·2 % agar) complemented with cycloheximide (25 µl ml–1) and amphotericin B (10 µl ml–1) at 28 °C. The pure culture was maintained as a glycerol suspension (10 % v/v) of spores and mycelial fragments at –70 °C. The reference strain K. putterlickiae MTCC 6643T was obtained from the Microbial Type Culture Collection (MTCC), Chandigarh, India.
Strain VT-36T was grown on Streptomyces agar for morphological and physiological characteristics. Morphological properties were examined according to the methods reported by Williams et al. (1983). Growth and sporulation were observed on standard media-like actinomycetes isolation agar (0·2 % sodium caseinate, 0·01 % asparagine, 0·4 % sodium propionate, 0·05 % dipotassium phosphate, 0·01 % magnesium sulphate, 0·0001 % ferrous sulphate and 1·5 % agar, pH 8·1) and other media as detailed in Table 1. Aerial spore mass colour, pigmentation of substrate mycelium and the production of diffusible pigments were examined on diagnostic media as described by Williams et al. (1989), following incubation at 28 °C for 10 days. Growth on sole carbon sources were examined on carbon utilization agar and melanin production was examined on peptone/yeast extract/iron agar (ISP-6) and tyrosine agar (ISP-7) (Shirling & Gottlieb, 1966; Williams et al., 1983, 1989). Tolerance to sodium chloride was examined on modified Bennett's agar at concentrations up to 10 %. Physiological tests and susceptibility to antibiotics were examined by the method described by Groth et al. (2004) by using antibiotic discs (HiMedia). For scanning electron microscopy, samples were processed as mentioned previously (Richard & Wilson, 1993).
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and whole-cell sugars were determined as described by Mikami & Ishida (1983). Polar lipids were extracted and analysed by methods described by Minnikin et al. (1984). Menaquinones were analysed as described by Collins et al. (1984). The N-acyl type of muramic acid in the cell wall was determined by the method of Uchida & Aida (1984). Absence of mycolic acid was demonstrated by TLC (Minnikin & Goodfellow, 1976). Fatty acid methyl esters were extracted and analysed as described by Pandey et al. (2002). Chemotaxonomic characters are given in the species description.
Genomic DNA extraction, amplification and sequencing of the 16S rRNA gene and phylogenetic analyses were performed as described earlier by Mayilraj et al. (2005). DNA–DNA hybridization was performed using the membrane filter method (Tourova & Antonov, 1987). The G+C content of genomic DNA was determined spectrophotometrically (Lambda 35; Perkin Elmer) using the thermal denaturation method (Mandel & Marmur, 1968).
The results of the polyphasic study placed isolate VT-36T in the genus Kitasatospora. The organism formed an extensively branched substrate and aerial mycelium with smooth-surfaced spores. The colours of the aerial and substrate mycelium on different media are given in Table 1
. The almost complete (1481 nt) 16S rRNA gene sequence of the strain VT-36T was determined and analysis revealed high similarity with other members of the genus Kitasatospora. It was evident from the phylogenetic tree (Fig. 1) that isolate VT-36T forms a cluster along with K. putterlickiae. It showed closest sequence similarity with K. putterlickiae (98·3 %) followed by K. arboriphila (97 %) and K. azatica (97 %). The 16S rRNA gene sequence similarity of strain VT-36T to the type strains of other Kitasatospora species was less than 97 % (95·1–96·7 %). Genomic relatedness as shown by DNA–DNA hybridization of strain VT-36T with the most closely related species K. putterlickiae was 54 %, which is distinctly lower than the 70 % cut-off point recommended for the delineation of bacterial species (Wayne et al., 1987). Determination of DNA–DNA relatedness between strain VT-36T and other type strains of Kitasatospora species was not carried out as the 16S rRNA gene sequence similarities did not exceed 97 %, the threshold value for the delineation of bacterial species (Stackebrandt & Goebel, 1994). Strain VT-36T differed from K. putterlickiae and other species of this genus in some phenotypic characters (Tables 1 and 2). The fatty acid methyl ester profile matches qualitatively that of the genus Kitasatospora. However, in the present study, under similar growth conditions (tryptic soy broth medium, 30 °C for 36 h), VT-36T showed quantitative differences from K. putterlickiae. Fatty acid content in VT-36T and K. putterlickiae was iso-C15 : 0, 20·1 and 14·3 %; iso-C16 : 0, 16·48 and 15·5; iso-C17 : 0, 12·5 and 10·2; iso-C16 : 0, 10·9 and 15·5; anteiso-C15 : 0, 9·6 and 4·8; anteiso-C17 : 0, 9·4 and 7·1; 9-methyl-C16 : 0, 6·3 and 8·8; C16 : 1
7c, 4·9 and 8·6 %, respectively. Based on the phenotypic and genotypic data, strain VT-36T could be clearly distinguished from its closest phylogenetic relative, K. putterlickiae. We, therefore, conclude that strain VT-36T should be assigned as a novel species of the genus Kitasatospora, for which we propose the name Kitasatospora sampliensis sp. nov.
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Aerobic, Gram-positive and non-acid-fast actinobacterium. Produces pale-grey, dark-grey or dark-grey–brown substrate mycelium and a pale-grey or dark-grey aerial mycelium on almost all ISP media. Dark-brown or dark-grey soluble pigments are formed on ISP-2, ISP-3, ISP-6, ISP-7, Streptomyces agar, actinomycetes isolation agar and Sabouraud dextrose agar. Melanin is produced on peptone/yeast extract/iron agar and tyrosine agar. Spore chain morphology is rectiflexibiles with 10 or more smooth-surfaced spores per chain. Nitrate is reduced to nitrite, casein is not degraded, starch is not hydrolysed and gelatin is not liquefied. Can grow in NaCl at a concentration up to 2·5 %, but not at 3·0 % or above. Optimum growth is observed at pH 7·0–8·0 and at 30 °C. Positive for utilization of L-arabinose, L-rhamnose, D-fructose, D-mannitol, D-raffinose, D-sucrose and D-xylose as sole carbon and energy sources. Cell wall contains both meso- and LL-diaminopimelic acid. The N-acyl type of muramic acid of the peptidoglycan is acetyl. Major fatty acids are C16 : 0 (16·48 %), iso-C15 : 0 (20·07 %), iso-C16 : 0 (10·94 %), iso-C17 : 0 (12·54 %), anteiso-C15 : 0 (9·55 %) and anteiso-C17 : 0 (9·36 %). Whole-cell sugars are galactose, glucose, mannose and ribose. The polar lipids are phosphatidylinositol, phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylinositolmannosides. The major menaquinone is MK-9 (H6). The G+C content of the DNA of the type strain is 76·5 mol%.
The type strain, VT-36T (=MTCC 6546T=DSM 44898T=JCM 13010T), was isolated from a sugar-cane field soil sample collected from Sampli village, Punjab state, India.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Groth, I., Schutze, B., Boettcher, T., Pullen, C. B., Rodriguez, C., Leistner, E. & Goodfellow, M. (2003). Kitasatospora putterlickiae sp. nov., isolated from rhizosphere soil, transfer of Streptomyces kifuensis to the genus Kitasatospora as Kitasatospora kifunensis comb. nov., and emended description of Streptomyces aureofaciens Duggar 1948. Int J Syst Evol Microbiol 53, 2033–2040.
Groth, I., Rodriguez, C., Schutze, B., Schmitz, P., Leistner, E. & Goodfellow, M. (2004). Five novel Kitasatospora species from soil: Kitasatospora arboriphila sp. nov., K. gansuensis sp. nov., K. nipponensis sp. nov., K. paranensis sp. nov. and K. terrestris sp. nov. Int J Syst Evol Microbiol 54, 2121–2129.
Liu, Z., Rodriguez, C., Wang, L., Cui, Q., Huang, Y., Quintana, E. T. & Goodfellow, M. (2005). Kitasatospora viridis sp. nov., a novel actinomycete from soil. Int J Syst Evol Microbiol 55, 707–711.
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