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Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0032, Japan
Correspondence
Bing Li
aa67047{at}mail.ecc.u-tokyo.ac.jp
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ABSTRACT |
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9c and 10-methyl C18 : 0, the presence of cell-wall chemotype IV and mycolic acids, were consistent with the properties of members of the genus Rhodococcus. The DNA G+C content was 64.5 mol%. On the basis of both phenotypic and genotypic evidence, strain DS472T represents a novel species of the genus Rhodococcus, for which the name Rhodococcus kyotonensis sp. nov. is proposed. The type strain is strain DS472T (=IAM 15415T=CCTCC AB206088T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain DS472T is AB269261.
The characteristics that distinguish strain DS472T from the closely related species of the genus Rhodococcus are presented in a supplementary table available with the online version of this paper.
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MAIN TEXT |
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, 1999). At the time of writing, the genus Rhodococcus was composed of 26 species with validly published names, including the recently described Rhodococcus imtechensis (Ghosh et al., 2006) and Rhodococcus kroppenstedtii (Mayilraj et al., 2006).
Strain DS472T was isolated from a soil sample in Kyoto city, Japan and grown on nutrient agar (Difco) medium. The plates were incubated at 27 °C for 3 weeks. Single colonies from the plates were purified by transferring them onto new plates. Strain DS472T was one of the isolated colonies and was used in this study. Strain DS472T was maintained on a trypticase soy agar (TSA; BBL) slant at 4 °C and as a glycerol suspension sample (20 %, v/v) at –20 °C. Cell morphology was observed by light microscopy (BX60; Olympus). For phenotypic properties, the growth range for temperature and pH were tested on yeast extract-malt extract ISP2 medium (Shirling & Gottlieb, 1966), the pH range was tested with pH values ranging from 5.0 to 11.0. For pH 5.0–7.5, the pH was adjusted by the addition of 1 M HCl after adding 1 g NaHCO3 l–1. For pH 8.0–11.0, different amounts of carbonate were used. Catalase activity was determined by 3 % H2O2, and bubble production was identified as a positive reaction. Oxidase was determined by cytochrome oxidase paper (Nissui Pharmaceutical). Tolerance of salinity was determined by inoculating the strains into ISP2 medium supplemented with 0–12 % (w/v) NaCl at 1 % intervals. API 50CH, API 20E and API ZYM strips (bioMérieux) were used to determine physiological and biochemical characteristics according to the manufacturer's instructions.
For cellular fatty acid analysis, the strain was grown on TSA (BBL) at 27 °C for 3 days, and fatty acid methyl esters were prepared and identified using the Microbial Identification System as described by Xie & Yokota (2003). For other chemotaxonomic analyses, freeze-dried cells were obtained from cultures grown in trypticase soy broth (TSB; BBL) at 27 °C for 5 days. The amino acid composition and isomers of diaminopimelic acid (DAP) in the cell walls were examined by two-dimensional TLC (Tokyo Kasei Co.) as described by Harper & Davis (1979), and by HPLC according to procedures described by Yokota et al. (1993). The alkaline methanolysis procedure was used to detect mycolic acids (Minnikin et al., 1980) and whole-cell sugars were analysed by TLC (Yokota & Takashima, 2001). Respiratory quinones were extracted from dried cells (300 mg) using chloroform/methanol (2 : 1) and were purified by TLC. The purified respiratory quinones were analysed by reverse-phase HPLC (Komagata & Suzuki, 1987). The DNA G+C content was determined by HPLC as described by Mesbah et al. (1989).
An approximately 1500 bp fragment of the 16S rRNA gene sequence was amplified by PCR and sequenced as described by Xie & Yokota (2003). The 16S rRNA gene sequence of strain DS472T was compared with the sequences obtained from GenBank and aligned by using the CLUSTAL_X software package (Thompson et al., 1997), evolutionary distances and the Knuc value (Kimura, 1980) were calculated. Alignment gaps and ambiguous bases were not taken into consideration in the calculations. A phylogenetic tree based on a comparison of 1210 bases was constructed using the neighbour-joining method (Saitou & Nei, 1987). The topology of the tree was evaluated by using the bootstrap resampling method of Felsenstein (1985) with 1000 replicates, whereas similarity values were calculated by using MEGA3 (Kumar et al., 2004). DNA–DNA hybridization was performed by using the photobiotin-labelled probes in microplate wells, as described by Ezaki et al. (1989), and using a multi-well plate reader (CytoFluoR; PerSeptive Biosystems). The experiment was performed with two replications for each sample and the DNA relatedness values were calculated as the mean of the two values.
Phylogenetic analysis suggests that the isolate belongs within the genus Rhodococcus and clustered with Rhodococcus fasciens and Rhodococcus yunnanensis (Fig. 1). Comparison of almost-complete 16S rRNA gene sequence (1420 nt) showed that strain DS472T shared the highest similarities with the sequences of R. yunnanensis YIM 70056T (98.2 %) and R. fascians DSM 20669T (97.8 %). DNA–DNA hybridization experiments revealed 36 and 29 % relatedness between the isolate and its phylogenetic relatives, R. yunnanensis and R. fasciens, respectively, which is below the 70 % cut-off point recommended for the delineation of bacterial species by Wayne et al. (1987).
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). These results are consistent with the properties of the genus Rhodococcus (Goodfellow, 1986). Moreover, strain DS472T could be distinguished from the other species of the genus Rhodococcus by various phenotypic data, as shown in Supplementary Table S1 (available in IJSEM Online) and Table 1. Other characteristics are given in the species description. On the basis of phenotypic, chemotaxonomic and genotypic characteristics, it is proposed that strain DS472T represents a novel species of the genus Rhodococcus, for which the name Rhodococcus kyotonensis sp. nov. is proposed.
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Gram-positive, non-motile, non-spore-forming and acid-fast actinomycete that forms non-branching hyphae which fragment into short rod-to-coccus elements. Colonies on TSA medium are yellow–orange-pigmented, smooth and opaque. Growth occurs well over a pH range (6.0–8.0) on the ISP2 medium; the optimal pH for growth is around 7.0. The temperature range for growth is 10–37 °C; with optimal growth temperature of 27–30 °C, growth cannot occur at 40 °C. The NaCl tolerance range is up to 9 %. Catalase activity is positive, whereas oxidase activity is negative. Hydrolyses Tweens 20, 40, 60 and 80. Gelatin hydrolysis, citrate utilization, activities of ONPG, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, production of hydrogen sulfide and indole, and reduction of nitrate and nitrite are negative. Positive results for the activities of esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, 
-glucosidase, urease, tryptophan deaminase, hydrolysis of aesculin and Voges–Proskauer test, but negative results for alkaline phosphatase, esterase (C4), lipase (C4), trypsin, chymotrypsin, naphthol-AS-BI-phosphohydrolase, -galactosidase, 
-galactosidase, -glucuronidase, -glucosidase, N-acetyl--glucosamidase, -mannosidase and -fucosidase. Utilizes glycerol, glucose, ribose, galactose, fructose, sorbose, rhamnose, myo-inositol, mannitol, sorbitol, sucrose, xylitol, D-arabitol, and gluconate as the sole carbon and energy sources, but erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, adonitol, methyl -D-xyloside, mannose, dulcitol, methyl -D-mannoside, methyl -D-glucoside, N-acetyl-D-glucosamine, amygdalin, arbutin, salicin, cellobiose, maltose, lactose, melibiose, trehalose, inulin, melezitose, raffinose, starch, glycogen, gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, L-arabitol, 2-keto-gluconate and 5-keto-gluconate are not utilized. Diamino acid in the peptidoglycan is meso-DAP; arabinose and galactose dominate in the whole-cell hydrolysates (wall type IV). The percentage of major menaquinone MK-8(H2) is 94.6 %, minor menaquinones MK-7(H2) and MK-9(H2) is 2.8 and 2.6 %, respectively. Mycolic acids are present, which co-migrate with those of the type strain of R. rhodochrous. The fatty acids are C16 : 0 (40.8 %), C18 : 19c (18.1 %), 10-methyl C18 : 0 (17.5 %), C14 : 0 (7.2 %), iso-C15 : 0 2-OH/C16 : 17c (5.5 %) and C15 : 0 (2.1 %). The DNA G+C content is 64.5 mol%.
The type strain, DS472T (=IAM 15415T=CCTCC AB206088T), was isolated from the soil in Kyoto city, Japan.
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REFERENCES |
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Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]
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Mayilraj, S., Krishnamurthi, S., Saha, P. & Saini, H. S. (2006). Rhodococcus kroppenstedtii sp. nov., a novel actinobacterium isolated from a cold desert of the Himalayas, India. Int J Syst Evol Microbiol 56, 979–982.
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Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
Xie, C.-H. & Yokota, A. (2003). Phylogenetic analyses of Lampropedia hyalina based on the 16S rRNA gene sequence. J Gen Appl Microbiol 49, 345–349.[CrossRef][Medline]
Yokota, A. & Takashima, M. (2001). Cell wall. In Classification and Identification of Microorganisms, pp. 97–117. Edited by K. Suzuki, A. Hiraishi & A. Yokota. Tokyo: Springer-Verlag (in Japanese).
Yokota, A., Tamura, T., Nishii, T. & Hasegawa, T. (1993). Kineococcus aurantiacus gen. nov., sp. nov., a new aerobic gram-positive, motile coccus with meso-diaminopimelic acid and arabinogalactan in the cell wall. Int J Syst Bacteriol 43, 52–57.
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