Rhodococcus kyotonensis sp. nov., a novel actinomycete isolated from soil Bing Li, Keiko Furihata, Lin-Xian Ding and Akira Yokota Correspondence Bing Li aa67047@mail.ecc.u-tokyo.ac.jp Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0032, Japan A polyphasic study was undertaken to establish the taxonomic position of an isolate, strain DS472 T , from soil in Kyoto, Japan. Phylogenetic analysis, based on the 16S rRNA gene sequences, revealed that this strain constitutes a new subline within the genus Rhodococcus, with Rhodococcus yunnanensis YIM 70056 T and Rhodococcus fascians DSM 20669 T as its nearest phylogenetic neighbours (98.2 and 97.8 % sequence similarity, respectively). DNA–DNA hybridization experiments revealed 36 and 29 % relatedness between the isolate and its phylogenetic relatives, R. yunnanensis and R. fascians, respectively. Chemotaxonomic characteristics, including the major quinone MK-8(H 2 ), predominant fatty acids C 16 : 0 ,C 18 : 1 v9c and 10-methyl C 18 : 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 DS472 T represents a novel species of the genus Rhodococcus, for which the name Rhodococcus kyotonensis sp. nov. is proposed. The type strain is strain DS472 T (5IAM 15415 T 5CCTCC AB206088 T ). The improved classification of the genus Rhodococcus provides a sound framework for the recognition and description of additional Rhodococcus species (Goodfellow et al., 1998, 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 DS472 T was isolated from a soil sample in Kyoto city, Japan and grown on nutrient agar (Difco) medium. The plates were incubated at 27 uC for 3 weeks. Single colonies from the plates were purified by transferring them onto new plates. Strain DS472 T was one of the isolated colonies and was used in this study. Strain DS472 T was maintained on a trypticase soy agar (TSA; BBL) slant at 4 u C and as a glycerol suspension sample (20 %, v/v) at 220 u C. Cell morphology was observed by light micro- scopy (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 NaHCO 3 l 21 . For pH 8.0–11.0, different amounts of carbonate were used. Catalase activity was determined by 3 % H 2 O 2 , 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 (bioMe ´rieux) 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 u 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 uC 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). Abbreviation: DAP, diaminopimelic acid. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain DS472 T is AB269261. The characteristics that distinguish strain DS472 T from the closely related species of the genus Rhodococcus are presented in a supplementary table available with the online version of this paper. International Journal of Systematic and Evolutionary Microbiology (2007), 57, 1956–1959 DOI 10.1099/ijs.0.64770-0 1956 64770 G 2007 IUMS Printed in Great Britain