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1 Department of Bioscience and Technology, School of Engineering, Hokkaido Tokai University, 5-1-1-1 Minamisawa, Minami-ku, Sapporo 005-8601, Japan
2 National Institute of Advanced Industrial Science and Technology, Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan
3 Japan Collection of Microorganisms, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama 351-0198, Japan
4 Research Laboratory, Higeta Shoyu Co., Choshi, Chiba 288-8680, Japan
Correspondence
Hidetoshi Matsuyama
matsuyama{at}db.htokai.ac.jp
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Published online ahead of print on 7 February 2003 as DOI 10.1099/ijs.0.02523-0.
The DDBJ/GenBank/EMBL accession number for the 16S rRNA sequence of strain Mb8T is AB067734.
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). Many bacterial enzymes that degrade EPS have been purified for use in analysing EPS structure. Bacteria that degrade EPS produced by M. kitamiense have been isolated in our studies. One strain, Mb8T, was considered to be a Rhodococcus-like strain. The genus Rhodococcus comprises strains that are metabolically versatile (Finnerty, 1992). Recently, species of the genus Rhodococcus have been assigned to four 16S rDNA subclades: Rhodococcus equi, Rhodococcus rhodnii, Rhodococcus rhodochrous and Rhodococcus erythropolis (McMinn et al., 2000). In this study, the physiological and biochemical features, chemotaxonomic characteristics and phylogeny of strain Mb8T were examined. DNA–DNA relatedness data showed that the strain should be classified as a novel species of the genus Rhodococcus.
Strain Mb8T was isolated by selective enrichment from soil in Sapporo City, Japan. The soil sample was inoculated in 50 ml minimal salts medium containing (l-1) 0·1 g KNO3, 10 ml trace element solution (Patel, 1984) and 3 g EPS produced by M. kitamiense. This medium was incubated at 25 °C on a horizontal shaker at 150 r.p.m. After being shaken for several days, a portion of the suspension was transferred into 50 ml fresh medium and the medium was re-incubated. After four successive transfers, the suspension was plated onto solid medium containing 0·3 % (w/v) EPS to isolate pure cultures. Of the strains found, Mb8T, which showed a good growth rate, was selected for further study. To investigate its morphological and physiological characteristics, strain Mb8T was cultivated aerobically at 25 °C in tryptic soy agar (TSA). Cell morphology was examined by light microscopy and SEM. For SEM, cells from the early growth phase (1 day incubation) and stationary phase (5 days incubation) were prepared by the method described by Kormendy (1975) and the specimens were examined with a model JSM 5200 SEM.
Strain Mb8T was also examined for a range of phenotypic properties using standard procedures (Gordon et al., 1974; Komagata, 1985). In addition, decomposition of aesculin and arbutin were examined according to Kurup & Fink (1975). NaCl tolerance and the temperature range for growth of strain Mb8T and representatives of Rhodococcus species were determined on yeast extract-malt extract agar (ISP medium no. 2) for 5 days at 30 °C, except for Rhodococcus marinonascens JCM 6241T, which was incubated on yeast extract-malt extract agar containing 3 % (w/v) NaCl for 10 days at 17 °C.
The isomer type of the diamino acid in the cell wall was analysed according to Schleifer & Kandler (1972) at NCIMB, Japan. Cell-wall sugars were analysed according to Yokota & Takashima (2001). Analysis of mycolic acids was performed as described by Tomiyasu (1982). Cells cultivated overnight in tryptic soy broth at 25 °C were used to determine quinone composition using the method of Komagata & Suzuki (1987). Cells cultivated overnight in tryptic soy broth at 25 °C were used for cellular fatty acid analysis. Cellular fatty acid composition was determined using the method of Suzuki & Komagata (1983). Chromosomal DNA was prepared from bacterial cells using the method of Marmur (1961). The G+C content of DNA was determined according to Tamaoka & Komagata (1984). Levels of DNA relatedness were determined using the method of Ezaki et al. (1989). A probe for DNA–DNA hybridization was prepared from strain Mb8T. Sequencing of 16S rRNA genes was carried out by the method described previously (Shida et al., 1997). Multiple alignments of the sequence were performed and the nucleotide substitution rate (Knuc value) was calculated. A phylogenetic tree was constructed by the neighbour-joining method (Kimura, 1980; Saitou & Nei, 1987) using the program CLUSTAL W (Thompson et al., 1994). Similarity values for sequences were calculated using the program GENETYX (Software Development).
Strain Mb8T was an aerobic, non-motile, non-acid-fast, non-spore-forming, Gram-positive bacterium. Cells were rod-shaped and formed filaments or showed elementary branching in the early growth phase. Most cells in the stationary phase were cocci (Fig. 1). Colonies were cream-coloured, opaque and with slightly irregular edges on TSA. Strain Mb8T showed catalase activity, but no oxidase activity. The pH range for growth was pH 5·5–8·5 and the temperature range for growth was 15–45 °C.
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). The major isoprenoid quinone was dihydrogenated menaquinone with eight isoprenoid units [MK-8(H2)]. Strain Mb8T contained large amounts of mycolic acids with 44–52 carbon atoms. Major cellular fatty acids were C16 : 0 (33·7 % total cellular fatty acids) and C18 : 1 (9) (18·6 % total cellular fatty acids). The G+C content of strain Mb8T was 66·0 mol%. It was evident from the polyphasic taxonomic study that strain Mb8T had properties typical of members of the genus Rhodococcus (Goodfellow et al., 1998).
16S rDNA data supported the results of morphological and chemotaxonomic analysis. The 16S rDNA sequence of strain Mb8T, which was determined directly after PCR amplification, was 1331 nt. Strain Mb8T clearly belonged to the R. erythropolis subclade (Fig. 2). The organism was most closely related to the type strain of Rhodococcus maanshanensis; the two strains shared 98·0 % 16S rDNA similarity, which corresponds to 27 nt differences over 1331 positions.
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). Strain Mb8T could also be distinguished from the type strains that form a coherent cluster in the R. erythropolis subclade by phenotypic properties (Table 1). However, the phenotypic properties of R. marinonascens were not examined because of its growth temperature range and NaCl requirement.
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Description of Rhodococcus tukisamuensis sp. nov.
Rhodococcus tukisamuensis (tu.ki.sa.mu.en'sis. N.L. masc. adj. tukisamuensis referring to Tukisamu, a town in Sapporo, Hokkaido, Japan, where the type strain was isolated).
Cells are Gram-positive, non-acid-fast and non-motile. They are rods, form filaments or show elementary branching in the early growth phase and are mostly cocci in the stationary phase. Colonies are cream-coloured, opaque and convex, with slightly irregular edges on TSA. Grows at pH 5·5–8·5 and 15–45 °C. Growth occurs in 1–3 % (w/v) NaCl, but not in 5 % (w/v) NaCl. Nitrate is reduced to nitrite. Oxidase-negative and catalase-positive. The pH in Voges–Proskauer broth is 7·6. Hydrogen sulfide and indole are not produced. Decomposes aesculin, arbutin, Tween 80 and testosterone. Does not decompose adenine, tyrosine, urea, uric acid, casein, elastin, hypoxanthine, starch or xanthine. D-Mannose, D-trehalose, glycerol, D-melibiose, D-raffinose, D-ribose (weak), D-fructose (weak), D-glucose, sucrose, D-cellobiose, adonitol (weak), dulcitol, D-galactose, L-rhamnose, maltose, melezitose, D-turanose, xylitol and inulin are utilized. Citrate, D-mannitol, D-sorbitol, D-arabinose, D-xylose, lactose, arabitol and myo-inositol are not utilized. Growth is inhibited by 0·02 % (w/v) NaN3. No acid is produced from glucose, arabinose, fructose, galactose, maltose, lactose, sucrose, xylose, trehalose, glycerol, mannitol, cellobiose, ribose, salicin, sorbitol, sorbose, mannose, melibiose, rhamnose, raffinose, inositol or adonitol. The DNA G+C content of the type strain, Mb8T (=JCM 11308T=NCIMB 13903T), is 66·0 mol%.
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ACKNOWLEDGEMENTS |
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