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1 Bio-resource Collection and Research Center, Food Industry Research and Development Institute, HsinChu, 300, Taiwan
2 Yunnan Institute of Microbiology, Yunnan University, Kunming, Yunnan, 650091, People's Republic of China
Correspondence
Min Tseng
tjm{at}firdi.org.tw
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 0345M-7T is DQ160215.
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MAIN TEXT |
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and at the time of writing comprises 27 recognized species. Members of the genus are aerobic, Gram-positive, non-acid-fast, non-motile organisms that form a branched substrate mycelium that fragments into square-shaped elements. Aerial hyphae may be sterile or differentiate into long chains of spore-like structures. Amycolatopsis species contain meso-diaminopimelic acid (meso-A2pm), arabinose and galactose in the cell wall (wall chemotype IV of Lechevalier & Lechevalier, 1970), are rich in iso- and anteiso-branched fatty acids and contain di-, tetra- and hexahydrogenated menaquinones with nine isoprene units as the predominant isoprenologue. Phosphatidylethanolamine (PE) and phosphatidylglycerol are major polar lipids with diphosphatidylinositol mannosides variably present. Members of the genus have DNA G+C contents in the range 66–73 mol%. During our investigation of the ecology of novel actinomycetes, we isolated several new strains from soils in Taiwan. In this study, we describe one isolate, 0345M-7T, as belonging to the genus Amycolatopsis. On the basis of the polyphasic data presented, we propose that strain 0345M-7T should be classified within a novel species of the genus Amycolatopsis.
Strain 0345M-7T was isolated from a soil sample collected from Yilan county, Taiwan, by using HV agar (Hayakawa & Nonomura, 1987), and incubated at 28 °C for 4 weeks. The strain was maintained on oatmeal agar and stored at –20 °C as a suspension of spores or mycelial fragments in 20 % (v/v) glycerol.
Morphological characteristics of cells of strain 0345M-7T were observed via scanning electron microscopy (S-420; Hitachi), following incubation on HV agar for 14 days at 28 °C and fixation in 4 % osmium tetroxide solution, dehydration through a graded ethanol series and acetone, followed by critical-point drying. Cultural characteristics were tested by using 14-day-old cultures grown at 28 °C on various agar media (Table 1). Colour designations of the substrate mycelium were according to Kelly (1964). All physiological tests were performed at 28 °C. Growth temperature, NaCl tolerance, hydrolysis of aesculin, casein, gelatin, hypoxanthine and xanthine and production of amylase, nitrate reductase and urease were tested following the methods and procedures of Gordon et al. (1974). Carbon source utilization was examined on ISP 9 as the basal medium (Shirling & Gottlieb, 1966) supplemented with a final concentration of 1 % of the tested carbon source.
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. The N-acyl types of muramic acid were determined by the method of Kawamoto et al. (1981). Presence of mycolic acids was examined via TLC following Minnikin et al. (1975) and phospholipids were extracted and identified following the method of Minnikin et al. (1984). Menaquinones were extracted and purified according to the method of Collins et al. (1977) and then analysed by HPLC (model 600; Waters) with a Nova-Pak C18 column. For quantitative analysis of the cellular fatty acid content, strain 0345M-7T was cultured using TSB medium at 28 °C on a shaking incubator at 125 r.p.m. for 7 days. Extracts of the methylated fatty acids were prepared according to the protocol provided by MIDI Inc.
For the extraction of DNA to be used for sequencing of the 16S rRNA gene, strain 0345M-7T was grown in YG broth at 28 °C for 7 days. Cells were removed from the broth using a pipette tip and total DNA was extracted by using the QIAGEN Genomic DNA kit. The G+C content of the DNA was determined by HPLC according to the method of Tamaoka & Komagata (1984). DNA was prepared by using the same method as above. The 16S rRNA gene was PCR-amplified according to the methods of Nakajima et al. (1999) and directly sequenced on an ABI model 3730 automatic DNA sequencer using the BigDye Terminator v3.1 kit (Applied Biosystems). Phylogenetic analysis was performed using the software packages PHYLIP (Felsenstein, 1993) and MEGA version 2.1 (Kumar et al., 2001) after multiple alignment of data by using CLUSTAL X (Thompson et al., 1997). Calculation of evolutionary distances (distance options according to the Kimura two-parameter model; Kimura, 1980, 1983) and clustering were with the neighbour-joining method (Saitou & Nei, 1987). Bootstrap analysis was used to evaluate the tree topology of the neighbour-joining data by performing 1000 resamplings (Felsenstein, 1985).
Strain 0345M-7T produced branched and non-fragmented substrate mycelia, borne on which were short spore chains. No aerial mycelia were found after growth on any of the test media. Spores were non-motile, oval and smooth-surfaced (Fig. 1). The cultural characteristics of strain 0345M-7T are detailed in Table 1
. Good growth and pale-yellow substrate mycelia were observed but sporulation was poor on most of the media tested. No soluble pigment was produced in all media tested. The physiological and biochemical characteristics of strain 0345M-7T are given in Table 1 and in the species description below.
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The almost-complete 16S rRNA gene sequence (1507 nt) of strain 0345M-7T was determined. Preliminary comparison of the sequence against the GenBank database revealed high sequence similarity values with members of the genus Amycolatopsis. The phylogenetic tree based on the 16S rRNA gene sequences of strain 0345M-7T and recognized members of the genus Amycolatopsis is shown in Fig. 2. Strain 0345M-7T had 16S rRNA gene similarity values ranging between 92.7 % (Amycolatopsis fastidiosa IMSNU 20054T) and 95.4 % (Amycolatopsis methanolica IMSNU 20055T). It is generally recognized that organisms displaying similarity values of <97 % do not belong to the same species (Stackebrandt & Goebel, 1994). The sequence divergence values of >4.0 % with recognized members of the genus Amycolatopsis clearly indicate that strain 0345M-7T represents a novel species.
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). However, chemotaxonomic data, including the predominant menaquinone, polar lipid and major fatty acids, indicate that strain 0345M-7T should be classified within the genus Amycolatopsis.
The distinctiveness of the isolate is also shown by phenotypic comparison with its nearest phylogenetic neighbours (Table 2). On the basis of the phenotypic and genotypic data presented, 0345M-7T should be classified as the type strain of a novel species of the genus Amycolatopsis, for which the name Amycolatopsis taiwanensis sp. nov. is proposed.
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Gram-positive, aerobic, non-acid-fast and mesophilic. No aerial mycelia are present. Short spore chains are formed on substrate mycelia; spores are oval with a smooth surface and are non-motile. The substrate mycelium is yellowish white to purple yellow. No soluble pigment is produced. Growth is good on most test media, but sporulation is very poor. Good growth occurs between 20 and 40 °C; no growth occurs above 1 % NaCl. Hydrolyses casein and aesculin and is positive for gelatin liquefaction. Able to reduce nitrate. Glucose, arabinose, cellulose and sucrose can be used as sole carbon sources for growth, but not fructose or salicin; use of rhamnose, inositol, raffinose, mannitol and xylose is questionable. The cell-wall peptidoglycan contains meso-A2pm; arabinose, galactose, glucose and ribose are detected in whole-cell hydrolysates. The predominant menaquinone is MK-9(H4). Mycolic acids are not detected. The diagnostic phospholipid is PE. Major cellular fatty acids are iso-C16 : 0 (38.1 %) and C17 : 1 (25.4 %). The G+C content of the DNA of the type strain is 68.9 mol%.
The type strain, 0345M-7T (=BCRC 16802T=KCTC 19116T), was isolated from a soil collected from Yilan county, Taiwan.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Chun, J., Kim, S. B., Oh, Y. K. & 7 other authors (1999). Amycolatopsis thermoflava sp. nov., a novel soil actinomycete from Hainan Island, China. Int J Syst Bacteriol 49, 1369–1373.
Collins, M. D., Pirouz, T., Goodfellow, M. & Minnikin, D. E. (1977). Distribution of menaquinones in actinomycetes and corynebacteria. J Gen Microbiol 100, 221–230.
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