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1 Yunnan Institute of Microbiology, Yunnan University, Kunming, Yunnan 650091, People's Republic of China
2 Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People's Republic of China
Correspondence
Wen-Jun Li
wjli{at}ynu.edu.cn
Li-Hua Xu
lihxu{at}ynu.edu.cn
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ABSTRACT |
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8c, 10-methyl C17 : 0 and C16 : 0. A comparative analysis of 16S rRNA gene sequences indicated that the organism formed a distinct clade within the evolutionary radiation of the family Thermomonosporaceae, and that it was closely associated with members of the genus Actinomadura. A broad range of phenotypic and genetic data supported the suggestion that this organism represents a novel species of the genus Actinomadura, for which the name Actinomadura alba sp. nov. is proposed, with YIM 45681T (=DSM 45045T =CCTCC AA206005T) as the type strain.
These authors contributed equally to this work. 
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain YIM 45681T is DQ985164.
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and its description has been emended by Zhang et al. (1998, 2001) and Miyadoh & Miyara (2001). Currently, the genus comprises 35 recognized species and two subspecies with validly published names. The genus Actinomadura accommodates aerobic, Gram-positive, non-acid-fast, non-motile actinomycetes that produce well-developed, non-fragmenting vegetative mycelia and aerial hyphae that differentiate into surface-ornamented spore chains. These chains are of various lengths and can be straight, hooked or spiral in form, with irregular, smooth, spiny or warty spores. Members of the genus Actinomadura are characterized chemotaxonomically by the presence of type III/B cell walls (i.e. containing meso-diaminopimelic acid and madurose) with peptidoglycan structures of the acetyl type, major proportions of hexahydrogenated menaquinones with nine isoprene units, complex fatty acid profiles, including hexadecanoic, 14-methylpentadecanoic and 10-methyloctadecanoic acids as predominant components, and diphosphatidylglycerol and phosphatidylinositol as major phospholipids (Kroppenstedt et al., 1990).
Strain YIM 45681T was isolated by using the following procedure. Soil samples were collected from the rhizosphere of Catharanthus roseus, at the surface and down to a depth of about 10 cm. Soil samples were air-dried for about 7 days. One gram of the soil sample was first suspended in 9 ml MOPS (10 mM) containing 1 % keratin and incubated for 2 h at 45 °C with vigorous shaking, in order to kill fast-growing bacteria and promote actinomycete spore germination. This culture was centrifuged and the supernatant was serially diluted to 10–3 and spread on a humic acid/vitamin/gellan gum (HVG; Suzuki et al., 1999) medium and incubated at 28 °C for 30 days.
Strain YIM 45681T was grown on ISP 2, ISP 3, ISP 4, ISP 5 (Shirling & Gottlieb, 1966) and Czapek agar plates (Pridham & Lyons, 1980) at 28 °C. Colour determination was performed with colour chips from the Inter-Society Color Council–National Bureau of Standards Color Name Charts (Standard Samples, no. 2106; Kelly, 1964). Spore chains were observed using the cover-slip technique of Kawato & Shinobu (1959). Morphological characteristics were examined by light microscopy with a model BH-2 microscope (Olympus) and scanning electron microscopy (XL30, ESEM-TMP; Philips). Morphological features were observed on ISP 4 medium at 28 °C. Growth was tested over a range of temperatures (4–45 °C) and pH (pH 6.0–12.0) on ISP 2 medium. The phenotypic properties of the isolate are also consistent with its classification in the genus Actinomadura. Strain YIM 45681T grew well on ISP 2, ISP 3 and ISP 4 media, but grew only moderately well on ISP 5 and Czapek agar plates. Vegetative hyphae were well developed on all of the media tested. The whitish aerial mycelium, which was produced only on ISP 3, ISP 4 and Czapek agar media, formed short, spiral chains of irregular-surfaced spores (Fig. 1). No diffusion pigments were produced on any of the media tested. The optimal temperature and pH for growth were 28 °C and pH 7.0–8.0.
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and the species description. ISP 9 (Shirling & Gottlieb, 1966) was used as the basic medium for testing carbohydrate utilization; each filter-sterilized compound was tested at a final concentration of 1 % (w/v). Urease activity was determined by assessing a colour change in Bacto urea broth (Difco). The production of H2S was tested on peptone iron agar (Difco). Nitrate reduction, gelatin liquefaction and the degradation of elastin and starch were investigated by using previously described methods (MacFaddin, 1980). The decomposition of adenine, hypoxanthine, casein, DL-tyrosine and xanthine was examined by using the methods of Gordon et al. (1974).
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. Menaquinones were extracted by using the method of Collins et al. (1977) and were analysed by HPLC, as described by Tamaoka et al. (1983). Polar lipids were extracted as described by Minnikin et al. (1979) and identified by two-dimensional TLC and spraying with specific reagents (Collins & Jones, 1980). Biomass for a quantitative fatty acid analysis of strain YIM 45681T was prepared by scraping growth from TSB agar plates that had been incubated for 7 days at 28 °C. Fatty acids were extracted, methylated and analysed using the MIDI (Microbial Identification) system. The wall diamino acid in the peptidoglycan layer of strain YIM 45681T was meso-diaminopimelic acid and the whole-cell sugars were ribose, madurose, xylose, galactose and glucose (cell-wall chemotype III, according to Lechevalier & Lechevalier, 1970a). The predominant menaquinones were MK-9(H4), MK-9(H6) and MK-9(H2) (ratio of peak areas, 64.6 : 18.2 : 11.5). The phospholipids included diphosphatidylglycerol, phosphatidylinositol, phosphatidylglycerol and phosphatidylinositol mannoside as the major polar lipids (phospholipid type I; Lechevalier et al., 1977), and the fatty acid profile is described in more detail in the species description. Genomic DNA for PCR amplification was prepared from cells lysed by microwave: a small amount of biomass was transferred from solid medium to a new Eppendorf tube. After washing of the cells with 1 ml PBS (pH 8.0) and 1 ml washing buffer (50 mM Tris/HCl, pH 7.7; 25 mM EDTA; 0.1 % SDS, 0.1 % polyvinylpyrrolidone), 50 µl lysis buffer (50 mM Tris/HCl, pH 8.0; 25 mM EDTA; 3 % SDS; 1.2 % polyvinylpyrrolidone) was added to resuspend the cells, which were then heated in a microwave oven at 700 W for 45 s. Four hundred microlitres warm extraction buffer (10 mM Tris/HCl, pH 8.0; 1 mM EDTA; 0.5 M sodium acetate; 1.2 % polyvinylpyrrolidone, pre-warmed at 65 °C) was then added immediately; after mixing, extraction was performed using the same volume of phenol/chloroform. The DNA was precipitated with isopropanol. After washing with 70 % ethanol, the DNA was dissolved using 20 µl TE solution.
PCR amplification and 16S rRNA gene sequencing were carried out as described previously (Cui et al., 2001). An almost-complete 16S rRNA gene sequence for strain YIM 45681T was determined by direct sequencing of the PCR-amplified gene. The G+C content of the DNA, determined by using the HPLC method (Mesbah et al., 1989), was found to be 66.5 mol%.
A preliminary comparison between the almost-complete 16S rRNA gene sequence of strain YIM 45681T (1479 bp) and sequences in the GenBank database indicated that the novel isolate was closely related to the members of the genus Actinomadura. A phylogenetic analysis was performed using the software packages PHYLIP (Felsenstein, 1993) and MEGA, version 2.1 (Kumar et al., 2001) after multiple alignment of the data using CLUSTAL_X (Thompson et al., 1997). Distances (using distance options according to the Kimura two-parameter model; Kimura, 1980, 1983) were calculated and clustering was performed with the neighbour-joining method (Saitou & Nei, 1987). A bootstrap analysis (1000 resamplings) was used to evaluate the tree topology of the neighbour-joining data (Felsenstein, 1985). The phylogenetic analysis (Fig. 2) indicated that isolate YIM 45681T formed a distinct clade within the radiation encompassing members of the family Thermomonosporaceae, and that it was closely associated with members of the genus Actinomadura. The levels of 16S rRNA gene sequence similarity between strain 45681T and its two closest neighbours, Actinomadura echinospora DSM 43163T and Actinomadura spadix JCM 3146T, were 96.6 and 95.9 %, respectively (see Fig. 2).
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). Support for the novel species status of the organism also comes from the phenotypic evidence shown in Table 1
. Therefore, on the basis of the phenotypic and molecular genetic data, it is evident that strain YIM 45681T represents a novel species of the genus Actinomadura, for which we propose the name Actinomadura alba sp. nov.
Description of Actinomadura alba sp. nov.
Actinomadura alba (al'ba. L. fem. adj. alba white, referring to the white aerial mycelium).
Aerobic, Gram-positive. Cells grow well on ISP 2, ISP 3 and ISP 4 media and show moderate growth on ISP 5 and Czapek agar plates, forming a well-developed, non-fragmenting substrate mycelium. No diffusion pigment is produced on any of the media tested. On ISP 3 and ISP 4 media, a moderate amount of white aerial mycelium is formed; this matures into short spiral spore chains, and the spore surface is irregular. The optimal temperature and pH for growth are 28 °C and pH 7.0–8.0. Catalase- and urease-positive. Nitrate is reduced to nitrite. H2S is not produced. Gelatin liquefaction is observed. D-Mannitol, D-xylose, adonitol, D-ribose, D-fructose, D-trehalose, methyl 
-D-glucoside, maltose, D-lactose, sucrose and D-arabitol can be utilized as carbon sources, but glycerol, D-galactose, D-mannose, D-mannitol, L-rhamnose, D-cellobiose, myo-inositol, D-arabinose, D-melibiose and D-raffinose are not utilized. Decomposes hypoxanthine, casein, aesculin, gelatin and starch. Does not decompose xanthine, DL-tyrosine or Tween 80. meso-Diaminopimelic is the diagnostic diamino acid, and the cell-wall sugars are ribose, xylose, madurose, galactose and glucose. The predominant menaquinones are MK-9(H4), MK-9(H6) and MK-9(H2). The phospholipids are diphosphatidylglycerol, phosphatidylinositol, phosphatidylglycerol and phosphatidylinositol mannoside. The major fatty acids (>10 %) are iso-C16 : 1 H (19.77 %), C17 : 18c (18.51 %), 10-methyl C17 : 0 (13.31 %) and C16 : 0 (11.03 %). The G+C content of the DNA of the type strain is 66.5 mol%.
The type strain, YIM 45681T (=DSM 45045T=CCTCC AA206005T), was isolated from a soil sample collected in a suburb of Kunming, Yunnan Province, south-west China.
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ACKNOWLEDGEMENTS |
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