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Institute of Tropical Bioscience and Biotechnology/State Key Laboratory of Tropical Crop Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, PR China
Correspondence
Shixiang Bao
bsxhhq{at}yahoo.com.cn
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain AM105T is AY561829.
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). Strain AM105T was isolated from mangrove sediment samples from the South China Sea. Isolation was carried out by the standard dilution-plating technique on modified Gause inorganic agar (Gause et al., 1983), which contained 20 g soluble starch, 1 g KNO3, 0.5 g K2HPO4, 0.5 g MgSO4 . 7H2O, 0.01 g FeSO4 . 7H2O, 15 g agar, 1 l old sea water (pH 7.2–7.4). Plates were incubated for 30 days at 28 °C. A pure culture was maintained in a glycerol suspension (20 %, w/v) at –70 °C.
Cultural features were observed on oatmeal agar (DSMZ medium 609), glycerol–asparagine agar (Shirling & Gottlieb, 1966), GYM agar (DSMZ medium 65), potato dextrose agar (DSMZ medium 129), Sauton's agar (Mordarska et al., 1972) and Gause inorganic agar after 7, 14 and 21 days incubation at 28 °C. Cell morphology and spore production were observed by light and scanning electron microscopy using 6- and 20-day-old cultures grown on various agar media. The ability to grow on sole carbon sources (1 %, w/v) was tested as described by Williams et al. (1983). NaCl tolerance (0–4.5 %, w/v) and temperatures (4–45 °C) for growth were tested on GYM agar. Methods and media for other physiological tests and the assays for enzymic activities were performed according to Wang (1986).
For chemotaxonomic studies, the strain was grown in GYM broth in a shaking incubator at 200 r.p.m. and 28 °C for 5 days. Biomass was harvested by centrifugation and washed with distilled water. Isomers of diaminopimelic acid (DAP) and sugars were determined in whole-cell hydrolysates by TLC on microcrystalline cellulose (Wang, 1986).
Genomic DNA was isolated as described by Pitcher et al. (1989). The DNA G+C content was determined using the thermal melting method (Mandel & Marmur, 1968). PCR amplification of the 16S rRNA gene and sequencing of the purified PCR product were done as described previously (Rivas et al., 2003). The sequence of isolate AM105T was aligned and compared with representative sequences of members of the genus Micromonospora obtained from GenBank using the CLUSTAL_X 1.8 program (Thompson et al., 1997). Phylogenetic analysis was performed with the MEGA version 2.1 program (Kumar et al., 2001). Phylogenetic trees were constructed using three tree-making algorithms, namely, neighbour-joining (Saitou & Nei, 1987), minimum-evolution (Kidd & Sgaramella-Zonta, 1971; Rzhetsky & Nei, 1993) and maximum-parsimony (Fitch, 1972); bootstrap analysis was also conducted. DNA–DNA relatedness experiments were performed by the method of Ezaki et al. (1989). DNA–DNA relatedness values were calculated according to Christensen et al. (2000).
Strain AM105T showed good growth and no diffusible pigments on all of the media used. Colonies were orange, but turned dark brown on sporulation after 14–21 days on oatmeal agar and Gause inorganic agar. Spores were smooth rod-shaped, ovoid or spherical. Spores were single or in short chains with two to five spores, which borne on short or long sporophores. Some spores were sessile (Fig. 1). Aerial mycelium, at times visible only on Sauton's agar and Gause inorganic agar, was scanty and milk-white. Other cultural and morphological characters are given in the species description.
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) were arabinose and xylose. The G+C content of the DNA was 71 %. The DNA–DNA relatedness between strain AM105T and M. matsumotoense DSM 44100T was 53.6 % (the means of triplicate analyses), which are below the threshold value of 70 % for the definition of a bacterial species according to Wayne et al. (1987).
Strain AM105T grew between 20 and 37 °C, but no growth was found at 4, 15 or 45 °C after 3 weeks. Tolerates NaCl up to 3 % and no growth occurred at 4.5 %. Among the carbohydrates tested, only D-glucose was utilized well. Other physiological and biochemical characters are listed in the species description; characters differentiating the strain from related species are given in Table 1.
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). A clear antimicrobial activity was revealed against Staphylococcus aureus CGMCC 1.89, Staphylococcus aureus OY84 (a meticillin-resistant clinical isolate), Bacillus subtilis CGMCC 1.88, but not against Escherichia coli CGMCC 1.747, Candida albicans CGMCC 2.538 or Piricularia oryzae CGMCC 3.3283. The active ethyl acetate extract from the supernatant was separated by silica gel G250 column, and the bioactive fraction was eluted with petroleum ether/ethyl acetate (3 : 1), concentrated, and further separated by TLC (chloroform/methanol, 98 : 2). The bioactive compound was then purified through Sephadex LH-20 (chloroform/methanol, 1 : 1) and analysed by HPLC and various spectroscopic methods (Traxler et al., 1981; Hermann & Zuordnung, 1973; Gallo et al., 1974; Cellai et al., 1982; Arora & Arjunan, 1992). In HPLC analysis, the compound showed one peak but two sets of proton and carbon signals were observed in the 1H- and 13C-NMR spectra. Further identification using 1H-NMR, 13C-NMR, heteronuclear multiple bond correlation, heteronuclear single quantum coherence, rotary overhauser effect spectroscopy, electrospray ionization mass spectrometry and IR spectroscopy showed that the active compound (purple amorphous powder) consisted of rifamycin S and its isomer (Huang et al., 2006), while M. carbonacea was reported to produce everninomicin (Luedemann & Brodsky, 1965). No data are available for antibiotic production by other related species, M. matsumotoense, M. mirobrigensis and M. siamensis. Thus, the data obtained in this study clearly show that strain AM105T merits description as a novel species of the genus Micromonospora, for which the name Micromonospora rifamycinica sp. nov. is proposed.
Description of Micromonospora rifamycinica sp. nov.
Micromonospora rifamycinica (rif.a.my.cin'i.ca. N.L. n. rifamycinum -i rifamycin; L. suff. icus -a -um related to; N.L. fem. adj. rifamycinica referring to the ability to produce rifamycin).
Aerobic, Gram-positive actinomycetes which form an extensively branched fine substrate mycelium. Hard colonies on GYM agar are 2–3 mm in diameter after 2 weeks. Substrate mycelium is orange, turning brown on sporulation on oatmeal agar and Gause inorganic agar. Spores are rod-shaped, ovoid or spherical, smooth, single or in short chains with two to five spores, and borne on short or long sporophores. Some spores are sessile. Aerial mycelium, scanty and milk-white, at times produced on Sauton's agar and Gause inorganic agar. Diffusible pigments are not produced. Growth occurs in 0–3 % NaCl and 20–37 °C. D-Glucose is utilized as a sole carbon source; L-arabinose, D-xylose, D-fructose, L-rhamnose, mannitol, sucrose, D-raffinose and inositol are not utilized. Able to degrade starch and cellulose, but not gelatin or casein. Nitrate reductase and tyrosinase are produced but hydrogen sulfide (H2S) is not. Produces rifamycin S and its isomer. Whole-cell hydrolysates contain meso-DAP and the main sugars xylose and arabinose. The DNA G+C content is 71 %.
The type strain, AM105T (=CGMCC 4.2495T=DSM 44983T), was isolated from mangrove sediment from the South China Sea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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