Research Paper. Streptomyces sp. FJ3 HPLC LC-MS. 16S rdna. Doskochilova HPLC LC-MS A Q935. Ochi

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1 Research Paper Acta Microbiologica Sinica July 2011 ISSN CN / Q http / / journals. im. ac. cn / actamicrocn Streptomyces sp. FJ3 * HPLC LC-MS 16S rdna FJ3 MIC 0. 5 μg / ml 110 μg / ml FJ Doskochilova HPLC LC-MS Q935 A Ochi Hosaka RNA Streptomyces mauvecolor Piperidamycin 1 RNA cryptic Ochi CSTC2009CB1010 CSTCAB1029 * Tel chhhu@ vip. sina. com haile6@ 163. com

2 Streptomyces sp. FJ3. / μg / ml GYM 28 5 d FJ3- MIC 2 FJ TH2-C 1. 3 QYZ FJ3 JA2003A d SW-CJ-1FD GRANT XB FJ3 16S rdna Myclear Thermalcycler PCR TGL-16M DYY-8C LC-20AT F /1495r 5' min 93 1 min min min 72 5 min 30 PCR 1% 1500 bp L g NaCl 0. 5 g KNO g FeSO 4 7H 2 O g KH 2 PO g MgSO 4 7H 2 O 0. 5 g g ml ph GYM 4. 0 g g 1. 0 g NaCl 2. 0 g g ml g 15. 0g 5. 0 g 2. 5 g CaCO g NaCl 5. 0 g ml ph g Ⅱ. 2% Ⅲ V / V 5 ml Ⅳ. 2% Ⅴ mol / L ph cm 10 6 ml - 1 MIC GYM 2 28 DNA 16S rdan PCR AGAGTTTGATCCTGGCTCAG-3' 5'- ACGGCTACCTTGTTACGA CT-3' 16S rdna GenBank BLAST Doskochilova 8 8 Ⅰ. Ⅵ. 2% Ⅶ mol / L ph 7. 0 Ⅷ ml 3% 5%

3 936 Le Hai et al. / Acta Microbiologica Sinica HPLC LC-MS 2 30 ml30 ml 20 ml 5 ml HPLC ODS-C mm 250 mm 5 μm / 10 / ml / min SPD-6A 267 nm L / min CDL 300 heat block 300 scan MIC staphylococcus aureus. 4 BD20 FJ3 WZ20 FJ5 MIC MIC Table1 The MIC of streptomycin and rifampicin for 4 inacitve actinomycete strains and the number of active mutant strain Strain MIC of str / μg / ml MIC of rif / μg / ml FJ FJ WZ BD Number of active mutant MIC MIC FJ3 FJ3-2 FJ FJ3 a FJ3-2 b FJ3-6 c d 10μg / ml Fig. 1 Inhibition activity of fermentation broth of strain str FJ3 a FJ3-2 b FJ3-6 c and streptomycin d 10μg / ml against 2. 3 FJ3 2 FJ3 2 FJ3 A FJ3-2 B GYM FJ3-2 FJ3-6 Fig. 2 Morphology of strain FJ3 A and streptomycin resistance mutant str FJ3-2 B on GYM medium.

4 Streptomyces sp. FJ3. / FJ3 16S rdna FJ3 16S rdna Ⅰ Ⅱ Ⅲ ⅦVIII bp BLAST FJ3 Rf 1. 0 Ⅴ Ⅵ Streptomyces sp. MV32 Accession FJ % Streptomyces sp. FJ3 strain GenBank Noumber JF Rf 3 FJ FJ3-2 Doskochilova Rf Fig. 3 The Rf curve of the fermentation broths of FJ3-2 and thiolutin in Doskochilova system. A The fermentation broths of FJ3-2 B Thiolutin HPLC LC-MS 4 FJ3-6 FJ3-2 FJ3 a 2 b c a b c 3 b 5 b ESI-MS m / z M- H C 8 H 8 N 2 O 2 S 2 FJ3-2 Fig. 4 4 FJ3 FJ3-6 FJ3-2 HPLC HPLC chromatogram of fermentation broths of strain FJ3 and the mutant FJ3-2FJ3-6.

5 938 Le Hai et al. / Acta Microbiologica Sinica Fig. 5 5 FJ3-2 b LC-MS The peak b of FJ3-2 determined by LC-MS FJ3-2 FJ MIC 9 FJ3 FJ3-2 FJ3-6 rsmg HPLC 2 MIC LC- 3 MIC MS MIC 1955 Celmer Solomon Streptomyces luteorectiuli 2010 rpsl rsmg 13 FJ rpsl

6 Streptomyces sp. FJ3. / Wang GHosaka TOchi K. Dramatic activation of antibiotic production in Streptomyces coelicolor by cumulative drug resistance mutations. Applied and 7 Hu HFZhang QOchi K. Activation of antibiotic biosynthesis by specified mutations in the rpob Gene encoding the RNA polymerase β subunit of Streptomyces lividans. Journal of Bacterioogyl Hosaka TOhnishi-Kameyama1 MMuramatsu H Murakami KTsurumi YKodani SYoshida MFujie AOchi K. Antibacterial discovery in actinomycetes strains with mutations in RNA polymeraseor ribosomal protein S12. Nature biotechnology Hesketh AOchi K. A novel method for improving Streptomyces coelicolor A3 2 for production of actinorhodin by introduction of rpsl encoding ribosomal protein S12 mutations conferring resistance to streptomycin. Journal of Antibiotics Ochi kokamto STozawa YInaoka THosaka TXu JKurosawa K. Ribosome engineering and secondary metabolite production. Advances in Applied Microbiology Hu HFOchi k. Novel approach for improving the productivity of antibioticp-roducing strains by inducing combined resistant mutations. Applied and Environmenalt Microbioogyl Ochi khosoya Y. Genetic mapping and characterization of novel mutations which suppress the effect of a relc mutation on antibiotic production in Streptomyces coelicolor A3 2. Journal of Antibiotics Eenvironmental Microbiology Shima J Hesketh A Okamoto SKawamoto SOchi K. Induction of actinorhodin production by rpsl encoding ribosomal protein S12 mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3 2. Journal of Bacteriology Yoshiko O HSusumu OKozo Ochi. Development of antibiotic-overproducing strains by site-directed mutagenesis of the rpsl gene in Streptomyces lividans. Applied and Environment Microbiology Tanaka YKomatsu MOkamoto STokuyama SKaji AIkeda HOchi K. Antibiotic overproduction by rpsl and rsmg mutants of various actinomycetes. Applied and Environmental Microbiology International Journal of Pharmaceutical Research Jia YFWu SLIsenberg JSDai SSipes JMField L Zeng BBandle RWRidnour LAWink DA Ramchandran RKarger BLRoberts DD. Thiolutin inhibits endothelial cell adhesion by perturbing Hsp27 interactions with components of the actin and intermediate filament cytoskeleton. Cell Stress and Chaperones

7 940 Le Hai et al. / Acta Microbiologica Sinica Ribosome engineering of Streptomyces sp. FJ3 from Three Gorges reservoir area and metabolic product of the selected mutant strain Le HaiYuqi HuangGuojian LiaoChanghua Hu * Southwest UniversityCollege of PharmacyChongqing China Abstract ObjectiveTo explore new resource from inactive actinomycete strainswe screened resistant mutant strains by ribosome engineeringand analyzed the products derived from the selected mutant strains. MethodsThree Gorges reservoir area-derived actinomycete strains including BD20 FJ3 WZ20 and FJ5 were used as initial strainswhich showed no-antibacterial activities. The streptomycin-resistant str R mutants and rifampicin-resistant rif R mutants were screened by single colony isolation on streptomycin-containing plates and rifampicin-containing plates according to the method for obtaining drug-resistant mutants in ribosome engineering. The four initial strains and their str R -mutants and rif R -mutants were fermented in a liquid medium with the same composition. Mutants with anti-staphylococcus aureus activity were obtained by paper chromatography. The components of fermentation broth were analyzed by high performance liquid chromatography HPLC and high performance liquid chromatography-mass spectrometry LC-MS. FurthermoreFJ3 strain was identified by 16S rdna and morphology. ResultsThe minimal inhibitory concentration MIC of streptomycin and rifampicin for FJ3 was 0. 5μg / ml and 110μg / mlrespectively. Twenty-four str R -mutant strains and 20 rif R -mutant strains of FJ3 mutant strains were selected for bioassay. The result of the antibacterial activity screening demonstrated that six strains inhibited bacteria. Two strains FJ3-2 and FJ3-6 were screened from the streptomycinresistance mutants of inactive strain FJ3. The result of bioassay showed that the fermentation broth of FJ3-2 and FJ3-6 exhibited obvious anti-staphylococcus aureus activity. The assay of paper chromatography showed that the active substance may be nucleic acid class antibiotic via using solvent system Doskochilova. Moreoverthe results of HPLC and LC-MS exhibited that this substance may be thiolutin. ConclusionRibosome engineering for changing the secondary metabolic function of the inactive wild-type actinomycete strains was a feasible method for the acquirement of active mutant strains which will be beneficial to exploit the new medical actinomycete strains. Keywords Three Gorges reservoir areaactinomycetesribosome engineeringstreptomycin resistanceanti- Staphylococcus aureus activity Supported by the Programs for Science and Technology Development of Chongqing CSTC2009AB1029CSTC2009CB1010 * Corresponding author. Tel chhhu@ vip. sina. com Received 28 December2010 / Revised 19 April 2011