Mass spectrometric screening of transcriptional regulators involved in antibiotic biosynthesis in Streptomyces coelicolor A3(2)
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DNA-affinity capture assay (DACA) coupled with liquid chromatography–tandem mass spectrometry analysis was applied to identify the transcriptional regulators involved in the biosynthesis of actinorhodin (Act) and undecylprodigiosin (Red) in Streptomyces coelicolor. The aim of this analysis was to determine the specific transcriptional regulators binding to the promoter region of actII-ORF4 or redD. The results of the DACA, as the first screening tool, identified eight proteins, including AdpA, as candidate regulators binding to those promoter regions. To show the direct physical relationship between the regulators and promoters, we purified four regulators over-expressed in soluble form in Escherichia coli and subjected these to an electrophoretic mobility shift assay (EMSA). The results of the EMSA appeared to be compatible with the DACA results for those regulators. A null mutant was also constructed for one of these regulators, SCO6008, which showed early Red production and quite delayed Act production in R5− medium. These observations suggest that DACA can be widely used to find new regulators and that the regulator SCO6008 may be involved in antibiotic production through its binding to the redD promoter.
KeywordsActinorhodin DNA affinity capture assay Streptomyces coelicolor Transcriptional regulator Undecylprodigiosin
The authors thank Prof. Jung-Hye Roe for providing S. coelicolor A3(2) strain M145 and cosmids, and Prof. Yeo Joon Yoon for providing the plasmid pSE34. We also thank Profs. Ji-Sook Hahn, Seung R. Paik, and Eung-Soo Kim for their comments on the manuscript. This work was supported by a grant funded by the Korea government (MOST) (No. R0A-2007-000-10007-0), Republic of Korea.
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