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Pure Appl. Chem., 2010, Vol. 82, No. 1, pp. 57-67

Published online 2010-01-03

An integrative approach for high-throughput screening and characterization of transcriptional regulators in Streptomyces coelicolor

Eunjung Song1,2, Yung-Hun Yang2, Bo-Rahm Lee1,2, Eun-Jung Kim1,2, Ji-Nu Kim1,2, Sung-Soo Park2, Kwangwon Lee2, Woo-Seong Kim2, Sungyong You3, Daehee Hwang3 and Byung-Gee Kim1,2*

1 Institute of Bioengineering, Seoul National University, Seoul, Korea
2 School of Chemical and Biological Engineering, Seoul National University, Seoul, Korea
3 School of Chemical Engineering, Pohang University of Science and Technology, Pohang, Korea

Abstract: In an age of burgeoning information on genomes and proteomes, determining the specific functions of a gene of interest is still a challenging task, especially genes whose functions cannot be predicted from their sequence information alone. To solve this problem, we have developed an integrative approach for discovering novel transcriptional regulators (TRs) playing critical roles in antibiotic production and decoding their regulatory networks in Streptomyces species which contain many regulatory genes for synthesis of secondary metabolites and cell differentiation to spores. The DNA affinity capture assay (DACA) coupled with clustering of DNA chip data was used to find new TRs controlling antibiotic biosynthetic gene clusters. Functions of these newly identified TRs were characterized using 96-well-based minimal media screening (antibiotic production mapping, APM), pH indicator method, comparative two-dimensional gel electrophoresis (2D-gel), reverse-transcription polymerase chain reaction (RT‑PCR), electrophoretic mobility shift assay (EMSA), and scanning electron microscopy (SEM). Using these techniques, we were able to reconstruct a regulatory network describing how these new TRs collectively regulate antibiotic production. This proposed approach providing additional key regulators and their interactions to an existing incomplete regulatory network can also be applied in studying regulators in other bacteria of interest.