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

Abstract

In an age of burgeoning information oil genomes and proteomes, determining the specific functions of a gene of interest is still a challenging task, especially gene, 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 oene clusters. Functions ol'these newly idcritified TRs were characterized using 96-well-based minimal media screening (antibiotic production mapping, APM), pH indicator tor method, comparative two-dimensional gel electrophoresis (2D-gel), rcverse-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 ail existing incomplete regulatory network, can also be applied ill Studying regulators in other bacteria of interest.