Title Differential roles of three regulatory proteins in IM-2 / FarA signaling cascade governing secondary metabolism in Streptomyces lavendulae

of Thesis Members of the Gram-positive, soil-dwelling filamentous bacterial genus Streptomyces have been extensively studied due to their complex life cycle of morphological differentiation and their ability to synthesize secondary metabolites of structural and biological diversity possessing medical and industrial significance. These secondary metabolites production are tightly regulated in a hierarchical manner at several layers involving higher-level regulators regarded as global regulators that mediates stimuli from environment, controlled the activity of the pathway specific regulators which directly control the activation of biosynthetic genes for secondary metabolites. In streptomyces, the most well known hierarchical regulation is the γ-butyrolactone (GBL) signaling cascade consisting of a GBL molecule and a cognate GBL receptor protein which situated at the highest level hierarchy in the regulatory cascade. In the absence of GBL, the GBL receptor protein binds to the promoter region of target genes and represses its transcription. GBL molecule will bind and dissociates the receptor from the promoter of target genes and thereby triggering transcription of target genes and allowing the onset of secondary metabolism and/or morphological development. The GBL signaling cascade in Streptomyces lavendulae FRI 5 composed of a GBL molecule IM-2 and cognate GBL receptor FarA. Unlike other GBL molecules which play positive roles in the regulation of secondary metabolite production, IM-2 exerts dual positive negative effects on the regulation of secondary metabolism; namely, switches on the production of blue pigment and nucleoside antibiotics and switches off the production of D-cycloserine. We previously found that the farA-flanking region has seven regulatory genes, including farX, an IM-2 biosynthetic gene, and comprises a far regulatory island. Two putative regulatory genes (farR3 and farR4) encoding the Streptomyces Antibiotic Regulatory Protein (SARP)-family protein are present in the far regulatory island together with two more putative transcriptional regulatory genes ( farR1 and farR2) all of which are considered to be the direct transcriptional targets of FarA. This study is aimed to clarify the regulatory function of farR2, farR3, and far4 in the IM-2/FarA signaling cascade. In chapter 2, two regulatory genes, farR3 and farR4, were characterized. The SARP family regulators are DNA-binding proteins transcriptional regulator and in general acts as activators for the production of secondary metabolites. farR3 is transcribed both as a monocistronic RNA and as a bicistronic farR4-farR3 mRNA, and the expression profile is tightly controlled by the IM-2/FarA system. Loss of farR3 delayed and decreased the production of blue pigment indigoidine without any changes in the transcriptional profile of other far regulatory genes, indicating that FarR3 positively controls the biosynthesis of blue pigment indigoidine, and is positioned in the downstream region of the IM-2/FarA signaling system. Meanwhile, loss of farR4 induced the early production of IM-2 by increasing transcription of an IM-2 biosynthetic gene, farX, indicating that FarR4 negatively controls the biosynthesis of IM-2. Thus, this study suggested differential contributions of the SARP-family regulators to the regulation of secondary metabolism in S. lavendulae FRI-5. This is the first report to show that a SARP-family regulator is involved in the biosynthesis of a signaling molecule functioning at the most upstream region of the regulatory cascade for Streptomyces secondary metabolism. In chapter 3, a regulatory gene of the GBL receptor homologue family, farR2, which is located in the