Fig. 5

A regulatory cascade of IsaF/J/S for the biosynthesis of isatropolone. (A, B, C) HPLC analysis was used to monitor the production of isatropolone A (ISA A) and isatropolone C (ISA C) in the wild type strain (CPCC 204095), the empty vector control strain (CPCC 204095/pSET152), the overexpression strain (CPCC 204095/pL-isaS), gene knockout strains (SKO and JKO), and genetic complementary strains (SKO/pL-isaS, JKO/pL-isaS). (D, E) Transcriptional analysis of isaF, isaJ, isaA and isaB in the overexpression strains (CPCC 204095/pL-isaJ, CPCC 204,095/pL-isaF), gene knockout strain (JKO, FKO), and genetic complementary strains (JKO/pL-isaJ, FKO/pL-isaF) was performed using RT-qPCR analysis. The relative mRNA level of the target genes was normalized to the principal sigma factor gene hrdB. The relative expressional level of each sample was represented as the value related to the control strain CPCC 204095/pSET152. Values are presented as mean ± SEM (two biological repeats for each strain). (F) Schematic representation for two overlapped probe fragments in EMSA. Arrows indicate possible forward repetitive binding sequences. (G) EMSA was used to analyze the interaction of the fragmented promoter regions with purified IsaF or IsaJ. The amounts of IsaF or IsaJ in the reaction were indicated above the lanes. For isaS-Tp-1, the “competitor” lane contains 10 µl of IsaF or IsaJ with a 200-fold excess of unlabeled specific competitor. For isaS-Tp-2, the “competitor” lane contains 5 µl of IsaF or IsaJ with a 200-fold excess of unlabeled specific competitor