FIG 9.

Effect of C-5–O-B1 on AveT binding to target promoter regions. (A) EMSAs of His₆-AveT (0.05 μM) with concentrated fermentation broth of WT and aco deletion mutant Δaco grown in FM-I for 10 days. The concentrated supernatant of fermentation medium FM-I was used as a medium control. (B) Structures of C-5–O-B1 and avermectin B1. The conversion of C-5–O-B1 to avermectin B1 is catalyzed by AveF, which reduces the keto group at position C-5 of C-5–O-B1 to a hydroxyl group. (C) EMSAs of His₆-AveT (0.05 μM) with C-5–O-B1 and avermectin B1. Lanes −, control reaction without protein; lanes +, EMSA reaction in the presence of protein. Avermectin and C-5–O-B1 were dissolved in methanol, and methanol was used as a solvent control. In all EMSAs, each reaction mixture contained 0.15 nM labeled probe.