Fig. 4.

Fur-regulated genes are induced in a similar stepwise manner as a function of iron depletion in strains producing bacillibactin. Overnight cell culture was inoculated with 1:100 ratio into fresh LB medium amended with 25 µM FeSO₄. After OD₆₀₀ reaches ∼0.25, 1 mM IPTG was added to induce expression of FrvA. Total RNA was extracted and subjected to cDNA synthesis followed by qPCR. Relative expression of each gene at different times was monitored by comparing gene expression in the IPTG-induced cells versus that in sfp⁺ WT cells. The percentage of derepression was evaluated based on the full derepression observed in sfp⁺ fur mutant versus sfp⁺ WT, which was set as 100% derepression.