Figure 2.

Substrate inhibition of 0F by KinA produces a negative feedback in the phosphorelay.

A. Network diagram of the sporulation phosphorelay network that controls the activity of the master regulator Spo0A (0A). The phosphorelay includes both post-translational and transcriptional regulatory interactions. Post-translationally, the kinases KinA-E (only KinA is shown) transfer phosphoryl groups to the master regulator 0A via the two phosphotransferases Spo0B (0B) and Spo0F (0F). Transcriptionally, 0A~P controls the expression of kinA, 0F and 0A forming multiple transcriptional feedback loops. Our model also includes a substrate inhibition interaction (red blunted arrow) whereby excess 0F can bind to un-phosphorylated KinA and block its auto-phosphorylation. This substrate inhibition creates a negative feedback loop wherein 0A~P activates 0F expression and 0F inhibits 0A activation by inhibiting KinA. B. Mathematical model predicts that, for a phosphorelay with substrate-inhibition of KinA by 0F (blue curve), 0A promoter activity is a non-monotonic functions of 0F concentrations and decrease ultrasensitively for [0F]>5μM. In contrast, for a phosphorelay without substrate-inhibition (orange curve), 0A promoter activity monotonically increase to saturated value. C. Predicted non-monotonic dependence of 0A activity on 0F levels is confirmed by engineering inducible 0F strain, i0F^amyE (Δ0F; amyE::P_hsp-0F) and measuring maximum 0A promoter activity in the at different levels of 0F induction. Gray empty circles show maximum P_0A promoter activity levels achieved by individual cell lineages over 25 hours in starvation conditions at each IPTG concentration. Blue filled circles and error-bars indicate the mean and standard deviations of these measurements at each IPTG concentration. Maximum P_0A promoter activity decreases at high 0F expression levels (IPTG>10μM) in agreement with the substrate-inhibition effect of 0F overexpression.