Cell cycle regulator phosphorylation stimulates two distinct modes of binding at a chromosome replication origin

In Caulobacter crescentus, the global response regulator CtrA controls chromosome replication and determines the fate of two different cell progenies. Previous studies proposed that CtrA represses replication by binding to five sites, designated [a–e], in the replication origin. We show that phosphorylated CtrA binds sites [a–e] with 35‐ to 100‐fold lower Kd values than unphosphorylated CtrA. CtrA phosphorylation stimulates two distinct modes of binding to the replication origin. Phosphorylation stimulates weak intrinsic protein–protein cooperation between half‐sites and does not stimulate CtrA–P binding unless protein–DNA contacts are made at both half‐sites. CtrA phosphorylation also stimulates cooperative binding between complete sites [a] and [b]. However, binding to each of the other CtrA‐binding sites [c], [d] and [e] is completely independent and suggests a modular organization of replication control by CtrA. We therefore propose a model where a phosphorelay targets separate biochemical activities inside the replication origin through both cooperative and independent CtrA‐binding sites.