The CtrA Response Regulator Essential for Caulobacter Crescentus Cell-cycle Progression Requires a Bipartite Degradation Signal for Temporally Controlled Proteolysis

The two-component signaling protein CtrA activates or represses the expression of one-quarter of the cell-cycle-regulated genes in Caulobacter crescentus, integrating DNA replication, morphogenesis, and cell division. The activity of this essential protein is controlled by a positive transcriptional feedback loop, cell-cycle-regulated phosphorylation, and rapid proteolysis as cells enter S-phase at the swarmer-to-stalked cell transition and in the stalked portion of the asymmetric predivisional cell. CtrA activity must be removed from cells at the onset of DNA replication, because phosphorylated CtrA binds to and silences the origin of replication. The ClpXP protease is required for CtrA proteolysis but is present throughout the cell-cycle, so the mechanism for activating and deactivating CtrA proteolysis is unknown. Here, we identify a bipartite proteolytic signal in the CtrA response regulator consisting of two determinants that are each necessary but not sufficient for regulated degradation. One determinant is present in the last 15 amino acid residues of CtrA, particularly the terminal Ala-Ala residues, and another is located within the first 56 residues of the CtrA receiver domain. A fusion of the receiver domain and last 15 residues of CtrA to YFP is properly degraded in living cells. Although the N-terminal 56 residues contain the conserved Asp51 phosphorylation site, mutant analyses show that cell-cycle-controlled CtrA proteolysis is insensitive to the CtrA phosphorylation state. The N-terminal proteolytic determinant is predicted to reside on the surface of the receiver domain in beta-sheet 2 and alpha-helix 2.