Dynamics of Two Phosphorelays Controlling Cell Cycle Progression in Caulobacter crescentus

In Caulobacter crescentus, progression through the cell cycle is governed by the periodic activation and inactivation of the master regulator CtrA. Two phosphorelays, each initiating with the histidine kinase CckA, promote CtrA activation by driving its phosphorylation and by inactivating its proteolysis. Here, we examined whether the CckA phosphorelays also influence the downregulation of CtrA. We demonstrate that CckA is bifunctional, capable of acting as either a kinase or phosphatase to drive the activation or inactivation, respectively, of CtrA. By identifying mutations that uncouple these two activities, we show that CckA''s phosphatase activity is important for downregulating CtrA prior to DNA replication initiation in vivo but that other phosphatases may exist. Our results demonstrate that cell cycle transitions in Caulobacter require and are likely driven by the toggling of CckA between its kinase and phosphatase states. More generally, our results emphasize how the bifunctional nature of histidine kinases can help switch cells between mutually exclusive states.Caulobacter crescentus is a tractable model system for understanding the molecular mechanisms underlying cell cycle progression and the establishment of cellular asymmetry in bacteria. Each cell division for Caulobacter produces two morphologically different daughter cells, a swarmer cell and a stalked cell, which also differ in their ability to initiate DNA replication. A stalked cell can immediately initiate DNA replication following cell division, whereas a swarmer cell cannot initiate until after differentiating into a stalked cell. The swarmer-to-stalked cell transition thus coincides with a G₁-S cell cycle transition. DNA replication occurs once and only once per cell cycle, resulting in distinguishable G₁, S, and G₂ phases.Progression through the Caulobacter cell cycle requires the precise temporal and spatial coordination of both morphological and cell cycle events. Previous genetic screens have uncovered numerous two-component signal transduction genes that help to regulate these events (10, 11, 17, 24, 25, 29, 33, 34, 42). Two-component signaling pathways are typically comprised of a sensor histidine kinase that, upon activation, autophosphorylates and subsequently transfers its phosphoryl group to a cognate response regulator, which can then effect changes in cellular physiology (35). One common variation of this signaling paradigm is called a phosphorelay (3). Such pathways also initiate with the autophosphorylation of a histidine kinase and subsequent phosphotransfer to a response regulator, but these steps often occur intramolecularly within a hybrid histidine kinase. The phosphoryl group on the receiver domain of a hybrid kinase is then passed to a histidine phosphotransferase, which subsequently phosphorylates a soluble response regulator to effect an output response. Relative to canonical two-component pathways, phosphorelays provide additional points of control and enable signal integration; they are often involved in regulating key cell fate decisions in processes such as sporulation, cell cycle transitions, and quorum sensing (1, 3, 8).The master regulator of the Caulobacter cell cycle is CtrA, an essential response regulator that directly activates the expression of at least 70 genes (19, 29). CtrA also regulates DNA replication by binding to and silencing the origin of replication (30). Progression through the Caulobacter cell cycle thus requires the precise control of CtrA activity. CtrA must be abundant and active thoughout most of the cell cycle to drive gene expression and to silence the origin but must be temporarily inactivated in stalked cells prior to S phase to permit the initiation of DNA replication (see Fig. ​Fig.88).Open in a separate windowFIG. 8.Regulation of the balance between CckA kinase and phosphatase activities controls the cell cycle. (A) Left, summary of regulatory pathway controlling CtrA activity. Right, schematic of Caulobacter cell cycle indicating temporal pattern of CtrA activity. (B) Summary of cell cycle phosphorelays. Net phosphate flow depends on the activity of CckA. As a kinase, CckA drives the phosphorylation of CtrA and CpdR. As a phosphatase, CckA drives the dephosphorylation of CtrA and CpdR. Cell cycle transitions and changes in CtrA activity are thus driven by changes in the kinase/phosphatase balance of CckA. DivK influences CckA''s switching between kinase and phosphatase states. P_i, inorganic phosphate.CtrA is regulated on at least three levels: transcription, proteolysis, and phosphorylation (4, 5). During G₁, CtrA is phosphorylated and proteolytically stable. At the G₁-S transition, CtrA is dephosphorylated and degraded, thereby freeing the origin of replication to fire. After DNA replication initiates, ctrA is transcribed and the newly synthesized CtrA is again phosphorylated and protected from proteolysis. Following septation of the predivisional cell, CtrA remains phosphorylated and stable in the swarmer cell but is dephosphorylated and degraded in the stalked cell to permit DNA replication initiation. Cells that constitutively transcribe ctrA are viable and display only a mild phenotype, indicating that regulated phosphorylation and proteolysis alone can ensure the periodicity of CtrA activity (4). Cells producing nondegradable, constitutively active CtrA arrest in G₁ because CtrA activity cannot be eliminated (4).The regulation of CtrA activity involves two phosphorelays. Each initiates with CckA, a hybrid histidine kinase, and ChpT, a histidine phosphotransferase. After receiving a phosphoryl group from CckA, ChpT can act as the phosphodonor for either CtrA or the single-domain response regulator CpdR (1). Phosphorylation of CpdR prevents it from triggering CtrA proteolysis (1, 14). Unphosphorylated CpdR triggers CtrA degradation by somehow influencing the polar localization of the protease ClpXP (14), although why the protease must be localized is unclear.The downregulation of CtrA prior to DNA replication involves the dephosphorylation of CtrA and CpdR such that CtrA is both dephosphorylated and, ultimately, degraded. These events coincide with the time in the cell cycle when CckA''s kinase activity is lowest (16). As the phosphoryl groups on phosphorylated CtrA (CtrA∼P) and CpdR∼P are relatively stable, at least in vitro (1), phosphatases are likely critical to eliminating CtrA activity prior to S phase. For some phosphorelays, inactivation of the top-level kinase leads to a siphoning of phosphoryl groups from the terminal regulator back to the hybrid kinase''s receiver domain. The bifunctional hybrid kinase may then act as a phosphatase, stimulating hydrolysis and loss of the phosphoryl group (7, 8). For other phosphorelays, there are separate and dedicated phosphatases (23, 26, 27).Here, we demonstrate that CckA is bifunctional and can act as both a kinase and a phosphatase, such that inactivation of CckA as a kinase stimulates the dephosphorylation of CtrA∼P and CpdR∼P. We provide evidence that CckA''s phosphatase activity contributes to the downregulation of CtrA in vivo but that other phosphatases may exist. Our results indicate that the periodic toggling of CckA between kinase and phosphatase states is crucial to cell cycle progression in Caulobacter.