Polar Localization of the CckA Histidine Kinase and Cell Cycle Periodicity of the Essential Master Regulator CtrA in Caulobacter crescentus

The phosphorylated form of the response regulator CtrA represses DNA replication initiation and regulates the transcription of about 100 cell cycle-regulated genes in Caulobacter crescentus. CtrA activity fluctuates during the cell cycle, and its periodicity is a key element of the engine that drives cell cycle progression. The histidine kinase CckA controls the phosphorylation not only of CtrA but also of CpdR, whose unphosphorylated form promotes CtrA proteolysis. Thus, CckA has a central role in establishing the cell cycle periodicity of CtrA activity by controlling both its phosphorylation and stability. Evidence suggests that the polar localization of CckA during the cell cycle plays a role in CckA function. However, the exact pattern of CckA localization remains controversial. Here, we describe a thorough, quantitative analysis of the spatiotemporal distribution of a functional and chromosomally produced CckA-monomeric green fluorescent protein fusion that affects current models of cell cycle regulation. We also identify two cis-acting regions in CckA that are important for its proper localization and function. The disruption of a PAS-like motif in the sensor domain affects the stability of CckA accumulation at the poles. This is accompanied by a partial loss in CckA function. Shortening an extended linker between β-sheets within the CckA catalysis-assisting ATP-binding domain has a more severe effect on CckA polar localization and function. This mutant strain exhibits a dramatic cell-to-cell variability in CpdR levels and CtrA cell cycle periodicity, suggesting that the cell cycle-coordinated polar localization of CckA may be important for the robustness of signal transduction and cell cycle progression.The alphaproteobacterium Caulobacter crescentus provides a model system that has been particularly useful for unraveling the bacterial cell cycle (2, 5, 7, 16, 20, 42). Its life cycle is characterized by an asymmetric cell division that yields two distinct daughter cells, the motile swarmer cell and the sessile stalked cell. The swarmer cell grows into a stalked cell, during which time DNA replication is initiated. The stalked cell then proceeds with cell elongation, polar morphogenesis, cell division, and daughter cell separation (Fig. ​(Fig.1A1A).Open in a separate windowFIG. 1.CckA signal transduction pathway and its role in cell cycle regulation in C. crescentus. (A) The C. crescentus cell cycle is driven by the periodicity of CtrA∼P. When CtrA∼P is present, the initiation of DNA replication is inhibited. During the swarmer-to-stalked (SW-ST) cell transition, CtrA is recruited to the old pole and degraded, allowing DNA replication to initiate. CtrA∼P reaccumulates later in the ST and predivisional (PD) cell stages due to transcriptional activation and ensuing phosphorylation. The black circle represents the chromosome, while the asterisk shows the late PD stage when cells are compartmentalized due to cytokinesis preceding daughter cell separation. (B) The CckA signal transduction pathway regulates cell cycle progression and polar morphogenesis. Curved arrows between proteins indicate the flow of phosphoryl groups, while straight lines indicate activation or inhibition.His-Asp phosphorelay systems, which are one of the predominant stimulus response systems in prokaryotic cells (50), play an essential role in coordinating cell cycle progression in C. crescentus (2, 5, 42). The membrane-bound hybrid histidine kinase CckA is involved in two interconnected phosphorelays that regulate the DNA-binding activity of the CtrA response regulator (Fig. ​(Fig.1B).1B). In its active phosphorylated form, CtrA∼P inhibits the initiation of DNA replication while regulating the expression of about 100 cell cycle genes, including genes involved in cell division and flagellar and pilus biosynthesis (9, 34, 43, 44). In the first phosphorelay, CckA, via the single-domain histidine phosphotransferase ChpT (1), mediates the phosphorylation of CtrA (25, 26). In the second phosphorelay, CckA, again through the intermediate ChpT, mediates the phosphorylation and thereby the inactivation of the single-domain response regulator CpdR (Fig. ​(Fig.1B)1B) (1, 23). In its unphosphorylated, active form, CpdR promotes the polar localization of the ClpXP protease, leading to the regulated proteolysis of CtrA (23, 28, 38, 45). Thus, the activation of CckA through autophosphorylation both activates CtrA through phosphorylation and prevents the targeted degradation of CtrA through the phosphorylation of CpdR.The response regulator CtrA is a key factor in driving the C. crescentus cell cycle, and as such its activity is tightly regulated at the transcriptional level and at the posttranslational level (2, 5), leading to the periodicity of active CtrA∼P during the cell cycle (Fig. ​(Fig.1A).1A). CtrA∼P is present in swarmer cells (8, 25), where it binds to five sites within the chromosomal origin region to inhibit the initiation of DNA replication (44). This inhibition is relieved later through the dephosphorylation and ClpXP-dependent proteolysis of CtrA∼P during the swarmer-to-stalked cell transition (8, 28). The proteolysis of CtrA is triggered by multiple proteins and polar events (10, 38, 41), including the localization of unphosphorylated CpdR to the stalked pole, where it helps recruit the ClpXP protease (23). The essential single-domain response regulator DivK also has been proposed to negatively regulate CtrA activity during this swarmer-to-stalked cell transition by affecting CckA and CpdR function (1, 22, 24, 56). CtrA∼P reaccumulates in predivisional cells through de novo transcription and then phosphotransfer by the CckA pathway (1, 9, 19, 25, 43, 49). The concomitant inactivation of CpdR by phosphorylation through CckA-ChpT promotes the stabilization of active CtrA∼P (1, 23), allowing the transcriptional control of multiple genes involved in cell cycle progression and polar morphogenesis (34). Following cytokinesis (i.e., the partitioning of the cytoplasm into distinct compartments), which occurs before cell separation (30), CtrA∼P is cleared from the stalked compartment through dephosphorylation and targeted proteolysis while remaining stable in the swarmer compartment (8) (Fig. ​(Fig.1A).1A). This yields differential cell fate expression programs between the two progeny.CckA is at the top of the phosphorelays that control the periodic nature of CtrA activity during the cell cycle. While the CckA protein remains at constant levels throughout the cell cycle (26), its subcellular distribution varies between fairly dispersed membrane distribution and polar localization during the cell cycle. The CckA cell cycle localization pattern in general and the timing of its polar accumulation in particular are, however, controversial (1, 26). A precise and accurate knowledge of the spatial distribution of CckA during the cell cycle is critical for our understanding of cell cycle regulation in C. crescentus. For instance, a recent report proposes that the polar localization of CckA may be correlated with the activation of CtrA∼P and the subsequent inhibition of DNA replication initiation, and conversely the dispersion of CckA from the pole is accompanied by the inactivation of CtrA∼P and the subsequent initiation of DNA replication (1). This led to a model in which DivK∼P causes the delocalization and inactivation of CckA from the old pole, and this displacement causes CtrA∼P inactivation (1). However, this sequence of cell cycle events is incompatible with the original description of the CckA localization pattern during the cell cycle (26).Here, we report a quantitative analysis of the spatial distribution of CckA during the cell cycle that affects the current models of cell cycle regulation. We also identify two regions within CckA that contribute to the proper polar localization of CckA and show that these regions are important for protein function. Finally, we present data suggesting that the proper polar localization of CckA may be critical for the cell cycle periodicity of CtrA levels, which drives cell cycle progression and polar morphogenesis.