Polar remodeling and histidine kinase activation, which is essential for Caulobacter cell cycle progression, are dependent on DNA replication initiation.

The Caulobacter cell cycle progresses through a series of consecutive stages: the differentiation of a swarmer cell into a stalked cell, the initiation of chromosome replication, the onset of remodeling of the new cell pole, segregation of the newly replicated chromosomes, flagellum biogenesis, and cell division (6, 27, 32, 38). Cell cycle and polar differentiation events are interdependent processes, and checkpoints are in place to ensure that both cell cycle and differentiation processes are completed before the next stage is initiated (27, 54). The orchestrated coordination of cell cycle events results in the formation of two distinct cell types, a motile swarmer cell and a sessile stalked cell. The swarmer cell has a single polar flagellum, several polar pili, and a polar chemotaxis complex. As Caulobacter moves through the cell cycle, the cell poles undergo critical remodeling (see Fig. ​Fig.6A).6A). The flagellum and pili are lost, and a stalk grows at that pole, thereafter maintaining a stalked pole identity (6, 26). Subsequently, the pole opposite the stalk eventually acquires a flagellum, pili, and chemotaxis proteins. FIG. 6. Initiation of DnaA replication controls new pole remodeling and CckA kinase activation required for cell cycle progression. (A) Schematic of the Caulobacter cell cycle in a wild-type strain. The flagellum-bearing swarmer cell possesses a single chromosome ... In Caulobacter, if the initiation of DNA replication is inhibited, many temporally controlled events fail to occur. These include flagellum biosynthesis, synthesis of the CcrM DNA methyltransferase, and cell division (10, 11, 47, 48, 54). The essential CtrA response regulator, in its phosphorylated state, is central to the regulation of these cell cycle events. Flagellar genes, grouped in a complex regulatory hierarchy, are expressed sequentially; the order of expression corresponds to the order of assembly of the gene products into the growing flagellum. The expression of the class IV flagellar genes requires the previous expression of the class III genes, and this requires the expression of the class II flagellar genes (26, 56). CtrA functions as a class I gene controlling the transcription of class II flagellar genes (39, 57). CtrA also controls cytokinesis by regulating the expression of genes encoding essential components of cell division apparatus, including FtsZ, FtsA, and FtsQ (30, 54). In addition, CtrA activates the transcription of genes involved in chemotaxis, chromosome methylation, and pilus formation (29, 41, 45). In the swarmer cell, CtrA is in the active phosphorylated state, where it binds to the origin of replication and blocks the initiation of replication (12, 40). Upon differentiation of a swarmer cell into a stalked cell (12), CtrA is cleared from the cell, allowing replication to begin. After DNA replication has initiated, CtrA accumulates and is activated by phosphotransfer from the essential CckA hybrid kinase through the ChpT phosphotransferase (3, 23). It has been shown that the accumulation of CtrA requires the initiation of DNA replication (54). The expression of ctrA is under the control of two promoters, P1 and P2. P1 is a weak promoter that is active only in the late stalked cell, following the initiation of DNA replication (13). In a positive feedback loop, phosphorylated CtrA activates the strong ctrA P2 promoter in the late predivisional cell (13). It was shown that if DNA replication is inhibited, CtrA is depleted due to the inhibition of the ctrA P2 promoter (54). Because the activation of the P2 promoter requires CtrA in its phosphorylated state, the activation of the CckA phospho-signaling pathway may be the primary responder to a DNA replication checkpoint. The subcellular localization of CckA changes during the cell cycle; it accumulates at the cell poles following DNA replication initiation (2, 24). We have recently shown that CckA is autophosphorylated and activated when localized at the new cell pole (20). In addition, we demonstrated that CckA autophosphorylation, activation, and localization at the new pole is dependent on the essential DivL protein kinase, which predominantly localizes at the new pole in the same protein complex with CckA (20). Because polar localization of DivL and CckA occurs following replication initiation, we asked if the initiation of DNA replication is required for the localization of DivL and CckA at the new cell pole and, consequently, for CckA phosphorylation and activation. Here, using three different methods to inhibit replication initiation, we demonstrate that the initiation of DNA replication is required for localizing both DivL and CckA at the new cell pole and for CckA autophosphorylation and activation. We ascertained that replication initiation, and not segregation, is the essential element for CckA new pole localization and autophosphorylation. These results argue that the initiation of DNA replication is an important cell cycle requirement for the activation of the CckA hybrid kinase and, consequently, via activated CtrA∼P, the expression of multiple cell cycle events, including flagellum and pilus formation, chemotaxis, DNA methylation, and cell division.