Caulobacter Cell Cycle Control

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Model expansion

A consensus picture of cell cycle controls in Caulobacter

In this section we describe those elements of the cell division regulatory system in Caulobacter that are important for formulating our model.

CtrA and GcrA: master regulatory proteins

Caulobacter crescentus has 3,767 protein-encoding genes (Nierman et al., 2001), of which 553 are cell-cycle regulated (Laub et al., 2000). Two master-regulator proteins control more than 25% of cell-cycle regulated genes: the transcription factor CtrA (Quon et al., 1998) directly regulates 95 genes (including divK, ccrM, podJ, pleC, ftsZ, ftsQ) (Domian et al., 1996; Laub et al., 2002), while GcrA controls 49 genes (Laub et al., 2000; Laub et al., 2002; Holtzendorff et al., 2004). There is also evidence (McAdams, 2005) that CtrA can upregulate dnaA, although available microarray data (Laub et al., 2002) do not provide convincing support for this interaction. In addition, DNA synthesis in C. crescentus is under direct control by CtrA (Gorbatyuk and Marczynski, 2005; Marczynski and Shapiro, 2002; Marczynski and Shapiro, 1992), which binds to the origin of DNA replication and inhibits initiation of DNA synthesis (Quon et al., 1998).

CtrA is present at a high level in swarmer cells, whereas in stalked cells it changes from low to high level during the cell cycle (Domian et al., 1999; Grunenfelder et al., 2001; Holtzendorff et al., 2004). The abundance and activity of CtrA protein is regulated through gene transcription, protein degradation and phosphorylation.

Expression of ctrA is under control of two promoters, ctrA-P1 and ctrA-P2 (Domian et al., 1996; Domian et al., 1997; Domian et al., 1999). The weaker ctrA-P1 promoter is activated in the early stalked cell (~35 minutes after the initiation of DNA replication (Stephens et al., 1996; Reisenauer and Shapiro, 2002)) by GcrA protein (Holtzendorff et al., 2004) and inhibited by high levels of CtrA itself (Domian et al., 1999). The stronger ctrA-P2 promoter is activated later, in predivisional cells, by CtrA protein itself (Domian et al., 1999). In addition, the ctrA-P1 promoter is only activated from a new strand of hemimethylated DNA (Domian et al., 1996; Reisenauer and Shapiro, 2002). The ctrA-P2 promoter is not active in swarmer cells, even though these cells have high levels of CtrA (Domian et al., 1999). Furthermore, expression from ctrA-P2 is inhibited in predivisional cells by conditions that inhibit DNA replication (Wortinger et al., 2000). These facts indicate that ctrA-P2 has regulators other than CtrA itself (Domian et al., 1999).

Proteolysis of CtrA (and CtrA~P) is significantly accelerated by the phosphorylated form of DivK protein, DivK~P, via the ClpXP protease pathway (Hung and Shapiro, 2002), or with the help of some other (yet unknown) histidine phosphotransferases (Wu et al., 1999). Recently, RcdA and CpdR proteins have been reported to be involved in CtrA degradation in combination with ClpXP (Chen and Stephens, 2007; Ebersbach and Jacobs-Wagner, 2007). When this proteolysis pathway is activated, the half-life of CtrA in vivo is 5 min or less (Domian et al., 1997).

CtrA is active when phosphorylated (Jacobs et al., 2003; Quon et al., 1996), a reaction carried out by a histidine kinase, CckA (Jacobs et al., 1999; Jacobs et al., 2003), and a histidine phosphotransferase, ChpT (Biondi et al., 2006). In addition, CtrA is also phosphorylated by a tyrosine kinase, DivL (Pierce et al., 2006). CtrA is rapidly dephosphorylated in vivo. The activity of CckA was shown recently to be downregulated by a DivK~P (Chen and Stephens, 2007; Ebersbach and Jacobs-Wagner, 2007; Biondi et al., 2006), thereby linking the phosphorylation and proteolysis pathways of CtrA. But, otherwise, how the kinase and phosphatase reactions are regulated to control the fraction of active CtrA is poorly understood.

GcrA is an activator of components of the replisome and of the segregation machinery (Holtzendorff et al., 2004) and also regulates genes like ctrA, pleC and podJ (Holtzendorff et al., 2004; Collier et al., 2006). GcrA protein concentration varies through the cell division cycle, peaking early in the cycle in stalked cells and reaching its minimum in a swarmer cell, after cell division. The DNA replication-initiating protein, DnaA, is required for gcrA expression (Hottes et al., 2005). In addition, transcription of gcrA is repressed by CtrA protein (Holtzendorff et al., 2004).

DNA replication

DNA replication proceeds in three phases: initiation, elongation and termination. The origin of DNA replication (C_ori) in C. crescentus has one potential binding site for DnaA, a protein involved in initiating DNA synthesis (Gorbatyuk and Marczynski, 2001). The DnaA binding site partially overlaps with five CtrA binding sites in C_ori (Gorbatyuk and Marczynski, 2005; Marczynski and Shapiro, 2002; Marczynski and Shapiro, 1992). CtrA represses initiation of DNA replication (Quon et al., 1998). Thus, DNA replication is only initiated when DnaA level is high and CtrA level is low. In addition, the origin site has to be fully methylated for DNA replication to re-license the initiation (Zweiger et al., 1994; Reisenauer et al., 1999). These conditions prevail during the swarmer-to-stalked cell transition, and just after division in the stalked cell compartment (Marczynski and Shapiro, 2002). Once initiated, DNA synthesis continues bidirectionally along the circular chromosome, with an average speed of ~20.5 kb/min in minimal broth, finishing in the late predivisional cell (Dingwall and Shapiro, 1989). Elongation of newly replicating DNA strands requires a complex machinery, many components of which are under GcrA control (Holtzendorff et al., 2004).

DNA methylation by CcrM

Several cell cycle-related genes (ctrA, gcrA, dnaA, ftsZ and ccrM) have GANTC methylation sites in their promoters (Collier et al., 2006; Domian et al., 1996; Reisenauer et al., 1999; Reisenauer and Shapiro, 2002; Zweiger et al., 1994; Zweiger and Shapiro, 1994; Collier et al., 2007). Hence, the expression of these genes may be sensitive to the methylation state of the promoter. DNA replication transforms a fully methylated gene (both strands methylated) into a pair of hemimethylated genes (only one strand methylated). At some later time, the unmethylated strands become methylated by the action of CcrM to return the genes to the fully methylated state (Reisenauer et al., 1999). These methylation transitions may affect the expression of cell cycle-related genes (Reisenauer et al., 1999). Methylation of C_ori is also necessary for initiating a new round of DNA synthesis (Marczynski and Shapiro, 2002). These methylation effects provide feedback from the progression of DNA replication to the cell cycle control system.

In C. crescentus and other α-proteobacteria, CcrM is the methyltransferase that accounts for methylation of newly synthesized DNA strands. ccrM transcription is activated by CtrA only from a hemimethylated chromosome for about 20 min, in a late predivisional cell (its expression peaks at ~105 min in the 150 min swarmer cell cycle) (Stephens et al., 1995). Lon protease is required for CcrM degradation (Wright et al., 1996). The half-life of CcrM is less than 10 min in vivo (Stephens et al., 1996). Thus, CcrM activity is limited to a short portion of the predivisional cell phase, just before cell division.

The septal of Z-ring

The multicomponent Z-ring organelle, which forms and constricts at the mid-cell plane, plays an important role in compartmentation of the predivisional cell and its subsequent division (Brun and Shimkets, 2000). Compartmentation lasts about 20 min (Judd et al., 2003). After the late predivisional cell is divided into two progeny cells, the Z-ring is disassembled and degraded.

The Fts proteins (FtsZ, FtsQ, FtsA, FtsW) have been identified as crucial elements of the Z-ring. ftsZ expression is positively and negatively regulated by CtrA (Jensen et al., 2002), and it may also by regulated by DNA methylation since the ftsZ promoter has a methylation site (Reisenauer and Shapiro, 2002; Reisenauer et al., 1999). The ftsQ gene is expressed only after CtrA-mediated activation in the late predivisional cell (Wortinger et al., 2000). The FtsQ protein localizes predominantly to the mid-cell plane of the predivisional cell, consistently with the appearance of the Z-ring (Martin et al., 2004; Ohta et al., 1997). The FtsA protein exhibits the time course similar to FtsQ (Martin et al., 2004).

Polar distribution of DivK and DivK~P

divK transcription is activated by CtrA in late predivisional cells, which results in a slight elevation of DivK protein, otherwise present throughout the cell cycle at a nearly constant level (Hung and Shapiro, 2002; Jacobs et al., 2001). The total amount of DivK~P, the form which promotes CtrA degradation, does not appear to undergo dramatic changes during the cell cycle. It is 50±20% lower in swarmer cells than in predivisional cells (Jacobs et al., 2001). However, DivK and DivK~P are dynamically localized during the cell division cycle (Jacobs et al., 2001; Lam et al., 2003; McGrath et al., 2004; Skerker and Laub, 2004; Wheeler and Shapiro, 1999; Matroule et al., 2004). Membrane-bound proteins DivJ and PleC, which localize at stalked and flagellated cell poles respectively, regulate this process (McGrath et al., 2004; Skerker and Laub, 2004) by having opposite effects on DivK phosphorylation. DivJ is a kinase that continuously phosphorylates DivK at the stalked cell pole, and PleC promotes the continuous dephosphorylation of DivK~P at the flagellated cell pole (McGrath et al., 2004; Matroule et al., 2004). Hence, opposing gradients of DivK and DivK~P are established between the two cell poles. Full constriction of the Z-ring disrupts the diffusion of DivK between the two poles (Judd et al., 2003; McGrath et al., 2004). As a result, DivK~P accumulates in the nascent stalked cell compartment and unphosphorylated DivK accumulates in the nascent swarmer cell compartment. High DivK~P promotes CtrA degradation in the stalked cell compartment (Hung and Shapiro, 2002; Wu et al., 1999), while high CtrA is maintained in the swarmer cell compartment (Ryan et al., 2004). The nonuniform distribution of DivK and DivK~P, and their corresponding effects on CtrA degradation, contribute largely to the different developmental programs of swarmer and stalked cells in C. crescentus. In addition, recent investigations indicate that CtrA phosphorylation is also at least partially under the control of DivK~P (as mentioned above), which shows that DivK~P not only controls the stability of CtrA, but also its activity (Chen and Stephens, 2007; Ebersbach and Jacobs-Wagner, 2007).