Simulations for wild-type stalked cell cycle
Figure 5. Simulated variations of model state variables during the wild-type cell cycle. Here and in subsequent figures, the simulation begins when the initiation of DNA replication has completed. Three cell cycles are presented.
(A) Here and in subsequent figures, the scale for [Ini] is on the left and the scales for [Elong] and [DNA] are on the right. DNA replication (green curve) takes 90 min, as observed (Dingwall and Shapiro, 1989). Initiation (red) is elevated only for a short period of time to start DNA replication. The DNA curve (green) differs from the Elongation curve (blue) only by an additive constant equal to the number of full chromosomes in the cell.
(B) The methylation states of Cori and of three genes. As replication starts, Cori(red) is hemimethylated (hCori = 1), and ccrM (blue), ctrA (green), fts (black) and dnaA (not shown) are fully methylated (h.. = 0). As the replication forks proceed along the chromosome, these genes become hemimethylated in order, according to their positions on the chromosome. At the end of the cycle, when CcrM is active, all these sites become fully methylated.
(C) Early in the cycle, GcrA (blue) is increasing and triggers production of CtrA (red). When CtrA is high, it represses synthesis of GcrA and activates its own degradation by upregulating DivK~P (green).
(D) When a cell enters the predivisional phase, high CtrA activates the expression of fts genes (blue), which promote formation and constriction of the Z-ring (red). DnaA (green) and CcrM (black) are required for DNA initiation (panel A) and DNA methylation (panel B), respectively.
Figure 5 illustrates how scaled protein concentrations and other variables of the model change during repetitive cycling of a stalked cell. The duration of a wild-type stalked-cell division cycle in our simulations is 120 min (~90 min for S phase and ~30 min for G2/M phase), as typically observed in experiments (Ryan and Shapiro, 2003; Ausmees and Jacobs-Wagner, 2003; Judd et al., 2003).
The main physiological events of the division cycle can be traced back to characteristic signatures of protein expression, as described in the Introduction. The division cycle starts with initiation of DNA replication (Figure 5A) from a fully methylated origin site by elevated DnaA, when CtrA is low and GcrA is sufficiently high (to induce production of required components of the replication machinery) (Figure 5C, D). Immediately after DNA replication starts, Cori is hemimethylated.
As DNA synthesis progresses, certain genetic loci become hemimethylated in order along the chromosome (Figure 5B). Consequently, the regulatory proteins are produced and reach their peak concentrations sequentially. By contrast, dnaA expression seems to be activated by full methylation (Zweiger and Shapiro, 1994), so its expression rate declines immediately after DNA replication starts. The effect of methylation on dnaA expression is minor compared to the regulatory signals coming from GcrA and CtrA. When the replication fork passes the ccrM locus, the gene becomes available for transcription but is not immediately expressed because CtrA level is low. In a predivisional cell, at ~35 min after start of DNA replication, the replication fork passes the ctrA gene (Figure 5B) and its expression is immediately activated by GcrA (Figure 5C) and then further upregulated by CtrA itself. Later on, when CtrA level becomes high, expression of the ccrM gene and later hemimethylated fts genes (at ~ 65 minutes), are expressed by the activation from high level CtrA (Figure 5D).
High CtrA downregulates gcrA expression. When DNA replication is finished, the new DNA strands are methylated by elevated CcrM in about 20 min. DNA methylation shuts down production of CtrA, CcrM and Fts proteins. Meanwhile, elevated Fts proteins promote Z-ring formation and constriction (Figure 5D), which separates the predivisional cell into two compartments, thereby restricting access of DivK and DivK~P to only one of the old poles of the cell. As a result, in the stalked cell compartment, most DivK is converted into DivK~P, accelerating CtrA proteolysis there (Figure 5C). In a nascent stalked cell, low CtrA concentration releases gcrA expression, and GcrA protein level rises. Then, low CtrA, high GcrA and high DnaA drive the nascent stalked cell into a new round of DNA synthesis from the fully methylated chromosome. These computed properties of the model agree reasonably well with what is known (or expected) about cell cycle progression in C. crescentus.
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