The relative abundance of CtrA in a daughter cell right after division determines the morphology of the cell: CtrA is elevated in the incipient swarmer cell, but it is degraded to very low level in the stalked cell compartment. At some later time, CtrA must be degraded in the swarmer cell during its transition to the stalked morphology. The level of CtrA in the cell is determined by the balance between protein synthesis and degradation, which processes are regulated in turn by the actions of other proteins. Production of CtrA is initiated by GcrA ^[40], promoted by CtrA itself ^[41], and is a subject to the methylation state of the ctrA gene ^[42].

Once synthesized, CtrA protein must be phosphorylated into an active form, CtrA?P, to perform its functions ^[29]. During the division cycle of wild type cells, CtrA?P follows closely the time course of CtrA (with less than 10 min delay) ^[27],^[29],^[31],^[40], in some mutant cells this synchrony is lost ^[36],^[37].

The CckA histidine kinase contributes to CtrA phosphorylation. The cckA gene is expressed only briefly during the swarmer-to-stalked cell transition ^[36]. Otherwise, the total CckA level is stable throughout the cell cycle ^[43]. CckA molecules become localized and phosphorylated during the cell cycle. When phosphorylated, CckA?P activates CtrA phosphorylation ^[29]. During the cell cycle, CckA?P concentration is tightly correlated with CtrA?P concentration (they show similar pattern of variation) ^[36],^[44]. Because CckA contributes to the phosphorylation of several cell-cycle control proteins, there may be competition for phosphate groups among its targets. (We do not attempt to model such effects at this time.) In addition, DivL is reportedly involved, to some extent, in the process of CtrA phosphorylation ^[29],^[45].

Degradation of CtrA and its phosphorylated form, CtrA?P, is also regulated. The phosphorylated protein, DivK?P, accelerates proteolysis of both forms of CtrA ^[46]. This effect was used in our previous model to define the time when CtrA starts being degraded. The mechanism of CtrA proteolysis is complicated. The protease ClpXP is directly involved in degrading CtrA and CtrA?P, with the help of another protein, RcdA, whose production is activated by CtrA?P ^[35],^[47]. Although ClpXP is stable throughout the cell cycle ^[48], even though its promoter activity varies ^[49], the localization of ClpXP is regulated by the CpdR protein. When CpdR is dephosphorylated and localized, it recruits ClpXP to the cell poles where CtrA/CtrA?P is degraded. Thus, the activation of CpdR by dephosphorylation can be viewed as another determining factor for CtrA and CtrA?P degradation ^[38].

Finally, it needs to be mentioned that the pathways of CtrA phosphorylation and proteolysis are interwoven. High DivK?P represses CckA phosphorylation ^[50], thus down-regulating indirectly CtrA phosphorylation and CtrA activity. CckA?P activates CpdR phosphorylation and affects CpdR localization, which represses indirectly the degradation CtrA and CtrA?P ^[35],^[50].

The histidine kinases PleC and DivJ are two proteins that facilitate dephosphorylation and phosphorylation of DivK respectively, during the cell cycle. According to microarray data, pleC expression is activated by GcrA ^[40], but total PleC protein level is relatively stable during the cell cycle ^[34]. The protein localizes to the flagellar cell pole once expressed. Localization of the protein is required for its function, and the full length PodJ_L affects PleC localization ^[51].

Expression of the polar organelle development gene podJ is upregulated by GcrA and DnaA and downregulated by CtrA?P ^[31],^[40],^[41],^[52],^[53]. The PodJ protein has two distinct isoforms: the full-length translation product PodJ_L and a truncated form PodJ_S. PodJ_L localizes to the incipient swarmer pole, where it helps to recruit factors required for polar morphogenesis, including PleC. The periplasmic domain of PodJ_L is degraded by a periplasmic protease PerP, giving rise to a truncated form of the protein PodJ_S. Lower abundance of PodJ_L leads to the release of PleC from flagellar cell pole ^[34]. During the swarmer-to-stalked cell transition, PodJ_S is cleared and PodJ_L is synthesized and localized again ^[51].

The protease PerP is required for efficient truncation of PodJ_L. Microarray analysis shows that perP expression is activated by CtrA?P ^[41],^[54]. In addition, polar PleC activity also affects, to some extent, perP expression ^[55].

Therefore, PodJ_L, PerP and PleC work together in regulating the truncation of PodJ_L and consequent release of PleC ^[55]. This network dynamically regulates the temporal distributions of PodJ_L and PodJ_S, and the spatial localization of PleC. PleC, in turn, activates the dephosphorylation of DivK in the swarmer and predivisional cells through the cell division cycle ^[51].

DivJ is present at low concentration in a swarmer cell but increases dramatically during the swarmer-to-stalked transition ^[33]. Then, it localizes to the stalked cell pole and stays steady during the rest of the cell cycle. DivJ's localization helps the phosphorylation of DivK. PleC directly or indirectly regulates localization of DivJ ^[33],^[56]. The SpmX protein was recently reported as a factor involved in the process of DivJ localization after the release of PleC from the flagellar cell pole ^[57]. Otherwise, little is known about the regulation of DivJ in cells. High localized DivJ functions as a kinase in the stalked cell, where it activates the phosphorylation of DivK. In addition, recent data show that DivL is involved in the phosphorylation of DivK, directly or indirectly ^[58].

Because of the opposite localization of PleC and DivJ in a predivisional cell, a concentration gradient of DivK phosphorylation state is formed in predivisional cells, with DivK predominant at the swarmer pole and DivK?P predominant at the stalked pole ^[33],^[57]. After division, PleC and DivJ are separated to different compartments (nascent cells). Therefore, in the nascent swarmer cell, DivK gets quickly dephosphorylated (driven by PleC), which preserves a high level of CtrA in this compartment. In the nascent stalked cells, all DivK becomes phosphorylated due to the action of DivJ, which accelerates the degradation of CtrA. During the swarmer-to-stalked cell transition (G1/S transition), release of PleC and consequent localization of DivJ at the same pole activates DivK phosphorylation ^[57] leading to rapid degradation of CtrA.

Cytokinesis in Caulobacter is regulated by a number of factors, including DNA replication and segregation, flagellum assembly, and the master regulators CtrA and DnaA ^[53],^[59].

CtrA?P regulates transcription of a number of proteins involved in the formation and closure of the septal Z-ring, including ftsZ, ftsQ, ftsA^[60]?^[62]. In a predivisional cell and in a swarmer cell, CtrA?P represses transcription of ftsZ, the gene whose product (bacterial counterpart of tubulin) is a building block of the Z-ring ^[62]. DnaA, on the other hand, is an activator for ftsZ expression ^[53]. FtsZ was measured at maximal level in an early predivisional cell that has a visible Z-ring, then it decreased as CtrA?P level rises ^[61]. Following the assembly of the Z-ring, ftsQ and ftsA are activated by CtrA?P in an orderly manner ^[61]. Importantly, FtsQ, a regulator of Z-ring constriction, is only produced in predivisional cell phase when DNA is being replicated ^[63]. It is not produced in a swarmer cell even though CtrA?P is high in this stage of the cell cycle.

A short DNA sequence, parS, locates close to the origin of replication (C_ori). The chromosome partitioning protein, ParB, binds to parS and colocalizes with the origin of replication at the flagellated pole in swarmer cells ^[64]?^[66]. Soon after the initiation of DNA replication, one copy of the origin moves to the opposite pole of cell and, as a result, ParB starts to exhibit a bipolar localization pattern. Another chromosome partitioning protein, ParA, also shows a bipolar localization pattern in the predivisional cell and may form a complex with ParB at the origin. Depletion of ParB or overexpression of ParA results in filamentous cells that lack Z-rings or form them in improper locations ^[67]. Total amounts of ParA and ParB stay constant during the cell cycle ^[68]. Correct localization of these proteins is a necessary condition for correctly positioning the Z-ring in the middle of a cell ^[67]. Moreover, nucleotide exchange between ParA-ATP and ParA-ADP is regulated by ParB ^[69]. An increased level of ParA-ADP (which is proposed to be regulated by high ParB in the cytoplasm) inhibits Z-ring assembly and cell division ^[69]. It has been suggested that ParA-ADP is the active form of ParA that inhibits cell division by preventing the formation and constriction of the Z-ring, directly or indirectly ^[67]. In summary, DNA replication and chromosome partitioning accompanied by ParB and ParA rearrangement results in a lower level of ParB and ParA-ADP in the cytoplasm, which releases the repression of Z-ring formation and allows Z-ring constriction in the center of the cell.

In addition, CtrA?P activates class II genes for flagellum assembly, including ?^54[35],^[59]. The flagellar regulatory protein FlbD is activated by ?⁵⁴, which lead to the activation of a gene required for optimal FtsZ ring assembly. Recently, in Caulobacter, a spatial regulator coordinating chromosome segregation, MipZ, was recognized as an element with functions similar to those of the MinCDE system in E.coli (which serves to repress Z-ring formation and constriction) ^[39],^[70]. MipZ, activated by DnaA ^[39],^[71], is also involved in communication between ParB and Z-ring assembly and constriction ^[47]. Thus, Z-ring assembly and constriction are elaborately regulated, directly or indirectly, by DnaA and CtrA?P proteins, even though the details of how it happens in the cell are as yet unclear.

In this mutant the CtrA phosphorylation site, aspartate 51, is replaced with a glutamate residue. This creates a form of the CtrA which cannot be phosphorylated but is, nevertheless, able to activate downstream genes ^[73]. When the ctrAD51E gene was expressed from a high-copy number plasmid in cells deleted for the wt chromosomal ctrA gene, the mutated cells were observed to have normal morphology and DNA content (Figure 5 in ^[37]).

Since in this mutant the constitutively expressed CtrA is active all the time, we simulated the mutant by turning off CtrA production from the original gene and by producing the active form of CtrA (phophorylated form in our model) constitutively. Our simulation shows (Figure 5) that an elevated level of CtrA?P does not block progression of cells through the cell cycle. The CtrA degradation machinery is able to lower the level of CtrA?P enough that the necessary conditions for DNA replication are satisfied, while the components for the Z-ring assembly and constriction are also available when needed. Elevated CtrA?P reduces to some extent GcrA, PodJ_L/PleC, FtsZ and Zring, and accelerates DNA methylation, but these changes do not have lethal effects on cell cycle progression.

It can be observed from our simulation (Figure 5) that Z-ring closing time is a bit longer in the mutant than in wt cells. We consider this an artifact of our model, due to the oversimplified representation of the role of FtsQ. In our model, FtsQ abundance controls both the onset and duration of Z-ring constriction, while in reality this dual role may not be the case. (This artifact may also appear in other mutants.)

When the final three amino acids of CtrA are deleted (or substituted with a peptide tag), the rate of CtrA degradation is decreased to ?10% of wt rate ^[37]. Nonetheless, these mutant cells exhibit normal cellular morphology and DNA content (Figure 5 in ^[37]).

In our simulation of this mutant (Figure 6), CtrA and CtrA?P fluctuate in a manner similar to wt cells. Notice that the steady state levels of CtrA and CtrA?P during DNA synthesis phase are about 10-fold higher in the mutant than in wt cells because the degradation rate of CtrA (and CtrA?P) protein 10-fold smaller in the mutants. Nevertheless, CtrA proteolysis is still sufficient to permit DNA synthesis. Later in the cell cycle, during the ?50 min when the ctrA gene is hemimethylated and CtrA proteolysis is minimal, both wt cells and mutant cells accumulate nearly the same amount of CtrA and CtrA?P. Hence, the mutant cell undergoes normal division cycle.

This double mutant, which combines the properties of ctrAD51E and ctrA?3?, is observed to be filamentous and arrested in G1 phase ^[37]. In our simulation (Figure 7), CtrA?P rises to such a high level that DNA replication cannot be initiated, consistent with G1 arrest and a filamentous morphology. In addition, based on the variation patterns of DivJ and DivK?P, our model predicts that, after the introduction of mutation, the swarmer cell will differentiate into a stalked cell and then become arrested without initiation of DNA replication.

When ctrA is constitutively expressed (the only copy of ctrA gene is on pJS14), the cell cycle is normal ^[37]. In our simulation (Figure 8), constitutive ctrA expression at mild level (20%?80% of wild-type ctrA promoter activity) does not affect the normal cell cycle. Insignificant deviations, similar to those for the ctrAD51E mutant, were observed for some proteins, Z-ring closing time, and DNA methylation. If ctrA is expressed constitutively at <20% of the wild-type level, then CtrA?P never increases high enough to prompt expression of other essential genes, and the cell cannot proceed through the division cycle normally. The simulation results for this case are similar to those of ?ctrA mutant (data not shown here; see figure at the website http://mpf.biol.vt.edu/research/caulobacter/SWST/pp/).

If ctrA is expressed constitutively at too high level (above 80% of wild-type ctrA promoter activity), then CtrA?P reaches quite a high level even though the proteolysis pathway works normally. The high level of CtrA?P restricts the initiation of DNA replication and it cannot start at a right time. The cell cycle thus cannot proceed normally in this case. In ^[37], ctrA is expressed from a strong promoter, but the extent of overproduction is unknown. Our model predicts that the level of CtrA protein in this viable mutant should be in the range 20?80% of the wild type level because, if ctrA is greatly overexpressed (as in the ctrA^op mutant described below), then cell cycle progression is blocked.

When the genomic copy of the ctrA gene is missing a coding sequence for the last three amino acids (ctrA?3?), the resulted mutated CtrA protein is more stable ^[37]. When this gene is introduced in cells on a high copy-number plasmid (ctrA⁺ (wild type)+P_xylX-ctrA?3) and the cells are grown on 0.2% xylose to overexpress the stable (mutated) form of CtrA, then the mutant cells become filamentous and arrested either in G1 phase (unreplicated DNA) or in G2 phase (replicated DNA) ^[74].

In our simulation (Figure 9), elevated levels of DivJ and DivK?P indicate that the swarmer cell may have differentiated into a stalked cell. However, the high level of CtrA?P represses initiation of DNA replication in the mutant cell, making it arrest in G1 phase. The simulation for this case is similar to the case of ctrA constitutive expression (>80% of maximum wt rate), as just described. For the stalked cell, the division cycle can be arrested either in G1 phase or G2 phase, based on our simulation reported in ^[25].

After a longer duration (?200 min in our simulation), however, a new round of DNA replication starts in our simulation. This is a side-effect of the overly simplified term in our model for the initiation of DNA replication.

divJ^cs[75], divJ_H338A^[76], ?divJ^[33], and divJ:: ?(Spc^R)^[77] are all described as divJ deletion mutants in experiments. In these mutant cells, DivK?P level is reduced and, as a result, cells contain several copies of chromosomes and became filamentous.

In our simulation of these mutants (Figure 10), DivK?P level drops as expected, and CtrA?P stays high, repressing a new round of DNA replication after cell division. The mutant cells arrest in G1 phase and become filamentous. Without DivJ and DivK?P, the cell should remain in the swarmer cell morphology.

It has been reported that DivJ is also involved in repressing the initiation of DNA replication ^[33], a feature that is not implemented in our model. This may be the reason why we do not see, in our simulation, accumulation of multiple chromosomes, as was reported experimentally.

Elevated levels of DivK?P are observed in ?pleC mutant cells (pleC::Tn5) ^[33],^[75],^[78],^[79]. ?podJ mutant cells (podJ::Tn5 or podJ-xylX) become filamentous, and their phenotype is similar to ctrA401 cells ^[51],^[80].

In our simulation (Figure 11), the lower level of PodJ_L/PleC causes accumulation of DivK?P, which in turn lowers CtrA?P. Consequently, DivJ is elevated, which indicates (in our model) that the cell has differentiated into a stalked morphology. DNA replication could be initiated in this case but cell division does not proceed due to insufficient FtsQ (caused by low level of CtrA?P). Thus, such a cell is arrested in G2 phase and become filamentous in the simulation.

The effects of overexpressing PleC or PodJ have not been reported in the literature. In our simulation of these mutants (Figure 12), the high level of PodJ_L/PleC leads to an elevated level of CtrA?P. If DNA replication has been initiated by this time, the cell may proceed successfully through another division cycle (as in Figure 12) despite a high level of CtrA?P. However, elevated CtrA?P decreases the levels of GcrA, DivK?P and DivJ in our simulation, which may cause problems for these mutant cells in subsequent generations. Cells may arrest in G1 with low GcrA, or they may proceed through another round of DNA replication and arrest in G2 with high CtrA?P. The expected phenotype depends sensitively on parameter values in the model and cannot be predicted precisely.

If CckA cannot be phosphorylated, then, according to our simulation (Figure 13), CtrA?P level will fall. As a result, DivJ rises, indicating that the cell has transformed into a stalked morphology, and a new round of DNA replication is initiated, but the Z-ring cannot constrict due to insufficient FtsQ. Thus our simulation for this mutant indicates that the cell is arrested in the G2 phase with replicated chromosomes and filamentous morphology.