Principal Investigator Michael Laub
Caulobacter crescentus is a powerful model for understanding the bacterial cell cycle as well as the mechanisms cells use to establish and enforce cellular asymmetry. Caulobacter cells are easily synchronized, cell cycle progression can be tracked by monitoring a series of morphological transitions, and a complete suite of genetic tools is available. Every cell division is asymmetric, producing two different daughter cells, a swarmer cell and a stalked cell, that differ morphologically and, importantly, with respect to replicative capacity. Whereas the sessile stalked cell can immediately initiate a new round of DNA replication, the swarmer cell is delayed in a G1 state and must differentiation into a stalked cell before initiating S phase.
At the heart of the circuitry controlling the asymmetric fates of swarmer and stalked cells is the master regulator CtrA. This essential transcription factor controls nearly 100 genes and also binds directly to the origin of replication to silence initiation. We have recently mapped, for the first time, an integrated regulatory circuit that can account for the cell cycle- and cell type-dependent changes in CtrA activity. Crucial to the operation of this circuit is the histidine kinase CckA which ultimately drives CtrA phosphorylation. How CckA is regulated has been a long-standing mystery, as has the observation that CckA is maximally active when localized to the swarmer pole of predivisional cells. Recently, we demonstrated that CckA activity requires the non-canonical kinase DivL, but that phosphorylated DivK can inhibit the DivL:CckA complex. Localization of CckA and DivL is important as it brings them in proximity to a DivK phosphatase. The cell pole is thus used to create a protected zone within the cell to activate CckA.
In addition to functioning as a kinase at one pole, CckA also functions as a phosphatase at the opposite pole. The opposing polar activities of CckA lead to a gradient of phosphorylated CtrA across the predivisional cell, despite a homogeneous distribution of CtrA-GFP.
We are also probing the feedback structure of the cell cycle regulatory network. Why is the circuit so complex? What is the role of specific feedback loops to the reliability or robustness of the system? Although the network can appear irreducibly complex, our recent work suggests that the network is comprised of simpler and genetically separable modules. Finally, we have begun to examine the evolution of the bacterial cell cycle, to try and understand how the Caulobacter regulatory circuit relates to that used in E. coli and other bacteria.