Principal Investigator Michael Laub
Caulobacter crescentus is a powerful model for studying questions of regulation as 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. Although many of the major regulators in Caulobacter are known, it remains a major challenge to identify their connectivity and to understand the complete circuit which accounts for cell cycle oscillations.
The major focus right now is understanding regulation by two-component signal transduction systems, one of the major classes of signaling molecules in bacteria. These systems are comprised of sensor histidine kinases and their response regulator substrates which execute changes in cellular physiology upon phosphorylation. The Caulobacter genome encodes 64 histidine kinases and 42 response regulators. At least 10 of these two-component genes are involved in cell cycle progression. This includes CtrA, the master regulator of the Caulobacter cell cycle, which is analogous to (although not homologous to) the eukaryotic cyclin-dependent kinases. CtrA is a transcription factor which directly regulates nearly 100 genes and which also binds to and represses the origin of replication. Hence, CtrA activity must be temporarily eliminated at the G1-S transition to permit DNA replication, but must rapidly reaccumulate afterwards to drive transcription in the late stages of the cell cycle.
We have recently mapped an integrated genetic circuit which can account for the changes in CtrA activity during cell cycle progression. This circuit incorporates all previous identified cell cycle regulators in Caulobacter and suggests a model for how oscillations are produced. Similar to other genetic oscillators, the circuit requires a delayed negative feedback loop. As CtrA accumulates it triggers its own destruction by inducing the down-regulation of its own upstream kinase, CckA, but is delayed in doing so until after cell division.
Crucial to the operation of this cell cycle circuit is the dynamic sub-cellular localization of several histidine kinases. This includes CckA which is normally located at one or both poles of the cell, but is temporarily dispersed throughout the membrane at the onset of S phase. We have identified several regulatory molecules which mediate this delocalization and are currently investigating the mechanism by which they control the localization of CckA.
We are also beginning to probe the dynamics and 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? Is cell division the key time-delay necessary for oscillations? We are using a combination of genetics and biochemistry, as well as fluorescence microscopy of individual cells, to address these questions.