UC Berkeley

Caulobacter crescentus is a powerful model organism for understanding cellular differentiation, cell polarity and cell cycle regulation in bacteria. An elaborate network of two-component signaling proteins works to orchestrate the developmental program that characterizes the Caulobacter cell cycle. The essential DNA-binding response regulator CtrA is at the center of this regulatory scheme and acts to control the transcription of >100 genes that are required for cell cycle progression, motility, DNA methylation, morphology and other processes. Because CtrA also inhibits chromosome replication at specific stages of the Caulobacter cell cycle, its activity must be temporarily eliminated in order for DNA replication to occur. Inactivation of CtrA is achieved though dephosphorylation and regulated degradation by the broadly conserved energy-dependent protease ClpXP.

In this dissertation, I analyze the roles of three proteins that are required for CtrA degradation in living cells. These are a single domain response regulator CpdR, a protein with no predicted function, RcdA, and a cyclic diguanylate (cdG)-binding protein, PopA. Structure-directed mutagenesis of RcdA was used to probe RcdA function. Results from these studies undermine the prevailing model for RcdA function, which suggest that RcdA does not participate directly in delivering CtrA to ClpXP, but instead acts simply as a localization factor increasing the concentration of CtrA at the cell pole where the protease is located. Additionally, I reconstituted the regulated proteolytic reaction in vitro and probed the role of all three accessory proteins and the small molecule cdG in promoting CtrA degradation. Although ClpXP alone is known to degrade CtrA in vitro, I observed a dramatic acceleration of proteolysis in the presence of the accessory proteins and cdG. This accelerated proteolysis was characterized by a nearly 10-fold reduction in the KM of the reaction, which is consistent with predictions for an adaptor mediated mechanism. I began to characterize protein-protein interactions within the proteolytic complex using in vivo and in vitro techniques. These experiments demonstrate that CtrA interacts directly with PopA in a cdG-dependent fashion. CtrA also interacts directly with RcdA and with ClpX. The CtrA-PopA(cdG) and CtrA-RcdA interactions are weakened or abolished by mutations in the receiver domain of CtrA that slow its proteolysis in vivo.

We propose a mechanism in which CtrA forms a ternary complex with PopA and RcdA in response to rising cdG concentrations in the cell. In this complex, PopA and RcdA act as a multi-protein adaptor complex to enhance the delivery of CtrA to the catalytic pore of ClpX. CpdR is required for accelerated CtrA proteolysis, but its precise role is still unknown. The accessory proteins were able to stimulate CtrA degradation even in the presence of a DNA fragment containing a CtrA binding site, which is known to inhibit CtrA proteolysis. Future work will determine if the accessory factors prevent the formation of inhibitory CtrA-DNA complexes or actively disassemble them. This dissertation alters the concept of proteolytic adaptors to include multi-protein complexes and expands the range of mechanisms by which proteolytic adaptors are controlled to include direct regulation by the small molecule cdG.