The core regulatory circuitry that drives and paces cell cycle progression in the bacterium Caulobacter crescentus constitutes a highly integrated system designed to ensure that multiple events take place in a spatiotemporally regulated manner. Although identification and characterization of various master regulators has offered extraordinary insight into the molecular basis of cell cycle control, the recent identification of novel, essential transcription factors and non-coding genetic elements in the Caulobacter genome strongly suggest an incomplete regulatory circuit and novel, undescribed regulatory modalities. The central goal of this project is to determine the roles of novel essential regulator elements in cell cycle control. Specifically, I propose to identify the regulatory functions of two novel cell-cycle-regulated Caulobacter transcription factors by using microarray analysis to identify genes regulated by each. I will employ ChIP-chip and bioinformatic analysis to experimentally and computationally identify the binding motif(s) for each transcription factor. In addition, I will conduct genetic and biochemical analysis to determine the requirement for essential non-coding chromosomal elements in cell cycle progression. Among the intergenic, non-coding "essential gap" sequences recently identified in the Caulobacter genome, over 60% lie immediately adjacent to cell-cycle-regulated genes, suggesting a role for these sequences in cell cycle control. Therefore, I will divide this subset into five categories based on the points during the cell cycle at which adjacent ORFs are induced and characterize representative sequences from each set through deletion, complementation, inversion, and transposition analysis. This will allow me to probe the essential nature of these sequences by manipulating various parameters such as length, position, and orientation. As preliminary evidence suggests that proteins may bind some of these sequences, I will use DNA sampling in order to crosslink and purify proteins that bind each sequence specifically. This broad approach will enable me to determine how the new essential elements of the Caulobacter genome are integrated into the core genetic circuit that controls cell growth and development.
The bacterium Caulobacter crescentus is an ideal model organism in which to investigate cell cycle regulation, the study of which is critically important not only for understanding how cells grow and develop but also for understanding the molecular basis of cancer, which is in essence a consequence of unchecked, uncontrolled cell cycle progression. Additionally, since Caulobacter is a bacterial species and since all cells must carefully regulate the cell cycle in order to survive and reproduce, understanding the genetic circuitry that regulates the bacterial cell cycle will allow identification of new antibiotic targes and rational design of potent drugs that short-circuit that bacterial cell cycle program, preventin growth and division. Investigations into Caulobacter cell cycle regulation have already led to the design of highly effective small molecule drugs that are currently in clinical trials for the treatment of bacterial infections in humans, and investigation into the functions of recently identified cell cycle factors have the potential to lead to development of new, promising antibioti targets and powerful antibacterial drugs.