Collaborative Research: Identifying and modeling the advantages of regulating protein abundance in Caulobacter crescentus

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The cell cycle is a series of cellular events leading to DNA duplication and cell division, and ultimately to the production of two daughter cells. This project will address broadly relevant questions about cell cycle regulation. Namely, what is the function of the regulated degradation of crucial cell cycle proteins and how does this regulatory mechanism integrate with other levels of regulation to control the cell cycle? By addressing these questions, this work has the potential to have a significant impact on our basic understanding of how the cell cycle is regulated. This project will expose students to interdisciplinary research and allow undergraduate students in summer laboratory courses to be active participants in research. The timed synthesis and degradation of specific proteins is a ubiquitous mechanism of cell regulation. However, some proteins can be regulated by covalent modification or binding to an allosteric effector. If a protein's activity can be controlled without the energetic cost of degradation and resynthesis, what circumstances would favor regulation by proteolysis? To address this question, the investigators are studying CtrA, a transcriptional regulator that promotes cell division in Caulobacter, yet also blocks chromosome replication. To reconcile these opposing functions, CtrA activity is temporarily eliminated from the cell just prior to chromosome replication by both dephosphorylation and proteolysis. Caulobacter strains expressing a non-degradable version of CtrA can still initiate chromosome replication, indicating that CtrA activity can be significantly reduced by dephosphorylation alone. However, cells with non-degradable CtrA have subtle defects that may compromise their competitive fitness. During each division cycle, ~9000 CtrA molecules are degraded and resynthesized. To understand the selective advantages conferred by this apparently wasteful strategy, experimental and modeling approaches will be used to predict and test the consequences of regulating CtrA only by phosphorylation. In particular, this study will investigate whether the periodic degradation and resynthesis of a neutral passenger protein confers a competitive disadvantage in Caulobacter, as in other bacteria. Caulobacter strains expressing steady levels of a non-degradable CtrA protein will be used to identify phenotypes that differ from cells with oscillating CtrA levels. Competitive fitness assays and quantitative microscopy approaches will test explicitly the predictions of existing mathematical models, e.g., that cells with non-degradable CtrA experience a delay in chromosome replication and a longer cell division cycle. Existing deterministic and stochastic models of the Caulobacter cell cycle will be revised, taking into account recent published findings. Stochastic spatiotemporal modeling will benefit from single-cell data on times of division, chromosome replication, and protein localization.