Dissection of two pole-specific regulatory mechanisms in Caulobacter crescentus

Abstract

Proper coordination of events during the cell cycle is crucial for differentiation, proliferation and dispersal in all cell types. For example, in certain free-living and pathogenic bacteria, the process of flagellar (locomotory organelle) biogenesis has been observed to be tightly coupled to cytokinesis. Bacterial cells could utilize such coordination to ensure the production of flagellated offspring, thereby ensuring the proper dispersal of newly produced cells to a new, colonizable niche. However, the signaling mechanisms that help bacterial cells coordinate flagellar biogenesis with cytokinesis have remained largely unknown. The first part of my work describes a mechanism that Caulobacter and other alphaproteobacteria utilize to coordinate cytokinesis in tune with flagellar biogenesis, at the new cell pole. Interestingly, the regulatory protein that initiates flagellar biogenesis is tightly controlled at the level of stability through the activity of the highly conserved bacterial protease ClpXP, which is localized at the old cell pole. Strikingly, in Caulobacter, ClpXP follows a hierarchical proteolysis principle wherein substrate preference is temporally different during the cell cycle. The hierarchical proteolysis of substrates by ClpXP is attributed to the differential binding of adaptors to the unfoldase ClpX. Studies, till now, have shown that the adaptors are essential to recognize and recruit specific substrates to ClpX and enable proteolysis by ClpP. Nevertheless, the role of ClpX itself in recognizing these adaptor-bound substrates has remained unclear. Our work has led to the identification of specific mutations in ClpX that enable ClpXP to proteolyze only a specific set of substrates, both in vitro and in vivo. Notably, these mutations in ClpX have rendered the ClpXP-mediated proteolysis substrate-independent for a subset of substrates in the hierarchy. The second part of my study describes the direct role of ClpX in regulating hierarchical proteolysis during the cell cycle in Caulobacter.