Clp-Mediated Regulation of the Mycobacterial Cell Cycle

Abstract

Tuberculosis is currently the most deadly infectious disease worldwide. Given the high rates of treatment failure, new antibiotics are desperately needed. Antibacterial drugs typically target DNA replication, cell growth, and cell division processes, yet regulators of the mycobacterial cell cycle remain undiscovered. In my thesis work, I sought to identify key players in mycobacterial cell cycle control, potentially leading to new antibacterial targets. Mycobacteria share a unique pattern of asymmetric growth and division with the model bacterium Caulobacter crescentus. C. crescentus initiates its cell cycle through proteolysis of key cell cycle regulators using the housekeeping protease complex ClpXP. Therefore, we postulated that a Clp-dependent mechanism might underlie cell cycle control in mycobacteria as well. In order to measure changes to the cell cycle brought about by modulations to Clp family proteins, I first developed a suite of quantitative microscopy tools to analyze each period of the cell cycle. During validation of this toolset, I discovered a post-division phase where the new pole—created at the site of division—does not initially grow. The identification of this “lag phase” clarified an outstanding question in the field as to the origin of asymmetric growth. To understand the role of Clp proteins (ClpC, ClpX, and ClpP) in the cell cycle, I systematically depleted each protein in vivo, and used my suite of image analysis tools to quantify changes. Loss of the ATPase ClpC and the protease ClpP lead to similar morphologies, notably loss of asymmetric growth and division. Conversely, depletion of the ATPase ClpX resulted in loss of cell division and DNA integrity. These cells eventually lyse and die, providing the first experimental evidence for ClpX’s predicted essentiality. To identify proteins interacting with ClpX, we designed a novel methodology and biochemically validated a top hit from this screen as an adaptor of ClpX. Our data suggest a link between ClpX and DNA replication. This is the first functional demonstration of a mycobacterial Clp adaptor. My work provides a framework for understanding the unique essentiality of multiple Clp proteins in mycobacteria: their non-redundant regulatory roles in DNA replication, cell growth, and division.