Maintaining Balance: Regulation of Cell Wall Hydrolase Activity in Vibrio cholerae. May 20, 2025 – 2:00 PM – Warren Hall B25.

Bacteria exist in a dizzying array of shapes and sizes, and have evolved to thrive in almost every environment on Earth. Bacteria face innumerable challenges to survival, from predation to chemical assaults to osmotic pressure equivalent to an inflated bicycle tire. The bacterial cell envelope is the main defense against these problems. The rigidity of the cell envelope comes from the peptidoglycan cell wall, a covalently linked meshwork of glycan strands and peptide crosslinks. While the cell wall is an excellent defense against high intracellular pressure, it can become a restrictive cage and prevent normal elongation and division unless it is remodeled properly. This process involves cutting the cell wall and inserting new material, a highly risky activity for cells that are essentially tiny balloons. Humans have studied bacteria for hundreds of years, but we still do not fully understand how bacteria exist – how can the cell wall be cut without the cell exploding?  In this work, I investigate the regulation of the enzymes that cut the cell wall, also known as cell wall hydrolases, and the relationship different classes of hydrolases may have to cell wall synthases. I examine the roles of two nearly identical endopeptidases, ShyA and ShyC, during changing growth conditions. I show that ShyA, but not ShyC, is essential for fast adaptation to low salinity environments, suggesting a critical role for this cell wall hydrolase in the aquatic lifestyle of the human pathogen Vibrio cholerae. Next, I probe the regulatory network involved in cell wall homeostasis by disrupting the balance between cell wall synthases and hydrolases. I describe an unbiased genetic screen in which point mutations near the active site of the cell wall synthase Penicillin Binding Protein 2 (PBP2) restore growth in cells lacking sufficient hydrolase activity. Overall, these studies suggest a possible role for the endopeptidase ShyA as a pressure release valve, and indicate that the residues near the binding pocket of PBP2 may modulate the essentiality of multiple classes of cell wall hydrolases. These findings offer insight into the complex machinery of cell wall homeostasis, and may represent a step toward novel antibiotic targets.