Regulation of Cell Wall Hydrolysis by the WalRK Two-Component System in Bacillus subtilis

Abstract

The cell envelope surrounds the bacterial cell, providing physical integrity, mediating contact with the surrounding environment, and acting as a barrier against external assaults. The cell wall forms the structural component of the cell envelope, and is composed of peptidoglycan (PG), a covalently-linked meshwork of glycan strands cross-linked together by short peptides. During cell growth, bacteria rely on the activity of PG-synthetic enzymes for the creation of new material and PG-hydrolytic enzymes to expand the existing meshwork. Notably, injury to the cell wall can arise from the action of PG hydrolases, whose function can be deadly if mis- or hyper-activated. Thus, bacteria must tightly control the activity of these enzymes and employ systems to sense and respond to envelope stress. WalRK is an essential and widely conserved two-component regulatory system (TCS) that regulates genes involved in cell wall metabolism, most notably PG hydrolases. Though WalRK was understood to be important for cell wall biogenesis, its precise function was not known. Here, I used the model Gram-positive organism Bacillus subtilis to investigate the role of the WalRK TCS in the regulation of cell wall hydrolases and elucidate strategies used by bacteria to control the synthesis and remodeling of their cell wall. I demonstrated that the essentiality of WalRK in B. subtilis is derived from its transcriptional activation of the cell wall hydrolases lytE and cwlO, and repression of the hydrolase inhibitor iseA and PG deacetylase pdaC. I provide evidence that the kinase WalK monitors the status of PG hydrolysis during cell growth by sensing PG cleavage products released by LytE and CwlO. When hydrolase activity is too high or too low, WalRK adjusts the transcription of these hydrolases accordingly to achieve homeostasis. Additionally, I revealed that WalRK also controls hydrolytic activity by modulating the acetylation state of the PG, which inhibits hydrolysis by CwlO and LytE. Altogether, my work demonstrates that WalRK maintains precise hydrolase activity during cell growth through both transcriptional regulation and posttranslational inhibition of two essential PG hydrolases. I propose that homologous WalRK systems in Gram-positive bacteria similarly function to homeostatically control PG hydrolase activity during cell growth.