Understanding the Role and Regulation of Cell Wall Hydrolases in Bacillus subtilis

Abstract

The majority of bacteria are surrounded by their cell wall, a crosslinked, protective shell that is often made of a material called peptidoglycan (PG). To grow, bacteria must continuously remodel their cell wall, inserting new material and breaking old bonds to allow the wall to expand. Enzymes called cell wall hydrolases catalyze the breakage of bonds in the PG meshwork. Consequently, cell wall hydrolase activity is essential, as bond breakage is necessary for cell growth. However, it can also be lethal: the cell wall serves to protect the cell from lysis, and so disruptions to its mechanical integrity will cause the cell to explode due to its internal turgor pressure. Thus, the activity of these hydrolases must be carefully regulated. Bacterial genomes can contain large numbers of cell wall hydrolase genes. This redundancy has impeded our understanding of the functions of each hydrolase and their modes of regulation: single hydrolase knockouts usually present no phenotype, as the other remaining hydrolases compensate for the loss. We first addressed the problems posed by extensive redundancy: we used an exhaustive multiple-knockout approach to determine the minimal set of hydrolases required for growth in Bacillus subtilis. Using PHMMER, we identified 42 candidate cell wall hydrolases and we were able to delete 40 of these genes in a single strain; this “∆40” strain has normal growth rate, indicating that these 40 hydrolases are dispensable for cell growth. Additionally, the ∆40 strain does not shed old cell wall as wild-type cells do in a process called cell wall turnover, suggesting that cell wall turnover is not essential for growth. LytE and CwlO are the remaining two hydrolases in the ∆40 strain. They have previously shown to be synthetically lethal, suggesting that their activity is necessary for cell growth. Either can be knocked out in ∆40, indicating that either hydrolase alone is sufficient for cell growth, suggesting that the only essential function of cell wall hydrolases in B. subtilis during exponential growth is to enable cell growth by expanding the wall. These experiments introduce the ∆40 strain as a tool to study hydrolase activity and regulation in B. subtilis. Next, we investigated how different classes of hydrolases are regulated. Mechanical stress in PG has been proposed to regulate hydrolase activity, and we sought to test this hypothesis experimentally. We first tested whether substrate stress could regulate the activity of the turnover hydrolases LytC and LytD. Using osmotic shocks to vary stress on the cell wall, we found that autolysis (lysis caused by LytC and LytD activity) rates varied with cell wall stress. Lysis was faster when stress on the wall was decreased, and slower when stress on the wall was increased, suggesting that LytC and LytD preferentially cleave material that is not under stress. Next, we investigated potential stress regulation of the growth hydrolases LytE and CwlO. Our results suggest that CwlO and LytE play a key role in determining the thickness of the cell wall, as the ∆40 strain did not show a change in cell wall thickness. According to a simple pressure vessel model, the stress placed on the cell wall by turgor pressure is proportional to the cell diameter and inversely proportional to the thickness of the wall. We found that if we experimentally increase cell diameter, cell wall thickness increases and vice versa, as would be expected if cell wall stress remains constant. This is consistent with mechanical stress as a homeostatic cue in the cell wall, and, given that LytE and CwlO are implicated in specifying cell wall thickness, suggests that these hydrolases might also be stress regulated. We thus propose a stress-based model for regulation of the hydrolases involved in both cell wall expansion and cell wall turnover.