An Exhaustive Multiple Knockout Approach to Understanding Cell Wall Hydrolase Function in Bacillus subtilis

Most bacteria are surrounded by their cell wall, a highly crosslinked protective envelope of peptidoglycan. To grow, bacteria must continuously remodel their wall, inserting new material and breaking old bonds. Bond cleavage is performed by cell wall hydrolases, allowing the wall to expand. Understanding the functions of individual hydrolases has been impeded by their redundancy: single knockouts usually present no phenotype. We used an exhaustive multiple-knockout approach to determine the minimal set of hydrolases required for growth in Bacillus subtilis. We identified 42 candidate cell wall hydrolases. Strikingly, we were able to remove all but two of these genes in a single strain; this “Δ40” strain shows a normal growth rate, indicating that none of the 40 hydrolases are necessary for cell growth. The Δ40 strain does not shed old cell wall, demonstrating that turnover is not essential for growth. The remaining two hydrolases in the Δ40 strain are LytE and CwlO, previously shown to be synthetically lethal. Either can be knocked out in Δ40, indicating that either hydrolase alone is sufficient for cell growth. Environmental screening and zymography revealed that LytE activity is inhibited by Mg2+ and that RlpA-like proteins may stimulate LytE activity. Together, these results demonstrate that the only essential function of cell wall hydrolases in B. subtilis is to enable cell growth by expanding the wall and that LytE or CwlO alone is sufficient for this function. These experiments introduce the Δ40 strain as a tool to study hydrolase activity and regulation in B. subtilis . IMPORTANCE In order to grow, bacterial cells must both create and break down their cell wall. The enzymes that are responsible for these processes are the target of some of our best antibiotics. Our understanding of the proteins that break down the wall – cell wall hydrolases – has been limited by redundancy among the large number of hydrolases many bacteria contain. To solve this problem, we identified 42 cell wall hydrolases in Bacillus subtilis and created a strain lacking 40 of them. We show that cells can survive using only a single cell wall hydrolase; this means that to understand the growth of B. subtilis in standard laboratory conditions, it is only necessary to study a very limited number of proteins, simplifying the problem substantially. We additionally show that the Δ40 strain is a research tool to characterize hydrolases, using it to identify 3 ‘helper’ hydrolases that act in certain stress conditions.