Bacillus subtilis YpeB holds SleB inactive preventing cortex peptidoglycan degradation during spore dormancy

Bacterial endospores are encased in a thick layer of specialized peptidoglycan called the cortex that is essential for core dehydration and heat resistance. Spore germination and outgrowth requires cortex degradation by enzymes that are deposited in the spore during sporulation. How these enzymes are held inactive during dormancy and activated during germination remains poorly understood. In Bacillus subtilis and many other endospore-forming bacteria, one of the lytic enzymes, SleB, is encoded in an operon with its putative regulator YpeB. The two proteins depend on each other for stability, but whether and how YpeB inhibits SleB were unknown. AlphaFold predicts a high-confidence interaction between the two proteins with YpeBs PepSY domains embracing SleBs catalytic domain. Here, we demonstrate that the two proteins can be co-purified when expressed in E. coli. Furthermore, in B. subtilis spores, amino acid substitutions at the predicted SleB/YpeB interface destabilized both proteins, resulting in impaired spore germination in the absence of the functionally redundant cortex lytic enzyme CwlJ. Selection for germination-competent suppressors identified general and allele-specific suppressors in sleB or, separately, ypeB that stabilized both proteins. Altogether, our data support a model in which YpeB inhibits SleB in the dormant spore through direct interaction. We propose that YpeB's PepSY domains function as chaperone-inhibitors of SleB, akin to the role of pro-domains in protease zymogens. Chaperone dependence ensures that SleB proteins that fail to interact with YpeB remain unfolded and are ultimately degraded, while YpeB-bound SleB enzymes persist but are inhibited, preventing inappropriate cortex degradation during dormancy.