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 YpeBs 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. IMPORTANCEBacterial spores are among the most resilient cell types in nature. Their ability to resist sterilization during dormancy yet rapidly germinate and resume growth are central to the transmission and pathogenesis of spore-forming pathogens. A key component of their resistance is a thick layer of specialized peptidoglycan called the cortex that encases them. The cortex maintains the spore core in a highly desiccated state by physically restricting its expansion. Germination and outgrowth requires the degradation of this essential envelope layer. The enzymes responsible for cortex degradation are deposited in the spore during sporulation. How these lytic enzymes are held in active during dormancy has been a longstanding question. One of these enzymes, SleB, is encoded in the same operon as its putative regulator YpeB. Here, we provide biochemical and genetic evidence that YpeB interacts with and inhibits SleB during dormancy. These findings lay the groundwork for elucidating how SleB is activated during germination.