YpeB stability affects germination possibly through delaying SleB activity in Bacillus subtilis

Bacterial endospores formed by Bacilli and Clostridia can persist for decades and survive a wide range of killing mechanisms but can rapidly germinate in permissive environments. SleB and CwlJ are partially redundant germination-specific lytic enzymes that degrade Bacillus spore cortex peptidoglycan, contributing to the transition to the vegetative state. SleB interacts with YpeB, which may hold it inactive until germination starts. YpeB is rapidly proteolyzed during germination, which may release SleB to degrade the cortex. HtrC cleaves YpeB, but YpeB is still degraded in the absence of HtrC, suggesting the contribution of other proteases that are associated with the inner membrane. We hypothesize that at least one additional protease serves a YpeB degradation role. A mutant strain lacking five spore-associated proteases had significantly delayed spore germination and YpeB degradation when compared to the wild type. It is hypothesized that delayed YpeB degradation might cause SleB to become active later during germination. To further characterize the role of YpeB stability on SleB activity, cortex muropeptides were analyzed using HPLC and mass spectrometry. A decrease in two SleB-associated muropeptides was seen in the protease mutant strain after 30 and 60 min of germination when compared to the WT, which might be the consequence of delayed SleB activity. Identifying protein-protein interactions during germination could guide manipulation of spores either to increase the germination efficiency, rendering the spores more susceptible to killing, or to prevent germination, making the spores inefficient in causing human disease.

Bacterial endospores are agents of some human and animal diseases and of food spoilage. While dormant spores can persist for decades and survive a wide range of killing mechanisms, they must germinate to exert negative effects. Previous studies have indicated that membrane-associated proteases may play a role in a key germination process of cell wall degradation. A Bacillus subtilis strain lacking multiple spore proteases was studied and found to exhibit delays in some germination processes. An understanding of, and ability to manipulate, important steps in spore germination can contribute to the development of improved methods for spore inhibition and inactivation.