Bacillus anthracis is the causative agent of the disease anthrax and poses a threat due to its potential to be used as a biological weapon. The spore form of this bacterium is an extremely resistant structure, making spore decontamination exceptionally challenging. During spore germination, nutrient germinants interact with Ger receptors, triggering a cascade of events. A crucial event in this process is degradation of the cortex peptidoglycan by germination-specific lytic enzymes (GSLEs), resulting in cells that are easily killed. This work investigated the regulation of the GSLE SleB by other proteins in the spore. A full understanding of how GSLEs are held inactive in the dormant spore and are activated during germination could lead to development of simplified spore decontamination strategies in which spore germination is the first step. It was found that SleB and YpeB are co-dependent. In the absence of one protein, the other is degraded during sporulation by an unidentified protease(s), although HtrC and SpoIVB are not likely responsible. Specific regions and residues of YpeB were also identified as being important to its relationship with SleB. While some evidence suggests that SleB and YpeB physically interact, a direct interaction was not observed in vivo or in vitro. YpeB was demonstrated to be proteolytically processed by HtrC during germination, resulting in stable products containing the YpeB C-terminus. The presence of inhibitory PepSY domains at the C-terminus of YpeB, coupled with YpeB degradation during germination, may suggest that YpeB processing results in SleB activation. Modification of the predominant YpeB cleavage sites or deletion of htrC reduced proteolysis, but cleavage at other sites still resulted in YpeB instability. Additionally, these changes did not have a significant impact on SleB activity. SleB regulation by other spore proteins was also examined. To test if SleB activation is Ger receptor-dependent, Bacillus subtilis strains lacking Ger receptors and/or GSLEs were germinated via non-nutrient means. Results indicated SleB can be activated independent of these proteins. B. anthracis homologs of the B. subtilis lipoproteins YlaJ and YhcN were also studied, but deletion of these genes did not result in significant changes in SleB stability or activity. / Ph. D.

Components of the bacterial spore germination apparatus are crucial for survival and for initiation of infection by some pathogens. While some components of the germination apparatus are well conserved in spore-forming species, such as the spoVA operon, each species may possess a different and possibly unique germinant recognition mechanism. The significance of several individual proteins in the germination process has been characterized. However, the mechanisms of how these proteins perform their functions and the network connecting these proteins in the complete germination process are still a mystery. In this study, we characterized a Bacillus subtilis superdormant spore population and investigated the abundance of 11 germination-related proteins. The relative quantities of these proteins in dormant, germinating and superdormant spores suggested that variation in the levels of proteins, other than germinant receptor proteins may result in superdormancy. Specifically, variation in the abundance of the GerD lipoprotein may contribute to heterogeneity of spore germination rates. Spore membrane proteomes of Bacillus anthracis and B. subtilis were characterized to generate a candidate protein list that can be further investigated. Proteins that were not previously known to be spore-associated were identified, and many of these proteins shared great similarity in both Bacillus species. A significant number of these proteins are implicated in functions that play major roles in spore formation and germination, such as amino acid or inorganic ion transport and protein fate determination. By analyzing the in vivo and in vitro activity of HtrC, we proved that the protease is responsible for YpeB proteolytic processing at specific sites during germination. However, without HtrC present in the spore, other proteases appear to degrade YpeB at a reduced rate. The activity of purified HtrC in vitro was stimulated by a relatively high concentration of Mn²⁺ or Ca²⁺ ions, but the mechanism behind the stimulation is not clear. We also demonstrated that YpeB and SleB, in the absence of their partner protein, were degraded by unknown proteases other than HtrC during spore formation. Identification and characterization of these unknown proteases would be a future direction for revealing the roles of proteases in spore germination. / Ph. D.