YpeB dimerization may be required to stabilize SleB for effective germination of Bacillus anthracis spores.

BACKGROUND: Bacillus cells faced with unfavorable environmental conditions undergo an asymmetric division process ultimately leading to the formation of the bacterial spore. In some instances the spore serves as an infectious agent; such is the case with the spore of Bacillus anthracis and the disease anthrax. Spores are resistant to a variety of environment conditions including traditional decontamination techniques due to the formation of specialized cellular structures. One such structure, the spore cortex, is a thick layer of modified peptidoglycan that contributes to spore dormancy through maintenance of the dehydrated state of the spore core. During spore germination, degradation of the cortex is required to facilitate complete hydration of the core and a return to vegetative growth. Degradation of the cortex is accomplished through the action of germination-specific lytic enzymes. One of these enzymes, SleB, has been previously shown to require the presence of the YpeB protein for its stable incorporation and subsequent function in spores of B. anthracis. The focus of the present study is to identify protein interactions of YpeB through in vivo chemical cross-linking and two-hybrid analysis. RESULTS: Conserved residues within YpeB PepSY domains were altered to facilitate implementation of a site-specific chemical cross-linker, 4-Azidophenacyl bromide. Analyses of crosslinked-spore extracts suggests that YpeB exists as a dimer or larger multimer within the spore, potentially mediated through interactions of the C-terminal domains. Spores expressing stable truncated forms of YpeB were crosslinked and corresponding truncated dimers were detected. Further characterization of individual YpeB domains using bacterial two-hybrid analysis indicated a possible role for both N-and C-terminal domains in YpeB oligomerization. CONCLUSIONS: The YpeB protein likely exists as dimer or higher-order multimer in the dormant spore. Both the N- and C-terminal YpeB domains contribute to multimerization. SleB likely also exists as an oligomer, and SleB and YpeB may be found together within a protein complex. Disassembly of this complex during spore germination likely allows SleB to become active in spore cortex degradation. Further study of this protein complex may contribute to the development of methods to inhibit or stimulate germination, allowing more effective spore decontamination.

Identification of L-Valine-initiated-germination-active genes in Bacillus subtilis using Tn-seq.

Bacterial endospores can survive harsh environmental conditions and long-term dormancy in the absence of nutrients, but can rapidly germinate under favorable conditions. In the present study, we employed transposon sequencing (Tn-seq) to identify genes with previously uncharacterized roles in spore germination. Identified genes that encoded spore inner membrane proteins were chosen for study of defined mutants, which exhibited delayed germination in several assays in response to varying germinants. Significantly slowed release of DPA indicated that mutants were affected in Stage I of germination. Several mutants exhibited phenotypic traits consistent with failure of a GerA germinant receptor-mediated response, while others appeared to have a more general loss of response to varied germinants. Use of a gerA-lacZ transcriptional fusion and quantitative western blotting of GerAC allowed mutants to be classified based upon normal or decreased gerA transcription and normal or reduced GerA accumulation. Fourteen genes were identified to have newly described roles within Bacillus spore germination. A more complete understanding of this process can contribute to the development of better spore decontamination procedures.

Clostridium difficile Lipoprotein GerS Is Required for Cortex Modification and Thus Spore Germination.

Clostridium difficile, also known as Clostridioides difficile, is a Gram-positive, spore-forming bacterium that is a leading cause of antibiotic-associated diarrhea. C. difficile infections begin when its metabolically dormant spores germinate to form toxin-producing vegetative cells. Successful spore germination depends on the degradation of the cortex, a thick layer of modified peptidoglycan that maintains dormancy. Cortex degradation is mediated by the SleC cortex lytic enzyme, which is thought to recognize the cortex-specific modification muramic-δ-lactam. C. difficile cortex degradation also depends on the Peptostreptococcaceae-specific lipoprotein GerS for unknown reasons. In this study, we tested whether GerS regulates production of muramic-δ-lactam and thus controls the ability of SleC to recognize its cortex substrate. By comparing the muropeptide profiles of ΔgerS spores to those of spores lacking either CwlD or PdaA, both of which mediate cortex modification in Bacillus subtilis, we determined that C. difficile GerS, CwlD, and PdaA are all required to generate muramic-δ-lactam. Both GerS and CwlD were needed to cleave the peptide side chains from N-acetylmuramic acid, suggesting that these two factors act in concert. Consistent with this hypothesis, biochemical analyses revealed that GerS and CwlD directly interact and that CwlD modulates GerS incorporation into mature spores. Since ΔgerS, ΔcwlD, and ΔpdaA spores exhibited equivalent germination defects, our results indicate that C. difficile spore germination depends on cortex-specific modifications, reveal GerS as a novel regulator of these processes, and highlight additional differences in the regulation of spore germination in C. difficile relative to B. subtilis and other spore-forming organisms.IMPORTANCE The Gram-positive, spore-forming bacterium Clostridium difficile is a leading cause of antibiotic-associated diarrhea. Because C. difficile is an obligate anaerobe, its aerotolerant spores are essential for transmitting disease, and their germination into toxin-producing cells is necessary for causing disease. Spore germination requires the removal of the cortex, a thick layer of modified peptidoglycan that maintains spore dormancy. Cortex degradation is mediated by the SleC hydrolase, which is thought to recognize cortex-specific modifications. Cortex degradation also requires the GerS lipoprotein for unknown reasons. In our study, we tested whether GerS is required to generate cortex-specific modifications by comparing the cortex composition of ΔgerS spores to the cortex composition of spores lacking two putative cortex-modifying enzymes, CwlD and PdaA. These analyses revealed that GerS, CwlD, and PdaA are all required to generate cortex-specific modifications. Since loss of these modifications in ΔgerS, ΔcwlD, and ΔpdaA mutants resulted in spore germination and heat resistance defects, the SleC cortex lytic enzyme depends on cortex-specific modifications to efficiently degrade this protective layer. Our results further indicate that GerS and CwlD are mutually required for removing peptide chains from spore peptidoglycan and revealed a novel interaction between these proteins. Thus, our findings provide new mechanistic insight into C. difficile spore germination.