Expression and localization of the germination-specific cortex-lytic enzymes of bacterial endospores during sporulation

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The cortex, a thick layer of peptidoglycan specific to bacterial spores, is responsible for the maintenance of dormancy and heat resistance of spores. Cortex hydrolysis during germination induced by specific nutrient germinants like L-alanine leads to a rapid loss of dormancy and thermostability of spores. Thus exploring the mechanisms of the expression, localization and processing of germination-specific cortex-lytic enzymes during sporulation and activation of the enzyme during germination is important to understand the molecular process of germination. We obtained the following results during the research aided by this grant.1. Using GFP fusion protein, we indicated that B.subtilis SleB, a homolog to the germination-specific cortex-lytic enzyme from B.cereus spores, is synthesized in forespore under the control of σ^G and translocated across the forespore's inner membrane by secretion signal to locate inside of coat layer. Furthermore, we indicated the results which suggested that t … More he proper assembly of spore coat is not essential for the deposition of SleB on the exterior side of cortex in the dormant spore, but the interaction between the direct repeat motif of the enzyme and muramic acid δ-lactam is required for the targeting to the destination of SleB.2. We indicated that a spore cortex-lytic enzyme, SleC, of Clostridium perfringens S40 is synthesized during sporulation as a precursor consisting of four domains. We also indicated that after cleavage of an N-terminal preregion and a C-terminal proregion, inactive proenzyme (termed C_<35>) is converted to active enzyme by processing of an N-terminal prosequence with germination-specific protease (GSP) during germination. We further demonstrated that the cleaved N-terminal prepeptide remained associated with C_<35>. After the isolated complex was denatured and dissociated in 6 M urea solution, removal of urea regenerated a prepeptide-C_<35> complex which produces active enzyme when incubated with GSP.However, isolated C_<35> alone could not be activated by GSP.The prepeptide-C_<35> complex was more heat stable than active enzyme. Thus, non-covalent attachment of the prepeptide to C_<35> is required to assist correct folding of C_<35> and stabilize its conformation, suggesting that the prepeptide functions as an intramolecular chaperone. Recombinant proteins, which have prepeptide covalently bonded to C_<35>, were processed by GSP as well as the in vivo prepeptide-C_<35> complex, and the full length of the N-terminal presequence was needed to fulfill its role. Although the C-terminal prosequence is present as an independent domain which is not involved in the activation process of the enzyme, it appears that the N-terminal prosequence contributes to the regulation of enzyme activity as an inhibitor of the enzyme. Less