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

皮层是细菌孢子特有的一层厚厚的肽聚糖，负责维持孢子的休眠和耐热性。在萌发过程中，由l -丙氨酸等特定营养萌发物诱导的皮层水解导致孢子的休眠和热稳定性迅速丧失。因此，探索芽孢形成过程中萌发特异性皮质裂解酶的表达、定位和加工机制以及萌发过程中酶的激活对了解萌发的分子过程具有重要意义。在这项资助的研究中，我们获得了以下结果。利用GFP融合蛋白，我们发现枯草芽孢杆菌SleB是在σ^G控制下在前孢子中合成的，与蜡样芽孢杆菌孢子中萌发特异性皮质裂解酶同源，并通过分泌信号在前孢子的内膜上转运，定位于前孢子的被膜内部。此外，我们的研究结果表明，在休眠孢子中，SleB在皮层外侧的沉积并不需要孢子外壳的正确组装，而是需要酶的直接重复基序与菌酸δ-内酰胺的相互作用才能靶向SleB.2的目的地。我们发现产气荚膜梭菌S40的孢子皮裂解酶SleC是在孢子形成过程中合成的一个由四个结构域组成的前体。我们还发现，在萌发过程中，经过n端预区和c端前区切割后，无活性的前酶（称为C_<35>）通过萌发特异性蛋白酶（萌发特异性蛋白酶，GSP）处理n端前区转化为活性酶。我们进一步证明，裂解的n端前肽仍然与C_<35>相关。分离的配合物在6 M尿素溶液中变性解离后，去除尿素再生一个前肽- c_ <35>的配合物，在GSP孵育下产生活性酶。而单独分离的C_<35>不能被GSP激活。预肽- c_ <35>复合物的热稳定性优于活性酶。因此，需要前肽与C_<35>的非共价附着才能帮助C_<35>的正确折叠并稳定其构象，这表明前肽具有分子内伴侣的功能。通过GSP和体内预肽-C_<35>复合物对C_<35>共价结合的重组蛋白进行加工，需要完整的n端预序列才能发挥其作用。虽然c端前序作为一个独立的结构域存在，不参与酶的激活过程，但n端前序似乎作为酶的抑制剂参与酶活性的调节。少

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