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

细菌内生孢子萌发特异性皮质裂解酶在孢子形成过程中的表达和定位

• 批准号: 11660085
• 负责人: MORIYAMA Ryuichi
• 金额: $ 2.3万
• 依托单位: NAGOYA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660085/
• 关键词: bacterial spores; cortex-lytic enzyme (s); germination; sporulation; precursor; シグマ因子; 前駆体

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

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，由特定的营养萌发剂如L-丙氨酸诱导的皮层水解导致孢子的休眠和热稳定性的迅速丧失。因此，探讨萌发特异性皮层裂解酶在孢子形成过程中的表达、定位和加工机制，以及萌发过程中酶的激活机制，对于理解萌发的分子过程具有重要意义。本课题组在研究过程中取得了以下成果：1.利用GFP融合蛋白，我们发现枯草B杆菌SleB是蜡状B杆菌孢子萌发特异性皮层裂解酶的同源物，它在σ^G的控制下在前孢子中合成，并通过分泌信号穿过前孢子的内膜，定位于外被层内部。此外，我们指出，结果表明， ...更多信息 SleB在休眠孢子皮层外侧的沉积并不依赖于孢子衣的正确组装，而是需要酶的直接重复基序与胞壁酸δ-内酰胺之间的相互作用才能靶向SleB的目的地.我们表明，产气荚膜梭菌S40的孢子皮层裂解酶，SleC，在产孢过程中合成的前体组成的四个域。我们还发现，在萌发过程中，N-端前区和C-端前区被切割后，<35>通过萌发特异性蛋白酶（GSP）加工N-端前序列，失活的酶原（称为C_）被转化为活性酶。我们进一步证明了切割的N-末端前肽仍然与C_结合<35>。分离的复合物在6 M尿素溶液中变性解离后，去除尿素，再生成前肽-<35>C_2复合物，该复合物与GSP共同孵育时产生活性酶，但单独的<35>C_2不能被GSP激活，前肽-<35>C_2复合物比活性酶更热稳定。因此，前肽与C_的非共价连接<35>是帮助C_正确折叠<35>并稳定其构象所必需的，这表明前肽具有分子内伴侣的功能。重组蛋白中前肽与C_2共价结合<35>，在体内前肽-C_2复合物也可被GSP加工<35>，需要全长的N端前序列才能发挥作用。虽然C-末端前序列是作为一个独立的结构域，不参与酶的活化过程，它似乎是N-末端前序列有助于调节酶的活性作为酶的抑制剂。少

项目成果

Sachiko Okamura: "the N-terminal prepeptide is required for the production of spore cortex-lytic enzyme from its inactive precursor during germination of Clostridium perfringens S40 spores."Molecular Microbiology. 37・4. 821-827 (2000)

Sachiko Okamura：“在产气荚膜梭菌 S40 孢子萌发过程中，N 末端前肽是从其无活性前体产生孢子皮层裂解酶所必需的。”《分子微生物学》37・4（2000 年）。

Ryuichi Moriyama: "Expression of germination-specific amidase, SleB, of Bacilli in the forespore compartment of sporulating cells and its localization on the exterior side of cortex in dormant spores."Journal of Bacteriology. 181・8. 2373-2378 (1999)

Ryuichi Moriyama：“芽孢细胞前孢子室中芽孢杆菌的萌发特异性酰胺酶 SleB 的表达及其在休眠孢子皮层外侧的定位。”细菌学杂志 181・8（1999）。

Shio Makino: "Germination of bacterial spores"Journal of Antibacterial and Antifungal Agents. 28(in Japanese). 243-254 (2000)

Shio Makino：“细菌孢子的萌发”抗菌和抗真菌剂杂志。

牧野志雄: "細菌胞子の発芽-その生化学的解析を中心として-"防菌防黴誌. 28. 243-254 (2000)

Shio Makino：“细菌孢子的萌发 - 重点关注其生化分析”《细菌和抗真菌预防杂志》28. 243-254 (2000)。