Regulation of the germination-specific cortex-lytic enzymes of bacterial endospores by protein-protein interaction 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，探索种子萌发过程中皮层裂解酶的调控机制，对了解种子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. SleB-ypeB操纵子编码YpeB基因，缺失ypeB的枯草芽孢杆菌与SleB突变体一样孢子萌发不完全。我们证实了先前报道的生化亚细胞分级检测在ypeB突变孢子的SleB不可检测性。然而，SleB-GFP融合蛋白被观察到位于孢子形成过程中在野生型和ypB缺陷型菌株的孢子的外部。此外，免疫电子 ...更多信息 显微镜观察表明，SleB通常位于突变孢子的外被层内部，与野生型的情况一样。这些结果需要进一步的研究来确定YpeB的功能，特别是SleB在休眠孢子中的存在方式和孢子萌发过程中SleB的激活.我们还研究了产气荚膜梭菌孢子皮层裂解酶SleC的活化过程。我们发现在种子萌发过程中，萌发特异性蛋白酶GSP将失活的酶原转化为活性酶。该蛋白酶是一种半胱氨酸依赖性丝氨酸蛋白酶和GSP可能是本地化与SleC在休眠孢子的皮层层的外部，皮层水解反应的级联的追求相关的位置。SleC可能是一种双功能酶，具有溶解性转糖基酶和N-乙酰胞壁酰-L-丙氨酸酰胺酶活性。这些结果表明，芽孢杆菌和C.产气荚膜杆菌少

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 regulation of germination-specific cortex-lytic enzymes during during germination is important to understand the molecular process of germination. We obtained the following results during the research aided by this grant.1. YpeB is encoded in sleB-ypeB operon, and spore germination of ypeB-deficient Bacillus subtilis is incomplete like that of sleB mutant. We confirmed previously reported undetectability of SleB in ypeB mutant spores by biochemical subcellular fractionation. However, SleB-GFP fusion proteins were observed to be located in the outside of spores during sporulation in both wild-type and ypB-deficient strains. Furthermore, immunoelectron … More microscopic observations indicated that SleB is normally localized inside of coat layer of the mutant spores as in the case of wild-type. These results require further investigations to determine the function of YpeB, especially in relation to the mode of existence of SleB in dormant spores and the activation of SleB during spore germination.2. We also studied the activation process of a SleC, a spore cortex-lytic enzyme of Clostridium perfringens. We found that inactive proenzyme is converted to active enzyme by germination-specific protease GSP during germination. The protease was a cysteine-dependent serine protease and GSP is likely localized with SleC on the exterior of the cortex layer in the dormant spore, a location relevant to the pursuit of a cascade of cortex hydrolytic reaction. SleC was most likely a bifunctional enzyme possessing lytic transglycosylase and N-acetylmuramoyl-L-alanine amidase activities. These results suggest that the mechanism on spore cortex hydrolysis during germination is not conserved between Bacillus species and C. perfringens. Less