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，探索萌发过程中萌发特异性皮质裂解酶的调控机制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。YpeB编码在sleB- YpeB操纵子中，与sleB突变体一样，缺乏YpeB的枯草芽孢杆菌孢子萌发不完全。我们证实了先前报道的通过生化亚细胞分离在乙型肝炎突变体孢子中检测不到SleB。然而，在野生型和ypb缺陷菌株的孢子形成过程中，发现SleB-GFP融合蛋白位于孢子外部。此外，免疫电镜和显微镜观察表明，SleB与野生型一样，通常位于突变孢子的外壳层内。这些结果需要进一步的研究来确定YpeB的功能，特别是与休眠孢子中SleB的存在方式和孢子萌发过程中SleB的激活有关。我们还研究了产气荚膜梭菌孢子皮裂解酶SleC的活化过程。我们发现，在萌发过程中，无活性的原酶被萌发特异性蛋白酶GSP转化为活性酶。该蛋白酶是一种依赖半胱氨酸的丝氨酸蛋白酶，GSP可能与SleC一起定位在休眠孢子皮层的外部，这一位置与追求皮层水解反应的级联有关。SleC很可能是一种双功能酶，具有裂解转糖基酶和n -乙酰muramyl - l-丙氨酸氨基酶活性。这些结果表明芽孢杆菌和产气荚膜芽胞杆菌在萌发过程中孢子皮质水解的机制并不保守。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