Molecular mechanism on a germination cascade reaction of spore-forming bacteria

为了在分子水平上阐明细菌萌发，我们研究了产气荚膜梭菌孢子皮裂解酶SleC的激活过程，SleC是产气荚膜梭菌孢子形成初期合成的一个由4个结构域组成的前体。在孢子成熟过程中，一个n端前序和一个c端前序被裂解后，在萌发过程中，一个n端前序被萌发特异性蛋白酶GSP加工转化为活性酶。该蛋白酶是一种依赖半胱氨酸的丝氨酸蛋白酶，这表明GSP可能与SleC一起定位在休眠孢子皮层的外部，这一位置与追求级联的皮层水解反应有关。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。大脑皮层的水解是由SleC和SleM共同作用完成的，研究表明，SleC只会引起大脑皮层结构的小而局部的变化，而这是SleM发挥作用所必需的。对萌发过程中释放的多肽进行分析发现，产气荚膜芽孢杆菌孢子皮层中没有残留的菌酸被单一的l -丙氨酸所取代，而l -丙氨酸是芽孢杆菌孢子肽聚糖的主要成分。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。

In order to elucidate bacterial germination in a molecular level, we studied the activation process of a SleC, a spore cortex-lytic enzyme of Clostridium perfringens which is synthesyzed at an early stage of sporulation as a precursor consisting of 4 domains. After cleavage of an N-terminal presequence and a C-terminal prosequence during spore maturation, inactive proenzyme is converted to active enzyme by processing of a N-terminal prosequence with germination-specific protease GSP during germination. The protease was a cysteine-dependent serine protease and it was suggested that GSP likely is 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. Cortex hydrolysis was performed by the cooperative action of SleC and SleM, and it was indicated that SleC causes merely small and local changes in the cortex structure, which are necessary before SleM can function. Analysis of muropeptides released during germination revealed that no muramic acid residue in the spore cortex of C. perfringens was substituted with a single L-alanine, which is a major constituent common to spore peptidoglycan of Bacillus species. These results suggest that the mechanism on spore cortex hydrolysis during germination is not conserved between Bacillus species and C. perfringens.