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

为了在分子水平上解释细菌萌发，我们研究了产气荚膜梭菌孢子皮层裂解酶SLEC的激活过程，该酶是产气荚膜梭菌孢子形成早期合成的前体，由4个结构域组成。在孢子成熟过程中，N端的前序列和C端的前序列被裂解后，在萌发过程中，通过用萌发特异的蛋白酶GSP处理N端的前序列，失活的前酶被转化为活性的酶。该酶是一种依赖半胱氨酸的丝氨酸蛋白酶，推测GSP可能与SLEC一起定位在休眠孢子的皮层外层，这一位置与追求一系列皮层水解反应有关。SLEC很可能是一种具有裂解转糖基酶和N-乙酰胞浆酰基-L-丙氨酸酰胺酶活性的双功能酶。在SLEC和SleM的协同作用下，大脑皮层发生了水解性变化，结果表明，SleM发挥功能之前，SLEC只能引起皮质结构的局部微小改变。对萌发过程中释放的神经肽的分析表明，产气荚膜梭菌孢子皮层中的壁壁酸残留量没有被单一的L-丙氨酸取代，这是芽孢杆菌孢子肽多糖的主要组成成分。这些结果表明芽孢杆菌和产气荚膜芽孢杆菌在萌发过程中对孢子皮质的降解机制并不保守。

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.