Mechanism of the molecular construction of germination apparatus of bacterial spores through self-assembly and subcellular localization of germiantion-related enzymes.

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。发芽是由一组预先存在的与发芽相关的酶（发芽器）的顺序激活控制的过程。因此，探讨孢子形成过程中萌发器的分子构建机制，对于理解萌发的分子过程具有重要意义。本研究获得以下结果：1.枯草芽孢杆菌基因组中YpeB以sleB-ypeB操纵子的形式编码，缺失YpeB的枯草芽孢杆菌孢子萌发与sleB突变体一样不完全，提示YpeB蛋白可能参与了枯草芽孢杆菌孢子萌发过程。我们证实sleB-ypeB操纵子在其他芽孢杆菌属物种中是保守的。我们发现，与枯草B芽孢杆菌孢子的情况一样，蜡状B芽孢杆菌Ype B在萌发过程中被加工成30 kDa片段并释放到萌发渗出物中。蜡样B和枯草芽孢杆菌Ype B的30 kDa片段N-末端序列基本相同，表明Ype B在萌发过程中的加工位点在芽孢杆菌中是保守的。枯草B杆菌YpeB的定点突变对枯草芽孢杆菌孢子萌发的影响。此外，我们还发现在B.cereus孢子萌发分泌物中存在蛋白酶活性，该酶可切割上述加工位点周围的GST-Ype B融合蛋白2。我们还发现SleC很可能是一种具有裂解性糖基转移酶和N-乙酰胞壁酰-L-丙氨酸酰胺酶活性的双功能酶，这表明芽孢杆菌和产气荚膜梭菌在萌发过程中对孢子皮层水解的机制并不保守。

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. Germination is a process controlled by the sequential activation of a set of pre-existing germination-related enzymes (germination apparatus). Thus exploring the mechanisms of molecular construction of germination apparatus during sporulation is crucial to understand the molecular process of germination. We obtained the following results during the research aided by this grant.1.YpeB is encoded as sleB-ypeB operon in Bacillus subtilis genome, and spore germination of ypeB-deficient B.subtilis is incomplete like that of sleB mutant, suggesting that YpeB protein may be involved in germination process of B.subtilis spores. We confirmed that sleB-ypeB operon is conserved in other Bacillus species. We found that, as in the case of B.subtilis spores, B.cereus YpeB is processed into 30 kDa fragment during germination and released into germination exudate. N-terminal sequence of 30 kDa fragment of B.cereus and Bacillus subtilis YpeB was virtually identical, suggesting that the processing site of YpeB during germination should be conserved among Bacillus species. The site-directed mutagenesis of B.subtilis YpeB affected to the spore germination of B.subtilis. Furthermore, we found the protease activity, which cleaves GST-YpeB fusion protein around the processing site indicated above, in germination exudate of B.cereus spores2.We also found that SleC is most likely a bifunctional enzyme possessing lytic transglycosylase and N-acetylmuramoyl-L-alanine amidase activities, suggesting that the mechanism on spore cortex hydrolysis during germination is not conserved between Bacillus species and C.perfringens.