Biochemical and molecular biological studies on germination of bacterial spores

从蜡状芽孢杆菌和产气荚膜梭菌完全萌发的孢子渗出液中分离纯化出具有活性的细菌孢子皮质裂解酶(SCLEs)，其作用为N-乙酰胞浆-L-丙氨酸酰胺酶。这些酶能降解降解的孢子，并使孢子变黑，但对分离的肽聚糖底物的活性很低。它们对温度、pH、离子强度和各种化学物质的敏感性相似。两种SCLE均位于孢子皮质层的外部。然而，对它们的基因分析显示了发芽专一性酰胺酶的结构多样性。它们的氨基酸序列没有同源性，除了一个参与酶与孢子肽聚糖结合的基序。合成的产气荚膜梭菌酰胺酶由N-末端前体区、N-末端前体区、成熟型和C-末端前体区组成，并将一个带有N-末端前体区的非活性前体进行处理以产生活性成熟酶。蜡样芽孢杆菌缺乏这样的激活过程，这种酶是作为具有可切割信号序列的前体产生的。在产气荚膜梭菌酰胺酶序列中没有发现这样的信号序列。这表明，酶的转位到达孢子上的最终目的地存在物种特异性的机制。因此，进化上的差异可能导致许多皮层裂解酶在发芽过程中的特异性和作用方式以及激活机制上的不同。此外，酶只有在从完整的孢子上移走时才是热敏感的，这一事实表明休眠孢子中较外围位置的酶存在某种热保护机制，这与孢子的核心环境无关。

Bacterial spore cortex-lytic enzymes (SCLEs), which act as N-acetylmuramyl-L-alanine amidase, were purified in an active form from the exudate of fully germinated spores of Bacillus cereus and Clostridium perfringens. The enzymes hydrolysed decoated spores and caused phase-darkening of the spores, but had minimal activity on isolated peptidoglycan substrates. They shared similar sensitivities to temperature, pH,ionic strenght, and a variety of chemicals. Both SCLEs were localized on the exterior of cortex layrs in the spore. However, analyzes of their genes showed a diversity of structures of germination-specific amidases. There was no homology in their amino acid alignments, except for a motif involved in binding of the enzymes to spore peptidoglycan. The C.perfringens amidase was synthesized in a form comprising N-terminal prepro-region, N-terminal pro-region, mature form and C-terminal pro-region, and an inactive precursor with N-terminal pro-region is precessed to produce an active mature enzyme. Such an activation process lacks in the B.cereus enzyme, and the enzyme was produced as a precursor posessing a cleavable signal sequence. Such a signal sequence was not found in the sequence of C.perfringens amidase. This indicates that there is species-spcific mechanism on translocation of the enzymes to reach final destination on spores. Thus, evolutionary divergence might lead to many cortex-lytic enzymes differing in the specificities and modes of action and in the mechanism of activation during germination. Furthermore, the fact that the enzymes are heat sensitive only when removed from intact spores indicate the presence of some heat protective mechanism for enzymes in rather peripheral location in the dormant spore, which is not dependent on the core environment of spores.