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Biochemical and molecular biological studies on germination of bacterial spores

细菌孢子萌发的生化和分子生物学研究

基本信息

批准号：
07660105
负责人：
MAKINO Shio
金额：
$ 1.41万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07660105/
关键词：
Bacterial spores Germination Cortex-lytic enzymes Amidase 細胞胞子

项目摘要

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.

以活性形式从蜡样芽孢杆菌和产气荚膜梭菌完全萌发的孢子渗出液中纯化出具有n -乙酰muramyl- l-丙氨酸酰胺酶作用的细菌孢子皮裂解酶（SCLEs）。这些酶能水解脱氢的孢子并使孢子变暗，但对分离的肽聚糖底物活性极低。它们对温度、pH值、离子强度和各种化学物质都有相似的敏感性。两个小鳞片都位于孢子皮层的外侧。然而，对它们基因的分析显示，萌发特异性氨基酶的结构存在多样性。除了酶与孢子肽聚糖结合的基序外，它们的氨基酸排列没有同源性。合成了含n端前区、n端前区、成熟型和c端前区的产气荚膜葡萄球菌酰胺酶，并将含有n端前区的失活前体预处理，生成了具有活性的成熟酶。蜡样芽孢杆菌酶缺乏这样的激活过程，该酶是作为具有可切割信号序列的前体产生的。该信号序列在产气荚膜菌酰胺酶序列中未发现。这表明这些酶在到达最终目的地孢子上的易位存在物种特异性机制。因此，进化差异可能导致许多皮质裂解酶在萌发过程中的特异性和作用方式以及激活机制上存在差异。此外，这些酶只有在从完整孢子中去除时才对热敏感，这表明酶在休眠孢子的外围位置存在一定的热保护机制，而不依赖于孢子的核心环境。