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Assembled structure and biological functions of the cell surface layer of Clostridium botulinum type E.

E型肉毒梭菌细胞表面层的组装结构和生物学功能。

基本信息

批准号：
62570193
负责人：
TAKUMI Kenji
金额：
$ 1.41万
依托单位：
Tokusima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

Surface layers of regularly arranged (RA) protein subunits are now recognized as a common feature of both eubacterial and archaebacterial cell walls. These RAs are noncovalently attached to the surface of the outer membrane or the peptidoglycan sacculus in linear, tetragonal, or hexahonal arragements, formimg in some bases, the primary barrier between the cell and the environment. Clostridium botulinum type E, which is one of the most important bacterial species for the toxigenicbacterial food-borne poisoning has also such RA layered structure. This study atempted to isolate the RA of the organism and to analyse its immunochemical properties by immunobloting together with its ultramicrostructure by using negatively stained thin sections or immunoelectron microscopy.The RA was extracted selectively from the cell wall by the tr.eatment with 4 M guanidin hydrochloride (GHCL) or 6 M urea. The extract was then submitted to SDS-PAGE, by which that two protein subunits with molecular weights of 900,000 (90K) and 600,000(6oK) are major constituents of the RA is firstly ascertained. Immunoblot with rabbit antiserum to the whole cells indicated that these proteins are surface-exposed components of the cell wall. This surface localization of the protein subunits also confermed by immunoelectraon microscopically by Con-A-gold particle conjugate method. On the other hand, the subunits of linearly ordered RA layers isolated by 4M GHCL were tested for their ability to reform the regular paterns in the absence of cell wall fragments. In negatively stained preparations the majority of the self-arrenged products of the linearly ordered subunits were observed:the diameter of 40-280nm; the length of about 1 um.

规则排列（RA）蛋白亚基的表层现在被认为是真细菌和古细菌细胞壁的共同特征。这些RAs以线性、四边形或六边形的排列方式非共价地附着在外膜表面或肽聚糖小囊上，在某些碱基中形成细胞与环境之间的主要屏障。E型肉毒杆菌是食源性毒素细菌中毒的重要菌种之一，也具有这种RA层状结构。本研究试图分离该生物的RA，并通过免疫印迹法分析其免疫化学性质，并通过阴性染色薄片或免疫电镜分析其超微结构。用4 M盐酸胍（GHCL）或6 M尿素处理，选择性地从细胞壁提取RA。然后将提取物提交到SDS-PAGE，通过SDS-PAGE首次确定了分子量为900,000 （90K）和600,000（6oK）的两个蛋白质亚基是RA的主要成分。兔抗血清全细胞免疫印迹表明，这些蛋白是细胞壁表面暴露的成分。这种蛋白亚基的表面定位也通过con - a -金粒子共轭法在免疫电镜下得到证实。另一方面，我们测试了4M GHCL分离的线性有序RA层的亚基在没有细胞壁碎片的情况下重组规则模式的能力。在阴性染色制剂中，观察到大多数线性有序亚基的自排列产物：直径为40-280nm；长度约为1微米。