Egelseer E, Schocher I, Sára M, Sleytr U B
Zentrum für Ultrastrukturforschung, Universität für Bodenkultur, Vienna, Austria.
J Bacteriol. 1995 Mar;177(6):1444-51. doi: 10.1128/jb.177.6.1444-1451.1995.
The S-layer lattice from Bacillus stearothermophilus DSM 2358 completely covers the cell surface and exhibits oblique symmetry. During growth of B. stearothermophilus DSM 2358 on starch medium, three amylases with molecular weights of 58,000, 98,000, and 184,000 were secreted into the culture fluid, but only the high-molecular-weight enzyme was found to be cell associated. Studies of interactions between cell wall components and amylases revealed no affinity of the high-molecular-weight amylase to isolated peptidoglycan. On the other hand, this enzyme was always found to be associated with S-layer self-assembly products or S-layer fragments released during preparation of spheroplasts by treatment of whole cells with lysozyme. The molar ratio of S-layer subunits to the bound amylase was approximately 8:1, which corresponded to one enzyme molecule per four morphological subunits. Immunoblotting experiments with polyclonal antisera against the high-molecular-weight amylase revealed a strong immunological signal in response to the enzyme but no cross-reaction with the S-layer protein or the smaller amylases. Immunogold labeling of whole cells with anti-amylase antiserum showed that the high-molecular-weight amylase is located on the outer face of the S-layer lattice. Because extraction of the amylase was possible without disintegration of the S-layer lattice into its constituent subunits, it can be excluded that the enzyme is incorporated into the crystal lattice and participates in the self-assembly process. Affinity experiments strongly suggest the presence of a specific recognition mechanism between the amylase molecules and S-layer protein domains either exposed on the outermost surface or inside the pores. In summary, results obtained in this study confirmed that the S-layer protein from B. stearothermophilus DSM 2358 functions as an adhesion site for a high-molecular-weight amylase.
嗜热脂肪芽孢杆菌DSM 2358的S层晶格完全覆盖细胞表面并呈现斜对称。在嗜热脂肪芽孢杆菌DSM 2358于淀粉培养基上生长期间，三种分子量分别为58,000、98,000和184,000的淀粉酶被分泌到培养液中，但仅发现高分子量的酶与细胞相关。细胞壁成分与淀粉酶之间的相互作用研究表明，高分子量淀粉酶与分离的肽聚糖没有亲和力。另一方面，该酶总是被发现与S层自组装产物或在用溶菌酶处理全细胞制备原生质体过程中释放的S层片段相关。S层亚基与结合的淀粉酶的摩尔比约为8:1，这相当于每四个形态亚基有一个酶分子。用针对高分子量淀粉酶的多克隆抗血清进行的免疫印迹实验显示，对该酶有强烈的免疫信号，但与S层蛋白或较小的淀粉酶没有交叉反应。用抗淀粉酶抗血清对全细胞进行免疫金标记表明，高分子量淀粉酶位于S层晶格的外表面。由于在不将S层晶格分解成其组成亚基的情况下就可以提取淀粉酶，因此可以排除该酶被纳入晶格并参与自组装过程。亲和力实验强烈表明，淀粉酶分子与暴露在最外表面或孔内的S层蛋白结构域之间存在特异性识别机制。总之，本研究获得的结果证实，嗜热脂肪芽孢杆菌DSM 2358的S层蛋白作为高分子量淀粉酶的粘附位点发挥作用。
