Evidence that an N-terminal S-layer protein fragment triggers the release of a cell-associated high-molecular-weight amylase in Bacillus stearothermophilus ATCC 12980
Evidence that an N-terminal S-layer protein fragment triggers the release of a cell-associated high-molecular-weight amylase in Bacillus stearothermophilus ATCC 12980
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DOI：
10.1128/jb.178.19.5602-5609.1996
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发表时间：
1996-10
影响因子：
3.2
通讯作者：
E. Egelseer;I. Schocher;U. Sleytr;M. Sára
中科院分区：
文献类型：
--
作者：
E. Egelseer;I. Schocher;U. Sleytr;M. Sára
在淀粉培养基上生长期间，携带S-层的嗜热脂肪芽孢杆菌ATCC 12980和S-层缺陷的变体各自向培养液中分泌三种淀粉酶，具有相同的分子量58，000、122，000和184，000。只有高分子量淀粉酶（hmwA）也被确定为细胞相关。提取和再结合实验表明，hmwA具有高水平的亲和力，含肽聚糖层和S-层表面，但与含肽聚糖层的相互作用强于与S-层表面。对于S-层缺陷的变体，在淀粉培养基上生长过程中可以观察到细胞相关和游离hmwA的量没有变化，而对于S-层携带菌株，hmwA的细胞相关性强烈依赖于细胞的生长阶段。最大量的细胞相关的hmwA观察接种后3小时，这对应于早期的指数增长。在持续生长过程中，细胞相关hmwA的稳定下降与十二烷基硫酸钠凝胶上表观分子量为60，000的蛋白质的外观和强度增加相关。该蛋白质对含肽聚糖的层具有高水平的亲和力，并被鉴定为N-末端S-层蛋白片段，其不是由整个S-层蛋白的蛋白水解裂解产生的，而是在某些培养条件下与hmwA共表达的S-层蛋白的截短拷贝。在淀粉培养基上生长过程中，N端S层蛋白片段整合到S层晶格中，导致其规则结构大范围丧失，淀粉酶结合位点丧失。在本研究中获得的结果提供的证据表明，N-末端部分的S-层蛋白是负责锚定的亚基的肽聚糖含层，而位于表面的C-末端的一半可以作为一个结合位点的hmwA。
During growth on starch medium, the S-layer-carrying Bacillus stearothermophilus ATCC 12980 and an S-layer-deficient variant each secreted three amylases, with identical molecular weights of 58,000, 122,000, and 184,000, into the culture fluid. Only the high-molecular-weight amylase (hmwA) was also identified as cell associated. Extraction and reassociation experiments showed that the hmwA had a high-level affinity to the peptidoglycan-containing layer and to the S-layer surface, but the interactions with the peptidoglycan-containing layer were stronger than those with the S-layer surface. For the S-layer-deficient variant, no changes in the amount of cell-associated and free hmwA could be observed during growth on starch medium, while for the S-layer-carrying strain, cell association of the hmwA strongly depended on the growth phase of the cells. The maximum amount of cell-associated hmwA was observed 3 h after inoculation, which corresponded to early exponential growth. The steady decrease in cell-associated hmwA during continued growth correlated with the appearance and the increasing intensity of a protein with an apparent molecular weight of 60,000 on sodium dodecyl sulfate gels. This protein had a high-level affinity to the peptidoglycan-containing layer and was identified as an N-terminal S-layer protein fragment which did not result from proteolytic cleavage of the whole S-layer protein but seems to be a truncated copy of the S-layer protein which is coexpressed with the hmwA under certain culture conditions. During growth on starch medium, the N-terminal S-layer protein fragment was integrated into the S-layer lattice, which led to the loss of its regular structure over a wide range and to the loss of amylase binding sites. Results obtained in the present study provide evidence that the N-terminal part of the S-layer protein is responsible for the anchoring of the subunits to the peptidoglycan-containing layer, while the surface-located C-terminal half could function as a binding site for the hmwA.