S-layer anchoring and localization of an S-layer-associated protease in Caulobacter crescentus
S-layer anchoring and localization of an S-layer-associated protease in Caulobacter crescentus
复制标题
DOI：
10.1128/jb.01690-06
复制
发表时间：
2007-03-01
影响因子：
3.2
通讯作者：
Smit, John
中科院分区：
文献类型：
--
作者：
Ford, Matthew J.;Nomellini, John F.;Smit, John
革兰氏阴性菌月牙根杆菌的s层由一种名为RsaA的蛋白质组成，这种蛋白质分泌并组装成覆盖整个生物体的六角形晶体阵列。尽管类似的细菌s层广泛存在，但对细胞表面的s层附着知之甚少，特别是对于革兰氏阴性菌。有初步迹象表明，RsaA的N端将单体锚定在细胞表面，我们开发了一种区分直接表面附着与亚基-亚基相互作用的试验，其中将小RsaA片段与s层阴性细胞孵育以评估片段重新附着的能力。在此过程中，我们发现RsaA锚定区位于第一个相似的225个氨基酸中，并且该RsaA锚定区需要在外膜中发现的光滑脂多糖种。通过在6个半随机位点进行突变，我们了解到，在第一个类似于RsaA的225个氨基酸中，相对较小的扰动会导致锚定的丧失。在其他研究中，我们证实只有这个n端区域在s层锚定中起直接作用。作为锚定研究的副产品，我们发现Sap， C crescent - us s层相关蛋白酶，识别截断的RsaA片段中的一个切割位点，而Sap在全长RsaA中没有检测到。这反过来又导致发现Sap是细胞外膜结合蛋白酶，而不是之前提出的细胞内蛋白酶。此外，Sap主要通过s层I型分泌器分泌到细胞表面。
The S-layer of the gram-negative bacterium Caulobacter crescentus is composed of a single protein, RsaA, that is secreted and assembled into a hexagonal crystalline array that covers the organism. Despite the widespread occurrence of comparable bacterial S-layers, little is known about S-layer attachment to cell surfaces, especially for gram-negative organisms. Having preliminary indications that the N terminus of RsaA anchors the monomer to the cell surface, we developed an assay to distinguish direct surface attachment from subunit-subunit interactions where small RsaA fragments are incubated with S-layer-negative cells to assess the ability of the fragments to reattach. In doing so, we found that the RsaA anchoring region lies in the first similar to 225 amino acids and that this RsaA anchoring region requires a smooth lipopolysaccharide species found in the outer membrane. By making mutations at six semirandom sites, we learned that relatively minor perturbations within the first similar to 225 amino acids of RsaA caused loss of anchoring. In other studies, we confirmed that only this N-terminal region has a direct role in S-layer anchoring. As a by-product of the anchoring studies, we discovered that Sap, the C crescentus S-layer-associated protease, recognized a cleavage site in the truncated RsaA fragments that is not detected by Sap in full-length RsaA. This, in turn, led to the discovery that Sap was an extracellular membrane-bound protease, rather than intracellular, as previously proposed. Moreover, Sap was secreted to the cell surface primarily by the S-layer type I secretion apparatus.