Ford Matthew J, Nomellini John F, Smit John
Department of Microbiology and Immunology, University of British Columbia, 2509-2350 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3.
J Bacteriol. 2007 Mar;189(6):2226-37. doi: 10.1128/JB.01690-06. Epub 2007 Jan 5.
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 approximately 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 approximately 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.
革兰氏阴性菌新月柄杆菌的S层由单一蛋白质RsaA组成，该蛋白质被分泌并组装成覆盖整个菌体的六边形晶体阵列。尽管类似的细菌S层广泛存在，但对于S层与细胞表面的附着情况却知之甚少，尤其是革兰氏阴性菌。鉴于初步迹象表明RsaA的N端将单体锚定在细胞表面，我们开发了一种检测方法，以区分直接的表面附着和亚基 - 亚基相互作用，即将小的RsaA片段与S层阴性细胞一起孵育，以评估片段重新附着的能力。通过这样做，我们发现RsaA的锚定区域位于最初大约225个氨基酸中，并且该RsaA锚定区域需要在外膜中发现的一种光滑的脂多糖种类。通过在六个半随机位点进行突变，我们了解到在RsaA最初大约225个氨基酸内相对较小的扰动会导致锚定丧失。在其他研究中，我们证实只有这个N端区域在S层锚定中具有直接作用。作为锚定研究的一个副产品，我们发现新月柄杆菌S层相关蛋白酶Sap识别截短的RsaA片段中的一个切割位点，而全长RsaA中的Sap未检测到该位点。这反过来又导致发现Sap是一种细胞外膜结合蛋白酶，而不是如先前提出的细胞内蛋白酶。此外，Sap主要通过S层I型分泌装置分泌到细胞表面。
