Zilhão Rita, Serrano Mónica, Isticato Rachele, Ricca Ezio, Moran Charles P, Henriques Adriano O
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2781-901 Oeiras Codex, Portugal.
J Bacteriol. 2004 Feb;186(4):1110-9. doi: 10.1128/JB.186.4.1110-1119.2004.
Spores formed by wild-type Bacillus subtilis are encased in a multilayered protein structure (called the coat) formed by the ordered assembly of over 30 polypeptides. One polypeptide (CotB) is a surface-exposed coat component that has been used as a vehicle for the display of heterologous antigens at the spore surface. The cotB gene was initially identified by reverse genetics as encoding an abundant coat component. cotB is predicted to code for a 43-kDa polypeptide, but the form that prevails in the spore coat has a molecular mass of about 66 kDa (herein designated CotB-66). Here we show that in good agreement with its predicted size, expression of cotB in Escherichia coli results in the accumulation of a 46-kDa protein (CotB-46). Expression of cotB in sporulating cells of B. subtilis also results in a 46-kDa polypeptide which appears to be rapidly converted into CotB-66. These results suggest that soon after synthesis, CotB undergoes a posttranslational modification. Assembly of CotB-66 has been shown to depend on expression of both the cotH and cotG loci. We found that CotB-46 is the predominant form found in extracts prepared from sporulating cells or in spore coat preparations of cotH or cotG mutants. Therefore, both cotH and cotG are required for the efficient conversion of CotB-46 into CotB-66 but are dispensable for the association of CotB-46 with the spore coat. We also show that CotG does not accumulate in sporulating cells of a cotH mutant, suggesting that CotH (or a CotH-controlled factor) stabilizes the otherwise unstable CotG. Thus, the need for CotH for formation of CotB-66 results in part from its role in the stabilization of CotG. We also found that CotB-46 is present in complexes with CotG at the time when formation of CotB-66 is detected. Moreover, using a yeast two-hybrid system, we found evidence that CotB directly interacts with CotG and that both CotB and CotG self-interact. We suggest that an interaction between CotG and CotB is required for the formation of CotB-66, which may represent a multimeric form of CotB.
野生型枯草芽孢杆菌形成的芽孢被包裹在一个由30多种多肽有序组装而成的多层蛋白质结构（称为芽孢衣）中。一种多肽（CotB）是一种暴露于表面的芽孢衣成分，已被用作在芽孢表面展示异源抗原的载体。cotB基因最初通过反向遗传学被鉴定为编码一种丰富的芽孢衣成分。预测cotB编码一种43 kDa的多肽，但在芽孢衣中占主导的形式分子量约为66 kDa（在此称为CotB-66）。我们在此表明，与其预测大小高度一致，cotB在大肠杆菌中的表达导致一种46 kDa蛋白质（CotB-46）的积累。cotB在枯草芽孢杆菌的芽孢形成细胞中的表达也产生一种46 kDa的多肽，该多肽似乎会迅速转化为CotB-66。这些结果表明，CotB在合成后不久会经历翻译后修饰。已表明CotB-66的组装依赖于cotH和cotG基因座的表达。我们发现CotB-46是在芽孢形成细胞制备的提取物或cotH或cotG突变体的芽孢衣制备物中发现的主要形式。因此，cotH和cotG都是将CotB-46有效转化为CotB-66所必需的，但对于CotB-46与芽孢衣的结合则不是必需的。我们还表明，CotG不会在cotH突变体的芽孢形成细胞中积累，这表明CotH（或一个受CotH控制的因子）稳定了原本不稳定的CotG。因此，形成CotB-66对CotH的需求部分源于其在稳定CotG方面的作用。我们还发现，在检测到CotB-66形成时，CotB-46与CotG存在于复合物中。此外，使用酵母双杂交系统，我们发现证据表明CotB直接与CotG相互作用，并且CotB和CotG都会自身相互作用。我们认为CotG与CotB之间的相互作用是形成CotB-66所必需的，CotB-66可能代表CotB的一种多聚体形式。
