Searching for Protein-Protein Interactions within the Bacillus subtilis Spore Coat
Searching for Protein-Protein Interactions within the Bacillus subtilis Spore Coat
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DOI：
10.1128/jb.01807-08
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发表时间：
2009-05-01
影响因子：
3.2
通讯作者：
Barak, Imrich
中科院分区：
文献类型：
--
作者：
Krajcikova, Daniela;Lukacova, Magda;Barak, Imrich
枯草芽孢杆菌的内孢子抵御极端环境条件的能力是由几个属性保证的。其中一种是包裹孢子的蛋白质外壳，被称为外壳，为孢子提供了对有毒化学物质、裂解酶和单细胞和多细胞真核生物捕食的特有抵抗力。尽管孢子被的大部分成分已经确定，但我们对这种复杂的结构是如何组装的只有一个模糊的了解。利用酵母双杂交系统，我们试图鉴定分配给孢子壳不溶部分的蛋白质之间的直接联系：CotV、Cow、CotX、Coty和Cotz。我们还研究了它们是否能与Cote相互作用，Cote是控制外皮形成的最关键的形态发生蛋白之一，也存在于不溶部分中。在我们测试的所有21个可能的交互中，有4个被发现是积极的。在这些相互作用中，我们证实了先前的观察结果，即Cote形成同源低聚物。此外，我们观察到Coty的同型相互作用，Cotz和Coty之间的强相互作用，以及CotV和CoTw之间相对较弱但显著的相互作用。重组外壳蛋白的大小排除层析和下拉实验证实了该酵母双杂交分析的结果。
The capability of endospores of Bacillus subtilis to withstand extreme environmental conditions is secured by several attributes. One of them, the protein shell that encases the spore and is known as the coat, provides the spore with its characteristic resistance to toxic chemicals, lytic enzymes, and predation by unicellular and multicellular eukaryotes. Despite most of the components of the spore coat having been identified, we have only a vague understanding of how such a complex structure is assembled. Using the yeast two-hybrid system, we attempted to identify direct contacts among the proteins allocated to the insoluble fraction of the spore coat: CotV, CotW, CotX, CotY, and CotZ. We also examined whether they could interact with CotE, one of the most crucial morphogenetic proteins governing outer coat formation and also present in the insoluble fraction. Out of all 21 possible interactions we tested, 4 were found to be positive. Among these interactions, we confirmed the previous observation that CotE forms homo-oligomers. In addition, we observed homotypic interactions of CotY, strong interactions between CotZ and CotY, and relatively weak, yet significant, interactions between CotV and CotW. The results of this yeast two-hybrid analysis were confirmed by size exclusion chromatography of recombinant coat proteins and a pull-down assay.