Characterization of periplasmic components of the type III secretion system from Xanthomonas

革兰氏阴性植物致病菌油菜黄单胞菌。毛囊病通过III型分泌（T3S）系统将细菌效应蛋白转运到植物细胞中。T3S系统是一个高度复杂的纳米机器，与细胞外菌和植物质膜中可预测的通道样转座子有关。T3S依赖于早期T3S底物HrpB2， HrpB2对菌毛组装至关重要，并与内膜蛋白HrcD以及预测的肽聚糖结合蛋白HrpB1相互作用。蛋白质研究表明，HrpB1和HrpB2形成复合物，并在细菌周质和外膜上共定位。有趣的是，HrpB2的质周定位与t3s无关，这表明它可以通过另一种运输途径穿过内膜。本文的目的是分析HrpB1和HrpB2的质周定位和复合物的形成，并表征它们对T3S的贡献。下拉实验和突变体研究将有助于确定HrpB1-和hrpb2相互作用的伙伴，并定位功能重要的蛋白质区域。在项目的第二部分，我们将研究HrpB2中T3S信号和预测的Sec信号对其质周定位的贡献。此外，我们将研究Sec系统中预测的ATPase SecA在HrpB2靶向细菌外周质中的潜在作用。提出的实验包括诱变方法，体外和体内相互作用以及在我的小组中建立的感染研究。对于表达结构的生成，我们将使用高效的金门克隆方法。在选定启动子的控制下，与表位编码序列融合表达基因的目的载体。和大肠杆菌都是现成的。电子显微镜和生化方法分析蛋白质复合物将与大学的其他部门合作进行。由于T3S系统的膜相关亚结构尚未在植物致病菌中得到研究，计划中的实验将显著提高我们对这一重要蛋白质注射机制的组装和结构的认识。

The Gram-negative plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to translocate bacterial effector proteins into plant cells. The T3S system is a highly complex nanomachine that is associated with an extracellular pilus and a predicted channel-like translocon in the plant plasma membrane. T3S depends on the early T3S substrate HrpB2 that is essential for pilus assembly and interacts with the inner membrane protein HrcD as well as with the predicted peptidoglycan-binding protein HrpB1. Protein studies suggest that HrpB1 and HrpB2 form complexes and colocalize to the bacterial periplasm and the outer membrane. Interestingly, the periplasmic localization of HrpB2 is T3S-independent, suggesting that it can traverse the inner membrane by an alternative transport route. The aim of this proposal is to analyze the periplasmic localization and complex formation of HrpB1 and HrpB2 and to characterize their contribution to T3S. Pull-down experiments and mutant studies will help to identify HrpB1- and HrpB2-interaction partners and to localize functionally important protein regions. In a second part of the project, we will study the contribution of the T3S signal and the predicted Sec signal in HrpB2 to its periplasmic localization. Furthermore, we will investigate a potential role of the predicted ATPase SecA of the Sec system to the targeting of HrpB2 to the bacterial periplasm. The proposed experimentals include mutagenesis approaches, in vitro and in vivo interaction as well as infection studies that are established in my group. For the generation of expression constructs, we will use the highly efficient Golden Gate cloning method. Respective destination vectors for the expression of genes in fusion with epitope-encoding sequences under control of selected promoters in X. campestris pv. vesicatoria or E. coli are already available. Electron microscopy and biochemical approaches for the analysis of protein complexes will be performed in collaboration with other departments of the university. As membrane-associated substructures of T3S systems have not yet been studied in plant pathogenic bacteria, the planned experiments will significantly improve our knowledge on the assembly and architecture of this essential protein injection machinery.