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

野油菜黄单胞菌（Xanthomonascampestris pv.）vesicatoria利用III型分泌（T3S）系统将细菌效应蛋白易位到植物细胞中。T3S系统是一种高度复杂的纳米机器，与细胞外菌毛和植物质膜中的预测通道样易位子相关。T3S依赖于早期T3S底物HrpB2，该底物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形成复合物并共定位于细菌周质和外膜。有趣的是，HrpB2的周质定位是T3S独立的，这表明它可以通过另一种运输途径穿过内膜。该建议的目的是分析HrpB1和HrpB2的周质定位和复合物形成，并表征其对T3S的贡献。下拉实验和突变体的研究将有助于确定HrpB1和HrpB2相互作用的合作伙伴和本地化功能重要的蛋白质区域。在该项目的第二部分中，我们将研究T3S信号和预测的Sec信号在HrpB2的周质定位的贡献。此外，我们将研究预测的ATPase SecA的Sec系统的HrpB2的细菌周质的靶向的潜在作用。本课题组拟开展的实验包括诱变方法、体外和体内相互作用以及感染研究。对于表达构建体的产生，我们将使用高效的Golden Gate克隆方法。用于在X中表达与在选定启动子控制下的表位编码序列融合的基因的相应目的载体。野油菜致病变种vesicatoria或E.大肠杆菌已经存在。电子显微镜和蛋白质复合物分析的生物化学方法将与大学的其他部门合作进行。由于尚未在植物病原菌中研究T3S系统的膜相关亚结构，因此计划的实验将显着提高我们对这种必需蛋白质注射机器的组装和架构的认识。

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.