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

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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.