Structural and functional studies of the Xanthomonas citri Type IV Secretion System

Abstract

The bacterial type IV secretion system (T4SS) is a large megaDalton protein complex involved in the transport of DNA and proteins across the cell envelope. Apart from its well known role in bacterial conjugation, many T4SSs are responsible for the transfer of virulence factors into host cells and are therefore important objects of study in the combat against disease in animals and plants. Bacteria of the Xanthomonadaceae family possess a distinctive T4SS, though its precise physiological function has not yet been described. Recently, our group has accumulated convincing experimental evidence that the physiological role of the Xanthomonas T4SS is to transfer toxins to other bacterias and our hypothesis is that this system may therefore be a decisive factor in the ability of Xac to compete with other Gram-negative bacteria in the environment. This post-doctoral project has two groups of complementary objectives:The first objective is to investigate signal transduction pathways that regulate the genetic expression and the post-transcriptional activation of the Xanthomonas citri (Xac) T4SS. These pathways will be investigated using Xac knockouts in genes involved in c-di-GMP signaling, "quorum sensing", two-component systems, alternative sigma factors and possible regulators of the Xac vir locus (many of these knockouts are already available in our laboratory). Expression levels of the Xac vir locus will be evaluated in these mutants as well as T4SS activation and function. The second objective of the project is to carry out structural studies of the Xac T4SS, specifically the VirB7-VirB9-VirB10 complex that forms the pore through both the inner and outer membrane layers of the bacterial envelope. These studies will begin by expressing the VirB7-VirB9-VirB10 complex in E. coli and submitting the purified complex to structural studies using X-ray crystallography, SAXS and NMR.