Structure and function of bacterial secretion systems

Abstract

Bacterial macromolecular protein secretion systems are large (multi-mega Dalton) multiprotein complexes that transport effector proteins through bacterial membranes to the extracellular milieu or directly into the cytoplasm of other cells. These systems also mediate the horizontal transfer of genetic material (conjugation) between bacterial cells and specialized homologous forms of these systems have evolved into bacterial pili and flagella. Therefore, macromolecular secretion systems are important determinants for bacterial adaptation, competition and survival in diverse environmental niches. During the course of the previous thematic project, we demonstrated the importance of type IV pili (T4P) in surface twitching motility and biofilm formation in Xanthomonas citri and characterized key regulators of T4P biogenesis. We also showed that the Xanthomonas citri Type IV secretion system (T4SS) provides these cells the capacity to kill other Gram-negative bacterial species in a contact-dependent manner. This was the first demonstration of the involvement of a T4SS in bacterial killing and points to this special class of T4SS as an important determinant for Xanthomonad survival during competitive encounters with other bacterial species in the environment. In this project, we propose to pursue several lines of research into the molecular structure, molecular function and physiological relevance of these two complex nano-machines in Xanthomonas citri as well as in other members of the Xanthomonadaceae family. Specifically, we plan to pursue two general lines of research. Project 1: studies on the Xanthomonadaceae Type IV secretion systems. We plan to i) determine the structure of individual T4SS components as well as multiprotein complexes; ii) perform structural and ezymological studies on the toxins secreted by this system as well as their cognate inhibitors; iii) study the molecular basis of recognition of toxins by the T4SS apparatus; iv) study the cellular localization and regulation of expression of the T4SS, and iv) extend our studies from the Xanthomonas citri system to homologous T4SSs found in other bacterial species including Stenotrophomonas, Lysobacter and in some species of the betaproteobacteria orders Burkholderiales and Neisseriales. Project 2: studies on the Xanthomonas Type IV pilus. We plan to investigate i) how PilB ATPase activity is regulated by PilZ, FimX and c-diGMP and other inner membrane (platform) components; ii) the structures of the PilB-PilZ and PilB-PilZ-FimX complexes; iii) the roles of the conserved minor pilins in Xanthomonadaceae species; iv) the role of PilU, the third and poorly understood ATPase; v) how specific environmental signals control T4P-dependent functions in Xanthomonas specie; vi) how T4P contribute to Xanthomonas competitiveness in antagonistic interactions with other bacterial species, and vii) the structure of the extracellular pilus (polymer of PilA subunits). Our studies will employ a variety of experimental methods including X-ray crystallography, Nuclear Magnetic Resonance (NMR), cryoelectron microscopy of single particles, Small-Angle X-ray Scattering (SAXS), molecular dynamics simulations, the characterization of gene knockouts, gene expression analysis, fluorescence microscopy, enzymology and bioinformatics. This project involves the training of undergraduate and graduate students, post-doctoral researchers and collaboration with researchers here in Brazil and overseas. (AU)