STRUCTURAL BASIS FOR SUBUNIT INTERACTIONS WITHIN THE TYPE IV SECRETION SYSTEM OF XANTHOMONAS CITRI

Bacteria use secretion systems to translocate macromolecules across the cellular envelope. These very large macromolecular complexes mediate a diverse range of functions, including DNA conjugation and the secretion of virulence factors. The Type IV Secretion System of the phytopathogen Xanthomonas citri (T4SS_XAC) is used to carry toxins to kill other gram-negative bacteria. While Cryo-EM have been providing crucial information regarding the architecture of these systems, structural details about presumably flexible regions are still missing. Furthermore, the core complex is the only T4SS_XAC sub-assembly that was structurally characterized to date. VirB8 is a key component of the T4SS periplasmic complex. The Xanthomonas citri VirB8 (VirB8_XAC) differs from the prototype VirB8 found in conjugation systems because it has a much larger and potentially disordered C-terminal tail. To obtain information on the structure of VirB8_XAC, we designed four constructs with and without the disordered C-terminal tail. We expressed and purified all of them. Preliminary characterization by 1D 1H NMR spectroscopy and gel filtration chromatography showed that VirB8 residues 102-245 might assume a well folded structure and that the presence of the C-terminal tail favors the formation of dimers and higher order VirB8 oligomers.