Isolation and structural studies on the Xanthomonas citri Type IV pilus and T4SS pilus

Abstract

The pili of T2SS, T3SS, T4SS and T4P mediate the attachment of bacteria to other structures which can be other cells, phages or inanimate surfaces. This attachment can often lead to the transfer of virulence factors, invasion by a phage or the formation of biofilms. Type IV pili are specialized versions type II secretion systems (T2SSs) that translocate folded proteins from the periplasm across the cellular envelope of Gram-negative bacteria. The secreted pilin subunits form an extracellular helical polymer that can extend to several times the length of the bacterial cell. Associated proteins (adhesins, minor pilins) often facilitate T4P attachment to surfaces. Pilus polymerization, attachment and retraction (termed twitching motility) allow bacteria to move across surfaces and orient themselves with respect to other bacteria, and so play a very important role in the formation of the highly organized and multicellular structures in bacterial biofilms. We have been able to identify conditions in which the X. citri T4P are expressed, as well as a protocol for their purification. We recently been able to visiualize T4P on the surface of X. citri cells by electron microscopy. We have also studied several of regulatory proteins responsible for pilus biogenesis. We therefore plan to undertake a project to determine the molecular structure of coponents of the X. citri T4P by X-ray crystallography and Cryo-EM. Finally, we have recently sequenced the genome of the bacteriophage Xcm4-11 that infects X. citri via attachement to its Type IV pilus. We threfore plan to determine the structure of this baceriophage by Cryo-EM.