Structural Studies of the T- and RP4-Pili using Cryo-EM

Abstract

Bacterial pili are a diverse group of filamentous appendages with a variety of functions including but not limited to the adhesion to host surfaces and the transfer of DNA from one cell to another. The T-pilus, expressed by Agrobacterium tumefaciens, transfers T-DNA from bacterial cells to plant cells, while the RP4 pilus, used to transfer DNA between bacterial cells, is encoded by the conjugative plasmid, RP4, and can be expressed by multiple species of gram-negative bacteria. These pili are composed of thousands of subunits called pilins that are arranged into macromolecular assemblies with helical symmetry. CryoEM is the gold-standard for determining the complex arrangement of subunits in macromolecular structures. Recently a variety of bacterial pili have been investigated using cryoEM resulting in electron density maps at near-atomic resolution. One such pilus is the F-pilus which, like the T- and Rp4-pili, transfers DNA from one cell to another. Previous studies have indicated that both the T-pilus and RP4-pilus are composed of cyclic subunits, which serve as initial evidence that the structure of these two macromolecules may prove to be different than F-pili, which is composed of linear subunits. Other than F family pili there is little structural information regarding pili with the ability to transfer DNA. Additionally, the structural similarities among DNA-transferring pili have yet to be investigated thoroughly. In this study we use cryoEM to elucidate the structures of these pili, providing novel insights into how they transfer their DNA.