Integrative Structure and Function of the Vibrio cholerae Competence Pilus Machine

Abstract

Type IV pili are hair-like, surface-exposed polymers involved in many fundamental biological processes. The competence pilus (CP) is responsible for natural transformation, the ability to acquire exogenous DNA. Here we purified the Vibrio cholerae CP and used electron cryo-microscopy (cryo-EM) to obtain a 3.3 [A] map of the fiber by helical reconstruction. We then used electron cryo-tomography (cryo-ET) to reveal the in-situ architecture of the competence pilus machine (CPM). Compared to other type IVa pilus machines, the CPM has unique characteristics including multiple conformational states, a ring-like density formed by PilQs C-terminal domain absent in the previously solved SPA structure, and the presence of a double ATPase ring in the cytoplasm. Using integrative modeling with the cryo-ET map as an envelope, we generated a full-length pseudoatomic model of the CPM. Molecular dynamics (MD) simulations of the entire CPM embedded within realistic bacterial membranes and the peptidoglycan cell wall revealed that the model demonstrates structural integrity under physiological conditions. Steered MD simulations of pilus extension and retraction through PilQ revealed gate-opening mechanisms and demonstrated the channels mechanical resistance and flexibility during translocation.