RELATING STRUCTURE WITH FUNCTION AND MECHANICS OF BACTERIAL POLYMERS

Abstract

Bacterial polymers are a diverse group of proteins involved in a wide range of functions including cell shape maintenance, subcellular localization, cell division, and plasmid segregation. These polymers are highly important to understand major cell-scale processes. The mechanical properties of bacterial polymers have been suggested to play important roles in their functions. However, there are relatively few examples available where the mechanics and structures of the same polymers are known, such that the biophysical relationships between these properties remains largely unclear. Here we study two different bacterial polymers and utilize single particle Cryo-Electron Microscopy (cryo-EM) to relate the structure of the polymers to their function and mechanics. We first studied the periplasmic copolymer CrvAB, found in Vibrio cholerae. CrvAB is comprised of two proteins CrvA and CrvB which assemble together and induce cell curvature. The curvature of V. cholerae cells improves the fitness, motility, and pathogenesis. Here we aimed to study the interactions between CrvA and CrvB and the structure of the copolymer. Using cryo-EM, we developed a 3D reconstruction of the CrvAB filaments in vitro and developed a model to fit the reconstruction. We utilized in vitro and in vivo techniques to validate the model and discovered key amino acid interactions necessary for proper functioning of the CrvAB copolymer. Excitingly, the CrvAB filaments have a unique hexagonal structure not seen in other bacterial polymers. The second bacterial polymers studied here are Type IV Pili (T4P). These bacterial polymers extend beyond the cell surface and are involved in a wide range of functions including DNA uptake, twitching motility, and virulence. We found that T4P have distinct mechanics by measuring the persistence lengths of the polymers. The sequence of the pilin subunits does not predict the measured mechanics. We instead hypothesized that the structure of the pilus filaments would dictate the mechanics of the fiber. To determine this, we prepared two distinct T4P samples and utilized cryo-EM to determine the structure of the filaments. We found that the pili that had tighter packing of monomers was less flexible compared to the more loosely packed pilus.