Cell shape and cell-envelope integrity of bacteria are determined by the peptidoglycan cell wall. In rod-shaped Escherichia coli, two conserved sets of machinery are essential for cell-wall insertion in the cylindrical part of the cell: the Rod complex and the class-A penicillin-binding proteins (aPBPs). While the Rod complex governs rod-like cell shape, aPBP function is less well understood. aPBPs were previously hypothesized to either work in concert with the Rod complex or to independently repair cell-wall defects. First, we demonstrate through modulation of enzyme levels that aPBPs do not contribute to rod-like cell shape but are required for mechanical stability, supporting their independent activity. By combining measurements of cell-wall stiffness, cell-wall insertion, and PBP1b motion at the single-molecule level, we then present evidence that PBP1b, the major aPBP, contributes to cell-wall integrity by repairing cell wall defects.

The shapes of most bacteria are imparted by the structures of their peptidoglycan cell walls, which are determined by many dynamic processes that can be described on various length-scales ranging from short-range glycan insertions to cellular-scale elasticity.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 Understanding the mechanisms that maintain stable, rod-like morphologies in certain bacteria has proved to be challenging due to an incomplete understanding of the feedback between growth and the elastic and geometric properties of the cell wall.3, 4, 12, 13, 14 Here we probe the effects of mechanical strain on cell shape by modeling the mechanical strains caused by bending and differential growth of the cell wall. We show that the spatial coupling of growth to regions of high mechanical strain can explain the plastic response of cells to bending4 and quantitatively predict the rate at which bent cells straighten. By growing filamentous E. coli cells in donut-shaped microchambers, we find that the cells recovered their straight, native rod-shaped morphologies when released from captivity at a rate consistent with the theoretical prediction. We then measure the localization of MreB, an actin homolog crucial to cell wall synthesis, inside confinement and during the straightening process and find that it cannot explain the plastic response to bending or the observed straightening rate. Our results implicate mechanical strain-sensing, implemented by components of the elongasome yet to be fully characterized, as an important component of robust shape regulation in E. coli.