Alanine-scanning mutagenesis library of MreB reveals distinct roles for regulating cell shape and viability

Abstract

The bacterial actin-homolog MreB is a crucial component of the rod-system (elongasome) that maintains rod shape in many bacteria. It is localized beneath the inner membrane where it organizes the elongasome complex. Depletion or deletion ofmreBresults in loss of rod shape and cell death; however, the mechanism of how MreB operates is not known, given that the protein cannot be purified in a functionally intact form. Past studies have reported mutations inmreBcause varying degrees of cell shape and size alterations based on the type and position of the substitution. To better understand the role of MreB in rod shape formation we have taken the first truly systematic approach by replacing the native copy ofmreBwith an alanine-scanning mutagenesis library. Surprisingly, we observed stably growing spherical mutants that have lost MreB’s function(s) for shape regulation without losing viability. Hence, MreB has vital functions related to growth in addition to shape maintenance that can be separated. In support of this, rod shape suppressor analysis of these spherical mutants only revealed reversions or intragenicmreBmutations, suggesting that MreB is indispensable for rod shape. Additionally, our results imply the elongasome is no longer active in these strains, suggesting a novel way for rod shaped bacteria to synthesize cell wall.