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

## TL;DR

This study uses a systematic approach to show that MreB has separate roles in cell shape and viability in bacteria.

## Contribution

The first systematic alanine-scanning mutagenesis of MreB reveals distinct roles in cell shape and viability.

## Key findings

- Spherical mutants show MreB's role in viability can be separated from shape regulation.
- MreB is absolutely required for rod shape maintenance.
- Mutants may use a novel cell wall synthesis pathway when MreB is nonfunctional.

## 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 cytoplasmic membrane, where it organizes the elongasome complex. Depletion or deletion of mreB results in loss of rod shape and cell death; however, the mechanism of how MreB operates is not known. Past studies have reported that mutations in mreB cause 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 of mreB with 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 intragenic mreB mutations, 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 Additionally, our results imply the elongasome is no longer active in these strains, suggesting a novel way for rod shaped bacteria to synthesize cell wall.

The actin homolog MreB is believed to be the main scaffolding protein of the Rod system. Previously it was shown MreB is essential for viability and shape regulation but our understanding of how it operates is limited due the difficulty of in vitro interrogations. Here, using a native-site alanine-scanning mutagenesis library, we show that the vital functions of shape maintenance and viability can be separated. Furthermore, we find that MreB is absolutely required for cell shape maintenance and that mutants that have lost rod shape maybe using a novel cell wall synthesis pathway.