Biology subjects

Morgenstein, R. M.

Publications and source records attributed to Morgenstein, R. M..

3 recordsLinked to original sources

bioRxiv2024

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

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 of mreB results 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 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 MreBs 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 Additionally, our results imply the elongasome is no longer active in these strains, suggesting a novel way for rod shaped bacteria to synthesize cell wall.

microbiology↗

bioRxiv2022

Role of EnvC and the phosphoenolpyruvate:sugar phosphotransferase system in resistance to MreB disruption

Cell wall synthesis in bacteria is determine by two protein complexes: the elongasome and divisome. The elongasome is coordinated by the actin homolog MreB while the divisome is organized by the tubulin homolog FtsZ. While these two systems must coordinate with each other to ensure that elongation and division are coregulated, this cross talk has been understudied. Using the MreB depolymerizing agent, A22, we found that multiple gene deletions result in cells exhibiting increased sensitivity to MreB depolymerization. One of those genes encodes for EnvC, a part of the divisome that is responsible for splitting daughter cells after the completion of cytokinesis through the activation of specific amidases. Here we show this increased sensitivity to A22 works through two known amidase targets of EnvC: AmiA and AmiB. In addition, suppressor analysis revealed that mutations in the phosphoenolpyruvate:sugar phosphotransferase system (PTS) can suppress the effects of A22 in both wild-type and envC deletion cells. Together this work helps to link elongation, division, and metabolism.

microbiology↗

bioRxiv2017

RodZ promotes MreB polymer formation and curvature localization to determine the cylindrical uniformity of E. coli shape

Cell shape in bacteria is determined by the cell wall, which is synthesized by a variety of proteins whose actions are coordinated by the actin-like MreB protein. MreB uses local geometric cues of envelope curvature to avoid the cell poles and localize to specific regions of the cell body. However, it remains unclear whether MreBs curvature preference is regulated by additional factors, and which features of MreB are essential for specific aspects of rod shape growth, such as cylindrical uniformity. Here we show that in addition to its previously-described role in mediating MreB motion, RodZ also modulates MreB polymer number and curvature preference. MreB polymer number and curvature localization can be regulated independently. Quantitative 3D measurements and a series of mutant strains show that among various properties of MreB, polymer number, total length of MreB polymers, and MreB curvature preference are the key determinants of cylindrical uniformity, a measure of the variability in radius within a single cell. Changes in the values of these parameters are highly predictive of the resulting changes in cell shape (r2=0.93). Our data suggest a model for rod shape in which RodZ promotes the assembly of multiple long MreB polymers that ensure the growth of a uniform cylinder.

microbiology↗