Super-Resolution Microscopy and Single-Molecule Tracking Reveal Distinct Adaptive Dynamics of MreB and of Cell Wall-Synthesis Enzymes.

Simon Dersch,Johanna Mehl,Alexander Rohrbach,Lisa Stuckenschneider,Benjamin Mayer,Peter L Graumann,Julian Roth

doi:10.3389/fmicb.2020.01946

Simon Dersch, Johanna Mehl + Show 5 more

Open Access

https://doi.org/10.3389/fmicb.2020.01946

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Abstract

The movement of filamentous, actin-like MreB and of enzymes synthesizing the bacterial cell wall has been proposed to be highly coordinated. We have investigated the motion of MreB and of RodA and PbpH cell wall synthesis enzymes at 500 ms and at 20 ms time scales, allowing us to compare the motion of entire MreB filaments as well as of single molecules with that of the two synthesis proteins. While all three proteins formed assemblies that move with very similar trajectory orientation and with similar velocities, their trajectory lengths differed considerably, with PbpH showing shortest and MreB longest trajectories. These experiments suggest different on/off rates for RodA and PbpH at the putative peptidoglycan-extending machinery (PGEM), and during interaction with MreB filaments. Single molecule tracking revealed distinct slow-moving and freely diffusing populations of PbpH and RodA, indicating that they change between free diffusion and slow motion, indicating a dynamic interaction with the PGEM complex. Dynamics of MreB molecules and the orientation and speed of filaments changed markedly after induction of salt stress, while there was little change for RodA and PbpH single molecule dynamics. During the stress adaptation phase, cells continued to grow and extended the cell wall, while MreB formed fewer and more static filaments. Our results show that cell wall synthesis during stress adaptation occurs in a mode involving adaptation of MreB dynamics, and indicate that Bacillus subtilis cell wall extension involves an interplay of enzymes with distinct binding kinetics to sites of active synthesis.

Highlights

Bacteria have evolved an enormous assortment of different shapes to adapt to vastly different niches (Yang et al, 2016)
We wished to gain a clearer view on the dynamics of the molecules using superresolution (SR) fluorescence microscopy, with the specific question whether MreB filament dynamics follow similar paths and have similar dynamics compared to RodA and PbpH cell wall synthesis enzymes, and if MreB, RodA, and PbpH behave at the single molecule level
Because we observed that osmotic stress conditions have a different effect on the diffusion of MreB and of RodA and PbpH being involved in PG-synthesis, we further investigated if the patterns of PG-synthesis and of MreB macrostructures are altered under these conditions

Summary

Introduction

Bacteria have evolved an enormous assortment of different shapes to adapt to vastly different niches (Yang et al, 2016). It has become clear that a multitude of proteins affect cell morphology: do enzymes that actively extend the existing peptidoglycan (PG) strands hugely impact cell shape (Cabeen and Jacobs-Wagner, 2005), and proteins that affect ionic conditions within the wall (i.e., proteins generating teichoic acids within the cell wall) (Schirner et al, 2009), proteins that form filamentous structures within the cell (actin-like MreB) (Graumann, 2007), and proteins that provide precursors of cell wall material. When cell wall synthesis is inhibited, the motion of MreB filaments becomes highly reduced (Dominguez-Escobar et al, 2011; Garner et al, 2011; van Teeffelen et al, 2011), which has led to the model that perpendicular movement of MreB filaments is driven by the polymerization activity of cell wall synthetases

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