Filament Structure, Organization, and Dynamics in MreB Sheets

David Popp,Akihiro Narita,Kayo Maeda,Tetsuro Fujisawa,Umesh Ghoshdastider,Mitsusada Iwasa,Yuichiro Maéda,Robert C Robinson

doi:10.1074/jbc.m109.095901

Abstract

In vivo fluorescence microscopy studies of bacterial cells have shown that the bacterial shape-determining protein and actin homolog, MreB, forms cable-like structures that spiral around the periphery of the cell. The molecular structure of these cables has yet to be established. Here we show by electron microscopy that Thermatoga maritime MreB forms complex, several mum long multilayered sheets consisting of diagonally interwoven filaments in the presence of either ATP or GTP. This architecture, in agreement with recent rheological measurements on MreB cables, may have superior mechanical properties and could be an important feature for maintaining bacterial cell shape. MreB polymers within the sheets appear to be single-stranded helical filaments rather than the linear protofilaments found in the MreB crystal structure. Sheet assembly occurs over a wide range of pH, ionic strength, and temperature. Polymerization kinetics are consistent with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation. Steady-state TIRF microscopy studies of MreB suggest filament treadmilling while high pressure small angle x-ray scattering measurements indicate that the stability of MreB polymers is similar to that of F-actin filaments. In the presence of ADP or GDP, long, thin cables formed in which MreB was arranged in parallel as linear protofilaments. This suggests that the bacterial cell may exploit various nucleotides to generate different filament structures within cables for specific MreB-based functions.

Highlights

Despite usually being constrained by a cell wall, bacterial shapes are highly diverse, reflecting the large phylogenetic range
We show that MreB from T. maritime forms novel sheets consisting of interwoven helical-like filaments, which may be related to the cables observed by light microscopy and the ribbons observed by cryoelectron tomography in vivo
Disruption of pole-to-pole MreB helically arranged cables by mutational inactivation resulted in failure of lateral murein synthesis and formation of spherical cells [26]

Summary

Introduction

Despite usually being constrained by a cell wall, bacterial shapes are highly diverse, reflecting the large phylogenetic range. We show that MreB from T. maritime forms novel sheets consisting of interwoven helical-like filaments, which may be related to the cables observed by light microscopy and the ribbons observed by cryoelectron tomography in vivo. At physiological salt concentrations around 350 mM KCl found in bacterial cells [16] and at pH values between 7.0 and 7.7 typical for most bacteria, including T. maritime [17], most sheets were about 30 –100 nm wide and up to several ␮m long (Fig. 1, E and H), consistent with values obtained from in vivo light microscopy observations of MreB [2].

Results

Conclusion