Polymerization of Bacillus subtilis MreB on a lipid membrane reveals lateral co-polymerization of MreB paralogs and strong effects of cations on filament formation

Simon Dersch,Peter L Graumann,Hervé Joel Defeu Soufo,Christian Reimold,Joshua Stoll,Thomas Heimerl,Hannes Breddermann

doi:10.1186/s12860-020-00319-5

Abstract

BackgroundMreB is a bacterial ortholog of actin and forms mobile filaments underneath the cell membrane, perpendicular to the long axis of the cell, which play a crucial role for cell shape maintenance. We wished to visualize Bacillus subtilis MreB in vitro and therefore established a protocol to obtain monomeric protein, which could be polymerized on a planar membrane system, or associated with large membrane vesicles.ResultsUsing a planar membrane system and electron microscopy, we show that Bacillus subtilis MreB forms bundles of filaments, which can branch and fuse, with an average width of 70 nm. Fluorescence microscopy of non-polymerized YFP-MreB, CFP-Mbl and mCherry-MreBH proteins showed uniform binding to the membrane, suggesting that 2D diffusion along the membrane could facilitate filament formation. After addition of divalent magnesium and calcium ions, all three proteins formed highly disordered sheets of filaments that could split up or merge, such that at high protein concentration, MreB and its paralogs generated a network of filaments extending away from the membrane. Filament formation was positively affected by divalent ions and negatively by monovalent ions. YFP-MreB or CFP-Mbl also formed filaments between two adjacent membranes, which frequently has a curved appearance. New MreB, Mbl or MreBH monomers could add to the lateral side of preexisting filaments, and MreB paralogs co-polymerized, indicating direct lateral interaction between MreB paralogs.ConclusionsOur data show that B. subtilis MreB paralogs do not easily form ordered filaments in vitro, possibly due to extensive lateral contacts, but can co-polymerise. Monomeric MreB, Mbl and MreBH uniformly bind to a membrane, and form irregular and frequently split up filamentous structures, facilitated by the addition of divalent ions, and counteracted by monovalent ions, suggesting that intracellular potassium levels may be one important factor to counteract extensive filament formation and filament splitting in vivo.

Highlights

MreB is a bacterial ortholog of actin and forms mobile filaments underneath the cell membrane, perpendicular to the long axis of the cell, which play a crucial role for cell shape maintenance
We wished to obtain a better understanding of MreB filaments from a Grampositive bacterium
A combination of overnight expression at low temperatures under weak osmotic pressure with 500 mM sorbitol and purification of the obtained cell pellets using a buffer containing 300 mM salt (Purification buffer: 100 mM Tris HCl, 300 mM NaCl, 1 mM Ethylenediaminetetraacetic acid (EDTA), 0.2 mM Adenosine triphosphate (ATP), 5% glycerol pH 7.5) resulted in a peak containing monomeric MreB (Fig. 1a) that could be isolated via size-exclusion chromatography

Summary

Introduction

MreB is a bacterial ortholog of actin and forms mobile filaments underneath the cell membrane, perpendicular to the long axis of the cell, which play a crucial role for cell shape maintenance. The filaments consist of two protofilaments that are arranged as a right-handed double helix [3] The dynamics of this structure are based on a polar growth at steady state with a net polymerization on one end (plus end) and depolymerization on the other end (minus end), giving rise to a treadmilling-like movement of subunits within the filament [4]. Monomeric actin (globular/G-actin) consists of four subdomains with five highly conserved motifs that enclose the nucleotide-binding site as the central core Homologous proteins harboring these conserved motifs are classified as members of the actin superfamily and are present in all domains of life with highly divergent functions [6,7,8,9]

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