Abstract
BACKGROUND: 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 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. RESULTS: Using 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. CONCLUSIONS: Our 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.