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

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.

Fig. 1

a Gel filtration of YFP-MreB after streptactin purification and expression under low salt (100 mM NaCl) or under osmotic stress (expression overnight, addition of 500 mM sorbitol and 1 mM betaine). Molecular standards are shown above the elution peaks. Fraction A2 = void volume, fraction A6 = monomeric YFP-MreB, as shown in the SDS-PAGE inserts (Purification buffer: 100 mM Tris HCl, 300 mM NaCl, 1 mM EDTA, 0.2 mM ATP, 5% Glycerol pH 7.5). b Monomer peaks of YFP-MreB, CFP-Mbl, mCherry-MreBH obtained under osmotic stress conditions (indicated by arrows), as outlined previously. c Sucrose gradient (5–15%) of isolated monomer peaks of YFP-MreB, CFP-Mbl, mCherry-MreBH. Biorad gel-filtration standard was used as a reference. (Marker proteins appearing in lane 1: Myoglobin, 2: Ovalbumin, 4: Gamma-Globulin, 10: Thyroglobulin; YFP-MreB, CFP-Mbl, mCherry-MreB appear in lane 2). d Mass photometric analysis of isolated YFP-MreB, CFP-Mbl, mCherry-MreBH monomers and embedded western blot with specific antibodies against the respective protein (V: void volume, M: isolated monomer peak, S: after dialysis to low salt polymerization buffer)

Fig. 2

Electron microscopy of negatively-stained MreB solution before and after induction of polymerization. a MreB monomers (2 μM) in polymerization buffer (5 mM TRIS-HCl, 0.1 mM CaCl₂, 0.2 mM ATP, pH 7.5) on EM grid. b-c Filaments formed from the same MreB solution as in A, after induction of polymerization with 10 mM MgCl₂. d YFP-MreB monomers (2 μM) in polymerization buffer on EM grid. e-f Filaments formed from the same YFP-MreB solution as in D, after induction of polymerization with 10 mM MgCl₂. Scaling is indicated below the black bars

Fig. 3

Assembly of YFP-MreB at a planar membrane in vitro. a Preparation of membrane vesicles, stained with FM4–64 (red fluorescent dye), b Calcium-induced fusion of vesicles establishes a flat membrane on top of a glass slide. Membrane stain in red, green channel shows background fluorescence in the YFP channel. c Diffuse localization of monomeric YFP-MreB on the membrane after washing with buffer. Note the small hole in the membrane in the upper left corner. d Addition of magnesium (10 mM) induces the formation of YFP-MreB filaments (2 μM) at the membrane. e STED images of individual YFP-MreB filaments (2 μM, 10 mM MgCl₂, no KCl), and examples of branching and fusion of filaments. f-i Different concentrations of YFP-MreB as indicated (10 mM MgCl₂, no KCl). j In vitro fluorescently labeled MreB carrying an additional cysteine residue integrated into the N-terminus of the protein (Cys-MreB) (2 μM, 10 mM MgCl₂), (k) low concentration of YFP-MreB mixed with Cys-MreB, (l) Mixture of a low concentration of YFP-MreB and a high concentration of non-fluorescence-tagged MreB. Note that all constructs carry a Strep-tag for purification. White bars 2 μm

Fig. 4

Formation of curved/helical YFP-MreB filaments extending from the planar membrane layer. a Z-stack through several planes away from the planar membrane, showing extension and branching of filaments. Apparent helical architecture is indicated by white triangles, different planes are indicated by white bar within rectangle. b-d Deconvoluted images of Z-stacks, B) single YFP-MreB filament, (c) single CFP-Mbl filament, (d) YFP-MreB filament in the presence of 100 mM potassium. Filament formation was induced through addition of 10 mM magnesium. White bars 1 μm

Fig. 5

Fluorescence microscopy showing the dependency of filament formation of MreB paralogs on protein concentration, on a planar membrane. a Different concentrations of isolated YFP-MreB monomers after addition of different concentrations MgCl₂, or of CaCl₂, as stated above and below the panels, (b) YFP-MreB (2 μM) after addition of 10 mM MgCl₂, in the presence of different concentrations of KCl as stated above the panels, (c) Different concentrations of CFP-Mbl or of mCherry-MreBH (after strep-purification) as stated above the panels after addition of 10 mM MgCl₂, white bars 2 μm

Fig. 6

Co-localization of MreB paralogs in vitro. a MreB paralogs prior to induction of polymerization on a planar membrane. Note that there is no spectral bleed through between the panels, and that there is an area lacking membrane coating that fluctuates between the acquisitions. b Co-polymerization of CFP-Mbl and mCherry-MreBH, each 5 μM, (c) Co-polymerization of CFP-Mbl and YFP-MreB, both 2 μM, (d) Co-polymerization of all three MreB paralogs, each 1 μM, using confocal microscopy, (e) Addition of 2 μM CFP-Mbl (green in overlay) to pre-polymerized YFP-MreB (2 μM, red in overlay), yellow triangles indicate common filamentous structures, green triangles CFP-Mbl structures that assembled independent of preexisting YFP-MreB filaments. f Addition of 2 μM mCherry-MreBH (green in overlay) to preassembled CFP-Mbl (2 μM, red in overlay), yellow triangles in overlay indicate mCherry-MreB filaments assembled at preexisting YFP-MreB filaments, green triangles independent structures. Scalebars 2 μm

Fig. 7

Polymerization of YFP-MreB within multilayered vesicles. a larger field of vesicles, (b) zoom into a multi layered vesicle, panels according to A): First panel overlay of fluorescence and DIC, second panel YFP fluorescence, third panel Nomarski DIC. c Z-stack zoom in into fluorescence channel of B). White bars 2 μm