The construction of the bacterial envelope is a fundamental topic in cell biology: it confers integrity upon bacteria and, as such, represents the target of numerous antibiotics. In most rod-shaped bacteria, this envelope relies on peptidoglycan, a three-dimensional polymeric network whose composition and biosynthetic pathway are now well established, but whose spatial organization and assembly mechanism remain poorly understood. The protein MreB, the bacterial homolog of actin, is regarded as an essential component of the machinery that controls cell elongation and the maintenance of this cellular shape. Although recognized for its highly dynamic behavior — moving along the short axis of the cell, presumably tracking the progression of cell wall synthesis machineries — MreB has nonetheless been the subject of controversy for two decades regarding the structure, length, and conditions of formation of the polymers it assembles. Descriptions range from long helical filaments spanning the entire cell to small discrete entities, and this uncertainty directly affects the models describing the coordination of cell wall machineries.
To settle this debate, the authors analyzed various strains of Bacillus subtilis, a model organism for Gram-positive bacteria, using advanced microscopy techniques: total internal reflection fluorescence microscopy (TIRF) and its structured illumination variant (TIRF-SIM), whose experimental resolution was estimated at approximately 114 nm. Single-particle tracking, two-dimensional Gaussian fitting to measure filament dimensions, and kymograph analysis for the more extended structures enabled a fine characterization of the size and dynamics of MreB assemblies. The choice among these measurement methods was rigorously validated, notably through simulation, in order to avoid the biases inherent to each approach.
The results show that, during active growth, MreB forms on average nanofilaments shorter than 200 nm, below the diffraction limit of light. The micrometer-scale filaments previously reported appear to be the consequence of artifactual overaccumulation of the protein. Moreover, filament size influences neither their speed, nor their orientation, nor their other dynamic properties, conferring upon B. subtilis a broad tolerance to MreB levels and thus to the length of its polymers. The authors further observe that the density of mobile filaments remains constant regardless of the MreB level across strains, suggesting that another factor determines this constant. These data reveal the size of native MreB filaments during growth and contradict models in which filament length would directly affect the rate of cell wall synthesis or in which MreB would coordinate distant machineries along the lateral wall.