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. 2018 Feb 22;7:e32471. doi: 10.7554/eLife.32471

Figure 1. Curved MreB filament motions do not follow an ordered template (A–C).

(A) The negative curvature of MreB filaments (arrowheads) aligns with the negative principal curvature of the liposome surface (arrow). Scale bar is 50 nm. (B) Angular distribution of GFP-MreB and GFP-Mbl trajectories relative to the long axis of B subtilis cells indicates that while the distributions have a mode of 90°, they are broad (Mean Deviation = 34°, n = 1041 for GFP-MreB and Mean Deviation = 26°, n = 1772 for GFP-Mbl). (C) Particle tracking of GFP-Mbl (left) and Halo-JF549-MreB (right) during 120 s (~1 rotation) indicates trajectories close in time frequently cross paths (white arrows). Scale bar is 1 μm. See corresponding Figure 1—video 1. (D) Strains with tagO under inducible control display a teichoic acid-dependent decrease in width. Strain BEG300 was grown in LB supplemented with 20 mM Mg2+. (E) Plot of cell width as a function of tagO induction in LB supplemented with 20 mM Mg2+, calculated from rod-shaped cells (error bars are Standard Error of the Mean (SEM), n = 33, 56, 104, 175, 228, 489, 119). Areas not plotted at lower xylose levels (red dashed rectangle) are regions where cells are round (no width axis). Color scheme for D-F: red indicates round cells (no width axis), blue indicates rods (measurable width axis), and green indicates intermediate regimes where both rods and round cells are observed. (F) BEG300 at an intermediate level of tagO induction (15 mM xylose) shows a Mg2+ dependent decrease in width. All scale bars are 5 μm. See also Figure 1—figure supplement 1.

Figure 1—source data 1. Figure 1B – Raw GFP-MreB and GFP-Mbl track angle values from the cell midline and their associated frequency distributions.
Figure 1E – Mean cell width, Standard Deviation (SD) and number of cells (n) analyzed at various xylose concentrations to create the graph in Figure 1E.
elife-32471-fig1-data1.xlsx (87.1KB, xlsx)
DOI: 10.7554/eLife.32471.006
Figure 1—source data 2. Figure 1—figure supplement 1B-left – Mean cell width, Standard Deviation (SD) and number of cells (N) analyzed at various magnesium and xylose concentrations.
Figure 1—figure supplement 1B-right – Tangential correlation along the cell contours at various xylose and magnesium concentrations. This data was used to calculate the correlation curves in Figure 1—figure supplement 1B.
elife-32471-fig1-data2.xlsx (44.1KB, xlsx)
DOI: 10.7554/eLife.32471.007

Figure 1.

Figure 1—figure supplement 1. Varying magnesium levels in the growth medium changes cell shape.

Figure 1—figure supplement 1.

(A) TagO inducible cells grown in LB supplemented with varying Mg2+ levels (0, 10 and 20 mM), show similar trends in cell shape across increasing xylose concentrations, with the appearance of more rod-shaped cells that become thinner as xylose levels increase. Exogenous Mg2+ reduces the amount of TagO induction needed for rod shape, evidenced by shift in the amount of xylose required to form rods as Mg2+ is increased. (Color Outlines: Blue = rods, Green = Mixed rods and non-rods, Red = non rods). (B) Left Plot of cell width as a function of TagO induction at different Mg2+ concentrations (error bars are SEM). Areas not plotted at lower xylose levels are regions where cells are round, with no width axis. Dotted rectangles mark conditions where both round cells and wide rods exist. Error bars are Standard Error of the Mean (SEM). Right At low xylose and magnesium levels, tangential correlation along the cell contours falls off faster, indicating loss of rod shape. Correlation of angles was calculated as described in methods. The curves shown are population averages of tangential correlations at selected xylose and magnesium concentrations. A cutoff of 3 μm is applied as this is the mean cell length of B. subtilis.

Figure 1—figure supplement 2. Schematic of cell contours and correlation.

Figure 1—figure supplement 2.

For straight rods, the contour angles on average remain highly correlated over larger distances (blue arrows), becoming uncorrelated at the cell pole (black arrows). In spherical cells the angles become uncorrelated at shorter distances.

Figure 1—video 1. Movie showing the trajectories taken by MreB and Mbl filaments frequently cross each other close in time.

Download video file (2.2MB, mp4)
DOI: 10.7554/eLife.32471.008
(First sequence) BDR2061, containing GFP-Mbl expressed at the native locus under a xylose-inducible promoter, was induced with 10 mM xylose and imaged with TIRFM. (Second sequence) bYS40 containing an endogenous HaloTag fusion to MreB at the native locus and promoter was labeled with 25 pm JF549. Frames are 1 s apart. Scale bar is 5 μm.