Super-Resolution Microscopy and Single-Molecule Tracking Reveal Distinct Adaptive Dynamics of MreB and of Cell Wall-Synthesis Enzymes

Abstract

The movement of filamentous, actin-like MreB and of enzymes synthesizing the bacterial cell wall has been proposed to be highly coordinated. We have investigated the motion of MreB and of RodA and PbpH cell wall synthesis enzymes at 500 ms and at 20 ms time scales, allowing us to compare the motion of entire MreB filaments as well as of single molecules with that of the two synthesis proteins. While all three proteins formed assemblies that move with very similar trajectory orientation and with similar velocities, their trajectory lengths differed considerably, with PbpH showing shortest and MreB longest trajectories. These experiments suggest different on/off rates for RodA and PbpH at the putative peptidoglycan-extending machinery (PGEM), and during interaction with MreB filaments. Single molecule tracking revealed distinct slow-moving and freely diffusing populations of PbpH and RodA, indicating that they change between free diffusion and slow motion, indicating a dynamic interaction with the PGEM complex. Dynamics of MreB molecules and the orientation and speed of filaments changed markedly after induction of salt stress, while there was little change for RodA and PbpH single molecule dynamics. During the stress adaptation phase, cells continued to grow and extended the cell wall, while MreB formed fewer and more static filaments. Our results show that cell wall synthesis during stress adaptation occurs in a mode involving adaptation of MreB dynamics, and indicate that Bacillus subtilis cell wall extension involves an interplay of enzymes with distinct binding kinetics to sites of active synthesis.

Keywords: Bacillus subtilis; MreB cytoskeleton; RodA; cell shape maintenance; single molecule dynamics.

FIGURE 1

TIRF-SIM analysis of exponentially growing cells expressing functional GFP-MreB YFP-RodA and YFP-PbpH fusions from ectopic sites on the chromosome. (A) Time-averaged intensity of 20 subsequent images (time projection) of MreB filament dynamics, (B) corresponding standard deviation (time projection) of MreB filaments, (C) overlay of time projection and standard deviation, revealing static (red) and dynamic (yellow) filaments. Note that there is a space window of L_max = 800 nm, due to the TIRF-illumination and a corresponding time window of t_max = L/v. (D) Average intensity of 20 subsequent images (time projection) of YFP-RodA dynamics, (E) corresponding standard deviation (time projection) for YFP-RodA, (F) overlay (see C), (G) average intensity of 20 subsequent images (time projection) of YFP-PbpH assemblies, (H) corresponding standard deviation (time projection), (I) overlay. Heat maps show fluorescence intensities as arbitrary units.

FIGURE 2

Comparison of velocity and trajectory time distributions of MreB, RodA, and PbpH On the right hand side the similar velocities of MreB, RodA, and PbpH can be seen. The histograms of the protein velocities are shown with their respective Gaussian fit. On the upper part of the figure the histograms of the trajectory times and their respective Gaussian fits are shown. While PbpH has very short trajectory times, RodA and MreB have longer trajectory times. Trajectory lengths (TL) longer than 800 nm were not observed (gray dotted line). Standard deviations of the binned measurements are shown with error bars, the shaded area around the graphs is the error of the Gaussian fit, which is calculated with the square root of the value.

FIGURE 3

Single molecule tracking of YFP-MreB. (A) Tracks (red, containing several steps) of linked sub-pixel localization events, identified by Gaussian-fitting, overlaid on a time-lapse of YFP-MreB (20 ms acquisition time), scale bar 2 μm; (B) Relative intensity of identified track as normalized arbitrary units over time; (C) Example of a chain of B. subtilis cells in exponential phase (pxyl-yfp-mreB::amyE,+ 0.01% Xyl) with applied polygonal meshes (white) overlaid with single-molecule tracks of expressed YFP-MreB (blue) and confined tracks (red, 200 nm radius), scale bar 2 μm; (D) Distribution of tracks in a single exemplary cell (normalized), single track is highlighted.

FIGURE 4

Comparison of MreB, RodA, and PbpH single-molecule motion. (A–C) Two-population Gaussian-mixture-model (GMM) fit of (A) MreB, (B) RodA, (C) PbpH displacement vs. probability density and corresponding heat map of the probabilistic distribution of tracks in a normalized cell (dark red: higher probability, white: lower probability); (D) Bubble-plot of the diffusive populations (relative fraction sizes, D [μm²s^{– 1}]) as identified by non-simultaneous GMM curve fit for MreB, RodA and PbpH; (E–H) Single-molecule dynamics of GFP-Mbl. expressed under control of the native promotor. Simultaneous two-population Gaussian-mixture-model (GMM) fit of displacement vs. probability density, (E) under normal growth, (F) with the addition of 0.5 M NaCl, (G) with the addition of 1M sorbitol; (H) Bubble-plot of the diffusive populations (relative fraction sizes, D [μm²s^{– 1}]) as identified by GMM curve fit.

FIGURE 5

Scatterplot of the relative fraction sizes and diffusion D [μm²s^{– 1}] as identified by non-simultaneous GMM fit for MreB, PbpH, and RodA under normal growth and with the addition of 0.5 M NaCl or 1 M Sorbitol.

FIGURE 6

Fluorescent D-amino acid stain reveals MreB localization and the pattern of peptidoglycan synthesis change under osmotic stress. (A) Bright field, YFP-MreB (green-channel), HADA (red-channel, 0.5 mM) and overlay images of B. subtilis cells in exponential phase, expressing YFP-MreB under the control of the xylose promotor (+0.01% xylose) in S750 media: 20 min HADA staining without stress and with added 0.5 M NaCl or 1 M Sorbitol, images taken after washing 3 times with PBS, scalebar 2 μm; (B) Demograph of the distribution of YFP-MreB and HADA signal throughout n = 100 cells during normal growth or with added 0.5 M NaCl or 1; (C) Brightfield, YFP-MreB (green-channel), HADA (red-channel, 0.5 mM) and overlay images of B. subtilis expressing YFP-MreB under the control of the xylose promotor (+0.01% Xyl) in S750 media: 20 min HADA staining with added 0.5 M NaCl after 1 and 2 h, images taken after washing three times with PBS, scalebar 2 μm; (D) Line-kymograph of the movement of an exemplary YFP-MreB macrostructure (red rectangle) 2 h after 0.5 M NaCl addition in x- (red); y-direction (blue) over 60 s; (E) Gated-STED image of YFP-MreB after 2 h of 0.5M NaCl addition (green), overlaid on a DIC image of the cells; scale bars 2 μm.

FIGURE 7

Dynamics and assembly/maintenance of B. subtilis MreB are affected by salt stress. SIM time lapse experiments. (A) Field of B. subtilis cells expressing GFP-MreB (from the ectopic amy-locus) during exponential growth (“steady state”) and 30 min after salt stress (“NaCl”). White bars 2 μm. (B) Frequency and density of displacements, indicating filament speed. (C) Box plots for integrated density and (D) area of fluorescence, indicating the extent of filament formation for GFP-MreB. Black horizontal lines indicate means, dots in pink diamonds indicate outliers. Data from n = 481 (steady state) or n = 471 (NaCl) cells. ***indicates p value of less than 0.001.