Bacillus subtilis cell diameter is determined by the opposing actions of two distinct cell wall synthetic systems

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Abstract

Rod-shaped bacteria grow by adding material into their cell wall via the action of two spatially distinct enzymatic systems: the Rod complex moves around the cell circumference, whereas class A penicillin-binding proteins (aPBPs) do not. To understand how the combined action of these two systems defines bacterial dimensions, we examined how each affects the growth and width of Bacillus subtilis as well as the mechanical anisotropy and orientation of material within their sacculi. Rod width is not determined by MreB, rather it depends on the balance between the systems: the Rod complex reduces diameter, whereas aPBPs increase it. Increased Rod-complex activity correlates with an increased density of directional MreB filaments and a greater fraction of directional PBP2a enzymes. This increased circumferential synthesis increases the relative quantity of oriented material within the sacculi, making them more resistant to stretching across their width, thereby reinforcing rod shape. Together, these experiments explain how the combined action of the two main cell wall synthetic systems builds and maintains rods of different widths. Escherichia coli Rod mutants also show the same correlation between width and directional MreB filament density, suggesting this model may be generalizable to bacteria that elongate via the Rod complex.

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Fig. 1: Rod width depends on the relative levels of widening aPBPs to the thinning Rod system.
Fig. 2: Effects of RodA–PBP2a on cell width and how each PG synthetic system affects growth.
Fig. 3: Increased mreBCD increases directional MreB filament density and the fraction of directional PBP2a molecules.
Fig. 4: Increased Rod activity increases both the amount of oriented material within sacculi and their mechanical anisotropy.
Fig. 5: Directional MreB filament density also correlates with the cell width of E. coli Rod mutants.

Data availability

All datasets and raw data generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Raw and proteomic data are available at https://garnerlab.fas.harvard.edu/Dion2019/Raw-MS-data-Dion2019.zip.

Code availability

All particle tracking was done with the Trackmate plugin within FIJI, then analysed using code available at https://bitbucket.org/garnerlab/hussain-2017-elife. Filament density calculations and filament simulations were performed with custom code available at https://bitbucket.org/garnerlab/dion-2018/src/.

Change history

  • 06 June 2019

    In the version of this Article originally published, Supplementary Video 1 was incorrectly linked to Supplementary Video 3, Supplementary Video 2 was incorrectly linked to Supplementary Video 1 and Supplementary Video 3 was incorrectly linked to Supplementary Video 2. The files have now been replaced to rectify this.

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Acknowledgements

We thank C. Wivagg, Y. J. Eun and L. Harris for their helpful discussions, G. Squyres for TIRF-SIM, L. Lavis for JF dyes and Z. Gitai and K. C. Huang for bacterial strains. TIRF-SIM was performed at the Advanced Imaging Center at the Janelia Research Campus, a facility jointly supported by the Gordon and Betty Moore Foundation and Howard Hughes Medical Institute. This work was funded by the National Institute of Health (grant nos R01GM114274 to R.O. and DP2AI117923 to E.C.G) and support from the Volkswagen Foundation to E.C.G. and S.v.T. Some work was performed at the Center for Nanoscale Systems at Harvard University, supported by NSF ECS-0335765.

Author information

B. subtilis strains were cloned by M.F.D., Y.S. and M.K. All width, length and bulk growth measurements of B. subtilis were performed by M.F.D. and E. coli widths by Y.S. E. coli CRISPRi strains were cloned by A.V., supervised by S.v.T. Single-cell growth rates were done by Y.S. and M.K. TIRFM and tracking of PBP2a was conducted by M.K. All TIRFM of MreB was done by Y.S. TIRF-SIM of MreB was performed by Y.S., E.C.G. and J.R. Proteomic preparations and analyses, and sacculi purifications were done by M.F.D. Y.S. wrote the code for the analysis of single-cell growth rates, filament density and simulations of data. Polarization microscopy and analysis was conducted by J.R., R.O. and E.C.G. S.W. did the FDAA synthesis, osmotic shocks and transmission electron microscopy. The paper was written by E.C.G., M.K., M.F.D. and Y.S.

Correspondence to Ethan C. Garner.

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Supplementary information

Supplementary Information

Supplementary Figures 1–7 and Supplementary Video legends.

Reporting Summary

Supplementary Data 1

Mass spectrometry data.

Supplementary Video 1

Example single molecule videos (taken with TIRF microscopy) of Halo–PBP2a (labelled with JF549) at different mreBCD induction levels in bMK385 (amyE::erm Pxyl-mreBCD, ΔmreBCD::spc, pbpA::cat HaloTag-11aa-pbpA). Xylose concentrations are indicated in each panel. Frames are 300 ms apart.

Supplementary Video 2

Z-stack of LC-PolScope images of sacculi purified from WT strain PY79. Left, Z-stack of the retardance of each plane. Right, colour shows slow axis orientation, intensity corresponds to retardance in that direction (reference, upper left colour wheel). Z-steps were taken in 100 nm increments.

Supplementary Video 3

Z-stack colour map of birefringence of sacculi purified from strain bMD620 (amyE::erm Pxyl-mreBCD, ΔmreBCD::spc, yhdG::cat Pspank-ponA, ΔponA::kan), where cells were induced to “Wide” (0.5 mM xylose, 0.1 mM IPTG) and “Normal” widths (5 mM xylose, 0.025 mM IPTG). Colour shows slow axis orientation, intensity corresponds to retardance in that direction (reference, upper left colour wheel). Z-steps were taken in 100 nm increments.

Supplementary Video 4

TIRF-SIM videos of different MreB-msfGFPsw mutants. Cells were grown in LB at 37 °C, then placed under an LB agarose pad and imaged at 37 °C. Frames are 1 s apart. Scale bars are 1 μm.

Supplementary Video 5

TIRF-SIM videos of strain KC717 (mreB::msfGFP-mreB, ProdZ<> (frt araC PBAD)). Cells were grown for 5 h in LB with the indicated arabinose concentrations (to induce different amounts of RodZ expression) then placed under an LB agarose pad and imaged at 37 °C. Frames are 1 s apart. Scale bars are 1 μm.

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