Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

This article has been updated

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

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.

References

  1. Vadia, S. & Levin, P. A. Growth rate and cell size: a re-examination of the growth law. Curr. Opin. Microbiol. 24, 96–103 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Sharpe, M. E., Hauser, P. M., Sharpe, R. G. & Errington, J. Bacillus subtilis cell cycle as studied by fluorescence microscopy: constancy of cell length at initiation of DNA replication and evidence for active nucleoid partitioning. J. Bacteriol. 180, 547–555 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Vollmer, W., Blanot, D. & de Pedro, M. A. Peptidoglycan structure and architecture. FEMS Microbiol. Rev. 32, 149–167 (2008).

    Article  CAS  PubMed  Google Scholar 

  4. Lebar, M. D. et al. Reconstitution of peptidoglycan cross-linking leads to improved fluorescent probes of cell wall synthesis. J. Am. Chem. Soc. 136, 10874–10877 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Banzhaf, M. et al. Cooperativity of peptidoglycan synthases active in bacterial cell elongation. Mol. Microbiol. 85, 179–194 (2012).

    Article  CAS  PubMed  Google Scholar 

  6. Meeske, A. J. et al. SEDS proteins are a widespread family of bacterial cell wall polymerases. Nature 537, 634–638 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Jones, L. J., Carballido-López, R. & Errington, J. Control of cell shape in bacteria: helical, actin-like filaments in Bacillus subtilis. Cell 104, 913–922 (2001).

    Article  CAS  PubMed  Google Scholar 

  8. van den Ent, F., Amos, L. & Löwe, J. Bacterial ancestry of actin and tubulin. Curr. Opin. Microbiol. 4, 634–638 (2001).

    Article  PubMed  Google Scholar 

  9. van den Ent, F., Izoré, T., Bharat, T. A., Johnson, C. M. & Lowe, J. Bacterial actin MreB forms antiparallel double filaments. eLife 3, e02634 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Salje, J., van den Ent, F., de Boer, P. & Lowe, J. Direct membrane binding by bacterial actin MreB. Mol. Cell 43, 478–487 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hussain, S. et al. MreB filaments align along greatest principal membrane curvature to orient cell wall synthesis. eLife 7, e32471 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Garner, E. C. et al. Coupled, circumferential motions of the cell wall synthesis machinery and MreB filaments in B. subtilis. Science 333, 222–225 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. van Teeffelen, S. et al. The bacterial actin MreB rotates, and rotation depends on cell-wall assembly. Proc. Natl Acad. Sci. USA 108, 15822–15827 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Domínguez-Escobar, J. et al. Processive movement of MreB-associated cell wall biosynthetic complexes in bacteria. Science 333, 225–228 (2011).

    Article  PubMed  Google Scholar 

  15. Turner, R. D., Mesnage, S., Hobbs, J. K. & Foster, S. J. Molecular imaging of glycan chains couples cell-wall polysaccharide architecture to bacterial cell morphology. Nat. Commun. 9, 1263 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  16. McPherson, D. C. & Popham, D. L. Peptidoglycan synthesis in the absence of class A penicillin-binding proteins in Bacillus subtilis. J. Bacteriol. 185, 1423–1431 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Cho, H. et al. Bacterial cell wall biogenesis is mediated by SEDS and PBP polymerase families functioning semi-autonomously. Nat. Microbiol. 1, 16172 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Ursell, T. S. et al. Rod-like bacterial shape is maintained by feedback between cell curvature and cytoskeletal localization. Proc. Natl Acad. Sci. USA 111, E1025–E1034 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Ouzounov, N. et al. MreB orientation correlates with cell diameter in Escherichia coli. Biophys. J. 111, 1035–1043 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Wang, S. & Wingreen, N. S. Cell shape can mediate the spatial organization of the bacterial cytoskeleton. Biophys. J. 104, 541–552 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Tropini, C. et al. Principles of bacterial cell-size determination revealed by cell-wall synthesis perturbations. Cell Rep. 9, 1520–1527 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Shi, H. et al. Deep phenotypic mapping of bacterial cytoskeletal mutants reveals physiological robustness to cell size. Curr. Biol. 27, 3419–3429 (2017).

    Article  CAS  PubMed  Google Scholar 

  23. Colavin, A., Shi, H. & Huang, K. C. RodZ modulates geometric localization of the bacterial actin MreB to regulate cell shape. Nat. Commun. 9, 1280 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Olshausen, P. V. et al. Superresolution imaging of dynamic MreB filaments in B. subtilis—a multiple-motor-driven transport? Biophys. J. 105, 1171–1181 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Shi, H., Bratton, B. P., Gitai, Z. & Huang, K. C. How to build a bacterial cell: MreB as the foreman of E. coli construction. Cell 172, 1294–1305 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Schirner, K. & Errington, J. Influence of heterologous MreB proteins on cell morphology of Bacillus subtilis. Microbiology 155, 3611–3621 (2009).

    Article  CAS  PubMed  Google Scholar 

  27. Harris, L. K., Dye, N. A. & Theriot, J. A. A Caulobacter MreB mutant with irregular cell shape exhibits compensatory widening to maintain a preferred surface area to volume ratio. Mol. Microbiol. 5, 988–1005 (2014).

  28. Bisson-Filho, A. W. et al. FtsZ filament capping by MciZ, a developmental regulator of bacterial division. Proc. Natl Acad. Sci. USA 112, E2130–E2138 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Formstone, A. & Errington, J. A magnesium-dependent mreB null mutant: implications for the role of mreB in Bacillus subtilis. Mol. Microbiol. 55, 1646–1657 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. Vigouroux, A., Oldewurtel, E., Cui, L., Bikard, D. & van Teeffelen, S. Tuning dCas9’s ability to block transcription enables robust, noiseless knockdown of bacterial genes. Mol. Sys. Biol. 14, e7899 (2018).

    Google Scholar 

  31. Henriques, A. O., Glaser, P., Piggot, P. J. & Moran, C. P. Jr Control of cell shape and elongation by the rodA gene in Bacillus subtilis. Mol. Microbiol. 28, 235–247 (1998).

    Article  CAS  PubMed  Google Scholar 

  32. Fraipont, C. et al. The integral membrane FtsW protein and peptidoglycan synthase PBP3 form a subcomplex in Escherichia coli. Microbiology 157, 251–259 (2010).

    Article  PubMed  Google Scholar 

  33. Taheri-Araghi, S. et al. Cell-size control and homeostasis in bacteria. Curr. Biol. 25, 385–391 (2015).

    Article  CAS  PubMed  Google Scholar 

  34. Zheng, H. et al. Interrogating the Escherichia coli cell cycle by cell dimension perturbations. Proc. Natl Acad. Sci. USA 113, 15000–15005 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Murray, T., Popham, D. L. & Setlow, P. Bacillus subtilis cells lacking penicillin-binding protein 1 require increased levels of divalent cations for growth. J. Bacteriol. 180, 4555–4563 (1998).

  36. Popham, D. L. & Setlow, P. Phenotypes of Bacillus subtilis mutants lacking multiple class a high-molecular-weight penicillin-binding proteins. J. Bacteriol. 178, 2079–2085 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Emami, K. et al. RodA as the missing glycosyltransferase in Bacillus subtilis and antibiotic discovery for the peptidoglycan polymerase pathway. Nat. Microbiol. 2, 16253 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Grimm, J. B. et al. A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat. Methods 12, 244–250 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Oldenbourg, R. Polarized light microscopy: principles and practice. Cold Spring Harb. Protoc. 2013, pdb.top078600 (2013).

  40. Probine, M. C. & Preston, R. D. Cell growth and the structure and mechanical properties of the wall in internodal cells of Nitella opacai. Wall structure and growth. J. Exp. Bot. 12, 261–282 (1961).

    Article  CAS  Google Scholar 

  41. Inoué, S. Polarization Microscopy. Curr. Protoc. Cell Biol. 13, 4.9.1–4.9.27 (2002).

  42. Verwer, R. W., Beachey, E. H., Keck, W., Stoub, A. M. & Poldermans, J. E. Oriented fragmentation of Escherichia coli sacculi by sonication. J. Bacteriol. 141, 327–332 (1980).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Yao, X., Jericho, M., Pink, D. & Beveridge, T. Thickness and elasticity of gram-negative murein sacculi measured by atomic force microscopy. J. Bacteriol. 181, 6865–6875 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Bratton, B. P., Shaevitz, J. W., Gitai, Z. & Morgenstein, R. M. MreB polymers and curvature localization are enhanced by RodZ and predict E. coli’s cylindrical uniformity. Nat. Commun. 9, 2797 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Kurita, K., Shin, R., Tabei, T. & Shiomi, D. Relation between rotation of MreB actin and cell width of Escherichia coli. Genes Cells 24, 259–265 (2018).

  46. Rojas, E. R., Huang, K. C. & Theriot, J. A. Homeostatic cell growth is accomplished mechanically through membrane tension inhibition of cell-wall synthesis. Cell Syst. 5, 578–590 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Leaver, M. & Errington, J. Roles for MreC and MreD proteins in helical growth of the cylindrical cell wall in Bacillus subtilis. Mol. Microbiol. 57, 1196–1209 (2005).

    Article  CAS  PubMed  Google Scholar 

  48. Kawai, Y., Daniel, R. A. & Errington, J. Regulation of cell wall morphogenesis in Bacillus subtilis by recruitment of PBP1 to the MreB helix. Mol. Microbiol. 71, 1131–1144 (2009).

    Article  PubMed  Google Scholar 

  49. Lai, G. C., Cho, H. & Bernhardt, T. G. The mecillinam resistome reveals a role for peptidoglycan endopeptidases in stimulating cell wall synthesis in Escherichia coli. PLoS Genet. 13, e1006934 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Lee, T. K., Meng, K., Shi, H. & Huang, K. C. Single-molecule imaging reveals modulation of cell wall synthesis dynamics in live bacterial cells. Nat. Commun. 7, 13170 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Agresti, A. & Coull, B. A. Approximate is better than ‘exact’ for interval estimation of binomial proportions. Am. Stat. 52, 119–126 (1998).

    Google Scholar 

  52. Ursell, T. et al. Rapid, precise quantification of bacterial cellular dimensions across a genomic-scale knockout library. BMC Biol. 15, 17 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Grimm, J. B. et al. A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat. Methods 12, 244–250 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Tinevez, J.-Y. et al. TrackMate: an open and extensible platform for single-particle tracking. Methods 115, 80–90 (2017).

    Article  CAS  PubMed  Google Scholar 

  55. Billaudeau, C. et al. Contrasting mechanisms of growth in two model rod-shaped bacteria. Nat. Commun. 8, 15370 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Kner, P., Chhun, B. B., Griffis, E. R., Winoto, L. & Gustafsson, M. G. L. Super-resolution video microscopy of live cells by structured illumination. Nat. Methods 6, 339–342 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Kall, L., Storey, J. D. & Noble, W. S. Non-parametric estimation of posterior error probabilities associated with peptides identified by tandem mass spectrometry. Bioinformatics 24, i42–i48 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  58. Morgenstein, R. M. et al. RodZ links MreB to cell wall synthesis to mediate MreB rotation and robust morphogenesis. Proc. Natl Acad. Sci. USA 112, 12510–12515 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

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

Authors and Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to Ethan C. Garner.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Dion, M.F., Kapoor, M., Sun, Y. et al. Bacillus subtilis cell diameter is determined by the opposing actions of two distinct cell wall synthetic systems. Nat Microbiol 4, 1294–1305 (2019). https://doi.org/10.1038/s41564-019-0439-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41564-019-0439-0

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing