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Verticalization of bacterial biofilms

Nature Physics volume 14, pages 954–960 (2018)Cite this article

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Abstract

Biofilms are communities of bacteria adhered to surfaces. Recently, biofilms of rod-shaped bacteria were observed at single-cell resolution and shown to develop from a disordered, two-dimensional layer of founder cells into a three-dimensional structure with a vertically aligned core. Here, we elucidate the physical mechanism underpinning this transition using a combination of agent-based and continuum modelling. We find that verticalization proceeds through a series of localized mechanical instabilities on the cellular scale. For short cells, these instabilities are primarily triggered by cell division, whereas long cells are more likely to be peeled off the surface by nearby vertical cells, creating an ‘inverse domino effect’. The interplay between cell growth and cell verticalization gives rise to an exotic mechanical state in which the effective surface pressure becomes constant throughout the growing core of the biofilm surface layer. This dynamical isobaricity determines the expansion speed of a biofilm cluster and thereby governs how cells access the third dimension. In particular, theory predicts that a longer average cell length yields more rapidly expanding, flatter biofilms. We experimentally show that such changes in biofilm development occur by exploiting chemicals that modulate cell length.

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Fig. 1: Development of experimental and modelled biofilms.
Fig. 2: Mechanics of cell reorientation in modelled biofilms.
Fig. 3: Two-component fluid model for verticalizing cells in biofilms.
Fig. 4: Global morphological properties of experimental and modelled biofilms.

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Acknowledgements

We thank B. Bratton, X. Chen, S. Jena, N. Pappireddi and J. Shaevitz for insightful discussions and encouragement, G. Laevsky for assistance with imaging and T. Bartlett and Z. Gitai for sharing the ΔcrvA strain and reagents. This work was supported by the NIH under grant no. R01 GM082938 (F.B., Y.M. and N.S.W.), the Howard Hughes Medical Institute (B.L.B.), NSF grant MCB-1713731 (B.L.B.), NSF grant MCB-1344191 (H.A.S., B.L.B. and N.S.W.), NIH grant 2R37GM065859 (F.B. and B.L.B.), the Max Planck Society-Alexander von Humboldt Foundation (B.L.B.) and the Eric and Wendy Schmidt Transformative Technology Fund from Princeton University (H.A.S., B.L.B. and N.S.W.). J.Y. holds a Career Award at the Scientific Interface from the Burroughs Wellcome Fund.

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Authors and Affiliations

  1. Joseph Henry Laboratories of Physics, Princeton University, Princeton, NJ, USA

    Farzan Beroz

  2. Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, NJ, USA

    Jing Yan, Benedikt Sabass & Howard A. Stone

  3. Department of Molecular Biology, Princeton University, Princeton, NJ, USA

    Jing Yan, Bonnie L. Bassler & Ned S. Wingreen

  4. Department of Physics, Ben-Gurion University of the Negev, Beer-Sheva, Israel

    Yigal Meir

  5. ICS-2/IAS-2, Forschungszentrum Jülich, Jülich, Germany

    Benedikt Sabass

  6. Howard Hughes Medical Institute, Chevy Chase, MD, USA

    Bonnie L. Bassler

  7. Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA

    Ned S. Wingreen

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Contributions

F.B., J.Y., and N.S.W. conceived the research. F.B. performed all simulations and analysis. J.Y. carried out all experiments, and J.Y. and F.B. contributed to the experimental design. All authors contributed to the manuscript preparation.

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Correspondence to Ned S. Wingreen.

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Supplementary information

Supplementary Information

Supplementary Information, Supplementary Figures 1–16, Supplementary Discussion, Supplementary References 1–20

Reporting Summary

Supplementary Video 1

Growth of a V. cholerae biofilm cluster, showing cross-sectional images of the bottom cell layer. The strain constitutively expresses mKO. The viewing window is 45 by 45 µm2 and the total duration is 8 hours with 10 min time steps.

Supplementary Video 2

Visualization of the surface layer of a modelled biofilm with initial cell cylinder length \({\ell }_{0}\) = 1 µm, showing positions and orientations of horizontal (blue) and vertical (red) surface-adhered cells as spherocylinders of radius R = 0.8 µm, with the surface shown at height z = 0 µm (brown). Cells with nz < 0.5 (>0.5) are considered horizontal (vertical), where \({{\bf{{n}}}}\) is the orientation vector. Scale bar, 3 µm. The total duration is 10 hours.

Supplementary Video 3

Visualization of the surface layer of a modelled biofilm with initial cell cylinder length \({\ell }_{0}\) = 2 µm, showing horizontal (blue) and vertical (red) cells as spherocylinders, the surface (brown), and cell-to-cell contact forces (yellow lines connecting the centres of cells, with thicknesses proportional to the force). Cells with nz < 0.5 (>0.5) are considered horizontal (vertical), where \({{\bf{{n}}}}\) is the orientation vector. The length of the scale bar is 3 µm, and its thickness corresponds to 300 pN.

Supplementary Video 4

Numerical simulation of the continuum model assuming no vertical cell transport, showing radial densities of horizontal cells (ρh, blue), vertical cells (ρv, red), and total density (\({\widetilde{\rho }}_{{\rm{tot}}}\)= ρh + ξρv, black), versus radial coordinate r for isobaric regime with \({\widetilde{\rho }}_{0}\) = 1 m−2, \({\widetilde{\rho }}_{{\rm{t}}}\) = 1.5 m−2, β = 2.5α, and ξ = 0.5. Dashed grey line shows \({\widetilde{\rho }}_{{\rm{t}}}.\)

Supplementary Video 5

Expansion of V. cholerae biofilm clusters grown with the drug A22 at a concentration of 0.4 µg mL–1 (left) and the drug Cefalexin at a concentration of 4 µg mL–1 (right). Cross-sectional images of the bottom cell layers are shown. The strain constitutively expresses mKO. Scale bar, 30 µm. The total duration is 10 hours with 30 min time steps.

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Beroz, F., Yan, J., Meir, Y. et al. Verticalization of bacterial biofilms. Nature Phys 14, 954–960 (2018). https://doi.org/10.1038/s41567-018-0170-4

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