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Emergence of three-dimensional order and structure in growing biofilms


Surface-attached bacterial biofilms are self-replicating active liquid crystals and the dominant form of bacterial life on Earth1,2,3,4. In conventional liquid crystals and solid-state materials, the interaction potentials between the molecules that comprise the system determine the material properties. However, for growth-active biofilms it is unclear whether potential-based descriptions can account for the experimentally observed morphologies, and which potentials would be relevant. Here, we have overcome previous limitations of single-cell imaging techniques5,6 to reconstruct and track all individual cells inside growing three-dimensional biofilms with up to 10,000 individuals. Based on these data, we identify, constrain and provide a microscopic basis for an effective cell–cell interaction potential, which captures and predicts the growth dynamics, emergent architecture and local liquid-crystalline order of Vibrio cholerae biofilms. Furthermore, we show how external fluid flows control the microscopic structure and three-dimensional morphology of biofilms. Our analysis implies that local cellular order and global biofilm architecture in these active bacterial communities can arise from mechanical cell–cell interactions, which cells can modulate by regulating the production of particular matrix components. These results establish an experimentally validated foundation for improved continuum theories of active matter and thereby contribute to solving the important problem of controlling biofilm growth.

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Fig. 1: Dynamics of V. cholerae biofilm formation.
Fig. 2: Biofilm architecture development is captured by an effective mechanical cell–cell interaction potential.
Fig. 3: Biofilm architecture is shaped by external shear flow.


  1. 1.

    Zhou, S., Sokolov, A., Lavrentovich, O. D. & Aranson, I. S. Living liquid crystals. Proc. Natl Acad. Sci. USA 111, 1265–1270 (2014).

    ADS  Article  Google Scholar 

  2. 2.

    Hagan, M. F. & Baskaran, A. Emergent self-organization in active materials. Curr. Opin. Cell Biol. 38, 74–80 (2016).

    Article  Google Scholar 

  3. 3.

    Doostmohammadi, A., Adamer, M. F., Thampi, S. P. & Yeomans, J. M. Stabilization of active matter by flow-vortex lattices and defect ordering. Nat. Commun. 7, 10557 (2016).

    ADS  Article  Google Scholar 

  4. 4.

    Volfson, D., Cookson, S., Hasty, J. & Tsimring, L. S. Biomechanical ordering of dense cell populations. Proc. Natl Acad. Sci. USA 105, 15346–15351 (2008).

    ADS  Article  Google Scholar 

  5. 5.

    Drescher, K. et al. Architectural transitions in Vibrio cholerae biofilms at single-cell resolution. Proc. Natl Acad. Sci. USA 113, E2066–E2072 (2016).

    Article  Google Scholar 

  6. 6.

    Yan, J., Sharo, A. G., Stone, H. A., Wingreen, N. S. & Bassler, B. L. Vibrio cholerae biofilm growth program and architecture revealed by single-cell live imaging. Proc. Natl Acad. Sci. USA 113, E5337–E5343 (2016).

    Article  Google Scholar 

  7. 7.

    Kragh, K. N. et al. Role of multicellular aggregates in biofilm formation. mBio 7, e00237 (2016).

    Article  Google Scholar 

  8. 8.

    Flemming, H.-C. et al. Biofilms: an emergent form of bacterial life. Nat. Rev. Microbiol. 14, 563–575 (2016).

    Article  Google Scholar 

  9. 9.

    Marchetti, M. C. et al. Hydrodynamics of soft active matter. Rev. Mod. Phys. 85, 1143–1189 (2013).

    ADS  Article  Google Scholar 

  10. 10.

    Persat, A. et al. The mechanical world of bacteria. Cell 161, 988–997 (2015).

    Article  Google Scholar 

  11. 11.

    Liu, J. et al. Coupling between distant biofilms and emergence of nutrient time-sharing. Science 356, 638–642 (2017).

    ADS  Article  Google Scholar 

  12. 12.

    Rodesney, C. A. et al. Mechanosensing of shear by Pseudomonas aeruginosa leads to increased levels of the cyclic-di-GMP signal initiating biofilm development. Proc. Natl Acad. Sci. USA 114, 5906–5911 (2017).

    Article  Google Scholar 

  13. 13.

    Grant, M. A. A., Waclaw, B., Allen, R. J. & Cicuta, P. The role of mechanical forces in the planar-to-bulk transition in growing Escherichia coli microcolonies. J. R. Soc. Interface 11, 20140400 (2014).

    Article  Google Scholar 

  14. 14.

    You, Z., Pearce, D. J. G., Sengupta, A. & Giomi, L. Geometry and mechanics of microdomains in growing bacterial colonies. Phys. Rev. X 8, 031065 (2018).

    Google Scholar 

  15. 15.

    Delarue, M. et al. Self-driven jamming in growing microbial populations. Nat. Phys. 12, 762–766 (2016).

    Article  Google Scholar 

  16. 16.

    Seminara, A. et al. Osmotic spreading of Bacillus subtilis biofilms driven by an extracellular matrix. Proc. Natl Acad. Sci. USA 109, 1116–1121 (2012).

    ADS  Article  Google Scholar 

  17. 17.

    Trejo, M. et al. Elasticity and wrinkled morphology of Bacillus subtilis pellicles. Proc. Natl Acad. Sci. USA 110, 2011–2016 (2013).

    ADS  Article  Google Scholar 

  18. 18.

    Maier, B. & Wong, G. C. L. How bacteria use type IV pili machinery on surfaces. Trends Microbiol. 23, 775–788 (2015).

    Article  Google Scholar 

  19. 19.

    Teschler, J. K. et al. Living in the matrix: assembly and control of Vibrio cholerae biofilms. Nat. Rev. Microbiol. 13, 255–268 (2015).

    Article  Google Scholar 

  20. 20.

    Berk, V. et al. Molecular architecture and assembly principles of Vibrio cholerae biofilms. Science 337, 236–239 (2012).

    ADS  Article  Google Scholar 

  21. 21.

    Fong, J. C. et al. Structural dynamics of RbmA governs plasticity of Vibrio cholerae biofilms. Elife 6, e26163 (2017).

    Article  Google Scholar 

  22. 22.

    Maestre-Reyna, M., Wu, W.-J. & Wang, A. H.-J. Structural insights into RbmA, a biofilm scaffolding protein of V. Cholerae. PLoS ONE 8, e82458 (2013).

    ADS  Article  Google Scholar 

  23. 23.

    Fong, J. C. N., Karplus, K., Schoolnik, G. K. & Yildiz, F. H. Identification and characterization of RbmA, a novel protein required for the development of rugose colony morphology and biofilm structure in Vibrio cholerae. J. Bacteriol. 188, 1049–1059 (2006).

    Article  Google Scholar 

  24. 24.

    Hellweger, F. L., Clegg, R. J., Clark, J. R., Plugge, C. M. & Kreft, J. U. Advancing microbial sciences by individual-based modelling. Nat. Rev. Microbiol. 14, 461–471 (2016).

    Article  Google Scholar 

  25. 25.

    Lardon, L. A. et al. iDynoMiCS: next-generation individual-based modelling of biofilms. Environ. Microbiol. 13, 2416–2434 (2011).

    Article  Google Scholar 

  26. 26.

    Marcos, Fu,H. C., Powers, T. R. & Stocker, R. Bacterial rheotaxis. Proc. Natl Acad. Sci. USA 109, 4780–4785 (2012).

    ADS  Article  Google Scholar 

  27. 27.

    Mitchell, W. H. & Spagnolie, S. E. A generalized traction integral equation for Stokes flow, with applications to near-wall particle mobility and viscous erosion. J. Comput. Phys. 333, 462–482 (2017).

    ADS  MathSciNet  Article  Google Scholar 

  28. 28.

    Cates, M. E. & Tjhung, E. Theories of binary fluid mixtures: from phase-separation kinetics to active emulsions. J. Fluid. Mech. 836, 1–68 (2018).

    ADS  MathSciNet  Article  Google Scholar 

  29. 29.

    Singh, P. K. et al. Vibrio cholerae combines individual and collective sensing to trigger biofilm dispersal. Curr. Biol. 27, 3359–3366 (2017).

    Article  Google Scholar 

  30. 30.

    Vidakovic, L., Singh, P. K., Hartmann, R., Nadell, C. D. & Drescher, K. Dynamic biofilm architecture confers individual and collective mechanisms of viral protection. Nat. Microbiol. 3, 26–31 (2017).

    Article  Google Scholar 

  31. 31.

    Smith, W. P. J. et al. Cell morphology drives spatial patterning in microbial communities. Proc. Natl Acad. Sci. USA 114, E280–E286 (2017).

    Article  Google Scholar 

  32. 32.

    Sambrook, J., Fritsch, E. F. & Maniatis, T. Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989).

  33. 33.

    Skorupski, K. & Taylor, R. K. Positive selection vectors for allelic exchange. Gene 169, 47–52 (1996).

    Article  Google Scholar 

  34. 34.

    Beyhan, S. & Yildiz, F. H. Smooth to rugose phase variation in Vibrio cholerae can be mediated by a single nucleotide change that targets c-di-GMP signalling pathway. Mol. Microbiol. 63, 995–1007 (2007).

    Article  Google Scholar 

  35. 35.

    Bartlett, T. M. et al. A periplasmic polymer curves Vibrio cholerae and promotes pathogenesis. Cell 168, 172–185 (2017).

    Article  Google Scholar 

  36. 36.

    Nadell, C. D., Drescher, K., Wingreen, N. S. & Bassler, B. L. Extracellular matrix structure governs invasion resistance in bacterial biofilms. ISME J. 9, 1700–1709 (2015).

    Article  Google Scholar 

  37. 37.

    Edelstein, A. D. et al. Advanced methods of microscope control using μManager software. J. Biol. Methods 1, e10 (2014).

    Article  Google Scholar 

  38. 38.

    Nyland, L., Harris, M. & Prins, J. Fast N-body simulation with CUDA. Simulation 3, 677–696 (2007).

    Google Scholar 

  39. 39.

    Woods, R. D. & Saxon, D. S. Diffuse surface optical model for nucleon–nuclei scattering. Phys. Rev. 95, 577–578 (1954).

    ADS  Article  Google Scholar 

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The authors thank L. Vidakovic for contributions to bacterial strain creation, N. Netter and E. Jelli for preparing the graphics processing unit-based simulations to be run on the Max Planck Computing and Data Facility cluster, and C. Nadell and all members of the Drescher lab for discussions. This work was supported by grants from the Max Planck Society, the Human Frontier Science Program (CDA00084/2015-C), the European Research Council (StG-716734), the Deutsche Forschungsgemeinschaft (DFG) via the SFB987 framework to K.D., a MIT OGE Chyn Duog Shiah Memorial Fellowship to R.M., a James S. McDonnell Foundation Complex Systems Scholar Award and an Edmund F. Kelly Research Award to J.D., and an MIT-Germany MISTI Seed Grant to K.D. and J.D.

Author information




K.D. and J.D. designed and supervised the study. R.H. and P.K.S. performed experiments. P.K.S. and F.D.-P. created bacterial strains. R.H. developed experimental and analysis software. P.P. developed continuum simulations. R.M. developed cell-based simulation framework. R.M., R.H. and B.S. performed cell-based simulations. R.M., R.H., P.P. and B.S. developed cell–cell potentials. R.H., with the help of P.P., J.D. and K.D., analysed the data. R.H., P.P., J.D. and K.D. wrote the manuscript, with the help of all authors.

Corresponding authors

Correspondence to Jörn Dunkel or Knut Drescher.

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The authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Information, Supplementary Data, Supplementary Figures 1–25, Supplementary Tables 1–7, Supplementary References 1–32

Life Sciences Reporting Summary

Supplementary Video 1

Biofilm formation of the WT* strain grown at low shear rate (γ = 2 s–1). Cells originating from the biofilm founder cell are labelled in blue

Supplementary Video 2

Biofilm formation of the ΔrbmA mutant grown at low shear rate (γ = 2 s–1). Cells originating from the biofilm founder cell are labelled in blue

Supplementary Video 3

Biofilm formation of the WT* strain grown at high shear rate (γ = 2,000 s–1). Cells originating from the biofilm founder cell are labelled in blue

Supplementary Video 4

Biofilm formation of the ΔrbmA mutant grown at high shear rate (γ = 660 s–1). Cells originating from the biofilm founder cell are labelled in blue

Supplementary Movie 5

​Simulated biofilm formation for the ΔrbmA mutant and the WT* strain using the best-fitting interaction potential for each case. Cells are colour-coded according to their local nematic order

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Hartmann, R., Singh, P.K., Pearce, P. et al. Emergence of three-dimensional order and structure in growing biofilms. Nat. Phys. 15, 251–256 (2019).

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