Abstract
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.
Similar content being viewed by others
References
Zhou, S., Sokolov, A., Lavrentovich, O. D. & Aranson, I. S. Living liquid crystals. Proc. Natl Acad. Sci. USA 111, 1265–1270 (2014).
Hagan, M. F. & Baskaran, A. Emergent self-organization in active materials. Curr. Opin. Cell Biol. 38, 74–80 (2016).
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).
Volfson, D., Cookson, S., Hasty, J. & Tsimring, L. S. Biomechanical ordering of dense cell populations. Proc. Natl Acad. Sci. USA 105, 15346–15351 (2008).
Drescher, K. et al. Architectural transitions in Vibrio cholerae biofilms at single-cell resolution. Proc. Natl Acad. Sci. USA 113, E2066–E2072 (2016).
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).
Kragh, K. N. et al. Role of multicellular aggregates in biofilm formation. mBio 7, e00237 (2016).
Flemming, H.-C. et al. Biofilms: an emergent form of bacterial life. Nat. Rev. Microbiol. 14, 563–575 (2016).
Marchetti, M. C. et al. Hydrodynamics of soft active matter. Rev. Mod. Phys. 85, 1143–1189 (2013).
Persat, A. et al. The mechanical world of bacteria. Cell 161, 988–997 (2015).
Liu, J. et al. Coupling between distant biofilms and emergence of nutrient time-sharing. Science 356, 638–642 (2017).
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).
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).
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).
Delarue, M. et al. Self-driven jamming in growing microbial populations. Nat. Phys. 12, 762–766 (2016).
Seminara, A. et al. Osmotic spreading of Bacillus subtilis biofilms driven by an extracellular matrix. Proc. Natl Acad. Sci. USA 109, 1116–1121 (2012).
Trejo, M. et al. Elasticity and wrinkled morphology of Bacillus subtilis pellicles. Proc. Natl Acad. Sci. USA 110, 2011–2016 (2013).
Maier, B. & Wong, G. C. L. How bacteria use type IV pili machinery on surfaces. Trends Microbiol. 23, 775–788 (2015).
Teschler, J. K. et al. Living in the matrix: assembly and control of Vibrio cholerae biofilms. Nat. Rev. Microbiol. 13, 255–268 (2015).
Berk, V. et al. Molecular architecture and assembly principles of Vibrio cholerae biofilms. Science 337, 236–239 (2012).
Fong, J. C. et al. Structural dynamics of RbmA governs plasticity of Vibrio cholerae biofilms. Elife 6, e26163 (2017).
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).
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).
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).
Lardon, L. A. et al. iDynoMiCS: next-generation individual-based modelling of biofilms. Environ. Microbiol. 13, 2416–2434 (2011).
Marcos, Fu,H. C., Powers, T. R. & Stocker, R. Bacterial rheotaxis. Proc. Natl Acad. Sci. USA 109, 4780–4785 (2012).
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).
Cates, M. E. & Tjhung, E. Theories of binary fluid mixtures: from phase-separation kinetics to active emulsions. J. Fluid. Mech. 836, 1–68 (2018).
Singh, P. K. et al. Vibrio cholerae combines individual and collective sensing to trigger biofilm dispersal. Curr. Biol. 27, 3359–3366 (2017).
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).
Smith, W. P. J. et al. Cell morphology drives spatial patterning in microbial communities. Proc. Natl Acad. Sci. USA 114, E280–E286 (2017).
Sambrook, J., Fritsch, E. F. & Maniatis, T. Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989).
Skorupski, K. & Taylor, R. K. Positive selection vectors for allelic exchange. Gene 169, 47–52 (1996).
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).
Bartlett, T. M. et al. A periplasmic polymer curves Vibrio cholerae and promotes pathogenesis. Cell 168, 172–185 (2017).
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).
Edelstein, A. D. et al. Advanced methods of microscope control using μManager software. J. Biol. Methods 1, e10 (2014).
Nyland, L., Harris, M. & Prins, J. Fast N-body simulation with CUDA. Simulation 3, 677–696 (2007).
Woods, R. D. & Saxon, D. S. Diffuse surface optical model for nucleon–nuclei scattering. Phys. Rev. 95, 577–578 (1954).
Acknowledgements
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
Authors and Affiliations
Contributions
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
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 Information, Supplementary Data, Supplementary Figures 1–25, Supplementary Tables 1–7, Supplementary References 1–32
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
Rights and permissions
About this article
Cite this article
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). https://doi.org/10.1038/s41567-018-0356-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41567-018-0356-9
- Springer Nature Limited
This article is cited by
-
The biophysical basis of bacterial colony growth
Nature Physics (2024)
-
Onwards and upwards
Nature Physics (2024)
-
Low-cost and scalable projected light-sheet microscopy for the high-resolution imaging of cleared tissue and living samples
Nature Biomedical Engineering (2024)
-
Unfolding the interaction of radioactive Cs and Sr with polyethylene-derived microplastics in marine environment
Discover Oceans (2024)
-
Biofilm self-organization arises from active boundary shaping
Nature Physics (2023)