Cheaters shape the evolution of phenotypic heterogeneity in Bacillus subtilis biofilms

Abstract

Biofilms are closely packed cells held and shielded by extracellular matrix composed of structural proteins and exopolysaccharides (EPS). As matrix components are costly to produce and shared within the population, EPS-deficient cells can act as cheaters by gaining benefits from the cooperative nature of EPS producers. Remarkably, genetically programmed EPS producers can also exhibit phenotypic heterogeneity at single-cell level. Previous studies have shown that spatial structure of biofilms limits the spread of cheaters, but the long-term influence of cheating on biofilm evolution is not well understood. Here, we examine the influence of EPS nonproducers on evolution of matrix production within the populations of EPS producers in a model biofilm-forming bacterium, Bacillus subtilis. We discovered that general adaptation to biofilm lifestyle leads to an increase in phenotypical heterogeneity of eps expression. However, prolonged exposure to EPS-deficient cheaters may result in different adaptive strategy, where eps expression increases uniformly within the population. We propose a molecular mechanism behind such adaptive strategy and demonstrate how it can benefit the EPS producers in the presence of cheaters. This study provides additional insights on how biofilms adapt and respond to stress caused by exploitation in long-term scenario.

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Fig. 1: Pellicle productivity and phenotypic heterogeneity.
Fig. 2: Evolutionary changes in phenotypic heterogeneity pattern and expression of eps.
Fig. 3: Effect of rsiX deletion on eps expression in pellicles.
Fig. 4: Performance of evolved WT and ΔrsiX in cocultures with Δepsanc.
Fig. 5: Effect of rsiX deletion on positioning of EPS producers in the pellicle.
Fig. 6: Changes in relative frequencies and eps expression pattern during evolution with and without cheaters.

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Acknowledgements

The authors thank James Gurney and other anonymous reviewers for their suggestions. This work was funded by the Deutsche Forschungsgemeinschaft (DFG) to ÁTK (KO4741/2.1) within the Priority Program SPP1617. MM was supported by a FEMS Research and Training Grant (FEMS-RG-2017-0054). This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 713683 (H.C. Ørsted COFUND to AD). Work in the laboratory of ÁTK is partly supported by the Danish National Research Foundation (DNRF137) for the Center for Microbial Secondary Metabolites.

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ÁTK conceived the project. MM, AD, SB, and DS performed the experiments. GM contributed with methods. MM, AD, and ÁTK wrote the paper, with all authors contributing to the final version.

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Correspondence to Ákos T. Kovács.

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Martin, M., Dragoš, A., Otto, S.B. et al. Cheaters shape the evolution of phenotypic heterogeneity in Bacillus subtilis biofilms. ISME J 14, 2302–2312 (2020). https://doi.org/10.1038/s41396-020-0685-4

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