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The collapse of cooperation during range expansion of Pseudomonas aeruginosa

Nature Microbiology (2024)Cite this article

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Abstract

Cooperation is commonly believed to be favourable in spatially structured environments, as these systems promote genetic relatedness that reduces the likelihood of exploitation by cheaters. Here we show that a Pseudomonas aeruginosa population that exhibited cooperative swarming was invaded by cheaters when subjected to experimental evolution through cycles of range expansion on solid media, but not in well-mixed liquid cultures. Our results suggest that cooperation is disfavoured in a more structured environment, which is the opposite of the prevailing view. We show that spatial expansion of the population prolongs cooperative swarming, which was vulnerable to cheating. Our findings reveal a mechanism by which spatial structures can suppress cooperation through modulation of the quantitative traits of cooperation, a process that leads to population divergence towards distinct colonization strategies.

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Fig. 1: Experimental evolution of P. aeruginosa led to reduced colony biomass.
Fig. 2: Phenotypic and genetic analysis identified three classes of strains in the evolved population.
Fig. 3: Wild type, cheaters and hyperswarmers represent different strategies of colonization.
Fig. 4: Constraining the multi-strain colony growth model by spatial patterns.
Fig. 5: Increased competitive advantage of cheaters during colony expansion due to prolonged cooperation.
Fig. 6: Evolutionary dynamics of P. aeruginosa colonies depended on nutrient availability.

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Data availability

The data that support the findings of this study are available on Zenodo (https://doi.org/10.5281/zenodo.10210659). Raw sequencing reads from whole-genome sequencing are deposited in the NCBI Sequence Read Archive (BioProject PRJNA875393).

Code availability

The MATLAB code used for data generation and/or analysis in the study is available on GitHub (https://github.com/youlab/MultistrainPatterns_NanLuo).

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Acknowledgements

We thank Duke Compute Cluster for assistance with high-throughput computation. We thank J. Xavier (Memorial Sloan Kettering Cancer Center) for sharing P. aeruginosa PA14 strains. This work was partially supported by grants from the Office of Naval Research (L.Y.: N00014-12-1-0631), National Science Foundation (L.Y.: MCB-1937259) and National Institutes of Health (L.Y.: R01GM098642).

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Author notes

  1. Nan Luo

    Present address: Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China

  2. These authors contributed equally: Nan Luo, Jia Lu, Emrah Şimşek.

Authors and Affiliations

  1. Department of Biomedical Engineering, Duke University, Durham, NC, USA

    Nan Luo, Jia Lu, Emrah Şimşek, Anita Silver, Yi Yao & Lingchong You

  2. School of Physics, Peking University, Beijing, China

    Xiaoyi Ouyang

  3. Department of Biology, University of Oxford, Oxford, UK

    Stuart A. West

  4. Center for Quantitative Biodesign, Duke University, Durham, NC, USA

    Lingchong You

  5. Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, USA

    Lingchong You

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Contributions

N.L. conceived the research, designed and performed the modelling and experiments, interpreted the results and wrote the paper. J.L. and E.Ş. designed and performed the experiments, interpreted the results and assisted with paper revisions. A.S. assisted with modelling, interpretation of results and paper revisions. Y.Y. assisted with the experiments. X.O. assisted with the modelling and experiments. S.A.W. assisted with the interpretation of results and paper revisions. L.Y. conceived the research, assisted in research design, interpreted the results and wrote the paper.

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Correspondence to Lingchong You.

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Extended data

Extended Data Fig. 1 Experimental evolution of P. aeruginosa led to biomass decline.

The total accumulation of biomass of P. aeruginosa PA14 colonies declined during experimental evolution. Cells of a colony were flushed off the plate after colony growth for 20 h and resuspended, and the biomass was determined by measuring OD600. The relative biomass of a colony was normalized by the average of wildtype colonies.

Extended Data Fig. 2 Nonswarmer or hyperswarmer did not emerge during experimental evolution of P. aeruginosa in liquid cultures.

a, The growth rate of P. aeruginosa increased during experimental evolution within liquid cultures. Growth curves were measured using cell cultures of the ancestral strain (Day 1) and the populations on Day 5 and Day 7 of the experimental evolution (n = 3 for each group; all replicates were shown). Both experimental evolution and the growth curve measurements were carried out using liquid swarming media with 8 g/L casamino acids. b, Experimental evolution in liquid cultures did not alter the colony phenotype of P. aeruginosa or cause biomass decline. Shown are colonies of wildtype and the evolved populations of three independent lineages. c, Comparison of the phenotypic characterizations of wildtype and isolates from populations evolved in liquid or on plates. Strains with nonswarming phenotype (low colony area with low branching index) or hyperswarming phenotype (high colony area with low branching index) did not emerge during evolution in liquid cultures.

Extended Data Fig. 3 Nonswarmers are defective in surfactant production.

Cells were grown in liquid swarming media with 8 g/L casamino acids for 20 h. The amount of surfactant in the supernatant was determined using the drop collapse assay. The droplet area (normalized by the negative control, blank medium) reflects the surfactant concentration. N = 36 for each group. Data are presented as mean values +/- SEM. Unpaired, two-sided t test with Welch’s correction and a 95% confidence interval was used to compare between wildtype and nonswarmer. **** P < 0.0001.

Extended Data Fig. 4 Intermediate nutrient level causes P. aeruginosa wildtype and hyperswarmers to produce the highest amount of surfactant.

Cells were grown in liquid swarming media with varying initial concentrations of casamino acids for 20 h. The amount of surfactant in the supernatant was determined using the drop collapse assay. The droplet area (normalized by the negative control, blank medium) reflects the surfactant concentration. N = 36 for each group. Data are presented as mean values +/- SEM. Brown-Forsythe and Welch ANOVA test (which does not assume equal variances) were used to compare between groups. **** P < 0.0001, ** P = 0.0014. Data in Extended Data Fig. 3 were reused for comparison.

Extended Data Fig. 5 Cheater reduced the biomass accumulation of multi-strain colonies.

The total biomass of colonies with different compositions (normalized by wildtype) was determined by measuring OD600. All data points were shown: n = 3-5 for each colony type. Data are presented as mean values +/- SEM. One-way ANOVA followed by Tukey’s multiple comparisons test (95% confidence intervals) was used to compare between groups. Significantly different groups are indicated by letters: groups with at least one common letter are not significantly different; otherwise, they are significantly different.

Extended Data Fig. 6 Model recaptured the competition dynamics between strains.

Pairwise competition between the three types of strains (a, cheater vs. wild type; b, hyperswarmer vs. wildtype; c, cheater vs. hyperswarmer) were carried out either in liquid cultures or on solid swarming plates. Competing strains with fluorescent labels were mixed in varying ratios and grown for 16 h. The final population compositions were determined by CFU counting. In simulations, identical parameters were used when comparing liquid and solid phases. Data in panel a was presented in Fig. 4a in a different format.

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Supplementary Note 1, Figs. 1–12 and Tables 1–3.

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Luo, N., Lu, J., Şimşek, E. et al. The collapse of cooperation during range expansion of Pseudomonas aeruginosa. Nat Microbiol (2024). https://doi.org/10.1038/s41564-024-01627-8

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