doi: 10.1073/pnas.1301428110. Epub 2013 Jul 15.
Cell-cell contacts confine public goods diffusion inside Pseudomonas aeruginosa clonal microcolonies
Affiliations
- PMID: 23858453
- PMCID: PMC3732961
- DOI: 10.1073/pnas.1301428110
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Cell-cell contacts confine public goods diffusion inside Pseudomonas aeruginosa clonal microcolonies
Proc Natl Acad Sci U S A. .
Abstract
The maintenance of cooperation in populations where public goods are equally accessible to all but inflict a fitness cost on individual producers is a long-standing puzzle of evolutionary biology. An example of such a scenario is the secretion of siderophores by bacteria into their environment to fetch soluble iron. In a planktonic culture, these molecules diffuse rapidly, such that the same concentration is experienced by all bacteria. However, on solid substrates, bacteria form dense and packed colonies that may alter the diffusion dynamics through cell-cell contact interactions. In Pseudomonas aeruginosa microcolonies growing on solid substrate, we found that the concentration of pyoverdine, a secreted iron chelator, is heterogeneous, with a maximum at the center of the colony. We quantitatively explain the formation of this gradient by local exchange between contacting cells rather than by global diffusion of pyoverdine. In addition, we show that this local trafficking modulates the growth rate of individual cells. Taken together, these data provide a physical basis that explains the stability of public goods production in packed colonies.
Keywords: biofilm; ecology; evolution; noise; variability.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Dynamics and variability of Pvd concentrations in P. aeruginosa (PAO1) microcolonies. (A) Time-lapse fluorescence images of a growing microcolony, with correlation images (Upper) and Pvd intrinsic fluorescence (Lower). (B) Genealogical tree of the colony. The level of Pvd in each cell c is color-coded along each lineage (for clarity, only a subset of the tree is displayed). (C) Dynamics of the mean level of Pvd 
in four different colonies. AU, arbitrary unit. (D) Distributions of Pvd concentrations in cells at various time points in a given microcolony (green), in a different microcolony (red), and in a medium supplemented with human transferrin (blue). (Inset) Distributions are normalized by the mean in the microcolony and the predicted distribution of our model (black). Error bars represent the SEM. (E) Dependence on the relative Pvd concentration in the cell 
and its neighbors 
of the variation 
. Color transparency indicates the uncertainty of measurements (light color means uncertain). The correlation between 
and 
is assessed with a P value less than 
.
in four different colonies. AU, arbitrary unit. (D) Distributions of Pvd concentrations in cells at various time points in a given microcolony (green), in a different microcolony (red), and in a medium supplemented with human transferrin (blue). (Inset) Distributions are normalized by the mean in the microcolony and the predicted distribution of our model (black). Error bars represent the SEM. (E) Dependence on the relative Pvd concentration in the cell and its neighbors of the variation . Color transparency indicates the uncertainty of measurements (light color means uncertain). The correlation between and is assessed with a P value less than .
Measurement of the model parameters. (A) Average change in relative Pvd concentration in a given cell is plotted (points with error bars) vs. its average relative concentration (relative to its nearest neighbors). (Insets) Observed linear correlation is explained by a simple model: Bacteria exchange Pvd with their neighbors in proportion to the difference of their concentrations. The proportionality coefficient λ is estimated by a linear fit to the data (continuous line). Error bars represent the SEM. (B) Temporal autocorrelation function of the noise for various values of the mean Pvd concentration in the colony, 
. This function can be approximated by 
, which we fit for each Pvd level. Because τ is small, the noise autocorrelation function can be approximated by a δ-function: 
, with 
. (Inset) 
increases quadratically with the concentration: 
(dashed line: variation of 
as predicted from the estimation of 
deduced from SI Appendix, Fig. S9 ).
. This function can be approximated by , which we fit for each Pvd level. Because τ is small, the noise autocorrelation function can be approximated by a δ-function: , with . (Inset) increases quadratically with the concentration: (dashed line: variation of as predicted from the estimation of deduced from 
Model predictions for the WT strain PAO1. (A) SD of local fluctuations in Pvd is proportional to the mean fluorescence in the colony, as predicted by the model, with coefficient 
(black line). Colors code for different colonies 
. Each point is calculated from the distribution of fluorescence in a colony measured at a given time. (B) Pvd gradient is established from the edge to the center of the colony. The black points are obtained by pooling the data from 10 microcolonies in discrete bins according to their distances to the edge (white arrow). The red curve is the model prediction. Error bars represent the SEM. (C) Comparison between the measured (black) and the predicted (blue) temporal autocorrelation functions. The shaded areas show the uncertainty in parameter estimation. Error bars represent SEM (N = 10). The yellow arrow points to the same cell at different time points. Note that in A–C, the predicted curves are not the result of a fit. (D) Level of Pvd in individual bacteria is only weakly related to its production rate assessed by the reporter strain PvdA-YFP (AU).
(black line). Colors code for different colonies . Each point is calculated from the distribution of fluorescence in a colony measured at a given time. (B) Pvd gradient is established from the edge to the center of the colony. The black points are obtained by pooling the data from 10 microcolonies in discrete bins according to their distances to the edge (white arrow). The red curve is the model prediction. Error bars represent the SEM. (C) Comparison between the measured (black) and the predicted (blue) temporal autocorrelation functions. The shaded areas show the uncertainty in parameter estimation. Error bars represent SEM (N = 10). The yellow arrow points to the same cell at different time points. Note that in A–C, the predicted curves are not the result of a fit. (D) Level of Pvd in individual bacteria is only weakly related to its production rate assessed by the reporter strain PvdA-YFP (AU).
Model predictions for a planktonic culture and for a mutant. (A) Model prediction for the internal Pvd concentration for well-mixed planktonic cultures vs. 2D growth on an agar plate. The distribution of Pvd in cells grown in liquid and solid conditions shows that cells from liquid cultures retain less Pvd in their periplasm, whereas the concentration in the environment is higher (Inset). This suggests higher leakage when cells are not in contact. Error bars represent the SEM (n = 10). (B) As expected by the model, a partial mutant of Pvd export (ΔpvdRTopmQ) exhibits a smaller Pvd exchange rate λ (evaluated as in Fig. 2A) than for the WT strain (PAO1). Error bars represent the SEM, and the number is indicated for each condition on the bar plot. The two samples are different with a P value of 0.04. The number of asterisks indicates the significance level (0.01<*<0.05; ***<0.001).
Local trafficking affects the individual fitness and stabilizes cooperation. (A) Mean relative growth rate of cells (black points) is plotted against the distance to the colony edge (normalized by the mean radius of the colony 
) and the recent history of Pvd concentration in the cell’s neighborhood 
(normalized by 
). The colored planes are bivariate linear fits to the data. No significant dependence is found when the level of iron is low (SMM) (SI Appendix, Fig. S11 ). By contrast, when iron is depleted (SMM + Tsf), the individual growth rates depend on both the position inside the colony and the Pvd history in the nearest neighbors. (B) Phase diagram of the sustainability of cooperation in an in silico competition experiment between defectors and cooperators. The result of the simulation after 4,000 generations is shown as a function of the local exchange rate of Pvd λ and the cost of production. Cooperators are found to dominate in a wide range of values around the measured exchange rate 
. The white line depicts the initial ratio. The black line depicts the measured value of λ, and the dashed lines depict the confidence interval. The time evolution of the proportion of nonproducers at the points (I, II and III) marked in B is shown in SI Appendix, Fig. S14 .
) and the recent history of Pvd concentration in the cell’s neighborhood (normalized by ). The colored planes are bivariate linear fits to the data. No significant dependence is found when the level of iron is low (SMM) (. The white line depicts the initial ratio. The black line depicts the measured value of λ, and the dashed lines depict the confidence interval. The time evolution of the proportion of nonproducers at the points (I, II and III) marked in B is shown in References
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