Spatial-temporal dynamics of a microbial cooperative behavior robust to cheating

The ability of single-celled microbes to integrate environmental signals and control gene expression enables calculated decisions on whether they should invest in a behavior in a specific environment. But how can the same mechanisms of gene expression control—resulting from individuals sensing, integrating and responding to diffusible queues in dynamic, densely populated microbial communities—enable the evolution and stability of cooperative behaviors that could easily be exploited by cheaters? Here we combine fluorescent imaging with computational analyses to investigate how the micro-environment experienced by cells in spatially-structured systems impacts cooperative behavior. We focus on swarming in the opportunistic human pathogen , a behavior that requires cooperative secretions of rhamnolipid surfactants to facilitate collective movement over surfaces. Our analysis shows that the expression of rhamnolipid synthesis varies across the colony and, counter to previous knowledge, peaks at tips of swarming tendrils. To dissect the contribution of competing diffusive inputs—quorum sensing signals and growth-limiting nutrients—we adapted the classic Colony Forming Unit (CFU) assay to record colony growth and gene expression dynamics across thousands of colonies. We found these cells capable of centimeter-scale communication in a pattern of gene expression previously undetected in liquid culture systems. Validation experiments where we manipulated gene expression by flooding the environment with quorum sensing signals could accelerate the onset of swarming, but the cooperative trait remained robust to cheaters. Taken together, these results shed new light on the integration of diffusible signals that stabilizes swarming motility, a cooperative microbial behavior.