ABSTRACT
Biofilm is a group of heterogeneously structured and densely packed bacteria with limited access to nutrients and oxygen. These intrinsic features can allow a mono-species biofilm to diversify into polymorphic subpopulations, determining the overall community's adaptive capability to changing ecological niches. However, the specific biological functions underlying biofilm diversification and fitness adaptation are poorly demonstrated. Here, we launched and monitored the experimental evolution of Pseudomonas aeruginosa biofilms, finding that two divergent molecular trajectories were adopted for adaptation to higher competitive fitness in biofilm formation: one involved hijacking bacteriophage superinfection to aggressively inhibit kin competitors, whereas the other induced a subtle change in cyclic dimeric guanosine monophosphate signaling to gain a positional advantage via enhanced early biofilm adhesion. Bioinformatics analyses implicated that similar evolutionary strategies were prevalent among clinical P. aeruginosa strains, indicative of parallelism between natural and experimental evolution. Divergence in the molecular bases illustrated the adaptive values of genomic plasticity for gaining competitive fitness in biofilm formation. Finally, we demonstrated that these fitness-adaptive mutations reduced bacterial virulence. Our findings revealed how the mutations intrinsically generated from the biofilm environment influence the evolution of P. aeruginosa.