Abstract
<jats:p>Environmental predation is a major driver of bacterial evolution and may indirectly influence virulence through coincidental selection. However, how sustained versus fluctuating predator pressure shapes long-term evolutionary trajectories remains poorly understood. Here, we used experimental evolution to investigate the genetic and phenotypic responses of Pseudomonas aeruginosa to continuous, absent, or fluctuating exposure to the protozoan predator Tetrahymena thermophila over 180 days. Whole-population and isolate-level shotgun metagenomic sequencing revealed fewer mutations over time but increasing frequencies of surviving mutations, consistent with selection, extensive gene-level parallel evolution, and signatures of both positive and purifying selection. Recurrently mutated genes encompassed diverse functional pathways, reflecting both shared and treatment-specific adaptive responses. Despite this parallelism, historical contingency was evident, with starting conditions influencing subsequent evolutionary trajectories. We also observed the emergence of hypermutator lineages, which are frequently recovered from chronic lung infections, suggesting that repeatedly evolving elevated mutation rates may represent a common adaptive strategy of P. aeruginosa across environmental and host-associated settings. Fluctuating predation repeatedly reshaped the adaptive landscape, leading to greater temporal turnover of mutations and a higher accumulation of mutations that ultimately reached fixation than in constant environments. Phenotypic assays revealed widespread divergence in fitness, motility, biofilm formation, siderophore production, protease activity, hemolysis, and cell size, whereas virulence in an invertebrate host model varied among treatments but did not differ significantly. Together, these findings demonstrate that variation in predator-mediated selection reshapes the dynamics and genetic targets of bacterial adaptation, highlighting the roles of ecological context, historical contingency, and hypermutability in driving the evolutionary trajectories of opportunistic pathogens.</jats:p>