Genomic and phenotypic diversification of Pseudomonas aeruginosa during sustained exposure to a ciliate predator

Opportunistic bacterial pathogens often encounter strong selective pressures outside their hosts, yet the evolutionary consequences of long-term predator exposure remain poorly understood. Here, we used experimental evolution to examine how sustained interaction with a eukaryotic predator shapes genomic adaptation, phenotypic diversification, and virulence-associated traits in Pseudomonas aeruginosa. Replicate populations of P. aeruginosa were evolved for 60 days in the presence or absence of the ciliate predator Tetrahymena thermophila, followed by whole-population metagenomic sequencing, isolate-level genome sequencing, and quantitative phenotypic assays. We observed extensive genetic diversification across all populations, with strong signatures of both positive and purifying selection and pervasive parallel evolution at gene and nucleotide levels. Predator-exposed populations accumulated mutations enriched in regulatory, metabolic, and virulence-associated pathways, revealing predictable genomic targets of selection. However, many parallel mutations were shared between predator-exposed and predator-free populations, indicating that adaptation to the abiotic environment represented a dominant selective force. Genotype-phenotype analyses revealed pleiotropic effects and trade-offs linking motility, growth, and virulence-associated traits. Despite pronounced genomic adaptation and coordinated phenotypic shifts, changes in virulence in an in vivo host model were modest and context dependent. Taken together, our results indicate that predator exposure can influence evolutionary trajectories in P. aeruginosa and highlight the value of extending such approaches across multiple ecological and host contexts. SignificanceMany bacterial pathogens spend much of their evolutionary history outside hosts, where they face intense ecological pressures such as predation. How these pressures shape pathogen evolution and disease potential remains unclear. Using experimental evolution, genomics, and phenotypic analyses, we show that prolonged exposure to a eukaryotic predator drives predictable genetic and phenotypic changes in the opportunistic pathogen Pseudomonas aeruginosa. Predator exposure altered regulatory, metabolic, and virulence-associated pathways, yet much adaptation was shared with predator-free populations, highlighting the dominant role of abiotic environments. Although predator-driven evolution reshaped traits linked to motility and growth, its effects on virulence were modest and context dependent. These findings clarify how environmental interactions influence pathogen evolution and underscore the importance of studying pathogens across diverse ecological settings.