Polygenic adaptation, clonal interference, and the evolution of mutators in experimental Pseudomonas aeruginosa populations

In bacterial populations, switches in lifestyle from motile, planktonic growth to surface-associated biofilm is associated with persistence both in the environment and infections. Studies have identified the first steps of adaptation to biofilm growth but have yet to replicate the extensive genetic diversity observed in chronic infections or in the natural environment. We conducted an evolution experiment with Pseudomonas aeruginosa PA14 in growth media that promotes biofilm formation for 90 days in either planktonic culture or in a biofilm bead model. Surprisingly, all populations evolved extensive genetic diversity with hundreds of mutations maintained at intermediate frequencies, while fixation events were rare. Instead of the expected few beneficial mutations rising in frequency through successive sweeps, we observe a remarkable 40 genes with parallel mutations spanning both environments and often on coexisting genotypes within a population. Additionally, the evolution of mutator genotypes (mutS or mutL mutator alleles) that rise to high frequencies in as little as 25 days contribute to the extensive genetic variation and strong clonal interference. Parallelism in several transporters (including pitA, pntB, nosD, and pchF) indicate probable adaptation to the arginine media that becomes highly alkaline during growth, whereas genes involved in signal transduction (including gacS, aer2, bdlA, and PA14_71750) reflect likely adaptations to biofilm-inducing conditions. This experiment shows how extensive genetic and phenotypic diversity can arise and persist in microbial populations despite strong selection that would normally purge diversity.