Within-host adaptation mediated by intergenic evolution in Pseudomonas aeruginosa

Bacterial pathogens evolve during the course of infection as they adapt to the selective pressures that confront them inside the host. Identification of adaptive mutations and their contributions to pathogen fitness remains a central challenge. Although mutations can either target intergenic or coding regions in the pathogen genome, studies of host adaptation have focused predominantly on molecular evolution within coding regions, whereas the role of intergenic mutations remains unclear. Here, we address this issue and investigate the extent to which intergenic mutations contribute to the evolutionary response of a clinically important bacterial pathogen, Pseudomonas aeruginosa, to the host environment, and whether intergenic mutations have distinct roles in host adaptation. We characterize intergenic evolution in 44 clonal lineages of P. aeruginosa and identify 77 intergenic regions in which parallel evolution occurs. At the genetic level, we find that mutations in regions under selection are located primarily within regulatory elements upstream of transcriptional start sites. At the functional level, we show that some of these mutations both increase or decrease transcription of genes and are directly responsible for evolution of important pathogenic phenotypes including antibiotic sensitivity. Importantly, we find that intergenic mutations facilitate essential genes to become targets of evolution. In summary, our results highlight the evolutionary significance of intergenic mutations in creating host-adapted strains, and that intergenic and coding regions have different qualitative contributions to this process.