Host adaptation mediated by intergenic evolution in a bacterial pathogen

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 remain 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 pathogens to host environments, and if intergenic mutations have distinct roles in host adaptation. We characterize intergenic evolution in 44 lineages of a clinically important bacterial pathogen, Pseudomonas aeruginosa, as they adapt to their hosts. We identify 88 intergenic regions in which parallel evolution occur. At the genetic level, we find that mutations in these regions under selection are located primarily within regulatory elements upstream of transcriptional start sites. At the functional level, we show that these mutations both create or destroy regulatory interactions in connection to transcriptional processes, and are directly responsible for evolution of important pathogenic phenotypes including antibiotic sensitivity. Importantly, we find that intergenic mutations are more likely to be selected than coding region mutations, and that intergenic mutations enable essential genes to become targets of evolution. In summary, our results highlight the evolutionary significance of intergenic mutations in creating host-adapted variants, and that intergenic and coding regions have different qualitative and quantitative contributions to this process.\n\nSignificancePathogens adapt to their host during infection, but the contribution and function of non-coding intergenic sequences to adaptation is poorly understood. Here, genome-wide identification of adaptive mutations within intergenic regions demonstrates that these sequences constitute an important part of the genetic basis for host adaptation. We find that intergenic mutations are abundant relative to adaptive mutations within coding sequences, and can contribute directly to evolution of pathogen relevant traits. Importantly, we find that intergenic mutations modify expression of essential genes and thus make contributions that are functionally distinct from coding mutations. These results improve our understanding of the evolutionary processes in vivo, and can potentially assist in refining predictions of pathogen evolution, disease outcome, and antibiotic resistance development.