Insertion sequences (IS) are key drivers of bacterial genome evolution, profoundly shaping genome plasticity in Escherichia coli. This study analyzed fourteen E. coli genomes from commensal and pathogenic strains to investigate the distribution and functional impact of IS are major drivers of bacterial genome evolution and genome plasticity in E. coli. This study analyzed fourteen commensal and pathogenic E. coli genomes to investigate the distribution and functional impact of IS on genomic diversity and virulence.

A total of 512 complete IS elements belonging to fourteen families were identified.

Comparative genomic and phylogenetic analyses showed that some IS families, including IS3, are conserved through vertical inheritance, whereas others, such as IS66, are mainly associated with pathogenic strains and likely acquired through horizontal gene transfer.

Intragenic IS insertions were more frequent in commensal strains and frequently disrupted genes involved in virulence and host interaction, including autotransporters and fimbriae assembly proteins. A novel IS insertion disrupting the yagA gene was identified, and an IS-rich region co-localized with the virulence-associated pap operon was detected in the pathogenic strain EC958 but absent from the commensal strain SE15. These observations suggest that IS insertions may reduce virulence in commensal strains by disrupting pathogenicity-related genes or regulatory elements, whereas in pathogenic strains they may enhance virulence through mobilization or activation of key loci. Overall, IS activity contributes substantially to E. coli genome plasticity, genomic rearrangements, horizontal gene transfer, and virulence evolution. The study also highlights limitations in current genome annotation pipelines, which frequently underdetect IS elements, emphasizing the need for improved IS-specific bioinformatics tools and experimental validation approaches.