Laboratory evolution of the bacterial genome structure through insertion sequence activation

The genome structure critically impacts bacterial physiology, ecology, and evolution. However, its evolution, driven by transposons called insertion sequences (IS), has been challenging to track in laboratories due to its slow pace. Here, we accelerated this process by introducing multiple copies of a high-activity IS into Escherichia coli . Mimicking bursts of IS copies in host-restricted endosymbionts and pathogens, we evolved the bacteria under relaxed neutral conditions. Within ten weeks, we observed a median of 24.5 IS insertions per genome, comparable to a decade of wild-type evolution. Long-read sequencing revealed extensive IS-mediated genome rearrangements, resulting in novel IS variants and genome size changes exceeding ±5 %. By achieving such drastic genome evolution under relaxed selection, our study establishes a baseline for assessing the fitness effects of IS insertions, genome size changes, and rearrangements. This work paves the way for experimentally studying bacterial genome structure evolution, complementing analyses of genome structures in nature.