Creating bacterial genomic diversity through large-scale reconfigurations reveals phenotype robustness to organizational genome change

Abstract The ability to generate genomic diversity expands opportunities for understanding and engineering biology. Here, we demonstrate on-demand generation of diversity in bacterial genome configurations and its application to probing physiology under altered genome organization. We engineered the fast-growing bacterium Vibrio natriegens to enable large-scale stochastic duplications, translocations, inversions, and deletions, producing populations with a wide range of genome arrangements. We investigated phenotypic robustness to genome reconfigurations and found that distinct genome organizations can support stable physiology, indicating that bacteria may tolerate chromosomal alterations more readily than previously appreciated. Our work provides a framework for advancing the understanding and engineering of bacterial genomes and suggests that genome reconfigurations may contribute to phenogenetic drift, allowing for evolutionary exploration while preserving phenotype.