Development of mass allelic exchange, a technique to enable sexual genetics in <i>Escherichia coli</i>.

Despite dramatic advances in genomics, connecting genotypes to phenotypes is still challenging. Sexual genetics combined with linkage analysis is a powerful solution to this problem but generally unavailable in bacteria. We build upon a strong negative selection system to invent mass allelic exchange (MAE), which enables hybridization of arbitrary (including pathogenic) strains of <i>Escherichia coli</i>. MAE reimplements the natural phenomenon of random cross-overs, enabling classical linkage analysis. We demonstrate the utility of MAE with virulence-related gain-of-function screens, discovering that transfer of a single operon from a uropathogenic strain is sufficient for enabling a commensal <i>E. coli</i> to form large intracellular bacterial collections within bladder epithelial cells. MAE thus enables assaying natural allelic variation in <i>E. coli</i> (and potentially other bacteria), complementing existing loss-of-function genomic techniques.