Highly parallel lab evolution of single gene Escherichia coli deletion strains under tightly controlled population size and antibiotic selection reveals epistasis can curb antibiotic resistance evolution.

The looming antibiotic resistance crisis calls for new ways of restricting the ability of bacteria to evolve spontaneous resistance. Genetic perturbations that affect resistance have been characterized genome-wide, but how do such perturbations interact with subsequent evolutionary adaptation to the drug? Here, we show that strong epistasis between resistance mutations and systematically identified genes can be exploited to control spontaneous resistance evolution. We evolved hundreds of genetically perturbed Escherichia coli K-12 populations in parallel using a robotic platform that tightly controls population size and selection pressure. We found a global diminishing-returns epistasis pattern: Strains that are initially more sensitive generally undergo larger resistance gains. However, we also identified specific functional perturbations that deviate from this general trend and drastically curtail the evolvability of resistance; these include membrane transport, LPS biosynthesis, and chaperones. Notably, perturbations of efflux pumps forced evolution on inferior mutational paths, not explored in the wild type and some essentially blocked resistance evolution. We demonstrate that this effect is due to strong negative epistasis with resistance mutations. The identified cellular functions provide potential candidates for adjuvants that can block spontaneous resistance evolution when combined with antibiotics. See also: BioProject ID PRJNA1010512