Genetic determinants of gene amplifications alter frequency and evolutionary trajectory of antibiotic resistance in .

Gene amplifications are thought to be common in bacterial populations, providing a rapid reversible mode of adaptation to diverse stresses, including the acquisition of antibiotic resistance. We previously showed that the opportunistic pathogen evolves resistance to the dual-targeting fluoroquinolone delafloxacin (DLX) that inhibits both the DNA gyrase and DNA topoisomerase IV via gene amplifications of an efflux pump encoding gene . However, the pathways that control the formation or selection of gene amplifications, and consequently adaptive trajectories, remain understudied, especially in gram-positive bacteria like . Here, we show that specific DNA repair and chromosomal separation pathways alter the frequency of formation and selection of gene amplifications in . Through a screen of 36 mutants deficient in various DNA processes, we found that while amplification was still the almost universal path to DLX resistance, other mutations that increased expression reduced the selection frequency of amplifications, demonstrating the critical role of in DLX resistance. We found that similar to other bacteria, the formation and loss of amplifications required a functional RecA recombinase, but multiple other mutants in pathways required for amplifications in other species still exhibited frequent amplifications, suggesting that may have alternate routes of amplification formation. Finally, mutants in the tyrosine recombinase XerC that is involved in chromosomal separation were deficient for amplifications, indicating that XerC is a novel modulator of amplification formation, maintenance, or selection. Thus, our work sheds light on genetic factors that alter gene amplification-mediated evolutionary trajectories to antibiotic resistance in and can potentially unlock mechanisms by which such evolution of resistance can be inhibited.