Modulators of gene amplification alter evolution of antibiotic resistance in Staphylococcus aureus

Gene amplification is thought to be common in bacterial populations, providing a rapid and reversible mode of adaptation to diverse stresses, including the acquisition of antibiotic resistance. We previously showed that the opportunistic pathogen Staphylococcus aureus evolves resistance to the dual-targeting fluoroquinolone delafloxacin (DLX) that inhibits both the DNA gyrase and DNA topoisomerase IV via amplification of an efflux pump encoding gene sdrM. However, the pathways that control gene amplification, and consequently adaptive trajectories, remain understudied, especially in gram-positive bacteria like S. aureus. Here, we show that specific DNA repair and chromosomal separation proteins alter the frequency of gene amplification and selection of amplified regions in S. aureus. Through a screen of 40 mutants deficient in various DNA processes, we determined that while sdrM amplification was still the almost universal path to DLX resistance, other mutations that increased sdrM expression reduced the selection frequency of sdrM amplification, demonstrating the critical role of sdrM in DLX resistance. We found that similar to other bacteria, both sdrM amplification and loss of amplified gene copies required a functional RecA recombinase, but multiple other mutants in pathways required for amplification in other species still exhibited frequent sdrM amplification, suggesting that S. aureus may have alternate routes of gene amplification. Finally, loss of function mutants of the tyrosine recombinase XerC, that is known to play a role in chromosomal separation, were deficient for sdrM amplification, indicating that XerC is a novel modulator of gene amplification, or the maintenance or selection of amplified gene copies. Thus, our work sheds light on genetic factors that alter gene amplification-mediated evolutionary trajectories to antibiotic resistance in S. aureus and can potentially unlock mechanisms by which such evolution of resistance can be inhibited.