Non-canonical double-stranded DNA break repair in Pseudomonas aeruginosa includes LigD-independent non-homologous end joining.

Pseudomonas aeruginosa is a highly adaptive Gram-negative pathogen that is commonly managed with fluoroquinolone (FQ) antibiotics. Even without heritable resistance mutations, FQ-susceptible cells of P. aeruginosa can survive treatment in a phenomenon termed antibiotic persistence. Current models of stationary-phase FQ persistence established with other bacterial species posit that persister survival is dependent on DNA double-stranded break (DSB) repair via RecA-mediated homologous recombination (HR). In P. aeruginosa, however, the effects of RecA loss on FQ persistence vary depending on the culture conditions. In this current work, we find that stationary-phase P. aeruginosa does not depend on either of its canonical DSB repair pathways-HR or non-homologous end joining (NHEJ)-for FQ persistence. By creating cross-pathway deletion strains, we found that RecA and Ku (but not LigD) are conditionally essential, which suggests that P. aeruginosa can conduct LigD-independent NHEJ. Using a chromosomally integrated DSB reporter assay, we further show that P. aeruginosa can repair DSBs using two non-canonical pathways: LigD-independent NHEJ and microhomology-mediated repair. Together, these findings demonstrate that P. aeruginosa does not adhere to the conventional requirement for DSB repair after FQ treatment and can engage in alternative DSB repair pathways than those previously described.