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The phenoxazine dye resazurin exhibits bactericidal activity against the Gram-negative pathogens <i>Francisella tularensis</i> and <i>Neisseria gonorrhoeae</i>. One resazurin derivative, resorufin pentyl ether, significantly reduces vaginal colonization by <i>Neisseria gonorrhoeae</i> in a mouse model of infection. The narrow spectrum of bacteria susceptible to resazurin and its derivatives suggests these compounds have a novel mode of action. To identify potential targets of resazurin and mechanisms of resistance, we isolated mutants of <i>F. tularensis</i> subsp. <i>holarctica</i> live vaccine strain (LVS) exhibiting reduced susceptibility to resazurin and performed whole genome sequencing. The genes <i>pilD</i> (FTL_0959) and <i>dipA</i> (FTL_1306) were mutated in half of the 46 resazurin-resistant (RZR) strains sequenced. Complementation of select RZR LVS isolates with wild-type <i>dipA</i> or <i>pilD</i> partially restored sensitivity to resazurin. To further characterize the role of <i>dipA</i> and <i>pilD</i> in resazurin susceptibility, a <i>dipA</i> deletion mutant, Δ<i>dipA</i>, and <i>pilD</i> disruption mutant, FTL_0959d, were generated. Both mutants were less sensitive to killing by resazurin compared to wild-type LVS with phenotypes similar to the spontaneous resazurin-resistant mutants. This study identified a novel role for two genes <i>dipA</i> and <i>pilD</i> in <i>F. tularensis</i> susceptibility to resazurin.