Evolution of Resistance to Phenazine Antibiotics in and Its Role During Coinfection with .

In the niches that and coinhabit, the later pathogen produces phenazine antibiotics to inhibit the growth of . Recently, a group of halogenated phenazines (HPs) has been shown to have potent antimicrobial activities against ; however, no HP-resistant mutant has been reported. Here, we demonstrate that develops HP-resistance via single amino acid change (Arg116Cys) in a transcriptional repressor TetR21. RNA-seq analysis showed that the TetR21 variation caused drastic up-regulation of an adjacent gene (halogenated phenazine resistance protein of ). Deletion of the in the TetR21 background restored bacterial susceptibility to HP, while overexpression in conferred HP-resistance. The expression of HprS is under tight transcriptional control of the TetR21 via direct binding to the promoter region of . The R116C mutation in TetR21 significantly reduced its DNA binding affinity. Moreover, natural phenazine antibiotics (phenazine-1-carboxylic acid and pyocyanin) and a HP analog (HP-22) are ligands for the TetR21, regulating its repressor activity. Combining homology analysis and LC-MS/MS assay we demonstrated that HprS is a phenazine efflux pump. To the best of our knowledge, we provide the first report of phenazine efflux pump in . Interestingly, the TetR21 variation has been found in some clinical isolates, and a laboratory strain of with TetR21 variation showed enhanced growth competitiveness toward and promoted coinfection with in the host environment, demonstrating significance of the mutation in host infections.