A Novel Stress Response Pathway Mediates Antibiotic Tolerance and Architecture in Pseudomonas aeruginosa Biofilms

Abstract Pseudomonas aeruginosa is a multidrug-resistant opportunistic pathogen, with chronic infections often associated with biofilm formation. Here, we investigate the previously uncharacterized gene PA3049, which is upregulated under biofilm conditions, to determine its role in infection, biofilm formation, and antimicrobial tolerance. We show that the small uncharacterised protein PA3049, renamed as Biofilm antibiotic tolerance Regulator (BatR), promotes biofilm establishment and enhances bacterial survival in sub-inhibitory concentrations of antibiotics. Proteomic analysis revealed that BatR influences the R2/F2 pyocin cluster, which drives explosive cell lysis and extracellular DNA (eDNA) release during biofilm development. We further identify a specific interaction between BatR and PA0486 (SrkA), an uncharacterised Ser/Thr protein kinase. We show that SrkA controls biofilm and pyocyanin production, and lysis-mediated eDNA release through regulation of the R2/F2 pyocin cluster and activation of bacteriophage Pf1. Our findings support a model in which SrkA directly regulates key biofilm-associated phenotypes, while BatR acts as a modulatory partner that tunes SrkA activity under specific conditions. Finally, BatR function was tested in high- validity infection models, including the ex vivo pig lung model of cystic fibrosis infection and a synthetic chronic-wound model. In these models, BatR contributes to biofilm architecture and antibiotic tolerance, and modulates pyocyanin production. Our study implicates the BatR/SrkA system in the response of P. aeruginosa biofilms to antibiotic challenge in lung infections. Competing Interest Statement The authors have declared no competing interest.