A novel stress response pathway mediates biofilm architecture in Pseudomonas aeruginosa

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 sensitivity. We show that the small uncharacterised protein PA3049, renamed as B iofilm a rchi t ecture R egulator (BatR), promotes biofilm establishment and enhances biofilm 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 Pf4. 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 resistance and modulates pyocyanin production. Our study implicates the BatR/SrkA system in the response of P. aeruginosa biofilms to antibiotic challenge in lung infections.