The <i>Pseudomonas aeruginosa sirB2</i> gene is a fitness determinant of anaerobic growth and its inactivation affects virulence and rugose small colony variants emergence

<i>Pseudomonas aeruginosa</i> is the leading cause of death in cystic fibrosis (CF) patients, yet the genetic mechanisms driving its fitness in the host remain poorly defined. Previously collected transcriptomic data of clinical samples showed that expression of the gene PA14_RS04555 (<i>sirB2</i>) is stimulated in the CF lung environment. In this work, we show that <i>sirB2</i> is regulated by the global transcriptional regulators Vfr and AmrZ. Loss of <i>sirB2</i> markedly enhanced <i>P. aeruginosa</i> pathogenicity, increasing virulence in <i>Galleria mellonella</i>, and promoting bacterial translocation and biofilm formation in a differentiated airway epithelial infection model. Deletion of <i>sirB2</i> triggered the emergence of biofilm-proficient rugose small colony variants (RSCVs), driven by elevated c-di-GMP and increased Pel polysaccharide production when cultures were grown in static conditions. The RSCV phenotype depends on suppressor mutations in the <i>wsp</i> operon, possibly as a response to redox imbalance caused by the lack of <i>sirB2</i> under oxygen-limited conditions. Indeed, the <i>sirB2</i> mutant exhibited impaired fitness during anaerobic respiration when nitrate was the sole electron acceptor, in a manner independent of the ubiquinone pool. Our findings show that <i>sirB2</i> inactivation promotes RSCV emergence and identify <i>sirB2</i> as a novel genetic determinant of metabolic fitness under host-relevant conditions, thereby underscoring the role of redox balance in chronic CF infections.