The <i>Pseudomonas aeruginosa</i> PrrF sRNAs and PqsA promote biofilm formation at body temperature

<i>Pseudomonas aeruginosa</i> is a gram-negative opportunistic pathogen that causes both acute and chronic infections in vulnerable populations. Treatment of <i>P. aeruginosa</i> infections is increasingly challenging due to multi-drug resistance, and biofilm formation during infection further increases antibiotic tolerance. Iron, which is sequestered by the host innate immune system, is also a key nutrient that is required for <i>P. aeruginosa</i> biofilm formation. The iron-responsive PrrF small regulatory RNAs (sRNAs) are key to <i>P. aeruginosa's</i> iron starvation response, promote the production of the <i>Pseudomonas</i> quinolone signal (PQS) quorum sensing molecule, and are required for virulence in murine lung infection. Prior work showed that the PrrF sRNAs are dispensable for biofilm formation; however, these studies were performed using flow-cell biofilms grown at room temperature. Here, we demonstrate a temperature dependency for PrrF in <i>P. aeruginosa</i> biofilm formation: the genes for these sRNAs are required for optimal biofilm formation at 37°C but not 25°C. We further show that a ∆<i>pqsA</i> mutant, which lacks production of PQS and related metabolites, phenocopies the ∆<i>prrF</i> mutant. These studies demonstrate the importance of the PrrF sRNAs in <i>P. aeruginosa</i> biofilm formation at body temperature and reveal a previously underappreciated role of temperature in iron homeostasis and <i>P. aeruginosa</i> biofilm physiology.IMPORTANCEBiofilm formation is a critical virulence trait for many microbial pathogens that confers tolerance to the host immune system and antimicrobials. <i>Pseudomonas aeruginosa</i> is an opportunistic pathogen that forms biofilms resulting in treatment failure. Iron is a known requirement for <i>P. aeruginosa</i> biofilm formation, yet the precise role of iron in biofilm physiology remains unclear. Here, we show that temperature alters the requirement for the PrrF small regulatory RNAs, key components of <i>P. aeruginosa's</i> iron starvation response, for biofilm formation. Specifically, PrrF is required for the optimal formation of flow-cell biofilms at 37°C but not at 25°C, yet most flow-cell biofilm studies are conducted at 25°C. These results demonstrate a previously underappreciated role of temperature in <i>P. aeruginosa</i> biofilm physiology.