FleQ finetunes the expression of a subset of BrlR-activated genes to enable antibiotic tolerance by <i>Pseudomonas aeruginosa</i> biofilms

The transcriptional regulator FleQ contributes to <i>Pseudomonas aeruginosa</i> biofilm formation by activating the expression and biosynthesis of matrix exopolysaccharides in a manner dependent on c-di-GMP. However, little is known about the role of FleQ in the antibiotic tolerance phenotype of <i>P. aeruginosa</i> biofilms. Inactivation of <i>fleQ</i> impaired biofilm formation and rendered biofilms susceptible to tobramycin and norfloxacin. The phenotypes were similar to biofilms inactivated in <i>sagS</i> encoding the orphan sensor SagS that promotes the switch from planktonic to biofilm growth via BfiSR and antibiotic tolerance via BrlR. While FleQ was found to contribute to biofilm formation independently of SagS and BfiSR, FleQ instead converged with SagS-dependent regulation at the level of BrlR. This was supported by multicopy expression of <i>sagS</i> failing to restore biofilm antibiotic tolerance by <i>ΔfleQ</i> to wild-type levels (and <i>vice versa</i>) and by biofilms formed by the <i>ΔfleQΔsagS</i> double mutant being as susceptible as <i>ΔfleQ</i> and <i>ΔsagS</i> biofilms. Increased antibiotic susceptibility was independent of BrlR abundance or BrlR DNA binding but coincided with significantly reduced transcript abundance of the BrlR-activated <i>mexCD-oprJ</i> and PA1874-77, encoding an ABC transporter previously shown to contribute to the tolerance of biofilms to tobramycin and norfloxacin. FleQ- dependent regulation of gene expression was indirect. Co-immunoprecipitation and BACTH assays indicated FleQ to interact with SagS via its HisKA-Rec domain, likely suggesting FleQ and SagS to likely work in concert to enable biofilm antibiotic tolerance<b>,</b> by finetuning the expression of BrlR activated genes.IMPORTANCEIn <i>P. aeruginosa</i>, FleQ inversely regulates the expression of genes encoding flagella and biofilm matrix components, including exopolysaccharide (Pel, Psl) in a manner dependent on the levels of c-di-GMP. Our findings expand on the role of FleQ from regulating the transition to the biofilm mode of growth to FleQ contributing to the antimicrobial tolerance phenotype of biofilms, by FleQ affecting the expression of PA1874-77, a downstream target of the SagS-dependent transcriptional regulator BrlR. Importantly, our findings suggest FleQ works in concert with SagS, likely via FleQ-SagS protein-protein interactions, to enable the formation of inherently tolerant <i>P. aeruginosa</i> biofilms.