The FleQ-FleN circuit balances flagellar number against fitness in Pseudomonas aeruginosa

Evolutionary constraints governing flagellar number in bacterial pathogens remain poorly understood. While related Pseudomonas species are hyperflagellated, Pseudomonas aeruginosa maintains strict monoflagellation through the FleQ-FleN regulatory circuit. Here, we demonstrate that FleN dosage is essential for maintaining monoflagellation and fitness. Confirming earlier reports, wild-type P. aeruginosa displayed unipolar monoflagellation, whereas ∆ fleN mutants developed multiple flagella, frequently more than four, in unipolar or bipolar arrangements. These hyperflagellated cells showed marked fitness defects, including reduced growth, attenuated virulence in a nematode infection model, and a competitive disadvantage in co-culture. The virulence defect reflected hyperflagellation rather than the loss of motility, since a non-flagellated ∆ fliC mutant retained full pathogenicity. Notably, ∆ fleN cells rapidly evolved suppressor mutations in fleQ that partially restored growth and motility without always restoring monoflagellation. Five independent suppressor alleles mapped to key FleQ domains, four in the AAA+ ATPase domain, and one in the DNA-binding domain, consistent with reduced FleQ activity that rebalances the circuit. Single-cell holographic tracking showed that suppressor strains swim with heterogeneous dynamics, including subpopulations that reach wild-type (WT) speeds, despite carrying multiple flagella. Quantitative proteomics indicated that the fitness burden extends beyond flagellar components, with protein-abundance changes across metabolism, stress responses, and signaling. Conversely, selection under high-viscosity conditions drove wild-type cells to acquire enhanced spreading through a recurrent fleN mutation, fleN ^V178G ; multiflagellation is therefore accessible. Together, these findings indicate that the FleQ-FleN circuit balances the flagellar number against fitness, holding P. aeruginosa at a single flagellum while retaining the latent capacity to reach multiflagellated states through single mutations.