Regulatory Plasticity and Metabolic Trade-offs Drive Adaptive Evolution of Alternative Flagellar Configurations in Pseudomonas aeruginosa

Evolutionary constraints governing flagellar number in bacterial pathogens remain poorly understood. While related Pseudomonas species are hyperflagellated, P. aeruginosa maintains strict monoflagellation through the FleQ-FleN regulatory circuit. Here, we demonstrate that FleN dosage is essential for maintaining monoflagellation and bacterial fitness. Wild-type P. aeruginosa consistently displayed unipolar monoflagellation, while {Delta}fleN mutants developed over two-to-five flagella per cell in uni- or bipolar arrangements. Hyperflagellated {Delta}fleN cells exhibited severe fitness defects including reduced growth rates, attenuated virulence in nematode infection models, and competitive disadvantages in co-culture experiments. Remarkably, {Delta}fleN cells rapidly evolved suppressor mutations in fleQ that partially restored growth and motility without always restoring monoflagellation. Five independent suppressor alleles mapped to critical FleQ functional domains (four in the AAA+ ATPase domain, one in the DNA-binding domain), suggesting reduced protein activity that rebalances the disrupted regulatory circuit. Single-cell motility analysis revealed that suppressor strains exhibit heterogeneous swimming dynamics, with subpopulations achieving wild-type speeds despite carrying multiple flagella. Proteomic analysis demonstrated that hyperflagellation triggers extensive cellular reprogramming beyond flagellar components, affecting metabolic pathways, stress responses, and signaling networks. While hyperflagellated cells suffered complete loss of pathogenicity in animal infection models, environmental selection under viscous conditions could drive wild-type cells to evolve enhanced motility through specific fleN mutations. These findings suggest that bacterial flagellar regulatory circuits function as evolutionary capacitors, normally constraining phenotypic variation but enabling rapid adaptation to alternative motility configurations when environmental pressures exceed the performance limits of standard monotrichous flagellation. SIGNIFICANCE STATEMENTBacterial flagella are extracellular appendages that rotate to propel the cell and enable swimming motility. While some bacteria have multiple flagella, many pathogenic species like Pseudomonas aeruginosa have just one. Surprisingly, mutants of P. aeruginosa with multiple flagella performed worse, i.e., they grew more slowly, were less infectious in laboratory animals, and were outcompeted by wild-type bacteria. Even when some mutant bacteria evolved compensatory changes, they still struggled compared to single-flagellum bacteria. This reveals an important evolutionary trade-off: while multiple flagella might seem advantageous for movement, having just one flagellum allows bacteria to grow faster and cause more severe infections. This plasticity likely explains why P. aeruginosa is so successful both in the environment and as a human pathogen.