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

Evolutionary constraints governing flagellar number in bacterial pathogens remain poorly understood. While related species are hyperflagellated, 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 consistently displayed unipolar monoflagellation, while Δ mutants developed over two-to-five flagella per cell in uni- or bipolar arrangements. Hyperflagellated Δ cells exhibited severe fitness defects including reduced growth rates, attenuated virulence in nematode infection models, and competitive disadvantages in co-culture experiments. Remarkably, Δ cells rapidly evolved suppressor mutations in 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 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.