Hypermotility impacts bacterial biofilm formation via proton cycling

The proton gradient is the core energy medium driving bacteria flagellar rotation. However, the proton cycling mechanisms in motile bacteria and their biophysical impacts on biofilm formation remain poorly understood. Here, single-cell-tracking microscopic imaging systems reveal that increased proton intensity boosts individual flagellar rotation and colony motility. Further trajectory mapping and causal mediation analysis demonstrate that hypermotility suppresses the motile-to-sessile transition, limiting biofilm initiation. Systematic energy metabolism profiling further reveals that a high-motility colony exhibits a 60.8% increase in proton motive force (PMF) and a 27.2% reduction in energy reserves to maintain continuous flagellar rotation. Mechanistically, a dual-engine proton cycling model based on metabolic signatures and transcriptomic evidence is proposed: protons flow through the MotA/MotB complex stators (MotAB) to generate torque during propulsion, while F-type ATP synthase reverses ATP hydrolysis to restore the proton gradient, maintaining PMF stability in a closed-loop system. This study enhances our understanding of proton-driven bacterial interface motility regulating microbial collective behavior, providing theoretical foundations for precise biofilm control.