Twitching motility suppressors reveal a role for FimX in type IV pilus extension dynamics

Abstract In Pseudomonas aeruginosa, retractable protein filaments called type IV pili (T4P) facilitate surface adherence, sensing, and directional movement known as twitching motility. T4P are necessary for the bacteria to engage in surface-associated behaviors, including establishing acute infections. Pilus extension is driven by the hexameric ATPase, PilB, at the base of the T4P nanomachine in coordination with various protein regulatory effectors. The cyclic-di-GMP binding protein, FimX, works with PilB to mediate normal extension processes, though how this effector controls pilus assembly remains unclear. To explore the role of FimX in T4P function, we leveraged the significant ΔfimX twitching motility deficit to screen for mutants capable of overcoming this phenotype. We identified suppressor mutations that increase twitching in ΔfimX background, mapping primarily to cyclic-AMP homeostatic machinery or to PilB, the FimX target. Distinct suppressor mutations in PilB increased ATP hydrolysis in vitro and this activity was subject to modulation by FimX. Using microscopy to monitor the extension dynamics of fluorescently labelled T4P, we showed that ΔfimX mutants produce slow-to-extend, short pili, a phenotype that is rescued by mutations enhancing PilB ATP hydrolysis and/or re-introduction of FimX. Together, these data implicate FimX as a regulator of PilB enzymatic function, potentially enabling P. aeruginosa to fine-tune pilus extension dynamics in response to environmental cues. Summary Type IV pili enable Pseudomonas aeruginosa to attach to surfaces, move (twitch), and form biofilms. Pilus extension is powered by the motor protein PilB, which is regulated by other factors, including FimX, a protein that binds cyclic-di-GMP. Although FimX is important for twitching, how it influences PilB was unclear. We deleted fimX, which severely reduces motility, and searched for mutants that regained movement. We identified two types: some had mutations in PilB that increased its ATPase activity, allowing it to function without FimX, while others affected the cyclic-AMP signaling pathway and increased overall production of pilus components, showing that motility can also be improved through changes in quantity versus quality. Our results suggest that FimX normally fine-tunes PilB enzymatic activity, enabling dynamic control of pilus extension in response to surface signals. This work helps explain how P. aeruginosa adapts to different environments, a process crucial for infection and biofilm development. Competing Interest Statement The authors have declared no competing interest. Footnotes This revision includes the supplementary information.