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

In , 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 Δ twitching motility deficit to screen for mutants capable of overcoming this phenotype. We identified suppressor mutations that increase twitching in Δ background, mapping primarily to cyclic-AMP homeostatic machinery or to PilB, the FimX target. Distinct suppressor mutations in PilB increased ATP hydrolysis and this activity was subject to modulation by FimX. Using microscopy to monitor the extension dynamics of fluorescently labelled T4P, we showed that Δ 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 to fine-tune pilus extension dynamics in response to environmental cues.