Pseudomonas aeruginosa orchestrates twitching motility by sequential control of type IV pili movements

Prokaryotes have the ability to walk on surfaces using type IV pili (TFP), a motility mechanism known as twitching1,2. Molecular motors drive TFP extension and retraction, but whether and how these movements are coordinated is unknown3. Here, we reveal how the pathogen Pseudomonas aeruginosa coordinates the motorized activity of TFP to power efficient surface motility. To do this, we dynamically visualized TFP extension, attachment and retraction events at high resolution in four dimensions using label-free interferometric scattering microscopy (iSCAT)4. By measuring TFP dynamics, we found that the retraction motor PilT was sufficient to generate tension and power motility in free solution, while its partner ATPase PilU may improve retraction only in high-friction environments. Using precise timing of successive attachment and retraction, we show that P. aeruginosa engages PilT motors very rapidly and almost only when TFP encounter the surface, suggesting contact sensing. Finally, measurements of TFP dwell times on surfaces show that tension reinforced the adhesion strength to the surface of individual pili, thereby increasing effective pulling time during retraction. The successive control of TFP extension, attachment, retraction and detachment suggests that sequential control of motility machinery is a conserved strategy for optimized locomotion across domains of life.