A bifunctional ATPase drives tad pilus extension and retraction

Abstract

Abstract Molecular motors convert chemical energy directly into mechanical work 1 and are found in all domains of life 2 . These motors are critical to intracellular transport 3 , motility 4,5 , macromolecular protein assembly 3,6 , and many essential processes 7 . A wide-spread class of related bacterial motors drive the dynamic activity of extracellular fibers, such as type IV pili (T4P), that are extended and retracted using so-called secretion motor ATPases. Among these, the tight ad herence (tad) pili are critical for surface sensing, surface attachment, and biofilm formation 8–10 . How tad pili undergo dynamic cycles of extension and retraction 8 despite lacking a dedicated retraction motor ATPase has remained a mystery. Here we find that a bifunctional pilus motor ATPase, CpaF, drives both activities through ATP hydrolysis. Specifically, we show that mutations within the ATP hydrolysis active site of Caulobacter crescentus CpaF result in a correlated reduction in the rates of extension and retraction. Moreover, a decrease in the rate of ATP hydrolysis directly scales with a decrease in the force of retraction and reduced dynamics in these CpaF mutants. This mechanism of motor protein bifunctionality extends to another genus of tad-bearing bacteria. In contrast, the T4aP subclass of pili possess dedicated extension and retraction motor ATPase paralogs. We show that these processes are uncoupled using a slow ATP hydrolysis mutation in the extension ATPase of competence T4aP of Vibrio cholerae that decreases the rate of extension but has no effect on the rate of retraction. Thus, a single motor ATPase is able to drive the bidirectional processes of pilus fiber extension and retraction.