The FliI ATPase couples ATP hydrolysis to substrate switching in bacterial flagellar type-III secretion

The ordered export of substrates by bacterial type-III secretion systems is essential for the assembly of complex surface structures such as the flagellum, yet the mechanisms that control the timing of substrate switching remain poorly understood. The flagellar type-III secretion system ATPase FliI has previously been implicated in coupling ATP hydrolysis to activation of the proton motive force-driven export apparatus, although its precise role in secretion remains incompletely understood. Here, we show that FliI contributes to the transition from early to late substrate export during flagellar biogenesis. Using mutants of the catalytic domain of FliI in <i>Salmonella</i>, we demonstrate that minimal ATPase activity is sufficient to support early export, whereas efficient substrate switching and late-stage flagellar assembly require near-wild-type activity levels. These findings highlight the multifaceted roles of FliI during flagellar assembly beyond its proposed function in activating the flagellar type-III secretion system and demonstrate that the ATPase is critical for coordinating the transition from early to late substrate export, linking ATPase activity to substrate switching in type-III secretion systems.