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

Bacterial flagella are assembled by a specialized type-III secretion system that exports structural subunits in a defined order. While the ATPase FliI is known to couple ATP hydrolysis to substrate translocation, its role in the transition between early and late secretion stages has remained unclear. Here, we systematically analyzed Salmonella enterica strains with mutations in the catalytic domain of FliI and found that early substrate export and hook-basal body formation can proceed with minimal FliI ATPase activity, whereas efficient triggering of the substrate specificity switch and late substrate export requires near-wild-type activity levels. Mutant strains showed delayed gene expression from class 3 promoters, prolonged early secretion, and impaired flagellar filament assembly, despite normal FliI localization and oligomerization. These findings support the involvement of FliI in controlling the temporal dynamics of flagellar assembly. We propose that FliI contributes to substrate switching, ensuring robust and orderly function of the flagellar type-III secretion system. This study highlights the multifaceted role of the type-III secretion system ATPase in optimizing the efficiency and robustness of flagellum assembly and establishes a mechanistic link between ATPase activity and substrate switching in type-III secretion systems. IMPORTANCE: 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 Salmonella, 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.