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 defined FliI point mutations and found that FliI activity is dispensable for early substrate export and hook-basal body formation but is important for triggering the substrate specificity switch and promoting late substrate export. 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 fT3SS function. This study highlights the multifaceted role of the fT3SS ATPase in optimizing the efficiency and robustness of flagellum assembly. SignificanceThe 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 bacterial flagellar ATPase FliI is best known for being involved in energizing the flagellar type III secretion system. Here, we show that FliI contributes to the transition from early to late substrate export during flagellar biogenesis. Using targeted FliI mutants in Salmonella, we show that ATPase activity is dispensable for early export but important for proper substrate switching and late-stage flagellar assembly. These findings highlight the multifaceted roles of FliI during flagellar assembly beyond activating the flagellar type III secretion system.