Active removal of inhibitory components drives the flagellar type 3 secretion-specificity switch

ABSTRACT Type 3 secretion (T3S) systems assemble bacterial nanomachines, including the flagellum and virulence-associated injectisomes, by exporting distinct classes of substrates in a defined temporal order. In both systems, completion of an early assembly intermediate triggers an irreversible switch from early to late substrate secretion. In the flagellar system, this switch is controlled by the secreted molecular ruler FliK, which acts on the core T3S component FlhB, but the molecular mechanism governing this transition has remained unclear. Here, we show that removal of two components, Fluke and the cleaved C-terminal domain of FlhB (FlhB CCD ), locks the secretion apparatus in a constitutive late secretion state. In these mutants, secretion specificity no longer requires completion of the hook-basal body or the FliK ruler, indicating that Fluke and FlhB CCD function to maintain the apparatus in early secretion mode. Consistent with this model, synchronized flagellar gene expression experiments reveal that FlhB CCD is retained during early assembly and is lost coincident with hook-basal body completion and activation of σ 28 -dependent late gene expression of flagellin and chemosensory genes. Mutants in fliK and the core secretion apparatus protein genes fliP , fliQ, and fliR that slow the rate of FliK secretion allow FliK to flip the secretion-specificity switch independent of the assembly of a hook-basal body structure. Structural modeling of the FliK C-terminal switch domain and FlhB CCD supports a mechanism in which secretion of FliK promotes destabilization and ejection of FlhB CCD from the secretion apparatus. Disruption of a folded region within the FliK switch domain uncouples secretion from switching, indicating that the timing of FliK unfolding during secretion is critical for activation of the specificity switch. These findings show that secretion-specificity switching is driven by FliK-dependent removal of inhibitory components, rather than passive sensing of assembly completion. IMPORTANCE Type III secretion (T3S) systems build complex bacterial nanomachines, including the flagellum and virulence-associated injectisomes, by exporting distinct classes of substrates in a defined temporal order. How these systems switch secretion specificity during assembly has remained a long-standing question. We demonstrate that the flagellar T3S-specificity switch requires removal of two inhibitory components that actively maintain the apparatus in an early secretion state. Their FliK-dependent ejection irreversibly triggers the transition to late substrate export, revealing that secretion switching is controlled by active inhibitory regulation rather than passive sensing of assembly completion. These results define a molecular mechanism for hierarchical control in bacterial secretion systems and provide insights likely relevant to other T3S nanomachines.