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bioRxiv · 10.64898/2026.05.20.726703

A single conserved residue in FlhA couples export gate activation to substrate specificity switching

Kinoshita, M.·Minamino, T.·Sakai, M.·Takekawa, N.·Uchihashi, T.·Kitao, A.·Imada, K.·Namba, K.
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Abstract

FlhA is a core component of the bacterial flagellar type III secretion system that functions as both an export engine and a switch regulator of substrate specificity. How these two processes are mechanistically coupled remains unclear. Here we identify a single conserved residue in FlhA, Arg-391, as a key molecular node that links export gate activation to substrate specificity switching. The R391A substitution blocks the transition from hook-type to filament-type secretion, resulting in polyhooks lacking filaments. An intermolecular salt-bridge formation of Arg-391 with Glu-351 of an adjacent FlhA subunit stabilizes the filament-type FlhA ring. Suppressor mutations in FlhA and the ruler protein FliK restore this interaction, indicating that FliK promotes the intermolecular salt-bridge formation during export switching. Moreover, Arg-391 is required for efficient ATPase-independent activation of the export gate. Together, these findings reveal an electrostatic mechanism by which FlhA coordinates export gate activation and substrate specificity switching during flagellar assembly.

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Kinoshita, M., Minamino, T., Sakai, M., Takekawa, N., Uchihashi, T., Kitao, A., Imada, K., Namba, K.. 2026-05-21. A single conserved residue in FlhA couples export gate activation to substrate specificity switching. https://doi.org/10.64898/2026.05.20.726703

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