Extended Data Figure 6: In vitro and in vivo head-to-tail linkage of flagellin subunits (in support of Fig. 2).

a, Isolation of flagellin linked (dimer) in vitro by site-specific cysteine–cysteine crosslinks. Flagellin (FliC) and its variants containing unique cysteines (S₁₁C, L₁₃C, L₁₈C, R₃₁C or K₁₇₈C; Extended Data Table 1) within and adjacent to the N-terminal helix predicted to generate a coiled-coil were incubated, with (+) or without (−) BMOE cross-linker (x-link), with a FliC variant containing a unique cysteine (N₄₈₉C) within the C-terminal helix (upper panel). Flagellin derivatives were engineered to lack either their C or N termini (FliC-Nt, FliC-Ct) to preclude self-interaction. Control crosslinking (lower panel) assay showing that FliC-Ct without engineered cysteines cannot form crosslinked complexes. b, Trapping of flagellin subunits linked head-to-tail in chains within flagella growing on S. typhimurium cells using in vivo site-specific cysteine–cysteine crosslinking. Cells expressing recombinant, export-competent full-length flagellin containing engineered cysteines (lanes from left to right: negative control L₁₈C; L₁₈C and Q₄₈₈C predicted to trap chain; negative control Q₄₈₈C alone; negative control wild type; negative control vector) were incubated with (+) or without (−) BMOE cross-linker (x-link). Flagellar filaments were then isolated, depolymerized and resolved (immunoblot, panel exposure times decrease from top to bottom) to reveal monomer (×1), dimer (×2) and higher order head-to-tail chains of flagellin. c, Depiction of the in vivo trapping of flagellin subunit chain in the transit channel of growing flagellar filaments. Panels show a series of magnifications of a flagellated S. typhimurium cell, flagellar filament containing flagellin subunit chain linked head-to-tail by N- and C-terminal helices (blue and red cylinders, respectively) and the flagellar transit channel containing the subunit–subunit link and figurative positions of engineered cysteine residues (yellow segment of cylinder). Engineered cysteine residues in C-terminal helices of assembled flagellin subunits that line the channel could form crosslinked dimers with subunits in transit. All experiments were carried out at least three times and were biological replicates.