Flagellin polymerisation control by a cytosolic export chaperone

Abstract

Assembly of the long helical filament of the bacterial flagellum requires polymerisation of ca 20,000 flagellin (FliC) monomeric subunits into the growing structure extending from the cell surface. Here, we show that export of Salmonella flagellin is facilitated specifically by a cytosolic protein, FliS, and that FliS binds to the FliC C-terminal helical domain, which contributes to stabilisation of flagellin subunit interactions during polymerisation. Stable complexes of FliS with flagellin were assembled efficiently in vitro, apparently by FliS homodimers binding to FliC monomers. The data suggest that FliS acts as a substrate-specific chaperone, preventing premature interaction of newly synthesised flagellin subunits in the cytosol. Compatible with this view, FliS was able to prevent in vitro polymerisation of FliC into filaments.

Figure 1

Effect of FliS loss on the secretion of flagellar filament subunit FliC. (a) Secreted proteins in culture supernatants of S. typhimurium SJW1103 wild-type (wt), the mutants fliT and fliST, and the fliST mutant transformed with plasmid pBAD-FliS (fliST + S), were precipitated with trichloroacetic acid, separated by SDS-12.5 % PAGE, and stained with Coomassie blue. Molecular mass markers are in kDa. (b) Secreted flagellar proteins in culture supernatants of SJW1103 (wt), the mutants SJW1368 (flhDC) and fliST, and the fliST mutant transformed with pBAD-FliT (fliST + T), were separated by SDS-15 % PAGE, and immunoblotted with anti-FlgK, anti-FliD, anti-FlgL and anti-FlgM antisera. The flhDC mutant, which expresses no flagellar proteins, was used as a negative control.

Figure 2

Cell location and size of FliS. (a) Left panel: Proteins from whole-cell lysates of S. typhimurium SJW1103 (wt) and the fliST mutant were separated by SDS-15 % PAGE and immunoblotted with anti-FliS antiserum. Right pannel: Cells from wt were fractionated and proteins from cytoplasmic (cyt), periplasmic (peri), membrane (mem) and supernatant (snt) fractions were separated by SDS-15 % PAGE and immunoblotted with either anti-FliS antiserum (FliS, upper) or anti-maltose-binding protein antiserum (MBP, lower) as a control for periplasm release. (b) Cytoplasmic cell extract was loaded onto a Superose 12 10/30 HR gel-filtration column. Eluted fractions were separated by SDS-15 % PAGE and immunoblotted with anti-FliS or anti-FliC antisera. Size markers (kDa) eluted as shown.

Figure 3

Affinity blot of FliS to proteins from cells and culture supernatants. Proteins from total cell lysates (cell) and culture supernatants (snt) of S. typhimurium SJW1103 (wt) and the derived fliC mutant SJW2536 (fliC) were separated by SDS-15 % PAGE and either immunoblotted with anti-FliC antiserum (anti-FliC, left panel) or affinity blotted with radiolabelled FliS (³⁵S-FliS, right panel). Molecular mass markers are in kDa.

Figure 4

In vitro complex assembly by FliS and FliC. Gel-filtration chromotography of purified FliS, FliC, and the FliS-FliC complex assembled in vitro. Protein samples were loaded separately onto a Superose 12 HR 10/30 column, and elution fractions were subjected to SDS-15 % PAGE and immunoblotted as indicated. Protein markers (kDa) for gel filtration are as for Figure 2. Upper panel: FliS alone (-C), and after incubation with FliC (+C), immunoblotted with anti-FliS antiserum. Lower panel: FliC alone (-S), and after incubation with FliS (+S), immunoblotted with anti-FliC antiserum. (note: the lower immunoblot in each shows the same fractions).

Figure 5

Affinity blot of FliS to truncated FliC derivatives. (a) Representation of full-length FliC and truncated derivatives, with N and C-terminal α-helical regions indicated as shaded boxes. (b) Proteins from E. coli BL21 (DE3) artificially overexpressing FliC or truncated derivatives (Δ411-495, Δ1-71/Δ411-495 and C186) were separated by SDS-15 % PAGE and either stained with Coomassie blue (left panel) or affinity blotted with ³⁵S-labelled FliS (right panel). Molecular mass markers are in kDa.

Figure 6

Effect of FliS on in vitro FliC polymerisation. In vitro polymerisation reactions containing flagellin seeds (5 μg) and monomeric flagellin (15 μg), in the absence (-) or presence (+) of increasing amounts of FliS (2.5 μg, 5 μg, 10 μg or 20 μg, corresponding to 0.5:1, 1:1, 2:1 or 4:1 FliS to FliC molar ratio, respectively, as indicated at the bottom) were centrifuged at 600,000 g for ten minutes, and the pellets (P) and supernatants (S) analysed by SDS-12.5 % PAGE and Coomassie blue staining. Molecular mass markers (M) are in kDa.

Figure 7

Inhibition of FliC filament polymerisation by FliS. Electron micrographs (6600×) of in vitro polymerisation reactions shown in Figure 6, i.e. reaction 1, FliC seeds; reaction 3, FliC monomers + seeds; reaction 5, FliC monomers + seeds + FliS (1:1 FliS to FliC molar ratio); reaction 6, FliC monomers seeds + FliS (2:1 FliS to FliC molar ratio).