Type III Secretion Systems (T3SS) deliver subunits from the bacterial cytosol to nascent cell surface flagella. Early flagellar subunits that form the rod and hook substructures are unchaperoned and contain their own export signals. A gate recognition motif (GRM) docks them at the FlhBc component of the FlhAB-FliPQR export gate, but the gate must then be opened and subunits must be unfolded to pass through the flagellar channel. This induced us to seek further signals on the subunits. Here, we identify a second signal at the extreme N-terminus of flagellar rod and hook subunits and determine that key to the signal is its hydrophobicity. We show that the two export signal elements are recognised separately and sequentially, as the N-terminal signal is recognised by the flagellar export machinery only after subunits have docked at FlhB_C via the GRM. The position of the N-terminal hydrophobic signal in the subunit sequence relative to the GRM appeared to be important, as a FlgD deletion variant (FlgD_short), in which the distance between the N-terminal signal and the GRM was shortened, ‘stalled’ at the export machinery and was not exported. The attenuation of motility caused by FlgD_short was suppressed by mutations that destabilised the closed conformation of the FlhAB-FliPQR export gate, suggesting that the hydrophobic N-terminal signal might trigger opening of the flagellar export gate.

Bacterial strains, plasmids and growth conditions

Strains containing chromosomally encoded FliP variants were constructed by aph-I-SceI Kanamycin resistance cassette replacement using pWRG730. Recombinant proteins were expressed in Salmonella from the isopropyl β-D-thiogalactoside-inducible (IPTG) inducible plasmid pTrc99a. Bacteria were cultured at 30–37°C in Luria-Bertani (LB) broth containing ampicillin (100 μg/ml).

Flagellar subunit export assay

Salmonella strains were cultured at 37°C in LB broth containing ampicillin and IPTG to mid-log phase (A_600nm 0.6-0.8). Cells were centrifuged (6000 x g, 3 min) and resuspended in fresh media and grown for a further 60 min at 37°C. The cells were pelleted by centrifugation (16,000 x g, 5 min) and the supernatant passed through a 0.2 μm nitrocellulose filter. Proteins were precipitated with 10% trichloroacetic acid (TCA) and 1% Triton X-100 on ice for 1 hour, pelleted by centrifugation (16,000 x g, 10 min), washed with ice-cold acetone and resuspended in SDS-PAGE loading buffer (volumes calibrated according to cell densities). Fractions were analysed by immunoblotting.

Motility assays

For swimming motility, cultures were grown in LB broth to A_600nm 1. Two microliters of culture were inoculated into soft tryptone agar (0.3% agar, 10 g/L tryptone, 5 g/L NaCl) containing ampicillin (100 μg/ml). Plates were incubated at 37°C for between 4 and 6 hours unless otherwise stated.

Isolation of motile strains carrying suppressor mutations

Cells of the Salmonella flgD null strain transformed with plasmids expressing FlgD variants (FlgDΔ2-5, FlgDΔ2-5-¹⁹GSGSMT²⁰-V₁₅A or FlgD_short) were cultured at 37°C in LB broth containing ampicillin (100 μg/ml) to mid-log phase and inoculated into soft tryptone agar (0.3% agar, 10 g/L tryptone, 5g/L NaCl) containing ampicillin (100 μg/ml). Plates were incubated at 30°C until motile ‘spurs’ appeared. Cells from the spurs were streaked to single colony and cultured to isolate the flgD encoding plasmid. Plasmids were transformed into the Salmonella flgD null strain to assess whether the plasmids were responsible for the motile suppressor phenotypes. Plasmids were sequenced to identify the suppressor mutations.

Quantification and statistical analysis

Experiments were performed at least three times. Immunoblots were quantified using Image Studio Lite. The unpaired two-tailed Student’s t-test was used to determine p-values and significance was determined as *p < 0.05. Data are represented as mean ± standard error of the mean (SEM), unless otherwise specified and reported as biological replicates.