DOI: 10.5061/dryad.66t1g1k3x
title: Recognition of discrete export signals in flagellar subunits during bacterial Type III secretion

description: 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 FlhBC 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
 (FlgDshort), 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 FlgDshort 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.
description: <strong>Bacterial strains, plasmids
 and growth conditions</strong>
 Strains
 containing chromosomally encoded FliP variants were constructed by
 aph-I-SceI Kanamycin resistance cassette replacement using
 pWRG730.
 Recombinant proteins were expressed in <em>Salmonella</em>
 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).
 <strong>Flagellar subunit export
 assay</strong> <em>Salmonella</em> strains were cultured at 37°C in LB broth containing ampicillin and IPTG to mid-log phase (A<sub>600nm</sub> 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. <strong>Motility assays</strong> For swimming motility, cultures were grown in LB broth to A<sub>600nm</sub> 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. <strong>Isolation of motile strains carrying suppressor mutations</strong> Cells of the <em>Salmonella flgD</em> null strain transformed with plasmids expressing FlgD variants (FlgDΔ2-5, FlgDΔ2-5-<sup>19</sup>GSGSMT<sup>20</sup>-V<sub>15</sub>A or FlgD<sub>short</sub>) 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 <em>flgD</em> encoding plasmid. Plasmids were transformed into the <em>Salmonella flgD</em> null strain to assess whether the plasmids were responsible for the motile suppressor phenotypes. Plasmids were sequenced to identify the suppressor mutations. <strong>Quantification and statistical analysis</strong> Experiments were performed at least three times. Immunoblots were quantified using Image Studio Lite. The unpaired two-tailed Student’s <em>t</em>-test was used to determine<em> p</em>-values and significance was determined as *<em>p</em> &lt; 0.05. Data are represented as mean ± standard error of the mean (SEM), unless otherwise specified and reported as biological replicates.