Subcellular localization of FliO revealed by structured illumination microscopy.

<p>The subcellular localization of FliO-HaloTag (A) or FliN-HaloTag (B) fusions expressed from their native chromosomal locus was analyzed in the wild-type (WT) and mutant backgrounds defective in MS-ring assembly (Δ<i>fliF</i>) or flagellar-specific type III secretion system (fT3SS) function (Δ<i>flhBAE</i>). WT FliO-HaloTag (EM1077), WT FliN-HaloTag (EM1081), Δ<i>fliF</i> FliO-HaloTag (EM6254), Δ<i>fliF</i> FliN-HaloTag (EM2640), Δ<i>flhBAE</i> FliO-HaloTag (EM6256), and Δ<i>flhBAE</i> FliN-HaloTag (EM6258). Strains were treated with 20 nM HaloTag ligands (HTL tetramethylrhodamine [TMR]) and observed using structured illumination microscopy (SIM). The autofluorescence of bacteria upon excitation with a 488 nm laser is shown in the middle panels. Scale bar 2 μm.</p

Single-particle tracking of FliO and colocalization with the flagellar basal body.

<p>(A) Strains expressing chromosomal FliN-HaloTag (EM1081) or FliO-HaloTag (EM1077) fusions were treated with 20 nM HaloTag ligands (HTL tetramethylrhodamine [TMR]) and analyzed by total internal reflection fluorescence (TIRF) microscopy. As described before, 500 frames were acquired with 5 mW laser power at the focal plane. The autofluorescence of bacteria upon excitation with a 488 nm laser is shown in the upper left corner. Scale bar 1 μm. (B) Single-molecule tracking (SMT) of TMR-labeled FliN and FliO. Selected frames from a series of 500 frames are shown, and frame numbers are indicated. (C) Mean square displacement (MSD) plots of pooled trajectories of at least 25 bacteria recorded under the same conditions. The diffusion coefficient <i>D</i> was calculated using the Jaqaman algorithm. (D) Dual-color direct stochastic optical reconstruction microscopy (dSTORM) of fixed bacteria expressing chromosomal FlgE-3×HA and FliO-HaloTag fusions (EM1214). Scale bar 1 μm.</p

FliP protein is unstable in the absence of FliO.

<p>(A) FliP protein stability in the presence and absence of FliO. Protein levels of chromosomally expressed FliP-3×FLAG were monitored at 0, 60, 120, and 180 min after arrest of de novo protein synthesis. Wild-type (WT) (EM2225), Δ<i>fliO</i> (EM3201). FliP protein levels were normalized to DnaK, and relative FliP levels report the mean ± SD, <i>n</i> = 6. (B) Stability of episomally expressed FliP-3×HA protein in Δ<i>fliP</i> and Δ<i>fliOP</i> mutants after arrest of de novo protein synthesis. Δ<i>fliP</i> + p<i>fliP</i> (TH17448), Δ<i>fliOP</i> + p<i>fliP</i> (EM1610). Relative FliP levels report the mean ± SD, <i>n</i> = 3. (C) Protein stability of chromosomally expressed FliP-3×HA in presence or absence of FliO in the WT (TH17323), Δ<i>fliO</i> (EM1274), Δ<i>clpXP</i> (EM4018), Δ<i>clpXP</i> Δ<i>fliO</i> (EM4019), Δ<i>lon</i> (EM4478), and Δ<i>lon</i> Δ<i>fliO</i> (EM4479) mutants.</p

Assembly of FliP subassemblies in core export apparatus mutants and model of the coordinated assembly of the flagellar-specific type III secretion system (fT3SS).

<p>Left: The assembly of stable FliP subassemblies is dependent on FliO and FliR but not on FliQ or FliF. Anti-FLAG Western blot of blue native PAGE (BN-PAGE) of crude membrane extracts prepared from the wild-type (WT) harboring untagged FliP (LT2, TH437) and mutant strains encoding for chromosomal FliP-3×FLAG: WT (EM6221), Δ<i>fliO</i> (EM6222), Δ<i>fliQ</i> (EM6223), Δ<i>fliR</i> (EM6224), Δ<i>fliF</i> (EM4859). Strains EM6221, EM6222, EM6223, and EM6224 additionally harbored a deletion of the proximal rod components <i>flgBC</i> in order to arrest flagellar synthesis after assembly of the core export apparatus. Right: Model of the coordinated assembly of the core flagellar export apparatus. Upon initiation of flagellum assembly, the flagellar type III secretion system (T3SS)-specific chaperone FliO facilitates formation of an oligomeric complex containing FliP and FliR. FliO then presumably dissociates from the stable FliP–FliR core complex. The FliP–FliR core complex forms the nucleus for the assembly of FliQ, FlhB, and FlhA [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002267#pbio.2002267.ref011" target="_blank">11</a>], followed by MS-ring (FliF) polymerization and formation of the completed protein export-competent flagellar T3SS.</p

Evidence for FliP–FliR complex formation revealed by 2D blue native PAGE (BN-PAGE).

<p>(A) Anti-FLAG Western blot of a 2D BN-PAGE of crude membrane extracts prepared from rod⁻ (Δ<i>flgBC</i>) strains encoding for chromosomal FliO-3×FLAG and FliP-3×FLAG. The strains additionally harbored a deletion of <i>rflP</i>, a negative regulator of the flagellar master regulator FlhDC and responsible for the phenotypic heterogeneity of flagellar gene expression in lysogeny broth (LB) medium [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002267#pbio.2002267.ref038" target="_blank">38</a>]. The deletion of <i>rflP</i> results in homogeneous flagella production for cultures grown in LB and thus facilitates detection of the chromosomally encoded epitope-tagged transmembrane export apparatus components, which are expressed at low levels. Wild-type (WT) (EM6229) and Δ<i>fliF</i> (EM6195). (B) Anti-FLAG Western blot of a 2D BN-PAGE of crude membrane extracts prepared from Δ<i>flgBC</i> Δ<i>rflP</i> strains encoding for chromosomal FliO-3×FLAG and FliR-3×FLAG. WT (EM6228) and Δ<i>fliF</i> (EM6196).</p