Assembly and stoichiometry of the core structure of the bacterial flagellar type III export gate complex

<div><p>The bacterial flagellar type III export apparatus, which is required for flagellar assembly beyond the cell membranes, consists of a transmembrane export gate complex and a cytoplasmic ATPase complex. FlhA, FlhB, FliP, FliQ, and FliR form the gate complex inside the basal body MS ring, although FliO is required for efficient export gate formation in <i>Salmonella enterica</i>. However, it remains unknown how they form the gate complex. Here we report that FliP forms a homohexameric ring with a diameter of 10 nm. Alanine substitutions of conserved Phe-137, Phe-150, and Glu-178 residues in the periplasmic domain of FliP (FliP_P) inhibited FliP₆ ring formation, suppressing flagellar protein export. FliO formed a 5-nm ring structure with 3 clamp-like structures that bind to the FliP₆ ring. The crystal structure of FliP_P derived from <i>Thermotoga maritia</i>, and structure-based photo-crosslinking experiments revealed that Phe-150 and Ser-156 of FliP_P are involved in the FliP–FliP interactions and that Phe-150, Arg-152, Ser-156, and Pro-158 are responsible for the FliP–FliO interactions. Overexpression of FliP restored motility of a ∆<i>fliO</i> mutant to the wild-type level, suggesting that the FliP₆ ring is a functional unit in the export gate complex and that FliO is not part of the final gate structure. Copurification assays revealed that FlhA, FlhB, FliQ, and FliR are associated with the FliO/FliP complex. We propose that the assembly of the export gate complex begins with FliP₆ ring formation with the help of the FliO scaffold, followed by FliQ, FliR, and FlhB and finally FlhA during MS ring formation.</p></div

Effect of lauryl maltose neopentyl glycol (LMNG) on the interactions of the FliP₆ ring with other export gate proteins.

<p>(A) Elution profiles of the FlhA/FlhB/FliO/FliP/FliQ/FliR complex from a Superdex 200 10/300 column equilibrated with 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 5% glycerol, and 0.01% LMNG. Membrane fractions were prepared from SJW1368 expressing FlhA, FlhB, FliO, His-FliP, HA-FliQ, and FliR-FLAG and were solubilized by 1% LMNG. Then, the protein complex was purified by Ni affinity chromatography, followed by size exclusion chromatography (SEC). (B) Immunoblotting of elution fractions from A, using anti-FliO (first row), anti-His (second row), anti-HA (third row), anti-FLAG (fourth row), anti-FlhB_C (fifth row), or anti-FlhA_C (sixth row) antibody. (Note: The C-terminal cytoplasmic domain of FlhB undergoes autocleavage between conserved Asp-269 and Pro270 residues [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002281#pbio.2002281.ref002" target="_blank">2</a>–<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002281#pbio.2002281.ref004" target="_blank">4</a>], and hence, the molecular size of the FlhB band recognized by polyclonal anti-FlhB_C antibody is smaller than that of full-length FlhB). These proteins treated with LMNG showed slightly distinct running behavior on SDS gels compared to those with n-dodecyl β-D-maltoside (DDM), presumably due to the detergent effect. The lane marked L represents the material loaded onto the SEC column.</p

Enlarged views of representative 2D class averages of the FliP₆ ring, the FliO complex, and the FliO/FliP complex.

<p>Reference-free 2D class average images were calculated by e2refine2d.py. All scale bars show 50 Å. The number of particles for each class is indicated in the top-right corner. FliP forms a ring structure with a diameter of about 10 nm (first row). The FliP ring has the 6-fold rotational symmetry as judged by autocorrelation analysis (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002281#pbio.2002281.s003" target="_blank">S3A</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002281#pbio.2002281.s003" target="_blank">S3B and S3C Fig</a>). FliO forms a ring structure of 5 nm in diameter with 3 flexible clamp-like structures (second row) that bind to the FliP ring with their ring axes perpendicular to the axis of the FliO ring (third row). Thus, the FliP rings in the first row are end-on views, and those in the third row are side views.</p

In vivo photo-crosslinking.

<p><i>E</i>. <i>coli</i> BL21 (DE3) cells coexpressing (A) FliP-FLAG with an amber mutation at indicated positions with FliO, FliQ, and FliR, (B) FliP-FLAG with an amber mutation with FliO, or (C) FliP-HA with an amber mutation with FliO-FLAG were grown in the presence of <i>p</i>-benzoyl-phenylalanine (pBPA) and then treated with (+) or without (−) UV irradiation. Wild-type FliP-FLAG (WT) was used as a negative control. Crude membrane fractions were prepared, followed by SDS-PAGE and finally immunoblotting with monoclonal anti-FLAG antibody. Red and blue dots indicate FliP-FliP and FliP-FliO photo-crosslinked products, respectively. Each cropped blot is shown within a box. (D) The residues selected for the photo-crosslinking experiment are mapped on the A–B dimer model of <i>St-</i>FliP_P. The residues that formed crosslinking products by the substitution with pBPA are shown in ball-and-stick with black labels, and those that did not are in stick with gray labels. Black arrowheads indicate possible interaction sites of <i>St-</i>FliP_P with FliO.</p

Model for the assembly process of the flagellar type III export apparatus.

<p>The export apparatus is composed of a transmembrane export gate complex made of FlhA, FlhB, FliP, FliQ, and FliR and a cytoplasmic ATPase ring complex consisting of FliH, FliI, and FliJ. The FliP dimers form a homohexamer with the help of the FliO complex, followed by the assembly of FliQ, FliR, and FlhB and finally of FlhA during MS ring formation in the cytoplasmic membrane. Then, the FliM/FliN complex binds to FliG to form the C ring on the cytoplasmic face of the MS ring. Finally, the FliH/FliI/FliJ ATPase ring complex is formed at the flagellar base through interactions of FliH with FlhA and FliN [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002281#pbio.2002281.ref002" target="_blank">2</a>–<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002281#pbio.2002281.ref004" target="_blank">4</a>], allowing export substrates to go into the central cavity of the FliP₆ ring complex.</p

Interactions of the FliP₆ ring with other export gate proteins.

<p>(A) SDS-PAGE of pooled fractions after size exclusion chromatography (SEC) with a Superdex 200 10/300 column. Membrane fractions were prepared from SJW1368 expressing FliO and His-FliP (lane 1); FliO, His-FliP, HA-FliQ, and FliR-FLAG (lane 2); FlhA, FlhB, FliO, His-FliP, HA-FliQ, and FliR-FLAG (lane 3); or FlhA, FlhB, FliF, FliG-His, FliO, FliP, HA-FliQ, and FliR-FLAG (lane 4) and solubilized by 1% n-dodecyl β-D-maltoside (DDM), followed by Ni affinity chromatography. For purification of the FliO/His-FliP/FliR-FLAG and FliO/His-FliP/FliR-FLAG/FlhB complexes, pooled fractions were subjected to FLAG affinity chromatography (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002281#pbio.2002281.s009" target="_blank">S9 Fig</a>), followed by SEC with a Superdex 200 10/300 column (lanes 2 and 3). For purification of the FlhA/FliF/FliG-His complex, pooled fractions were subjected to SEC (lane 4). (B) Representative negatively stained electron microscopy (EM) images of purified FlhA/FliF/FliG-His complexes. Scale bar shows 100 nm.</p

Structure of FliP_P.

<p>(A) Ribbon diagram of the <i>Tm</i>-FliP_P tetramer in the crystal (Protein Data Bank [PDB] ID: 5H72). Two different views are shown. Mol A, Mol B, Mol C, and Mol D are colored in cyan, yellow green, magenta, and green, respectively. Each subunit of the <i>Tm-</i>FliP_P tetramer is related by D2 symmetry. (B) Cα ribbon drawing of the <i>Tm</i>-FliP_P monomer. The secondary structure elements are labeled with α for α-helix. (C) Structure-based sequence alignment of <i>Salmonella</i> FliP_P (<i>St</i>FliP_P) and <i>Tm-</i>FliP_P. The secondary structure of <i>Tm-</i>FliP_P is shown below the sequence. Identical residues are highlighted in red. Uniprot accession numbers: <i>Salmonella</i> (P54700) and <i>Thermotoga</i> (Q9WZG2). (D) Homology model of the A–B dimer of <i>St-</i>FliP_P. (E) Homology model of the A–C dimer of <i>St-</i>FliP_P. (F) The model of the A–B dimer connected to the TM-3 helices. Both C-termini of the A–B dimer can be directly connected to the TM-3 helices. CM, cytoplasmic membrane. Residues selected for mutational analyses are mapped and labeled in (D), (E), and (F). The residues whose substitution affected the FliP function are shown in ball-and-stick with black labels, and those that did not are in stick with gray labels.</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

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

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