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 Salmonella enterica. 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 FliP 6 ring formation, suppressing flagellar protein export. FliO formed a 5-nm ring structure with 3 clamp-like structures that bind to the FliP 6 ring. The crystal structure of FliP P derived from Thermotoga maritia, 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 ∆fliO mutant to the wild-type level, suggesting that the FliP 6 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 FliP 6 ring formation with the help of the FliO scaffold, followed by FliQ, FliR, and FlhB and finally FlhA during MS ring formation.

The bacterial flagellar type III export gate complex is a membrane-embedded nanomachine responsible for flagellar protein export and exits in a patch of membrane within the central pore of the basal body MS ring. In this work, we investigate how formation of the export gate complex is initiated. The export gate complex is composed of 5 highly conserved transmembrane proteins: FlhA, FlhB, FliP, FliQ, and FliR. Each subunit protein assembles into the gate during MS ring formation in a well-coordinated manner. The transmembrane protein FliO is required for efficient assembly of the export gate complex in S. enterica but is not essential for flagellar protein export. Here we carry out biochemical and structural analyses of FliP and provide direct evidence suggesting that FliP forms a trimer-of-dimer structure with a diameter of 10 nm. The assembly of the export gate complex begins with FliP 6 ring formation with the help of the FliO scaffold, followed by FliQ, FliR, and FlhB and finally FlhA during MS ring formation. Given the structural and functional similarities between the flagellar and the virulence-factor-delivering injectisome machineries, we propose that the periplasmic domain of FliP homologues of the injectisome could be a good target for novel antibiotics.

Funding: This research has been supported in part by JSPS KAKENHI Grant Numbers JP26650021 (to TK), JP15H02386 (to KI), JP25000013 (to KN) and JP26293097 (to TM) from the Japan Society for the Promotion of Science ( http://www.jsps.go.jp/english/index.html ) and MEXT KAKENHI Grant Numbers JP23115008 (to K.I.) and JP24117004, JP25121718 and JP15H01640 (to TM) from the Ministry of Education, Culture, Sports, Science and Technology of Japan ( http://www.mext.go.jp/english/index.htm ) and by the Deutsche Forschungsgemeinschaft (DFG) as part of the Collaborative Research Center (SFB) 766 “Bacterial cell envelope”, project B14 (to SW) ( http://www.dfg.de ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: All relevant data are within the paper and its Supporting Information files. The atomic coordinates of the periplasmic domain of FliP derived from Thermotoga maritia have been deposited in Protein Data Bank under the accession code 5H72. Amino acid sequences of FliP derived from Salmonella and Thermotoga are available from the UniProt databse. Uniprot accession numbers are P54700 for Salmonella FliP and Q9WZG2 for Thermotoga FliP.

FliP is a 25-kDa transmembrane protein that has a cleavable N-terminal signal peptide, 4 transmembrane (TM) helices, and a relatively large periplasmic domain (FliP P ) between TM-2 and TM-3 ( S1 Fig ) [ 36 ]. The number of FliP molecules has been estimated to be 4 to 5 per basal body in Salmonella [ 32 ]. FliP P of T. maritia (Tm-FliP P ) forms a homotetramer in solution [ 37 ], raising the possibility that Salmonella FliP (St-FliP) forms an oligomer through interactions between FliP P domains. To study the oligomeric structure of FliP, we purified St-FliP from the membrane fraction by solubilizing it with 1% n-dodecyl β-D-maltoside (DDM) and analyzed it by electron microscopy (EM) and image analysis. We show that FliP forms a homohexameric ring with a diameter of about 10 nm. We also determined the structure of Tm-FliP P at 2.4 Å resolution and carried out structure-based photo-crosslinking experiments. We will discuss the assembly mechanism of the transmembrane export gate complex.

The export gate complex is located inside the basal body MS ring formed by a transmembrane protein, FliF [ 13 , 14 ]. FlhA forms a homononamer [ 8 , 13 ] and acts as an energy transducer along with the cytoplasmic ATPase complex [ 15 – 19 ]. The C-terminal cytoplasmic domains of FlhA and FlhB form a docking platform for the ATPase complex, flagellar type III export chaperones, and export substrates [ 20 – 22 ] and coordinate flagellar protein export with assembly [ 23 – 26 ]. Genetic analyses have suggested possible interactions of the N-terminal transmembrane domain of FlhA (FlhA TM ) with FliF [ 27 ], FliR [ 28 ] and FlhB [ 29 ]. Since a FlhB–FliR fusion protein is partially functional in Salmonella, FlhB presumably associates with FliR in a 1-to-1 fashion [ 30 ]. FliP and FliR are incorporated into the basal body at the earliest stage of MS ring formation [ 31 , 32 ]. The transmembrane export gate complex of the Salmonella SPI-1 T3SS is composed of SpaP (FliP homologue), SpaQ (FliQ homologue), SpaR (FliR homologue), SpaS (FlhB homologue), and InvA (FlhA homologue) in a 5:1:1:1:9 stoichiometry [ 33 ]. Recently, it has been shown that 5 copies of SpaP and 1 copy of SpaR form a donut-shaped structure with a diameter of about 8 nm [ 34 ]. Since the assembly of the export apparatus begins with SpaP, SpaQ, and SpaR, followed by the assembly of SpaS and finally of InvA in the Salmonella SPI-1 T3SS [ 34 , 35 ], the assembly of the flagellar export gate complex is postulated to occur in a way similar to the Salmonella SPI-1 T3SS [ 8 ].

The flagellar type III export apparatus utilizes ATP and proton motive force across the cytoplasmic membrane to drive protein export [ 2 , 3 ]. Recently, it has been shown that ATP hydrolysis by the FliI ATPase and the following rapid protein translocation by the export gate complex are both linked to efficient proton translocation through the gate, suggesting that the export apparatus acts as a proton/protein antiporter to couple the proton flow through the gate with protein export [ 9 ]. Interestingly, the structure of the cytoplasmic ATPase complex looks similar to those of F- and V-type rotary ATPases [ 10 – 12 ].

The bacterial flagellum is supramolecular motility machinery consisting of basal body rings and an axial structure consisting of the rod, the hook, the hook-filament junction, the filament, and the filament cap. Flagellar axial proteins are translocated across the cytoplasmic membrane by a type III protein export apparatus and assemble at the distal end of the growing structure. The export apparatus consists of an export gate complex formed by 5 highly conserved transmembrane proteins (FlhA, FlhB, FliP, FliQ, and FliR) and a cytoplasmic ATPase complex consisting of FliH, FliI, and FliJ [ 1 – 4 ]. These flagellar proteins are evolutionarily related to the components of the type III secretion system (T3SS) of pathogenic bacteria, also known as the injectisome [ 5 ]. The transmembrane protein, FliO, which is not conserved in flagellar and virulence-associated T3SS family, is required for efficient assembly of the export gate complex in S. enterica (hereafter referred to as Salmonella) but is not essential for flagellar protein export [ 6 – 8 ].

Results

Interaction between FliP and FliO Previous genetic analyses of a Salmonella ∆fliO mutant have suggested possible interactions between FliO and FliP [6,7]. To clarify this, we coexpressed FliO with His-FliP and purified them by Ni affinity chromatography and finally by SEC. In agreement with a previous report [6], FliO was expressed as 2 forms: FliO L and FliO S (Fig 1A). Both forms copurified with His-FliP from a SEC column (Fig 1B, second row). EM observation of the FliO/FliP complex revealed that 2 to 3 FliP 6 rings are connected to each other through an interaction between FliP and FliO (Fig 2 and S2B and S4 Figs). Since the inner diameter of the M ring of the flagellar basal body is about 20 nm [3] and is too small to accommodate such multiring complexes of FliO and FliP, it is likely that only 1 FliP 6 ring exists in the final structure of the export gate complex. To test this, we investigated whether overexpression of St-FliP restores motility of a Salmonella ∆fliO mutant. To monitor the expression level of St-FliP, we inserted a HA tag between Gln-22 and Leu-23 of St-FliP (HA-St-FliP). The motility of the ∆fliO mutant overexpressing HA-St-FliP was essentially the same as that of the ∆fliO mutant transformed with a pTrc99A-based plasmid encoding FliO (S5 Fig). This indicates that FliO is not essential for flagellar protein export. We next tested whether FliO itself forms an oligomer. We found that the FliP 6 ring dissociates from FliO during storage of the purified FliO/FliP complexes at 4°C (S6A Fig). Thus, we ran purified FliO/His-FliP complex samples on a Ni– nitrilotriacetic acid (NTA) column to remove His-FliP 6 rings and the FliO/His-FliP complex, followed by SEC to purify FliO (S6B Fig). EM observation and image analysis showed that FliO forms a 5-nm ring structure with 3 flexible clamp-like structures (Fig 2 and S2 and S4 Figs). These observations led us to conclude that the FliO ring complex is not incorporated into the MS ring.

Mutational analysis of St-FliP P The FliP(R143H) mutation, which is located in St-FliP P , can bypass the FliO defect to some extent [6,7], raising the possibility that St-FliP P is required for FliP 6 ring formation. To test this, we selected relatively well-conserved residues of FliP, Pro-115, Glu-125, Phe-137, Phe-150, Leu-170, Phe-172, Ala-173, Ser-177, Glu-178, Leu-179, Ala-182, and Phe-183 (S1B Fig); replaced each residue with alanine, except for Ala-173 and Ala-182, which we replaced with serine; and then analyzed the motility of mutant strains in soft agar (Fig 1C, upper panel). These substitutions did not significantly affect the steady cellular level of FliP as judged by immunoblotting with monoclonal HA-tag antibody (Fig 1C, lower panel). HA-St-FliP fully restored the motility of a ∆fliP mutant. The L170A mutant variant complemented the ∆fliP mutant to the wild-type level. The P115A, E125A, P172A, A173S, S177A, L179A, A182S, and F183A mutant variants restored the motility to a considerable degree, although not to the wild-type level. The F137A and F150A mutant variants complemented the ∆fliP mutant to some degree, and the E178A mutant variant did not at all. In agreement with these results, the F137A and F150A mutations in FliP significantly reduced the secretion levels of the hook-capping protein FlgD, the hook protein FlgE, and the hook-filament junction proteins FlgK and FlgL, and the E178A substitution inhibited the export of these flagellar proteins (Fig 1D). These results indicate that highly conserved Phe-137, Phe-150, and Glu-178 residues of FliP P are critical for the protein export activity. To investigate whether the F137A, F150A, and E178A mutations affect the FliP–FliO interaction, we carried out copurification assays by Ni-NTA affinity chromatography. FliO coeluted with His-FliP(F137A), His-FliP(F150A), and His-FliP(E178A) from a Ni-NTA column (Fig 1A, Output), indicating that they retain the ability to bind to FliO. To test whether these FliP mutations inhibit FliP 6 ring formation, we ran FliO/His-FliP(F137A), FliO/His-FliP(F1507A), and FliO/His-FliP(E178A) complexes on a SEC column and then analyzed the pooled fractions by EM. His-FliP(F137A), His-FliP(F150A), and His-FliP(E178A) dissociated from the FliO complex during SEC and eluted at the same position as peak 4 of wild-type FliP (Fig 1B and S2A Fig), indicating that these mutations reduced the binding affinity of FliP for FliO. The FliO ring structures were seen in their peak 3 fractions, but neither FliP(F137A), FliP(F150A), nor FliP(E178A) formed the homohexamer ring (S2B Fig, Peak 4). These results suggest that Phe-137, Phe-150, and Glu-178 in FliP P contribute to the FliP–FliP interactions in the 6-fold rotational symmetry ring as well as the FliO–FliP interaction.

Crystal structure of Tm-FliP P To clarify the role of FliP P in FliP 6 ring formation, we determined the crystal structure of FliP P . Although no St-FliP P crystal was obtained, the Tm-FliP P crystals were grown [37], and its structure was solved at 2.4 Å resolution. Tm-FliP P formed a homotetramer in the crystal (Mol A, Mol B, Mol C, and Mol D) related by pseudo D2 symmetry (Protein Data Bank [PDB] ID: 5H72) (Fig 3A). There are 2 tetramers in the asymmetric unit, and their structures are essentially identical. The 8 Tm-FliP P molecules in the asymmetric unit show no significant structural difference (root mean square distances for Cα atoms are less than 0.46 Å for the 8 molecules). Tm-FliP P monomer consists of 3 α-helices: α1, α2 and α3 (Fig 3B). The N-terminal 13 residues are invisible in the electron density map presumably because of their conformational flexibility. Therefore, the atomic model of Tm-FliP P contains residues from Thr-122 to Lys-188. Since each subunit of the Tm-FliP P tetramer is related by D2 symmetry, we studied 3 possible intermolecular interactions: between Mol A and Mol B (Mol C and Mol D), between Mol A and Mol C (Mol B and Mol D), and between Mol A and Mol D (Mol B and Mol C) (Fig 3A). The A–B interaction is hydrophobic, and Tyr-124, Phe-128, Met-154, Leu-155, Pro-176, and Leu-180 are involved in this interaction (S7A, S7C, S7D and S7E Fig). The A–C interaction contains both hydrophilic and hydrophobic nature, and Met-127, Arg-130, Val-131, Arg-134, Phe-138, Glu-142, Glu-182, Val-185, Ala-186, and Phe-187 are responsible (S7B, S7F, S7G and S7H Fig). Arg-134 forms a salt bridge with Glu-142 and Glu-182. Ala-186 and Phe-187 make hydrophobic interactions with Met-127, Val-131, and the side chain arm of Arg-130. There is no direct contact between Mol A and Mol D. Since sedimentation equilibrium analytical ultracentrifugation measurements have revealed that Tm-FliP P forms a homotetramer in solution [37], we conclude that the tetramer structure observed in the crystal appears to be equivalent to that in solution. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. Structure of FliP P . (A) Ribbon diagram of the Tm-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 Tm-FliP P tetramer is related by D2 symmetry. (B) Cα ribbon drawing of the Tm-FliP P monomer. The secondary structure elements are labeled with α for α-helix. (C) Structure-based sequence alignment of Salmonella FliP P (StFliP P ) and Tm-FliP P . The secondary structure of Tm-FliP P is shown below the sequence. Identical residues are highlighted in red. Uniprot accession numbers: Salmonella (P54700) and Thermotoga (Q9WZG2). (D) Homology model of the A–B dimer of St-FliP P . (E) Homology model of the A–C dimer of St-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. https://doi.org/10.1371/journal.pbio.2002281.g003