Role of the cytoplasmic C terminus of the FliF motor protein in flagellar assembly and rotation

Twenty-six FliF monomers assemble into the MS ring, a central motor component of the bacterial flagellum that anchors the structure in the inner membrane. Approximately 100 amino acids at the C terminus of FliF are exposed to the cytoplasm and, through the interaction with the FliG switch protein, a component of the flagellar C ring, are essential for the assembly of the motor. In this study, we have dissected the entire cytoplasmic C terminus of the Caulobacter crescentus FliF protein by high-resolution mutational analysis and studied the mutant forms with regard to the assembly, checkpoint control, and function of the flagellum. Only nine amino acids at the very C terminus of FliF are essential for flagellar assembly. Deletion or substitution of about 10 amino acids preceding the very C terminus of FliF resulted in assembly-competent but nonfunctional flagella, making these the first fliF mutations described so far with a Fla(+) but Mot(-) phenotype. Removal of about 20 amino acids further upstream resulted in functional flagella, but cells carrying these mutations were not able to spread efficiently on semisolid agar plates. At least 61 amino acids located between the functionally relevant C terminus and the second membrane-spanning domain of FliF were not required for flagellar assembly and performance. A strict correlation was found between the ability of FliF mutant versions to assemble into a flagellum, flagellar class III gene expression, and a block in cell division. Motile suppressors could be isolated for nonmotile mutants but not for mutants lacking a flagellum. Several of these suppressor mutations were localized to the 5' region of the fliG gene. These results provide genetic support for a model in which only a short stretch of amino acids at the immediate C terminus of FliF is required for flagellar assembly through stable interaction with the FliG switch protein.     

Structures of bacterial flagellar motors from two FliF-FliG gene fusion mutants

Flagella purified from Salmonella enterica serovar Typhimurium contain FliG, FliM, and FliN, cytoplasmic proteins that are important in torque generation and switching, and FliF, a transmembrane structural protein. The motor portion of the flagellum (the basal body complex) has a cytoplasmic C ring and a transmembrane M ring. Incubation of purified basal bodies at pH 4.5 removed FliM and FliN but not FliG or FliF. These basal bodies lacked C rings but had intact M rings, suggesting that FliM and FliN are part of the C ring but not a detectable part of the M ring. Incubation of basal bodies at pH 2.5 removed FliG, FliM, and FliN but not FliF. These basal bodies lacked the C ring, and the cytoplasmic face of the M ring was altered, suggesting that FliG makes up at least part of the cytoplasmic face of the M ring. Further insights into FliG were obtained from cells expressing a fusion protein of FliF and FliG. Flagella from these mutants still rotated but cells were not chemotactic. One mutant is a full-length fusion of FliF and FliG; the second mutant has a deletion lacking the last 56 residues of FliF and the first 94 residues of FliG. In the former, C rings appeared complete, but a portion of the M ring was shifted to higher radius. The C-ring-M-ring interaction appeared to be altered. In basal bodies with the fusion-deletion protein, the C ring was smaller in diameter, and one of its domains occupied space vacated by missing portions of FliF and FliG.     

Interaction of the C-Terminal Tail of FliF with FliG from the Na+-Driven Flagellar Motor of Vibrio alginolyticus

Rotation of the polar flagellum of Vibrio alginolyticus is driven by a Na⁺-type flagellar motor. FliG, one of the essential rotor proteins located at the upper rim of the C ring, binds to the membrane-embedded MS ring. The MS ring is composed of a single membrane protein, FliF, and serves as a foundation for flagellar assembly. Unexpectedly, about half of the Vibrio FliF protein produced at high levels in Escherichia coli was found in the soluble fraction. Soluble FliF purifies as an oligomer of ∼700 kDa, as judged by analytical size exclusion chromatography. By using fluorescence correlation spectroscopy, an interaction between a soluble FliF multimer and FliG was detected. This binding was weakened by a series of deletions at the C-terminal end of FliF and was nearly eliminated by a 24-residue deletion or a point mutation at a highly conserved tryptophan residue (W575). Mutations in FliF that caused a defect in FliF-FliG binding abolish flagellation and therefore confer a nonmotile phenotype. As data from in vitro binding assays using the soluble FliF multimer correlate with data from in vivo functional analyses, we conclude that the C-terminal region of the soluble form of FliF retains the ability to bind FliG. Our study confirms that the C-terminal tail of FliF provides the binding site for FliG and is thus required for flagellation in Vibrio, as reported for other species. This is the first report of detection of the FliF-FliG interaction in the Na⁺-driven flagellar motor, both in vivo and in vitro.     

Structural insight into the rotational switching mechanism of the bacterial flagellar motor

The bacterial flagellar motor can rotate either clockwise (CW) or counterclockwise (CCW). Three flagellar proteins, FliG, FliM, and FliN, are required for rapid switching between the CW and CCW directions. Switching is achieved by a conformational change in FliG induced by the binding of a chemotaxis signaling protein, phospho-CheY, to FliM and FliN. FliG consists of three domains, FliG(N), FliG(M), and FliG(C), and forms a ring on the cytoplasmic face of the MS ring of the flagellar basal body. Crystal structures have been reported for the FliG(MC) domains of Thermotoga maritima, which consist of the FliG(M) and FliG(C) domains and a helix E that connects these two domains, and full-length FliG of Aquifex aeolicus. However, the basis for the switching mechanism is based only on previously obtained genetic data and is hence rather indirect. We characterized a CW-biased mutant (fliG(ΔPAA)) of Salmonella enterica by direct observation of rotation of a single motor at high temporal and spatial resolution. We also determined the crystal structure of the FliG(MC) domains of an equivalent deletion mutant variant of T. maritima (fliG(ΔPEV)). The FliG(ΔPAA) motor produced torque at wild-type levels under a wide range of external load conditions. The wild-type motors rotated exclusively in the CCW direction under our experimental conditions, whereas the mutant motors rotated only in the CW direction. This result suggests that wild-type FliG is more stable in the CCW state than in the CW state, whereas FliG(ΔPAA) is more stable in the CW state than in the CCW state. The structure of the TM-FliG(MC)(ΔPEV) revealed that extremely CW-biased rotation was caused by a conformational change in helix E. Although the arrangement of FliG(C) relative to FliG(M) in a single molecule was different among the three crystals, a conserved FliG(M)-FliG(C) unit was observed in all three of them. We suggest that the conserved FliG(M)-FliG(C) unit is the basic functional element in the rotor ring and that the PAA deletion induces a conformational change in a hinge-loop between FliG(M) and helix E to achieve the CW state of the FliG ring. We also propose a novel model for the arrangement of FliG subunits within the motor. The model is in agreement with the previous mutational and cross-linking experiments and explains the cooperative switching mechanism of the flagellar motor.     

Assembly of the switch complex onto the MS ring complex of Salmonella typhimurium does not require any other flagellar proteins.

The cytoplasmic portion of the bacterial flagellum is thought to consist of at least two structural components: a switch complex and an export apparatus. These components seem to assemble around the MS ring complex, which is the first flagellar basal body substructure and is located in the cytoplasmic membrane. In order to elucidate the process of assembly of cytoplasmic substructures, the membrane localization of each component of the switch complex (FliG, FliM, and FliN) in various nonflagellated mutants was examined by immunoblotting. It was found that all these switch proteins require the MS ring protein FliF to associate with the cell membrane. FliG does not require FliM and FliN for this association, but FliM and FliN associate cooperatively with the membrane only through FliG. Furthermore, all three switch proteins were detected in membranes isolated from fliE, fliH, fliI, fliJ, fliO, fliP, fliQ, fliR, flhA, flhB, and flgJ mutants, indicating that the switch complex assembles on the MS ring complex without any other flagellar proteins involved in the early stage of flagellar assembly. The relationship between the switch complex and the export apparatus is discussed.     

Structure of the rotor of the bacterial flagellar motor revealed by electron cryomicroscopy and single-particle image analysis

The FliF ring is the base for self-assembly of the bacterial flagellum and the FliF/FliG ring complex is the core of the rotor of the flagellar motor. We report the structures of these two ring complexes obtained by electron cryomicroscopy and single-particle image analysis at 22A and 25A resolution, respectively. Direct comparison of these structures with the flagellar basal body made by superimposing the density maps on the central section reveals many interesting features, such as how the mechanically stable connection between the ring and the rod is formed, how directly FliF domains are involved in the near axial density of the basal body forming the proximal end of the central channel for a potential gating mechanism, some indication of flexibility in the connection of FliF and FliG, and structural and functional similarities to the head-to-tail connectors of bacteriophages.     

Assembly and stoichiometry of FliF and FlhA in Salmonella flagellar basal body

The bacterial flagellar export apparatus is required for the construction of the bacterial flagella beyond the cytoplasmic membrane. The membrane‐embedded part of the export apparatus, which consists of FlhA, FlhB, FliO, FliP, FliQ and FliR, is located in the central pore of the MS ring formed by 26 copies of FliF. The C‐terminal cytoplasmic domain of FlhA is located in the centre of the cavity within the C ring made of FliG, FliM and FliN. FlhA interacts with FliF, but its assembly mechanism remains unclear. Here, we fused yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) to the C‐termini of FliF and FlhA and investigated their subcellular localization by fluorescence microscopy. The punctate pattern of FliF–YFP localization required FliG but neither FliM, FliN, FlhA, FlhB, FliO, FliP, FliQ nor FliR. In contrast, FlhA–CFP localization required FliF, FliG, FliO, FliP, FliQ and FliR. The number of FlhA–YFP molecules associated with the MS ring was estimated to be about nine. We suggest that FlhA assembles into the export gate along with other membrane components during the MS ring complex formation in a co‐ordinated manner.     

Overproduction of the bacterial flagellar switch proteins and their interactions with the MS ring complex in vitro.

The flagellar switch proteins (FliG, FliM, and FliN) of Salmonella typhimurium were overproduced in Escherichia coli and partially purified in soluble form. They were mixed with purified MS ring complexes (which consist of subunits of FliF protein) to examine their interactions in vitro. The degree of interaction was estimated by ultracentrifugation, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From the band density on the gel, we estimated that FliG bound to the MS ring complex at an approximately 1:1 molar ratio (FliG:FliF), whereas FliM did so only at a 1:5 molar ratio (FliM:FliF). FliN did not bind to the MS ring complex by itself or in the presence of the other switch proteins. A possible configuration of the switch proteins is discussed.     

The three-dimensional structure of the flagellar rotor from a clockwise-locked mutant of Salmonella enterica serovar Typhimurium

Three-dimensional reconstructions from electron cryomicrographs of the rotor of the flagellar motor reveal that the symmetry of individual M rings varies from 24-fold to 26-fold while that of the C rings, containing the two motor/switch proteins FliM and FliN, varies from 32-fold to 36-fold, with no apparent correlation between the symmetries of the two rings. Results from other studies provided evidence that, in addition to the transmembrane protein FliF, at least some part of the third motor/switch protein, FliG, contributes to a thickening on the face of the M ring, but there was no evidence as to whether or not any portion of FliG also contributes to the C ring. Of the four morphological features in the cross section of the C ring, the feature closest to the M ring is not present with the rotational symmetry of the rest of the C ring, but instead it has the symmetry of the M ring. We suggest that this inner feature arises from a domain of FliG. We present a hypothetical docking in which the C-terminal motor domain of FliG lies in the C ring, where it can interact intimately with FliM.     

Mutational analysis of the flagellar protein FliG: sites of interaction with FliM and implications for organization of the switch complex

The switch complex at the base of the bacterial flagellum is essential for flagellar assembly, rotation, and switching. In Escherichia coli and Salmonella, the complex contains about 26 copies of FliG, 34 copies of FliM, and more then 100 copies of FliN, together forming the basal body C ring. FliG is involved most directly in motor rotation and is located in the upper (membrane-proximal) part of the C ring. A crystal structure of the middle and C-terminal parts of FliG shows two globular domains connected by an alpha-helix and a short extended segment. The middle domain of FliG has a conserved surface patch formed by the residues EHPQ(125-128) and R(160) (the EHPQR motif), and the C-terminal domain has a conserved surface hydrophobic patch. To examine the functional importance of these and other surface features of FliG, we made mutations in residues distributed over the protein surface and measured the effects on flagellar assembly and function. Mutations preventing flagellar assembly occurred mainly in the vicinity of the EHPQR motif and the hydrophobic patch. Mutations causing aberrant clockwise or counterclockwise motor bias occurred in these same regions and in the waist between the upper and lower parts of the C-terminal domain. Pull-down assays with glutathione S-transferase-FliM showed that FliG interacts with FliM through both the EHPQR motif and the hydrophobic patch. We propose a model for the organization of FliG and FliM subunits that accounts for the FliG-FliM interactions identified here and for the different copy numbers of FliG and FliM in the flagellum.     

FliG subunit arrangement in the flagellar rotor probed by targeted cross-linking

FliG is a component of the switch complex on the rotor of the bacterial flagellum. Each flagellar motor contains about 25 FliG molecules. The protein of Escherichia coli has 331 amino acid residues and comprises at least two discrete domains. A C-terminal domain of about 100 residues functions in rotation and includes charged residues that interact with the stator protein MotA. Other parts of the FliG protein are essential for flagellar assembly and interact with the MS ring protein FliF and the switch complex protein FliM. The crystal structure of the middle and C-terminal parts of FliG shows two globular domains joined by an alpha-helix and a short extended segment that contains two well-conserved glycine residues. Here, we describe targeted cross-linking studies of FliG that reveal features of its organization in the flagellum. Cys residues were introduced at various positions, singly or in pairs, and cross-linking by a maleimide or disulfide-inducing oxidant was examined. FliG molecules with pairs of Cys residues at certain positions in the middle domain formed disulfide-linked dimers and larger multimers with a high yield, showing that the middle domains of adjacent subunits are in fairly close proximity and putting constraints on the relative orientation of the domains. Certain proteins with single Cys replacements in the C-terminal domain formed dimers with moderate yields but not larger multimers. On the basis of the cross-linking results and the data available from mutational and electron microscopic studies, we propose a model for the organization of FliG subunits in the flagellum.     

Deletion analysis of the flagellar switch protein FliG of Salmonella

The flagellar motor/switch complex, consisting of the three proteins FliG, FliM, and FliN, plays a central role in bacterial motility and chemotaxis. We have analyzed FliG, using 10-amino-acid deletions throughout the protein and testing the deletion clones for their motility and dominance properties and for interaction of the deletion proteins with the MS ring protein FliF. Only the N-terminal 46 amino acids of FliG (segments 1 to 4) were important for binding to FliF; consistent with this, an N-terminal fragment consisting of residues 1 to 108 bound FliF strongly, whereas a C-terminal fragment consisting of residues 109 to 331 did not bind FliF at all. Deletions in the region from residues 37 to 96 (segments 4 to 9), 297 to 306 (segment 30), and 317 to 326 (segment 32) permitted swarming, though not at wild-type levels; all other deletions caused paralyzed or, more commonly, nonflagellate phenotype. Except for those near the N terminus, deletions had a dominant negative effect on wild-type cells.     

Cell cycle-controlled proteolysis of a flagellar motor protein that is asymmetrically distributed in the Caulobacter predivisional cell.

Flagellar biogenesis and release are developmental events tightly coupled to the cell cycle of Caulobacter crescentus. A single flagellum is assembled at the swarmer pole of the predivisional cell and is released later in the cell cycle. Here we show that the MS-ring monomer FliF, a central motor component that anchors the flagellum in the cell membrane, is synthesized only in the predivisional cell and is integrated into the membrane at the incipient swarmer cell pole, where it initiates flagellar assembly. FliF is proteolytically turned over during swarmer-to-stalked cell differentiation, coinciding with the loss of the flagellum, suggesting that its degradation is coupled to flagellar release. The membrane topology of FliF was determined and a region of the cytoplasmic C-terminal domain was shown to be required for the interaction with a component of the motor switch. The very C-terminal end of FliF contains a turnover determinant, required for the cell cycle-dependent degradation of the MS-ring. The cell cycle-dependent proteolysis of FliF and the targeting of FliF to the swarmer pole together contribute to the asymmetric localization of the MS-ring in the predivisional cell.     

Rusty, jammed, and well-oiled hinges: Mutations affecting the interdomain region of FliG, a rotor element of the Escherichia coli flagellar motor

The FliG protein is a central component of the bacterial flagellar motor. It is one of the first proteins added during assembly of the flagellar basal body, and there are 26 copies per motor. FliG interacts directly with the Mot protein complex of the stator to generate torque, and it is a crucial player in switching the direction of flagellar rotation from clockwise (CW) to counterclockwise and vice versa. A primarily helical linker joins the N-terminal assembly domain of FliG, which is firmly attached to the FliF protein of the MS ring of the basal body, to the motility domain that interacts with MotA/MotB. We report here the results of a mutagenic analysis focused on what has been called the hinge region of the linker. Residue substitutions in this region generate a diversity of phenotypes, including motors that are strongly CW biased, infrequent switchers, rapid switchers, and transiently or permanently paused. Isolation of these mutants was facilitated by a "sensitizing" mutation (E232G) outside of the hinge region that was accidentally introduced during cloning of the chromosomal fliG gene into our vector plasmid. This mutation partially interferes with flagellar assembly and accentuates the defects associated with mutations that by themselves have little phenotypic consequence. The effects of these mutations are analyzed in the context of a conformational-coupling model for motor switching and with respect to the structure of the C-terminal 70% of FliG from Thermotoga maritima.     

Crystal structure of the flagellar rotor protein FliN from Thermotoga maritima

FliN is a component of the bacterial flagellum that is present at levels of more than 100 copies and forms the bulk of the C ring, a drum-shaped structure at the inner end of the basal body. FliN interacts with FliG and FliM to form the rotor-mounted switch complex that controls clockwise-counterclockwise switching of the motor. In addition to its functions in motor rotation and switching, FliN is thought to have a role in the export of proteins that form the exterior structures of the flagellum (the rod, hook, and filament). Here, we describe the crystal structure of most of the FliN protein of Thermotoga maritima. FliN is a tightly intertwined dimer composed mostly of beta sheet. Several well-conserved hydrophobic residues form a nonpolar patch on the surface of the molecule. A mutation in the hydrophobic patch affected both flagellar assembly and switching, showing that this surface feature is important for FliN function. The association state of FliN in solution was studied by analytical ultracentrifugation, which provided clues to the higher-level organization of the protein. T. maritima FliN is primarily a dimer in solution, and T. maritima FliN and FliM together form a stable FliM(1)-FliN(4) complex. Escherichia coli FliN forms a stable tetramer in solution. The arrangement of FliN subunits in the tetramer was modeled by reference to the crystal structure of tetrameric HrcQB(C), a related protein that functions in virulence factor secretion in Pseudomonas syringae. The modeled tetramer is elongated, with approximate dimensions of 110 by 40 by 35 Angstroms, and it has a large hydrophobic cleft formed from the hydrophobic patches on the dimers. On the basis of the present data and available electron microscopic images, we propose a model for the organization of FliN subunits in the C ring.     

Identification of the protease and the turnover signal responsible for cell cycle-dependent degradation of the Caulobacter FliF motor protein

Flagellar ejection is tightly coupled to the cell cycle in Caulobacter crescentus. The MS ring protein FliF, which anchors the flagellar structure in the inner membrane, is degraded coincident with flagellar release. Previous work showed that removal of 26 amino acids from the C terminus of FliF prevents degradation of the protein and interferes with flagellar assembly. To understand FliF degradation in more detail, we identified the protease responsible for FliF degradation and performed a high-resolution mutational analysis of the C-terminal degradation signal of FliF. Cell cycle-dependent turnover of FliF requires an intact clpA gene, suggesting that the ClpAP protease is required for removal of the MS ring protein. Deletion analysis of the entire C-terminal cytoplasmic portion of FliF C confirmed that the degradation signal was contained in the last 26 amino acids that were identified previously. However, only deletions longer than 20 amino acids led to a stable FliF protein, while shorter deletions dispersed over the entire 26 amino acids critical for turnover had little effect on stability. This indicated that the nature of the degradation signal is not based on a distinct primary amino acid sequence. The addition of charged amino acids to the C-terminal end abolished cell cycle-dependent FliF degradation, implying that a hydrophobic tail feature is important for the degradation of FliF. Consistent with this, ClpA-dependent degradation was restored when a short stretch of hydrophobic amino acids was added to the C terminus of stable FliF mutant forms.     

Organization of FliN subunits in the flagellar motor of Escherichia coli

FliN is a major constituent of the C ring in the flagellar basal body of many bacteria. It is present in >100 copies per flagellum and together with FliM and FliG forms the switch complex that functions in flagellar assembly, rotation, and clockwise-counterclockwise switching. FliN is essential for flagellar assembly and switching, but its precise functions are unknown. The C-terminal part of the protein is best conserved and most important for function; a crystal structure of this C-terminal domain of FliN from Thermotoga maritima revealed a saddle-shaped dimer formed mainly from beta strands (P. N. Brown, M. A. A. Mathews, L. A. Joss, C. P. Hill, and D. F. Blair, J. Bacteriol. 187:2890-2902, 2005). Equilibrium sedimentation studies showed that FliN can form stable tetramers and that a FliM1FliN4 complex is also stable. Here, we have examined the organization of FliN subunits by using targeted cross-linking. Cys residues were introduced at various positions in FliN, singly or in pairs, and disulfide cross-linking was induced by oxidation. Efficient cross-linking was observed for certain positions near the ends of the dimer and for some positions in the structurally uncharacterized N-terminal domain. Certain combinations of two Cys replacements gave a high yield of cross-linked tetramer. The results support a model in which FliN is organized in doughnut-shaped tetramers, stabilized in part by contacts involving the N-terminal domain. Electron microscopic reconstructions show a bulge at the bottom of the C-ring whose size and shape are a close match for the hypothesized FliN tetramer.     

Crystal structure of the middle and C-terminal domains of the flagellar rotor protein FliG

The FliG protein is essential for assembly, rotation and clockwise/counter-clockwise (CW/CCW) switching of the bacterial flagellum. About 25 copies of FliG are present in a large rotor-mounted assembly termed the 'switch complex', which also contains the proteins FliM and FliN. Mutational studies have identified the segments of FliG most crucial for flagellar assembly, rotation and switching. The structure of the C-terminal domain, which functions specifically in rotation, was reported previously. Here, we describe the crystal structure of a larger fragment of the FliG protein from Thermotoga maritima, which encompasses the middle and C-terminal parts of the protein (termed FliG-MC). The FliG-MC molecule consists of two compact globular domains, linked by an alpha-helix and an extended segment that contains a well-conserved Gly-Gly motif. Mutational studies indicate that FliM binds to both of the globular domains, and given the flexibility of the linking segment, FliM is likely to determine the relative orientation of the domains in the flagellum. We propose a model for the organization of FliG-MC molecules in the flagellum, and suggest that CW/CCW switching might occur by movement of the C-terminal domain relative to other parts of FliG, under the control of FliM.     

Structure of Flagellar Motor Proteins in Complex Allows for Insights into Motor Structure and Switching

The flagellar motor is one type of propulsion device of motile bacteria. The cytoplasmic ring (C-ring) of the motor interacts with the stator to generate torque in clockwise and counterclockwise directions. The C-ring is composed of three proteins, FliM, FliN, and FliG. Together they form the “switch complex” and regulate switching and torque generation. Here we report the crystal structure of the middle domain of FliM in complex with the middle and C-terminal domains of FliG that shows the interaction surface and orientations of the proteins. In the complex, FliG assumes a compact conformation in which the middle and C-terminal domains (FliG_MC) collapse and stack together similarly to the recently published structure of a mutant of FliG_MC with a clockwise rotational bias. This intramolecular stacking of the domains is distinct from the intermolecular stacking seen in other structures of FliG. We fit the complex structure into the three-dimensional reconstructions of the motor and propose that the cytoplasmic ring is assembled from 34 FliG and FliM molecules in a 1:1 fashion.     

C‐ring requirement in flagellar type III secretion is bypassed by FlhDC upregulation

The cytoplasmic C‐ring of the flagellum consists of FliG, FliM and FliN and acts as an affinity cup to localize secretion substrates for protein translocation via the flagellar‐specific type III secretion system. Random T‐POP transposon mutagenesis was employed to screen for insertion mutants that allowed flagellar type III secretion in the absence of the C‐ring using the flagellar type III secretion system‐specific hook–β‐lactamase reporter ( Lee and Hughes, 2006 ). Any condition resulting in at least a twofold increase in flhDC expression was sufficient to overcome the requirement for the C‐ring and the ATPase complex FliHIJ in flagellar type III secretion. Insertions in known and unknown flagellar regulatory loci were isolated as well as chromosomal duplications of the flhDC region. The twofold increased flhDC mRNA level coincided in a twofold increase in the number of hook‐basal bodies per cell as analysed by fluorescent microscopy. These results indicate that the C‐ring functions as a nonessential affinity cup‐like structure during flagellar type III secretion to enhance the specificity and efficiency of the secretion process.