Abstract
The bacterial flagellar type III secretion system (fT3SS) exports structural subunits required for flagellar assembly by coupling protein translocation to ion motive force across the cytoplasmic membrane. Efficient activation of the transmembrane export gate depends on a cytoplasmic ATPase complex composed of FliH, FliI, and FliJ, which are also involved in substrate delivery. However, how these proteins mechanistically integrate substrate delivery with gate activation remains unclear. Here, we uncoupled these two functions by cross-complementation analyses using ATPase components from the Na+-driven polar flagellum of Vibrio and the H+-driven flagellum of Salmonella. Despite low sequence identity, Vibrio FliJ complemented a Salmonella ΔfliJ mutant and restored Na+-independent protein export to a substantial extent, demonstrating a highly conserved mechanism of export gate activation. In contrast, Vibrio FliH and FliI exhibited interspecies incompatibility when expressed individually, and their co-expression in a Salmonella ΔfliH-fliI mutant supported protein export only under Na+-coupled conditions, consistent with the failure to activate the H+-driven export gate. Biochemical analyses revealed species-specific interactions between FliH and FliI, while high-speed atomic force microscopy showed that the Vibrio FliH-FliI complex retains the ability to assemble into ring-shaped structures. Together, these findings demonstrate that ATPase ring assembly and substrate delivery are mechanistically separable from export gate activation, revealing distinct and differentially conserved roles of the flagellar ATPase complex in coupling ATP hydrolysis to ion-driven protein export.
Introduction
The bacterial flagellum is a supramolecular motility machine that allows bacterial cells to migrate toward more favorable environments. It consists of at least three distinct functional parts: the basal body, which functions as a rotary motor powered by ion motive force across the cytoplasmic membrane; the filament, which acts as a helical propeller to generate thrust; and the hook, which functions as a universal joint connecting the basal body and filament. For construction of the flagellum beyond the cell membranes, flagellar structural subunits are transported by the flagellar type III secretion system (fT3SS) located at the base of the flagellum and assemble at the distal end of the growing structure. The fT3SS consists of a transmembrane export gate complex composed of FlhA, FlhB, FliP, FliQ, and FliR, and a cytoplasmic ATPase complex composed of FliH, FliI, and FliJ (Figure 1) (). These component proteins share sequence and functional similarities with the virulence-associated type III secretion system, also known as the injectosome. This system delivers various effector proteins directly into eukaryotic host cells during the infection process (Wagner and Diepold, 2020).
Figure 1
In recent years, significant progress has been made in understanding the structural and functional basis of the transmembrane export gate complex (). The export gate is embedded within a central pore of the basal body MS-ring (). FliP, FliQ, and FliR form a protein-export channel that allows export substrates to be translocated across the cytoplasmic membrane (; ). FlhB associates with the FliPQR complex () and regulates dynamic opening and closing of the cytoplasmic gate (Kinoshita et al., 2021). FlhA surrounds the FliPQR-FlhB complex () and serves as a dual-ion channel that conducts proton (H+) and sodium ion (Na+) (). Thus, the export gate functions as an ion-protein antiporter that couples inward-directed ion flow with outward-directed protein transport.
The C-terminal cytoplasmic of FlhA (FlhAC) forms a nonameric ring structure that projects into the central cavity of the basal body C-ring (). Structural and biochemical analyses have revealed that flagellar export chaperones and their substrates dock onto this FlhA ring structure prior to secretion, providing a mechanistic basis for substrate recognition and ordered export (; ; Xing et al., 2018).
The cytoplasmic ATPase ring complex is composed of 12 copies of FliH, six copies of FliI, and a single copy of FliJ () and functions as an ATP-dependent activator that converts the export gate complex into a highly efficient H+-driven protein transporter (). This complex is structurally similar to the cytoplasmic portions of FOF1-ATP synthase and VOV1-ATPase (; ; ). FliI forms a homo-hexamer that hydrolyzes ATP at subunit interfaces (; ), while FliJ penetrates the central pore of the FliI6 ring and acts as a central stalk that activates the export gate through interaction with FlhA (). FliH forms a homodimer that functions as a peripheral stalk, anchoring the FliI6-FliJ ring complex to C-ring via interactions with FliN (; ; ; ; ; ; ; ). Furthermore, recent structural studies of type III ATPases have highlighted conserved features of hexameric ATPase assemblies and their interactions with central stalk proteins such as FliJ, suggesting a common mechanism of energy coupling across secretion systems ().
When the ATPase ring complex is functional, the export gate preferentially uses proton motive force over a wide range of environmental conditions (; ; ). In contrast, when the ATPase complex is non-functional, the export gate switches to Na+-driven export (; ). Moreover, when the membrane potential exceeds a critical threshold, an otherwise inactive export gate complex autonomously converts into a H+-driven transporter even in the absence of external Na+ (). These observations suggest that ATP hydrolysis by the ATPase complex switches the export gate from a dual-fuel mode to a highly efficient H+-driven mode; however, the molecular mechanism underlying this functional coupling remains unclear.
In addition to forming the ring complex for gate activation, FliH and FliI form a heterotrimeric FliH2-FliI complex diffusing around in the cytoplasm (; ; ). This complex is thought to act as a dynamic carrier that delivers FliJ, export substrates, and chaperone-substrate complexes to the substrate-docking platform, thereby ensuring the strict order of flagellar assembly (; ; Thomas et al., 2004; ; ; ; ; Rossi et al., 2023; ). Thus, the cytoplasmic ATPase complex appears to play at least two mechanistically distinct roles in flagellar protein export: activation of the export gate and delivery of export substrates. How these two functions are coordinated, and to what extent they can be mechanistically separated, remains unknown.
To address these questions, we performed cross-complementation analyses using ATPase components from the fT3SSs of the Na+-driven polar flagellum of Vibrio alginolyticus (hereafter referred to as Vibrio) and the H+-driven flagellum of Salmonella enterica serovar Typhimurium (hereafter referred to as Salmonella). We show that Vibrio FliJ is functional within the Salmonella fT3SS in the presence of native FliH and FliI, whereas Vibrio FliH and FliI exhibit interspecies incompatibility when expressed individually. Co-expression of Vibrio FliH and FliI partially restores function, and high-speed atomic force microscopy reveals that the Vibrio FliH-FliI complex retains the ability to assemble into ring-like structures. These findings reveal evolutionary constraints on the functional integration of the flagellar ATPase complex and provide insights into the mechanistic separation of its roles in gate activation and substrate delivery.
Materials and methods
Salmonella strains, plasmids, DNA manipulations, and media
Salmonella strains and plasmids used in this study are listed in Table 1. DNA manipulations were performed using standard protocols. The cloned DNA fragments were confirmed by DNA sequencing (Eurofins Genomics). L-broth contained 1% (w/v) tryptone, 0.5% (w/v) yeast extract, and 0.5% (w/v) NaCl. Soft tryptone agar plates contained 1% (w/v) tryptone, 0.5% (w/v) NaCl, and 0.35% (w/v) agar. Ampicillin was added as needed at a final concentration of 100 μg ml−1.
Table 1
| Strain/Plasmid | Relevant characteristics | References |
|---|---|---|
| Salmonella | ||
| SJW1368 | ∆(cheW-flhD); master operon mutant | |
| MKM11 | ∆fliH | |
| MKM30 | ∆fliI | |
| MKM40 | ∆fliJ | |
| MMHI001 | ∆fliH-fliI | |
| MMHIJ001 | ∆fliH-fliI-fliJ | |
| MMHIJ0117 | ∆fliH-fliI-fliJ flhB(P28T) | |
| Plasmids | ||
| pTrc99A | Expression vector | GE Healthcare |
| pTrc99AFF4 | Expression vector | |
| pMM309 | pTrc99AFF4/StFliH | |
| pMM404 | pTrc99AFF4/StFliJ | |
| pMM1702 | pTrc99A/His-StFliI | |
| pMM1719 | pTrc990117AFF4/StFliI | |
| pMMHI001 | pTrc99AFF4/ StFliH + StFliI | |
| pMKM1702iH | pTrc99A/ His-StFliI + StFliH | |
| pMKM1702Va | pTrc99A/His-VaFliI | This study |
| pMKM2001Va | pTrc99AFF4/VaFliH | This study |
| pMKM2002Va | pTrc99AFF4/VaFliI | This study |
| pMKM2003Va | pTrc99AFF4/VaFliJ | This study |
| pMKM2004Va | pTrc99AFF4/VaFliH + VaFliI | This study |
| pMKM2005Va | pTrc99A/ His-StFliI + VaFliH | This study |
| pMKM2006Va | pTrc99A/ His-VaFliI + StFliH | This study |
| pMKM2007Va | pTrc99A/ His-VaFliI + VaFliH | This study |
Salmonella strains and plasmids used in this study.
Sequence alignment
Sequence alignment was carried out using CLUSTAL-Ω1.
Structural modeling
Structural models were generated using the AlphaFold3 prediction server2. Structural comparisons between Salmonella and Vibrio flagellar ATPase components were conducted by UCSF ChimeraX (). All figures including structural models of the flagellar ATPase components were prepared using UCSF ChimeraX.
Soft-agar motility assays
Fresh colonies were inoculated onto soft agar plates supplemented with ampicillin and incubated at 30 °C. The assay was performed at least seven times to confirm the reproducibility of the results.
Secretion assays
Salmonella cells were grown in 5 mL L-broth supplemented with ampicillin at 30 °C with shaking until the cell density reached an OD600 of approximately 1.2–1.4. Cultures were then centrifuged to separate cell pellets and culture supernatants. Proteins in the whole-cell and culture supernatant fractions were normalized to the OD600 of each culture to ensure equivalent cell numbers.
Cell pellets were resuspended directly in SDS loading buffer [62.5 mM Tris–HCl (pH 6.8), 2% (w/v) sodium dodecyl sulfate (SDS), 10% (w/v) glycerol, and 0.001% (w/v) bromophenol blue] supplemented with 1 μL of 2-mercaptoethanol. Proteins in the culture supernatants were precipitated with 10% (w/v) trichloroacetic acid on ice for 1 h, centrifuged (20,000 g, 20 min, 4 °C), and resuspended in Tris-SDS loading buffer (one volume of 1 M Tris–HCl mixed with nine volumes of SDS loading buffer) containing 1 μL of 2-mercaptoethanol.
After heating at 95 °C for 3 min, protein samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to immunoblotting using polyclonal anti-FlgD antibody. Immunoblotting was performed with an iBind Flex Western Device according to the manufacturer’s instructions (Thermo Fisher Scientific). Chemiluminescent signals were detected using Amersham ECL Prime Western Blotting Detection Reagent (Cytiva) and captured with a Luminoimage Analyzer LAS-3000 (GE Healthcare). Image data were processed using Photoshop software (Adobe). The assay was performed at least three times to confirm the reproducibility of the results.
Pull-down assays by Ni-affinity chromatography
The Salmonella SJW1368 strain carrying pTrc99A-based plasmids co-expressing of untagged FliH and His-FliI was grown overnight at 30 °C in 100 mL L-broth supplemented with ampicillin. Cells were harvested by centrifugation, suspended in 25 mL of TN buffer [20 mM Tris–HCl (pH 8.0), 500 mM NaCl] containing 25 mM imidazole, and disrupted by sonication. After centrifugation to remove undisrupted cells and cell debris, the clarified cell lysates were loaded onto a nickel-nitrilotriacetic acid (Ni-NTA) agarose column (QIAGEN). After washing the column with the TN buffer containing 50 mM imidazole, bound proteins were eluted with the same buffer containing increasing concentrations of imidazole (100, 250, and 500 mM) in a stepwise manner. Eluted fractions were analyzed by SDS-PAGE, followed by Coomassie Brilliant Blue (CBB) staining.
Size exclusion chromatography
Salmonella FliH-His-FliI complex, Vibrio His-FliH, and Vibrio FliH-His-FliI complex, purified by Ni-affinity chromatography, were applied to a Superdex 200 16/60 column (GE Healthcare) equilibrated with buffer containing 50 mM Tri-HCl (pH 8.0), 150 mM NaCl and 1 mM EDTA at a flow rate of 1.0 mL min−1. Elution fractions were analyzed by SDS-PAGE, followed by CBB staining.
Analytical size exclusion chromatography was performed with a Superdex 200 Increase 10/300 column (GE Healthcare). Purified samples were applied to the column equilibrated with buffer containing 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 1 mM DTT at a flow rate of 0.5 mL min−1. Unless otherwise stated, size exclusion chromatography was performed in the absence of nucleotide.
Chemical cross-linking
Purified protein samples were dialyzed against 20 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl at 4 °C, and then glutaraldehyde was added to 50 μL aliquots of each protein sample (10 μM final protein concentration) to a final concentration of 0.1% (v/v). The cross-linking reaction was allowed to proceed for 30 min at room temperature and was quenched by the addition of Tris–HCl to a final concentration of 100 mM. Following quenching, 60 μL of 2 × SDS loading buffer [125 mM Tris–HCl (pH 6.8), 4% (w/v) SDS, 20% (w/v) glycerol, and 0.002% (w/v) bromophenol blue] supplemented with 1 μL of 2-mercaptoethanol was added to each sample. Samples were heated at 95 °C for 5 min, and proteins were subsequently separated by SDS-PAGE using a 10–20% gradient polyacrylamide gel and analyzed by CBB staining and immunoblotting using anti-His-tag mAb-HRP-DirecT (MBL, Japan). All image data were processed using Photoshop software (Adobe). Each experiment was performed at least three times independently.
High-speed atomic force microscopy
High-speed atomic force microscopy (HS-AFM) imaging was performed in solution using a laboratory-built HS-AFM system (; ). The purified FliH-FliI complex was diluted to a final concentration of 2 μM in observation buffer containing 25 mM Tris–HCl (pH 8.0), 75 mM NaCl, 5 mM MgCl2, and 5 mM ADP-AlF4− (a transition-state analog). After incubation at room temperature for a few minutes, a 2 μL aliquot was deposited onto a freshly cleaved mica surface and incubated for 5 min. Unbound molecules were removed by washing the surface with 100 μL of the observation buffer. HS-AFM imaging was performed in 70 μL of the observation buffer at room temperature.
HS-AFM imaging was carried out in tapping mode using small cantilevers (AC7, Olympus) with a resonant frequency of approximately 800 kHz and a spring constant of approximately 0.2 N/m. An amorphous carbon pillar was grown on the cantilever tip by electron beam deposition (EBD) and sharpened by argon gas plasma etching to serve as a probe. HS-AFM images were processed by applying a low-pass filter to reduce random noise and a line-by-line background subtraction filter to correct the xy-plane background using a laboratory-built Python-based viewer software (pyNuD).
Results
Sequence and structural comparison of FliH, FliI, and FliJ
To assess the degree of conservation between the flagellar ATPase components of Vibrio and Salmonella, we first compared the amino acid sequences of FliH, FliI, and FliJ. Pairwise sequence alignments revealed that Vibrio FliI is relatively well conserved, showing 56.2% identity and 68.2% similarity to Salmonella FliI, whereas Vibrio FliH and FliJ exhibit much lower sequence identity (21 and 18%, respectively), despite moderate similarity (38.1 and 49.3%, respectively) (Supplementary Figures 1A–3A).
To examine whether these sequence differences translate into major structural divergence, we compared the available crystal structures of the Salmonella FliH-FliI complex (PDB ID: 5B0O) and Salmonella FliJ (PDB ID: 3AJW) with AlphaFold-predicted structures of their Vibrio homologs. Despite the low sequence identity, the predicted structures of Vibrio FliH, FliI, and FliJ closely resemble the corresponding Salmonella crystal structures, exhibiting highly conserved overall folds (Supplementary Figures 1B–3B).
These observations indicate that although the primary sequences of FliH and FliJ have diverged substantially between Vibrio and Salmonella, their overall structures remain highly conserved. This raises the possibility that species-specific incompatibility arises not from global structural differences but from subtle variations at protein–protein interaction interfaces, and this hypothesis is tested in the functional analyses described below.
Functional compatibility of Vibrio FliH, FliI, and FliJ in Salmonella fT3SS
To examine the functional compatibility of the Vibrio ATPase components with those of Salmonella fT3SS, we performed cross-complementation assays using Salmonella ΔfliH, ΔfliI, and ΔfliJ mutant strains expressing the corresponding Vibrio proteins. Flagella-driven motility and fT3SS-mediated protein secretion were assessed by soft-agar motility assays and immunoblotting using a polyclonal antibody against FlgD, a representative export substrate of the fT3SS, respectively.
Expression of Vibrio FliJ substantially restored motility (Figure 2A and Supplementary Figure 4) and flagellar protein export (Figure 2B) in the Salmonella ΔfliJ mutant, indicating that Vibrio FliJ is functionally compatible with the Salmonella fT3SS. In contrast, expression of Vibrio FliH or Vibrio FliI alone failed to rescue the motility defects of the Salmonella ΔfliH or ΔfliI mutants, respectively (Supplementary Figure 4), demonstrating that these components are not individually interchangeable between the two species.
Figure 2
We next tested whether the function of Vibrio FliJ depends on the presence of Salmonella FliH and FliI. Because the flhB(P28T) mutation substantially increases the probability of flagellar formation in the absence of FliH and FliI (; ), we analyzed both a Salmonella ΔfliH-fliI-fliJ mutant and a ΔfliH-fliI-fliJ flhB(P28T) mutant. In a Salmonella background lacking both FliH and FliI, Vibrio FliJ failed to restore motility (Figure 2C and Supplementary Figure 5) or flagellar protein export (Figure 2D), indicating that FliJ-mediated activation of the export gate requires the native Salmonella FliH-FliI complex.
Because Vibrio FliH exhibits much lower sequence identity to Salmonella FliH than for FliI, we next investigated whether Vibrio FliH and FliI together can function as the flagellar ATPase complex in the Salmonella fT3SS. Simultaneous expression of Vibrio FliH and FliI in a Salmonella ΔfliH-fliI mutant partially restored motility (Figure 2E) and protein export activity (Figure 2F). This result suggests that the Vibrio FliH-FliI complex can function as a cytoplasmic ATPase complex together with native FliJ in Salmonella but is less efficient at activating the transmembrane export gate than the native Salmonella FliH-FliI complex.
Together, these results reveal that while Vibrio FliJ is compatible with the Salmonella export system, functional integration of Vibrio FliH and FliI requires their co-expression and remains incomplete, highlighting species-specific constraints in ATPase-export gate coupling.
3.3 Vibrio FliJ activates the H+-driven export gate of Salmonella fT3SS independently of Na+
The transmembrane export gate complex intrinsically functions as a dual-fuel export engine that exploits both H+ and Na+ as coupling ions to drive flagellar protein export (). In the absence of FliH and FliI, however, the export gate preferentially utilizes the sodium motive force across the cytoplasmic membrane over a wide range of external pH values (, ). As a result, Salmonella strains lacking FliH and FliI display weak but significant motility in the presence of Na+ but are essentially non-motile in its absence (Supplementary Figure 6). Once ATP is hydrolyzed by the FliI6 ring complex, FlhA turns an inefficient dual-fuel engine into a highly efficient H+-driven export engine through the FliJ-FlhA interaction (; ). Consequently, the export gate preferentially uses proton motive force as its primary energy source ().
To examine whether Vibrio FliJ can activate the Salmonella export gate in a manner comparable to native FliJ, we analyzed the Na+ dependence of flagellar protein export in a Salmonella ΔfliJ mutant expressing Vibrio FliJ. Export of the hook-capping protein FlgD was monitored in the presence of either 100 mM Na+ or 100 mM K+ (Figure 3A).
Figure 3
In the absence of FliJ, the export gate remains inactive even in the presence of Na+ (Supplementary Figure 6), and no FlgD export therefore occurred, consistent with previous observations (, ). In contrast, expression of Vibrio FliJ restored robust FlgD export regardless of Na+ availability, closely resembling the phenotype observed upon expression of native Salmonella FliJ (Figure 3A).
These results demonstrate that Vibrio FliJ efficiently activates the H+-driven export mode of the Salmonella flagellar export gate. Thus, despite low sequence identity, FliJ retains a conserved ability to couple the ATPase complex to gate activation across species boundaries.
3.4 Species-specific interaction between FliH and FliI underlies interspecies incompatibility
To determine whether the observed interspecies incompatibility of FliH and FliI reflects impaired physical interactions, we examined protein–protein interactions between Vibrio and Salmonella ATPase components using pull-down assays by Ni-affinity chromatography. When Salmonella His-FliI was used as a bait, it efficiently pulled down untagged Salmonella FliH (Figure 4A), in agreement with previous observations (; ). In contrast, Salmonella His-FliI failed to pull down untagged Vibrio FliH under the same experimental condition (Figure 4B). Reciprocally, Vibrio His-FliI interacted with untagged Vibrio FliH (Figure 4D) but not with untagged Salmonella FliH (Figure 4C), indicating that native FliH-FliI interactions are preserved within each species but not across species boundaries.
Figure 4
These results demonstrate that FliH-FliI interactions are species-specific and that incompatibility between Vibrio and Salmonella ATPase components arises from the failure to form heterologous FliH-FliI complexes. This explains why co-expression of Vibrio FliH and FliI is required to partially restore function in the Salmonella ΔfliH-fliI mutant.
3.5 The Vibrio FliH-FliI complex functions as a dynamic carrier but fails to activate the H+-driven export gate
To assess whether the Vibrio FliH-FliI complex can activate the Salmonella export gate, we examined the ion dependence of flagellar protein export in the Salmonella ΔfliH-fliI mutant expressing Vibrio FliH and FliI. Export of the hook-capping protein FlgD was analyzed in the presence of either 100 mM Na+ or 100 mM K+ (Figure 3B).
Expression of native Salmonella FliH and FliI restored robust FlgD export independently of Na+, as consistent with efficient activation of the H+-driven export gate. In contrast, co-expression of Vibrio FliH and FliI supported FlgD export only in the presence of Na+, but not when Na+ was replaced with K+ (Figure 3B). These results indicate that the Vibrio FliH-FliI complex can support substrate delivery to the export gate as a dynamic carrier but is incapable of activating the H+-driven transport mode of the Salmonella export gate.
3.6 Vibrio FliI alone fails to function as a dynamic carrier in Salmonella T3SS
Overexpression of Salmonella FliI alone can bypass the requirement for FliH to a considerable extent (; ). We therefore examined whether overexpression of Vibrio FliI could similarly restore flagella-driven motility of the Salmonella ΔfliH-fliI mutant. In contrast to Salmonella FliI, overexpression of Vibrio FliI failed to restore motility under any conditions tested (Supplementary Figure 7A), indicating that Vibrio FliI requires its cognate FliH to exert export activity in the Salmonella fT3SS.
We next examined whether Salmonella FliI alone can act as the ATP-driven activator of the export gate in the absence of FliH by performing soft-agar motility assays in the presence of either 100 mM Na+ or 100 mM K+ (Supplementary Figure 7B). Overexpression of Salmonella FliI enhanced motility only slightly in the presence of 100 mM K+, indicating that FliI alone is insufficient to fully activate the H+-driven export gate. In contrast, FliI overexpression substantially improved motility in the presence of 100 mM Na+, suggesting that FliI alone may function as the substrate carrier for Na+-driven export gate. Notably, deletion of fliJ abolished motility in the ΔfliH-fliI mutant overexpressing Salmonella FliI (Supplementary Figure 7A), indicating that FliJ is required for FliI-mediated motility.
Together, these results suggest that Salmonella FliI can function as a dynamic carrier that delivers FliJ and export substrates to the export gate, whereas Vibrio FliI alone lacks this capability in the Salmonella fT3SS.
3.7 Oligomerization state of Vibrio FliH and FliH-FliI complex
Salmonella FliH predominantly forms a homodimer in solution, and Salmonella FliH and FliI forms a hetero-trimeric FliH2-FliI complex (). To determine whether the functional defect of the Vibrio FliH-FliI ATPase complex arises from improper assembly, we purified Vibrio His-FliH and FliH-His-FliI complex by size exclusion chromatography (Figure 5A) and analyzed their oligomeric states by chemical cross-linking (Figure 5B).
Figure 5
Given that the deduced molecular mass of Vibrio His-FliH is approximately 32.6 kDa, chemical cross-linking analysis revealed that purified His-FliH predominantly formed dimers in solution. Moreover, a cross-linked product corresponding to a tetramer was also detected albeit at low frequency. Because no cross-linked product corresponding to a trimer was observed, this tetramer is likely a dimer of the dimer. In contrast, cross-linking of the purified Vibrio FliH-His-FliI complex yielded two major species: one corresponding to the FliH dimer and another corresponding to a heterotrimeric FliH2-FliI assembly with a deduced molecular mass of approximately 109.3 kDa. These results indicate that the inability of the Vibrio FliH-FliI complex to activate the export gate does not arise from gross defects in heterotrimer formation.
3.8 Vibrio FliH-FliI complex assembles into a ring-shaped structure as visualized by HS-AFM
Salmonella FliI efficiently forms a homohexamer in the presence of Mg2+ and the non-hydrolysable ATP analog ADP-AlF4− (). Consistently, EscN, a FliI homologue of the virulence-associated type III secretion system, form a homohexamer, and this EscN hexameric structure has been solved by cryo-electron microscopy image analysis (). AlphaFold predictions suggest that Vibrio FliI can form a similar homohexameric structure (Supplementary Figure 8). To examine whether the failure of the Vibrio FliH2-FliI complex to activate the Salmonella export gate results from its defect in higher-order assembly, we purified Salmonella and Vibrio FliH2-FliI heterotrimeric complexes by size exclusion chromatography (Supplementary Figure 9), incubated them with 5 mM Mg2+ and 5 mM ADP-AlF4−, and visualized them by HS-AFM.
HS-AFM imaging revealed that the Vibrio FliH2-FliI complex assembles into a ring-shaped structure on mica surfaces (Figure 6). The overall dimensions and ring morphology were comparable to those observed for the Salmonella FliH2-FliI complex under the same condition, and this ring-shaped structure was stably maintained during HS-AFM imaging.
Figure 6
Together with the biochemical and functional data, these observations indicate that the Vibrio FliH2-FliI complex retains the intrinsic capacity to assemble into the ATPase ring-like structure. Thus, formation of the ATPase ring is not sufficient for the activation of the H+-driven export gate, highlighting functional separation between ATPase ring assembly, substrate delivery, and export gate activation within the flagellar ATPase complex. It should be noted that the FliI hexameric structures observed in this study were assembled in vitro under defined experimental conditions, and their exact structural and functional states in vivo remain to be further investigated.
4 Discussion
In this study, we dissected the functional integration of the flagellar ATPase complex through cross-complementation between the Vibrio and Salmonella fT3SS. Our analyses demonstrate that the ATPase complex fulfills at least two mechanistically distinct roles during flagellar protein export: delivery of export substrates to the export gate and activation of the H+-driven export engine. While Vibrio FliJ was functional within the Salmonella fT3SS, the Vibrio FliH2-FliI complex could only partially substitute for its Salmonella counterpart (Figure 2). Despite retaining the ability to assemble into a ring-shaped complex (Figure 6), the Vibrio FliH2-FliI complex can support only the Na+-dependent flagellar protein export but failed to activate the H+-driven export engine (Figure 3B). These findings reveal distinct evolutionary constraints acting on the different functions of the flagellar ATPase complex.
One of the most striking observations in this study is the functional compatibility of Vibrio FliJ in the Salmonella fT3SS. Expression of Vibrio FliJ restored Na+-independent export of FlgD in a Salmonella ΔfliJ mutant (Figure 3A), indicating that it can activate the H+-driven mode of the export gate. This level of functional conservation is particularly remarkable given the low sequence identity between the two FliJ proteins (Supplementary Figure 3A). Structural comparisons between the Salmonella crystal structure and an AlphaFold-predicted model of Vibrio FliJ revealed a high degree of structural similarity (Supplementary Figure 3B), suggesting that key interaction surfaces required for export gate activation are strongly conserved. In fact, two highly conserved, surface-exposed residues, Phe-72 and Leu-76 of FliJ, are directly involved in the interaction with FlhA (Supplementary Figure 3A). Mutational analyses have shown that the FliJ(F72A) and FliJ(L76A) variants require the support of FliH and FliI to exert their export function to a considerable degree (). These observations suggest that FliJ plays a central role in activating the export gate through its interaction with FlhA. The high cross-species compatibility of FliJ, despite its low sequence identity, further supports the idea that FliJ functions as a universal activator of the export gate and interacts with a structurally conserved and evolutionarily constrained component of the export machinery, most likely the FlhA platform. In contrast, FliH and FliI appear to have co-evolved with species-specific regulatory networks, allowing fine-tuning of export gate activation in response to distinct physiological and energetic environments.
In contrast to FliJ, Vibrio FliH and FliI exhibited clear interspecies incompatibility when expressed individually in Salmonella. Pull-down assays demonstrated that FliH-FliI interactions are strictly species-specific: Vibrio FliH interacted with Vibrio FliI but not with Salmonella FliI, and vice versa (Figure 4). These findings indicate that species-specific physical interactions between FliH and FliI underlie the observed functional incompatibility. Although overall folds of FliH and FliI are conserved (Supplementary Figures 1B, 2B), subtle differences at their interaction interfaces likely prevent the formation of stable heterologous complexes. Thus, unlike FliJ, the FliH-FliI module appears to be subject to tighter evolutionary tuning, limiting its functional interchangeability across species.
Our ion-dependence analyses provide direct experimental evidence that the two major functions of the flagellar ATPase complex—substrate delivery and export gate activation—are mechanistically separable. In the Salmonella ΔfliH-fliI mutant expressing Vibrio FliH and FliI, FlgD export occurred only in the presence of Na+, a condition known to support protein export when the ATPase complex is non-functional. This phenotype indicates that the Vibrio FliH-FliI complex can function as the dynamic carrier that delivers substrates to the export gate but is insufficient to activate the H+-driven transport mode. By contrast, the native Salmonella ATPase complex supports efficient export independently of Na+. These results demonstrate that the ATP hydrolysis-driven gate activation represents a specialized function that is uncoupled from substrate delivery.
High-speed atomic force microscopy revealed that the Vibrio FliH-FliI complex assembles into a ring-shaped structure that closely resembles that formed by the Salmonella FliH-FliI complex (Figure 6). In addition, biochemical cross-linking experiments showed that Vibrio FliH forms a dimer and that the FliH-FliI complex assembles into a heterotrimeric unit, consistent with the canonical architecture of the ATPase complex (Figure 5B). These observations rule out gross defects in complex assembly as the primary cause of impaired gate activation. Instead, they suggest that dynamic or transient interactions between the ATPase complex and the export gate, rather than static structural features, are critical for coupling ATP hydrolysis to H+-driven protein transport.
ATP hydrolysis by the FliI ATPase is required for efficient and robust gate activation via specific interactions between FliJ and FlhA (; ). Vibrio FliJ requires Salmonella FliH and FliI to efficiently serve as an activator of the H+-driven export engine in the Salmonella fT3SS export gate. Based on our results, we propose a model in which FliJ functions as a universally conserved activator of the export gate, while the FliH-FliI complex serves as a species-specific regulator that fine-tunes the interaction between FliJ and FlhA, thereby enabling the export gate to become an active H+-driven protein transporter. The cytoplasmic ATPase ring complex is anchored to the basal body C-ring through interactions between the extreme N-terminal region of FliH, including Trp-7 and Trp-10, and FliN (; ; ). Although Vibrio FliH and FliI assembled into a ring structure (Figure 6B), they failed to activate the H+-driven export engine (Figure 3B). Notably, the N-terminal region of Vibrio FliH is 29 residues longer than that of Salmonella FliH (Supplementary Figure 1A), suggesting that proper FliH-FliN interactions are additionally required for precise functional coupling between ATP hydrolysis by the ATPase complex and gate activation. Future structural and biophysical studies focusing on the dynamic interactions among the FliH-FliI complex, FliJ, and FlhA will be essential to elucidate the molecular basis of this coupling. Together, our study provides a conceptual framework for understanding how the flagellar ATPase complex has evolved to integrate environmental ion availability with efficient and ordered protein export. More broadly, our findings suggest that functional specialization within conserved molecular machines can arise from divergence in dynamic coupling mechanisms rather than from changes in core architecture.
These findings are consistent with previous structural studies showing that export chaperone-substrate complexes interact with the FlhA platform prior to secretion (Xing et al., 2018), and that FliJ can associate with chaperone complexes (Rossi et al., 2023). In addition, structural analyses of homologous type III ATPases, such as the EscN hexamer (), have provided important insights into the conserved architecture and function of ATPase assemblies, supporting the relevance of our observations.
The Na+-driven flagellar motor in marine Vibrio is known to exhibit higher rotational speed than the H+-driven motor in Salmonella (), likely reflecting differences in ion flux rates and stator unit dynamics. Our findings raise the possibility that similar principles may apply to the export gate, where Na+-coupled protein export is favored under conditions in which ATPase-mediated activation is compromised. This suggests that the dual-fuel capability of the export gate provides functional flexibility to adapt to different energetic environments.
Despite these advances, how substrate delivery by the ATPase complex is mechanistically coupled to export gate activation remains unclear. Beyond its mechanistic implications, our findings may have relevance for bacterial pathogenicity and the development of alternative antimicrobial strategies. Flagellar assembly and motility contribute critically to host colonization, tissue invasion, and biofilm formation in many pathogenic bacteria. The fT3SS therefore represents a key regulatory node in virulence rather than in bacterial viability. Our demonstration that substrate delivery and export gate activation are mechanistically separable identifies the ATPase-gate coupling as a previously unrecognized vulnerability in the flagellar export apparatus. Importantly, disruption of this coupling without abolishing ATPase assembly itself may selectively impair flagellar biogenesis and virulence while imposing reduced selective pressure for resistance compared with conventional bactericidal antibiotics. Thus, the species-specific tuning of ATPase-gate interactions revealed here would provide a conceptual framework for targeting energy-coupling mechanisms of bacterial secretion systems as anti-virulence strategies, opening new avenues for antimicrobial intervention that exploit biophysical constraints rather than essential metabolic functions.
Statements
Data availability statement
The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author/s.
Author contributions
TM: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. MK: Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing – original draft. YT: Data curation, Formal analysis, Investigation, Validation, Visualization, Writing – original draft. TU: Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft. KN: Conceptualization, Funding acquisition, Supervision, Validation, Writing – review & editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This work was supported in part by JSPS KAKENHI Grant Numbers JP19H03182, JP22H02573, and JP22K19274 (to TM) and JP20K15749 and JP22K06162 (to MK) and MEXT KAKENHI Grant Numbers JP20H05532, and JP22H04844 (to TM). This work has also been supported by Research Support Project for Life Science and Drug Discovery (BINDS) from AMED under Grant Number JP23am121003, JP24am121003 and JP25am121003 (to KN), by the Cyclic Innovation for Clinical Empowerment (CiCLE) from AMED under Grant Number JP17pc0101020 (to KN), and by JEOL YOKOGUSHI Research Alliance Laboratories of The University of Osaka (to KN).
Acknowledgments
We thank Michio Homma and Seiji Kojima for providing chromosomal DNA of Vibrio alginolyticus 138-2 and DNA sequence information for the Vibrio FliH, FliI, and FliJ proteins of the Na+-driven polar flagellar system. We also thank Yasuyo Abe for technical assistance.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that Generative AI was not used in the creation of this manuscript.
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Supplementary material
The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2026.1841110/full#supplementary-material
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Summary
Keywords
ATPase complex, flagellar type III secretion system, FliH, Flii, FliJ, ion coupling, protein export
Citation
Minamino T, Kinoshita M, Tajimi Y, Uchihashi T and Namba K (2026) Uncoupling substrate delivery from export gate activation reveals distinct roles of the flagellar ATPase complex. Front. Microbiol. 17:1841110. doi: 10.3389/fmicb.2026.1841110
Received
28 March 2026
Revised
26 April 2026
Accepted
08 May 2026
Published
21 May 2026
Volume
17 - 2026
Edited by
Jun Xu, University of the Ryukyus, Japan
Reviewed by
Wangbiao Seven Guo, Yale University, United States
Stanley Moore, University of Saskatchewan, Canada
Updates
Copyright
© 2026 Minamino, Kinoshita, Tajimi, Uchihashi and Namba.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Tohru Minamino, minamio.tohru.fbs@osaka-u.ac.jp
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.