Type III Secretion Systems (T3SS) are multi-component molecular machines that deliver protein cargo from the bacterial cytosol either to their site of assembly in cell surface flagella or virulence factor injectisomes, or directly to their site of action in eukaryotic target cells or the extracellular environment (Evans et al., 2014a; Deng et al., 2017; Büttner and He, 2009; Konkel et al., 2004; Dongre et al., 2018). The flagellar T3SS (fT3SS) directs the export of thousands of structural subunits required for the assembly and operation of flagella, rotary nanomotors for cell motility that extend from the bacterial cell surface (Evans et al., 2014a; Evans et al., 2014b). Newly synthesised subunits of the flagellar rod, hook and filament are targeted to the fT3SS, where they are unfolded and translocated across the cell membrane, powered by the protonmotive force and ATP hydrolysis, into an external export channel that spans the length of the nascent flagellum (Minamino and Namba, 2008; Paul et al., 2008). During flagellum biogenesis, when the rod/hook structure reaches its mature length, the fT3SS switches export specificity from recognition of ‘early’ rod/hook subunits to ‘late’ subunits for filament assembly (Williams et al., 1996; Fraser et al., 2003). This means that early and late flagellar subunits must be differentiated by the fT3SS machinery to ensure that they are exported at the correct stage of flagellum biogenesis. This is achieved, in part, by targeting subunits to the export machinery at the right time using a combination of export signals in the subunit mRNA and/or polypeptide. T3SS substrates contain N-terminal signals for targeting to the export machinery, however they do not share a common peptide sequence (Kuwajima et al., 1989; Minamino and Macnab, 1999; Kornacker and Newton, 1994; Evans et al., 2013; Végh et al., 2006). In addition, some substrates are piloted to the T3SS machinery by specific chaperones (Wattiau et al., 1994; Fraser et al., 1999; Thomas et al., 2004; Akeda and Galán, 2005; Bange et al., 2010).

The core export components of the fT3SS are evolutionarily related to those of the virulence injectisome, with which they share considerable structural and amino acid sequence similarity (Kuhlen et al., 2018; Abrusci et al., 2012; Eichelberg et al., 1994; Johnson et al., 2019). The flagellar export machinery comprises an ATPase complex (FliHIJ) located in the cytoplasm, peripheral to the membrane. Immediately above the ATPase is a nonameric ring formed by the cytoplasmic domain of FlhA (FlhA_C), which functions as a subunit docking platform (Bange et al., 2010; Kinoshita et al., 2013; Bryant et al., 2021; Xing et al., 2018). A recent cryo-ET map indicates that the FlhA family have a sea-horse-like structure, in which FlhAc forms the ‘body’ and the FlhA N-terminal region (FlhA_N) forms the ‘head’, which is fixed in the plane of the membrane (Butan et al., 2019). FlhA_N wraps around the base of a complex formed by FliPQR and the N-terminal sub-domain of FlhB (FlhB_N), and together these form the FlhAB-FliPQR export gate that connects the cytoplasm to the central channel in the nascent flagellum, which is contiguous with the extracellular environment (Kuhlen et al., 2018; Abrusci et al., 2012; Eichelberg et al., 1994; Johnson et al., 2019; Bryant et al., 2021; Xing et al., 2018; Butan et al., 2019; Mizuno et al., 2011). FlhB_N is connected via a linker (FlhB_CN) to the cytoplasmic domain of FlhB (FlhB_C), which is thought to sit between the FlhA_N and FlhA_C rings, where it functions as a docking site for early flagellar subunits (Evans et al., 2013; Kuhlen et al., 2018; Butan et al., 2019).

The ‘early’ flagellar subunits that assemble to form the rod and hook substructures are not chaperoned: instead, the signals for targeting and export are found within the early subunits themselves. We have shown that of one of these signals is a small hydrophobic sequence termed the gate recognition motif (GRM), which is essential for early subunit export (Evans et al., 2013). This motif binds a surface exposed hydrophobic pocket on FlhBc (Evans et al., 2013). Once subunits reach the export machinery, they must be unfolded before they can pass through the narrow channel formed by FliPQR-FlhB_N into the central channel of the nascent flagellum, through which the subunits transit until they reach the tip and fold into the structure (Evans et al., 2014b; Kuhlen et al., 2018). Structural studies suggest that FliPQR-FlhB_N adopts an energetically favourable closed conformation, possibly to maintain the membrane permeability barrier (Kuhlen et al., 2018; Johnson et al., 2019; Ward et al., 2018; Kuhlen et al., 2020). This suggests that there must be a mechanism to trigger opening of the export gate when subunits dock at cytosolic face of the flagellar export machinery.

Here, we sought to identify new export signals within flagellar rod/hook subunits, using the hook-cap subunit FlgD as a model export substrate. We show that the extreme N-terminus of rod/hook subunits contains a hydrophobic export signal and investigate its functional relationship to the subunit gate recognition motif (GRM).

T3SS substrates contain N-terminal export signals, but these have not been fully defined and how they promote subunit export remains unclear. Here, we characterised a new hydrophobic N-terminal export signal in early flagellar rod/hook subunits and showed that the position of this signal relative to the known subunit gate recognition motif (GRM) is key to subunit export.

Loss of the hydrophobic N-terminal signal in the hook cap subunit FlgD had a stronger negative effect on subunit export than deletion of the GRM that enables subunit docking at FlhB_C, suggesting that the hydrophobic N-terminal signal may be required to trigger an essential export step. A suppressor screen showed that the export defect caused by deleting the hydrophobic N-terminal signal could be overcome by mutations that either reintroduced small non-polar amino acids positioned 3–7 residues from the subunit N-terminus (e.g. M₇I), or introduced additional residues between V₁₅ and the GRM. In such ‘gain of function’ strains containing insertions, changing V₁₅ to alanine abolished subunit export, which was rescued by re-introduction of small non-polar residues close to the N-terminus. These data point to an essential export function for small non-polar residues close to the N-terminus of rod/hook subunits.

It was fortuitous that we chose FlgD as the model for early flagellar subunit. All early subunits contain small hydrophobic residues close to the N-terminus, but FlgD is unique in that only four (I₃, A₄, V₅ and V₁₅) of its first 25 residues are small and non-polar (Figure 1—figure supplement 1). Indeed, there are only three other small hydrophobic residues between the FlgD N-terminus and the GRM (Figure 1, Figure 1—figure supplement 1, Figure 6—figure supplement 2). While deletion of residues 2–5 in FlgD removes the critical hydrophobic N-terminal signal, similar deletions in the N-terminal regions of other rod/hook subunits reposition existing small non-polar residues close to the N-terminus (Figure 1—figure supplement 1, Figure 6—figure supplement 2). This is perhaps why previous deletion studies in early flagellar subunits have failed to identify the hydrophobic N-terminal signal (Evans et al., 2013; Hirano et al., 2005; Minamino et al., 1999).

The finding that subunits lacking the hydrophobic N-terminal export signal, but not the GRM, stalled during export suggested that these two signals were recognised by the flagellar export machinery in a specific order. Mutant variants of other flagellar export substrates or export components have been observed to block the export pathway. For example, a FlgN chaperone variant lacking the C-terminal 20 residues stalls at the FliI ATPase (Thomas et al., 2004), while a GST-tagged FliJ binds FlhA but is unable to associate correctly with FliI so blocking wild type FliJ interaction with FlhA (Ibuki et al., 2012). These attenuations can be reversed by further mutations that disrupt the stalling interactions. This was also observed for FlgD∆2–5. Loss of the hydrophobic N-terminal signal resulted in a dominant-negative effect on motility and flagellar export, but this was abolished by subsequent deletion of the GRM. This indicates that FlgD∆2–5 stalls in the export pathway at FlhB_C, blocking the binding site for early flagellar subunits. These data are consistent with sequential recognition of the two export signals: the GRM first docking subunits at FlhB_C, and positioning the hydrophobic N-terminal signal to trigger the next export step.

The position of the subunit hydrophobic N-terminal export signal relative to the GRM appears critical for export. Engineering of flgD to encode variants in which the region between the N-terminus and the GRM was replaced with polypeptide sequences of varying lengths showed that these signals must be separated by a minimum of 19 residues for detectable export, with substantial export requiring separation by 30 residues (Figure 3). When subunits dock at FlhB_C, which is likely situated within or just below the plane of the inner membrane, the hydrophobic N-terminal signal is positioned close to the FlhAB-FliPQR export gate (Figure 6B). Subunits in which the GRM and N-terminal signal are brought closer together stall at FlhB_C, suggesting that the hydrophobic N-terminal signal is unable to contact its recognition site on the export machinery (Figures 5, 6A and B). In all flagellar rod/hook subunits, the GRM is positioned at least 30 residues from the N-terminus (Figure 1—figure supplement 1, Figure 6—figure supplement 2). The physical distance between the two signals will depend on the structure adopted by the subunit N-terminus (Figure 6A). The N-terminal region of flagellar subunits is often unstructured in solution (Kuwajima et al., 1989; Minamino and Macnab, 1999; Kornacker and Newton, 1994; Evans et al., 2013; Végh et al., 2006; Vonderviszt et al., 1992), and such disorder may be an intrinsic feature of flagellar export signals (Kuwajima et al., 1989; Minamino and Macnab, 1999; Kornacker and Newton, 1994; Evans et al., 2013; Végh et al., 2006; Weber-Sparenberg et al., 2006; Aizawa et al., 1990), as they are typical in other bacterial export N-terminal substrate signals such as those of the Sec and Tat systems (Tsirigotaki et al., 2017; Palmer and Berks, 2012). Unstructured signals may facilitate multiple interactions with different binding partners during export, and in the case of export systems that transport unfolded proteins they may aid initial entry of substrates into narrow export channels (Tsirigotaki et al., 2017).

As yet, nothing is known about the structure of the subunit N-terminal domain upon interaction with the flagellar export machinery. Signal peptides in TAT pathway substrates switch between disordered and α-helical conformations depending on the hydrophobicity of the environment (San Miguel et al., 2003). It therefore seems likely that local environments along the flagellar export pathway will influence the conformation of subunit export signals (Kuhlen et al., 2018; Erhardt et al., 2017). Interestingly, FlgD variants that contain additional sequence that position the N-terminal export signal and GRM further apart far above the required threshold did not impede export, which argues that the sequence between both export signals is unfolded (Figure 2).

If the region between the hydrophobic N-terminal signal and the GRM is unstructured and extended, this would correspond to a polypeptide contour length of approximately 72–105 Å (where the length of one amino acid is ~3.6 Å). If the same region were to fold as an α-helix, its length would be approximately 30–36 Å (where one amino acid rises every ~1.5 Å). Placement of the AlphaFold predicted structure of full length FlhB into a tomogram of the T3SS suggests that FlhB_C is positioned below the plane of the inner membrane but above the nonameric ring of FlhA_C (Figure 6—figure supplement 3). Without further structural information on subunit interactions with the flagellar export machinery and the precise position of FlhB_C within the machinery, it is difficult to determine precisely where the hydrophobic N-terminal signal contacts the machinery.

We speculate that one function of the subunit hydrophobic N-terminal signal might be to trigger opening of the FlhAB-FliPQR export gate, which rests in an energetically favourable closed conformation to maintain the permeability barrier across the bacterial inner membrane (Kuhlen et al., 2018; Butan et al., 2019; Ward et al., 2018; Kuhlen et al., 2020; Bryant and Fraser, 2021). The atomic resolution structure of FliPQR showed that it contains three gating regions (Kuhlen et al., 2018). FliR provides a loop (the R-plug) that sits within the core of the structure. Below this, five copies of FliP each provides three methionine residues that together form a methionine-rich ring (M-gasket) under which ionic interactions between adjacent FliQ subunits hold the base of the structure shut (Q-latch). Mutational and evolutionary analyses have shown that the R-plug, M-gasket, and Q-latch stabilize the closed export gate conformation to maintain the membrane barrier, preventing the leakage of small molecules whilst allowing the passage of substrates into the export channel (Bryant and Fraser, 2021; Hüsing et al., 2021). We have also shown that the FliPQR export gate opens and closes in response to export substrate availability, indicating that the export gate reverts to a closed conformation in the absence of export substrates, thereby maintaining the integrity of the cell membrane (Bryant and Fraser, 2021). These data indicate that the export gate must be opened in response to substrate docking at the export machinery (Bryant and Fraser, 2021; Hüsing et al., 2021).

If the function of the subunit hydrophobic N-terminal signal was to trigger opening of this gate, we hypothesised that mutations which destabilised the gate’s closed conformation would suppress the motility defect associated with FlgD_short, in which the distance between the N-terminal signal and the GRM is reduced. Introduction of the FliP-M₂₁₀A mutation, which partially destabilises the gate’s closed state, did indeed partly suppress the FlgD_short motility defect. We did not find export gate variants that completely destabilised gate closure, but it may be that such mutations disrupt the membrane permeability barrier (Ward et al., 2018). This could also explain why in screens for suppressors of FlgD∆2–5 or FlgD_short we did not isolate mutations in genes encoding export gate components (data not shown).

The surface-exposed hydrophobic GRM-binding pocket on FlhB_C is well conserved across the T3SS SctU family, to which FlhB belongs (Evans et al., 2013; Zarivach et al., 2008; Lountos et al., 2009). Furthermore, the GRM is conserved in all four injectisome early subunits (SctI, SctF, SctP, OrgC) and is located at least 30 residues away from small hydrophobic residues near the subunit N-terminus (Figure 6—figure supplement 4). It therefore seems plausible that the ‘dual signal’ mechanism we propose for early flagellar export operates in all T3SS pathways.

In many other pathways, the presence of a substrate triggers opening or assembly of the export channel. The outer membrane chitin transporter in Vibrio adopts a closed conformation in which the N-terminus of a neighbouring subunit acts as a pore plug (Aunkham et al., 2018). Chitin binding to the transporter ejects the plug, opening the transport channel and allowing chitin transport (Aunkham et al., 2018). In the Sec pathway, interactions of SecA, ribosomes or pre-proteins with SecYEG can induce conformational changes that promote channel opening (Tsirigotaki et al., 2017; Ge et al., 2014; Zimmer et al., 2008; Voorhees et al., 2014). In the TAT system, which transports folded substrates across the cytoplasmic membrane, substrate binding to the TatBC complex triggers association with, and subsequent polymerisation of, TatA, which is required for substrate translocation (Palmer and Berks, 2012; Mori and Cline, 2002). All of these mechanisms serve both to conserve energy and prevent disruption of the membrane permeability barrier. Our data suggest that in a comparable way the signal of non-polar residues within the N-termini of early rod/hook subunits trigger export gate opening.

Data generated or analysed during this study are included in the manuscript and supporting files or have been submitted to Dryad (https://doi.org/10.5061/dryad.66t1g1k3x).

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Author details

1. Owain J Bryant

   Department of Pathology, University of Cambridge, Cambridge, United Kingdom

   Present address

   Department of Biochemistry, University of Oxford, Oxford, United Kingdom

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2683-038X

2. Paraminder Dhillon

   Department of Pathology, University of Cambridge, Cambridge, United Kingdom

   Present address

   The FEBS Journal Editorial Office, Cambridge, United Kingdom

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

3. Colin Hughes

   Department of Pathology, University of Cambridge, Cambridge, United Kingdom

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

4. Gillian M Fraser

   Department of Pathology, University of Cambridge, Cambridge, United Kingdom

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing – review and editing

   For correspondence

   gmf25@cam.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4874-8734

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