Decision letter: Recognition of discrete export signals in early flagellar subunits during bacterial type III secretion
2021-04-17

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Type III Secretion Systems (T3SS) deliver subunits from the bacterial cytosol to nascent cell surface flagella. Early flagellar subunits that form the rod and hook substructures are unchaperoned and contain their own export signals. A gate recognition motif (GRM) docks them at the FlhBc component of the FlhAB-FliPQR export gate, but the gate must then be opened and subunits must be unfolded to pass through the flagellar channel. This induced us to seek further signals on the subunits. Here, we identify a second signal at the extreme N-terminus of flagellar rod and hook subunits and determine that key to the signal is its hydrophobicity. We show that the two export signal elements are recognised separately and sequentially, as the N-terminal signal is recognised by the flagellar export machinery only after subunits have docked at FlhBC via the GRM. The position of the N-terminal hydrophobic signal in the subunit sequence relative to the GRM appeared to be important, as a FlgD deletion variant (FlgDshort), in which the distance between the N-terminal signal and the GRM was shortened, ‘stalled’ at the export machinery and was not exported. The attenuation of motility caused by FlgDshort was suppressed by mutations that destabilised the closed conformation of the FlhAB-FliPQR export gate, suggesting that the hydrophobic N-terminal signal might trigger opening of the flagellar export gate. Editor's evaluation Herein, the authors show that the flagellar Type III Secretion System recognize sequentially two discrete export signals, 1. initial docking and 2. subsequent opening of the export gate, for bacterial flagella biogenesis. This important and elegantly designed study elucidates a key step in solving the long-standing question of how export substrates are recognized by the type III secretion system. https://doi.org/10.7554/eLife.66264.sa0 Decision letter eLife's review process Introduction 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 (FlhAC), 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 (FlhAN) forms the ‘head’, which is fixed in the plane of the membrane (Butan et al., 2019). FlhAN wraps around the base of a complex formed by FliPQR and the N-terminal sub-domain of FlhB (FlhBN), 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). FlhBN is connected via a linker (FlhBCN) to the cytoplasmic domain of FlhB (FlhBC), which is thought to sit between the FlhAN and FlhAC 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-FlhBN 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-FlhBN 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). Results Identification of a hydrophobic export signal at the N-terminus of FlgD The N-terminal region of flagellar rod and hook subunits is required for their export (Minamino and Macnab, 1999; Evans et al., 2013). Using the flagellar hook-cap protein FlgD as a model rod/hook subunit, we sought to identify specific export signals within the N-terminus. A screen of ten FlgD variants containing internal five-residue scanning deletions in the first 50 residues (although FlgD∆2–5 is a four-residue deletion, retaining the initial methionine) identified just two variants defective for export into culture supernatant (Figure 1A). Loss of residues 2–5 caused a significant reduction in export, as did deletion of residues 36–40, although to a lesser extent (Figure 1A; Evans et al., 2013). We have shown that FlgD residues 36–40 are the gate recognition motif (GRM) required for transient subunit docking at the FlhBC component of the export gate (Evans et al., 2013). The results suggest that the extreme N-terminus might also be important for interaction with the export machinery. Figure 1 with 5 supplements see all Download asset Open asset Screening for export-defective FlgD variants. (a) Whole cell (cell) and supernatant (sec) proteins from late exponential phase cultures of a Salmonella flgD null strain expressing plasmid-encoded wild type FlgD (FlgD) or its variants (Δ2–5, Δ6–10, Δ11–15, Δ16–20, Δ21–25, Δ26–30, Δ31–35, Δ36–40, Δ41–45 or Δ46–50) were separated by SDS (15%)-PAGE and analysed by immunoblotting with anti-FlgD polyclonal antisera. (b) A schematic displaying all intragenic suppressor mutations within amino acids 1–40 of FlgD isolated from the FlgDΔ2–5 variant. Small non-polar residues are highlighted in orange. All suppressor mutations were located between the gate-recognition motif (GRM, blue) and the extreme N-terminus, and can be separated into two classes: insertions or duplications that introduced additional sequence between valine-15 and the gate-recognition motif, or missense mutations that re-introduce small non-polar residues at the N-terminus. All intragenic suppressors isolated from FlgDΔ2–5 are displayed in this figure. (c) Whole cell (cell) and supernatant (sec) proteins from late exponential-phase cultures of Salmonella flgD null strains expressing plasmid-encoded: suppressor mutants isolated from the FlgDΔ2–5 variant (FlgDΔ2–5-N8I or FlgDΔ2–5-T11I), FlgDΔ2–5 variant (-) or wild type FlgD (FlgD) were separated by SDS (15%)-PAGE and analysed by immunoblotting with anti-FlgD polyclonal antisera. (d) Whole cell (cell) and supernatant (sec) proteins from late exponential phase cultures of Salmonella flgD null strains expressing plasmid-encoded wild type FlgD (labelled FlgD), FlgDΔ2–5 (labelled as DΔ2–5) or variants of FlgDΔ2–5 containing between residues 19 and 20 a six-residue insertion of either small non-polar (AGAGAG) residues (labelled as 3 x(AG)), polar (STSTST) residues (labelled as 3 x(ST)), or the sequence from an isolated insertion suppressor mutant (GSGSMT) (labelled as GSGSMT), were separated by SDS (15%)-PAGE and analysed by immunoblotting with anti-FlgD polyclonal antisera. Figure 1—source data 1 Full-length protein western blot of secreted proteins relating to Figure 1A. https://cdn.elifesciences.org/articles/66264/elife-66264-fig1-data1-v2.pdf Download elife-66264-fig1-data1-v2.pdf Figure 1—source data 2 Full-length western blot of cellular proteins relating to Figure 1A. https://cdn.elifesciences.org/articles/66264/elife-66264-fig1-data2-v2.pdf Download elife-66264-fig1-data2-v2.pdf Figure 1—source data 3 Full-length western blot of cellular and secreted proteins relating to Figure 1C (bottom). https://cdn.elifesciences.org/articles/66264/elife-66264-fig1-data3-v2.pdf Download elife-66264-fig1-data3-v2.pdf Figure 1—source data 4 Full-length western blot of cellular proteins relating to Figure 1D (low exposure, bottom). https://cdn.elifesciences.org/articles/66264/elife-66264-fig1-data4-v2.pdf Download elife-66264-fig1-data4-v2.pdf Figure 1—source data 5 Full-length western blot of cellular proteins relating to Figure 1D (high exposure, bottom). https://cdn.elifesciences.org/articles/66264/elife-66264-fig1-data5-v2.pdf Download elife-66264-fig1-data5-v2.pdf To gain insight into the putative new signal, we screened for intragenic suppressor mutations that could restore export of the FlgD∆2–5 variant. A Salmonella flgD null strain expressing flgD∆2–5 in trans was inoculated into soft-tryptone agar and incubated until ‘spurs’ of motile cell populations appeared. Sequencing of flgD∆2–5 alleles from these motile populations identified ten different intragenic gain-of-function mutations. These could be separated into two classes (Figure 1B, Figure 1—figure supplement 1). The first class of motile revertants carried flgD∆2–5 alleles with missense mutations that introduced small non-polar residues at the extreme N-terminus of FlgD∆2–5 (Figure 1B). Deletion of residues 2–5 (2SIAV5) had removed all small non-polar amino acids from the first ten residue region of FlgD, effectively creating a new N-terminus containing a combination of polar, charged or large non-polar residues (Figure 1—figure supplement 2). Analysis of other flagellar rod and hook subunit primary sequences revealed that in every case their native N-terminal regions contain small non-polar residues positioned upstream of the gate recognition motif (GRM residues 36–40; Figure 1—figure supplement 2), indicating that hydrophobicity may be key to the function of the N-terminal export signal. Removal of non-polar residues from the extreme N-terminus of the secreted hook-length control protein, FliK, attenuated its export, indicating that the hydrophobic N-terminal signal is required for export of early subunits (Figure 1—figure supplement 3). Export assays performed with two representative motile revertant strains carrying flgD∆2–5 variants with gain-of-function point mutations, those encoding FlgD∆2–5-N8I and FlgD∆2–5-T11I, revealed that export of these subunits had recovered to ~50% of the level observed for wild type FlgD (Figure 1C, Figure 1—figure supplements 4–5). The second class of motile revertants carried flgD∆2–5 alleles that had acquired duplications or insertions introducing at least six additional residues between the FlgD∆2–5 N-terminus and the gate recognition motif (GRM; Figure 1B). It seemed possible that these insertions/duplications might have restored subunit export either by insertion of amino acids that could function as a ‘new’ hydrophobic export signal, or by restoring the position of an existing small hydrophobic residue or sequence relative to the GRM. To assess these possibilities, we tested whether export of FlgD∆2–5 could be recovered by inserting either polar (19STSTST20) or small non-polar (19AGAGAG20) residues in the FlgD∆2–5 N-terminal region at a position equivalent to one of the suppressing duplications (19GSGSMT20; Figure 1B and D, Figure 1—figure supplements 4–5). We reasoned that if suppression by the additional sequence had been caused by repositioning an existing small hydrophobic amino acid relative to the GRM, then any insertional sequence (polar or non-polar) would restore export, while if suppression had resulted from insertion of a ‘new’ export signal, then either the polar STSTST or non-polar AGAGAG, but not both, could be expected to restore export. We found that both the engineered FlgD variants (FlgD∆2-5-19AGAGAG20 and FlgD∆2-5-19STSTST20) were exported from a Salmonella flgD null strain as effectively as the gain-of-function mutant FlgD∆2-5-19GSGSMT20 isolated from the suppressor screen (Figure 1D). This suggests that the insertions had repositioned a sequence in the FlgD∆2–5 N-terminus relative to the GRM to overcome the loss of small hydrophobic residues. The position of the hydrophobic export signal relative to the gate recognition motif is critical for Rod and Hook subunit export The intragenic suppressor experiments indicated that FlgD export requires a hydrophobic export signal towards the N-terminus and that the position of this hydrophobic signal relative to the previously described GRM is important. Sequence analysis of the gain-of-function FlgD∆2–5 insertion variants revealed that the insertions were all located between the GRM and valine15 (V15; Figure 1B). We reasoned that they repositioned valine15 relative to the GRM, such that it could perform the function of the N-terminal hydrophobic signal lost in FlgD∆2–5. To test this view, we replaced V15 by alanine in the gain-of-function variant FlgD∆2-5-19(GSGSMT)20 and assayed its export in the Salmonella flgD null (Figure 2A, Figure 2—figure supplement 1). Unlike the flgD null strain producing either the parental FlgD∆2-5-19(GSGSMT)20 or wild type FlgD, the flgD null carrying variant FlgD∆2-5-19(GSGSMT)20-V15A was non-motile, reflecting the variant’s failure to export (Figure 2A, Figure 2—figure supplement 1). This suggests that the V15 residue had indeed compensated for the missing N-terminal hydrophobic signal. Figure 2 with 2 supplements see all Download asset Open asset Export of FlgD variants in which the position of the hydrophobic export signal is varied relative to the gate recognition motif (GRM). Whole cell (cell) and supernatant (sec) proteins from late exponential-phase cultures of a Salmonella flgD null strain expressing plasmid encoded suppressor mutants isolated from the FlgDΔ2-5-19(GSGSMT)20 V15A variant (V15A-M7I, V15A-D9A, V15A-T11I, V15A-G14V), their parent FlgD variant FlgDΔ2-5-19(GSGSMT)20 V15A (labelled as V15A), FlgDΔ2-5-19(GSGSMT)20 (labelled as -) or wild type FlgD (FlgD) were separated by SDS (15%)-PAGE and analysed by immunoblotting with anti-FlgD polyclonal antisera. Swimming motility (bottom panel; 0.25% soft tryptone agar) of the same strains were carried out at 37 °C for 4–6 hours. (b). Whole cell (cell) and supernatant (sec) proteins from late exponential-phase cultures of a Salmonella flgD null strain expressing plasmid-encoded wild type FlgD (labelled as FlgD), FlgDΔ9–32 or its variants in which residues 9–32 were replaced by between one and four six-residue repeats of Gly-Ser-Thr-Asn-Ala-Ser (GSTNAS): (Δ9–32 4xRpt, Δ9–32 3xRpt, Δ9–32 2xRpt or Δ9–32 1xRpt) were separated by SDS (15%)-PAGE and analysed by immunoblotting with anti-FlgD polyclonal antisera. Swimming motility (bottom panel; 0.25% soft tryptone agar) of the same strains were carried out at 37 °C for 4–6 hours. (c). Whole cell (cell) and supernatant (sec) proteins from late exponential-phase cultures of a Salmonella flgD null strain expressing plasmid-encoded wild type FlgD (labelled as FlgD), a FlgD variant in which residues 9–32 were replaced by two repeats of a six-residue sequence Gly-Ser-Thr-Asn-Ala-Ser (labelled as 2xRpt) or its variants containing between one and five additional residues inserted directly after the two repeats (labelled as 2xRpt + 1, 2xRpt + 2, 2xRpt + 3, 2xRpt + 4 or 2xRpt + 5) were separated by SDS (15%)-PAGE and analysed by immunoblotting with anti-FlgD polyclonal antisera. Swimming motility (bottom panel; 0.25% soft tryptone agar) of the same strains were carried out at 37 °C for 4–6 hr. (d). Whole cell (cell) and supernatant (sec) proteins from late exponential-phase cultures of a Salmonella flgD null strain expressing plasmid-encoded wild type FlgD (labelled as FlgD), a FlgD variant in which residues 9–32 were replaced by two repeats of a six-residue sequence Gly-Ser-Thr-Asn-Ala-Ser (labelled as FlgDshort) or suppressor mutants isolated from this strain (labelled as rev1, rev2 or rev3) were separated by SDS (15%)-PAGE and analysed by immunoblotting with anti-FlgD polyclonal antisera. Swimming motility (bottom panel; 0.25% soft tryptone agar) of the same strains were carried out at 37 °C for 4–6 hr. (e). N-terminal sequences of wild type FlgD and its variants aligned to their gate-recognition motif (GRM; blue). The following sequence features or residues are displayed: The N-terminal hydrophobic signal (residue 2–5; orange), the Gly-Ser-Gly-Ser-Met-Thr (GSGSMT) insertion (green) isolated from the FlgDΔ2–5 suppressor screen, the valine-15 to alanine mutation (grey), small non-polar mutations (M7I, D9A, T11I, G14V; orange) isolated from the FlgDΔ2–5, 19GSGSMT20 suppressor screen, FlgDΔ9–32 and its variants in which residues 9–32 are replaced with one, two, three or four repeats of a six-residue sequence Gly-Ser-Thr-Asn-Ala-Ser (GSTNAS; yellow), FlgDΔ9–32, 2xrpt (hereafter termed FlgDshort) containing five, four, three, two, or one additional residues (underlined) inserted between the GRM and N-terminal hydrophobic signal, and suppressor mutants (Rev 1–7) isolated from FlgDshort that introduced additional residues (bold) between the N-terminal hydrophobic signal (orange) and the gate-recognition motif (blue). Figure 2—source data 1 Full-length protein western blot of cellular and secreted proteins relating to Figure 2A. https://cdn.elifesciences.org/articles/66264/elife-66264-fig2-data1-v2.pdf Download elife-66264-fig2-data1-v2.pdf Figure 2—source data 2 Full-length protein western blot of cellular and secreted proteins relating to Figure 2B (bottom). https://cdn.elifesciences.org/articles/66264/elife-66264-fig2-data2-v2.pdf Download elife-66264-fig2-data2-v2.pdf Figure 2—source data 3 Full-length protein western blot of cellular proteins relating to Figure 2C (bottom). https://cdn.elifesciences.org/articles/66264/elife-66264-fig2-data3-v2.pdf Download elife-66264-fig2-data3-v2.pdf Figure 2—source data 4 Full-length protein western blot of secreted proteins relating to Figure 2C (bottom). https://cdn.elifesciences.org/articles/66264/elife-66264-fig2-data4-v2.pdf Download elife-66264-fig2-data4-v2.pdf Figure 2—source data 5 Full-length protein western blot of cellular proteins relating to Figure 2D (bottom). https://cdn.elifesciences.org/articles/66264/elife-66264-fig2-data5-v2.pdf Download elife-66264-fig2-data5-v2.pdf Figure 2—source data 6 Full-length protein western blot of secreted proteins relating to Figure 2D (bottom, left). https://cdn.elifesciences.org/articles/66264/elife-66264-fig2-data6-v2.pdf Download elife-66264-fig2-data6-v2.pdf By screening for intragenic suppressors of the motility defect associated with FlgD∆2-5-19(GSGSMT)20-V15A, four gain-of-function missense mutations were identified, M7I, D9A, T11I and G14V. All these had introduced small hydrophobic residues, all positioned at least 27 residues upstream of the GRM. These FlgD∆2-5-19(GSGSMT)20-V15A gain-of-function variants restored motility to the Salmonella flgD null strain and were exported at levels similar to wildtype FlgD and FlgD∆2-5-19(GSGSMT)20 (Figure 2A, Figure 2—figure supplement 1). These data confirm the importance of small non-polar residues positioned upstream of the GRM. Our results so far had indicated that the position of the FlgD N-terminal hydrophobic export signal relative to the GRM was critical and suggested that, for export to occur efficiently, at least 26 residues must separate the hydrophobic signal and the GRM (Figure 1C, Figure 1—figure supplement 4). In the primary sequences of all Salmonella flagellar rod/hook subunits the GRM is positioned ≥30 amino acids downstream of the subunit N-terminus (Figure 1—figure supplement 4), suggesting that separation of the two signals by a minimum number of residues might be a common feature among early flagellar subunits. To test this, a suite of engineered flgD alleles was constructed that encoded FlgD variants in which wildtype residues 9–32 were replaced with between one and four repeats of the six amino acid sequence Gly-Ser-Thr-Asn-Ala-Ser (GSTNAS). Swimming motility and export assays revealed that the minimum number of inserted GSTNAS repeats that could support efficient FlgD export was three, equivalent to separation of the hydrophobic N-terminal signal and the GRM by 24 residues (Figure 2B). Below this threshold, FlgD export and swimming motility were strongly attenuated (Figure 2B). A further set of recombinant flgD alleles was constructed, which encoded FlgD∆9–32 variants carrying two GSTNAS repeats (hereafter termed FlgDshort) directly followed by between one and five additional residues (Figure 2E). Motility and FlgD export increased incrementally with the addition of each amino acid (Figure 2C, Figure 2—figure supplement 1). The data indicate that a low level of FlgD export is supported when the hydrophobic N-terminal signal (2SIAV5) and the GRM (36FLTLL40) are separated by 19 residues, with export efficiency and swimming motility increasing as separation of the export signals approaches an optimal 24 residues. To further establish the requirement for a minimum number of residues between the hydrophobic N-terminal signal and the GRM, we screened for intragenic suppressor mutations that could restore swimming motility in a flgD null strain producing FlgDshort. Sequencing of flgDshort alleles from motile revertant strains identified seven gain-of-function mutations that introduced additional residues between the hydrophobic N-terminal signal and the GRM (Figure 2—figure supplement 1). Swimming motility and FlgD export was assessed for three flgD null strains expressing representative flgDshort gain-of-function variants and all showed increased FlgD subunit export and swimming motility compared to the flgD null expressing flgDshort (Figure 2D, Figure 2—figure supplement 1). The data confirm that the position of the hydrophobic N-terminal signal relative to the GRM is critical for efficient FlgD subunit export. To establish that this is a general requirement for the export of other rod and hook subunits, engineered alleles of flgE (hook) and flgG (rod) were constructed that encoded variants in which FlgE residues 9–32 or FlgG residues 11–35 were either and or replaced with four repeats of the sequence GSTNAS (Figure 3). had been observed for export of the and variants was attenuated compared to FlgE and FlgG (Figure 3, Figure 2—figure supplement 2). insertion of four GSTNAS repeats into and recovered subunit export to indicating that the minimum separation of the hydrophobic N-terminal signal and the GRM is a feature rod and hook subunits (Figure 3). Figure 3 Download asset Open asset of the relative position of the N-terminus and GRM on the export of other rod and hook subunits. (a) of a subunit (labelled as a subunit containing a deletion of sequence from between the N-terminus and GRM (labelled as and a subunit in which the sequence was replaced by four repeats of a six-residue sequence Gly-Ser-Thr-Asn-Ala-Ser as (b). Whole cell (cell) and supernatant (sec) proteins from late exponential-phase cultures of a Salmonella flgE null strain expressing plasmid-encoded wild type FlgG (labelled as a FlgG variant in which residues 11–35 were (labelled as or a FlgG variant in which residues 11–35 were replaced by four repeats of a six-residue sequence Gly-Ser-Thr-Asn-Ala-Ser (labelled as + 4 All FlgG variants were engineered to contain an internal for were separated by SDS (15%)-PAGE and analysed by immunoblotting with antisera. (c). Whole cell (cell) and supernatant (sec) proteins from late exponential-phase cultures of a Salmonella flgD null strain expressing plasmid-encoded wild type FlgE (labelled as a FlgE variant in which residues 9–32 were (labelled as or a FlgE variant in which residues 9–32 were replaced by four repeats of a six-residue sequence Gly-Ser-Thr-Asn-Ala-Ser (labelled as + 4 All FlgE variants were engineered to contain an internal for were separated by SDS (15%)-PAGE and analysed by immunoblotting with antisera. Figure data 1 Full-length protein western blot of cellular and secreted proteins relating to Figure (low Download Figure data 2 Full-length protein western blot of cellular and secreted proteins relating to Figure (high Download Figure data 3 Full-length protein western blot of secreted proteins relating to Figure (low exposure, Download Figure data 4 Full-length protein western blot of cellular proteins relating to Figure (high exposure, bottom). Download of the subunit GRM and hydrophobic N-terminal export signal by the flagellar export machinery identified a new hydrophobic N-terminal export signal and that its position relative to the GRM was we to determine the in which the signals were by the export machinery. The signals might be recognised with both required for initial of rod/hook subunits into the export they might be recognised this were the then a subunit variant that the signal but was for the signal might the export but to at a specific step to the and export of wild type subunits. To test if FlgD∆2–5 or in the export recombinant encoding these variants or wild type FlgD were introduced into a Salmonella strain that is wild type for flagellar export (Figure 4). We could then assess whether the variant FlgD could in trans with the wildtype flagellar export. To do this, we assessed the export of an early flagellar FliK, which the length of the flagellar hook and of the late export