Recognition of discrete export signals in early flagellar subunits during bacterial type III secretion
- Department of Pathology, University of Cambridge, United Kingdom
Figures
Figure 1 with 5 supplements
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.
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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
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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
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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
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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
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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
Figure 1—figure supplement 1
Relative amounts of FlgD secreted into culture supernatants from a recombinant Salmonella flgD null strain expressing plasmid-based FlgDΔ2–5 suppressor mutants and wild type FlgD.
Relative amounts of FlgD secreted into culture supernatants were quantified using image Studio Lite software and normalised to the level of wild type FlgD in culture supernatants. Error bars represent the standard error of the mean calculated from at least three biological replicates.
Figure 1—figure supplement 2
Hydrophobicity of N-terminal sequences of flagellar rod and hook subunits.
(a) N-terminal sequences of all Salmonella flagellar rod and hook subunits aligned to their gate-recognition motif (GRM, blue). Small non-polar residues upstream from the gate-recognition motif are highlighted (yellow). (b). Hydrophobicity plots for the N-terminal 60 residues of each Salmonella flagellar rod and hook subunit were generated by ExPASy tools using the Kyte and Doolittle method (Kyte and Doolittle, 1982). The x axis of the plot indicates the amino acid position, starting from the N terminus. The y axis of the plot indicates the hydrophobicity of the amino acid sequence, where higher values represent higher hydrophobicity. Amino acid sequence corresponding to the gate-recognition motif of each subunit is highlighted in blue.
Figure 1—figure supplement 3
Deletion of the FliK N-terminal signal attenuates export.
Whole cell (cell) and supernatant (secreted) proteins from late exponential-phase cultures of Salmonella flgD null strains expressing wild type FliK containing a C-terminal myc tag for immunodetection (FliK) or its variant in which residues two to eight are deleted (FliKΔ2–8) were separated by SDS (15%)-PAGE and analysed by immunoblotting with anti-myc monoclonal antisera (top), anti-FlhA (middle), or anti-FlgN (bottom) polyclonal anti-sera.
Apparent molecular weights are in kilodaltons (kDa).
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Figure 1—figure supplement 3—source data 1
Full-length protein western blot of cellular and secreted proteins relating to Figure 1—figure supplement 1 (anti-myc).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig1-figsupp3-data1-v2.pdf
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Figure 1—figure supplement 3—source data 2
Full-length protein western blot of cellular and secreted proteins relating to Figure 1—figure supplement 1 (anti-FlhA).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig1-figsupp3-data2-v2.pdf
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Figure 1—figure supplement 3—source data 3
Full-length protein western blot of cellular and secreted proteins relating to Figure 1—figure supplement 1 (anti-FlgN).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig1-figsupp3-data3-v2.pdf
Figure 1—figure supplement 4
Swimming motility of FlgDdelta2-5 suppressor mutant strains.
(a) Swimming motility of a Salmonella ΔrecA strain expressing: suppressor mutants isolated from the FlgDΔ2–5 variant (labelled as FlgDΔ2–5 N8I or FlgDΔ2–5 T11I), the parent FlgDΔ2–5 variant (labelled as DΔ2–5) or wild type FlgD (labelled as FlgD). Motility was assessed in 0.25% soft-tryptone agar containing 100 μg/ml ampicillin and 50 μM IPTG and incubated 4–6 hours at 37 °C. (b). Swimming motility of a Salmonella ΔrecA strain expressing wild type FlgD (labelled as FlgD), FlgDΔ2–5 (labelled as DΔ2–5) or its variants containing a six-residue insertion between residues 19 and 20 of either small non-polar (AGAGAG) residues (labelled as DΔ2–5 3 x(AG)), polar (STSTST) residues (labelled as DΔ2–5 3 x(ST)), or the sequence from an isolated insertion suppressor mutant (GSGSMT) (labelled as DΔ2–5 GSGSMT). Motility was assessed in 0.25% soft-tryptone agar containing 100 μg/ml ampicillin and 50 μM IPTG and incubated 4–6 hr at 37 °C.
Figure 1—figure supplement 5
Control blots for Figure 1C-D showing that membrane embedded FlhA and cytoplasmic FlgN proteins are expressed (cell) but absent from supernatant fractions (sec).
(a) Whole cell (cell) and supernatant (secreted) proteins from late exponential-phase cultures of Salmonella flgD null strains expressing: 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-FlhA or FlgN polyclonal antisera. Apparent molecular weights are in kilodaltons (kDa). (b). Whole cell (cell) and supernatant (secreted) proteins from late exponential-phase cultures of Salmonella flgD null strains expressing wild type FlgD (FlgD), FlgDΔ2–5 (labelled as DΔ2–5) or its variants containing a six-residue insertion between residues 19 and 20 of either small non-polar (AGAGAG) residues (labelled as DΔ2–5 3 x(AG)), polar (STSTST) residues (labelled as DΔ2–5 3 x(ST)), or the sequence from an isolated insertion suppressor mutant (GSGSMT) (labelled as DΔ2–5 GSGSMT) were separated by SDS (15%)-PAGE and analysed by immunoblotting with anti-FlgD polyclonal antisera. Apparent molecular weights are in kilodaltons (kDa).
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Figure 1—figure supplement 5—source data 1
Full-length protein western blot of cellular and secreted proteins relating to Figure 1—figure supplement 5A (anti-FlhA (top) and anti-FlgN (bottom)).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig1-figsupp5-data1-v2.pdf
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Figure 1—figure supplement 5—source data 2
Full-length protein western blot of cellular and secreted proteins relating to Figure 1—figure supplement 5B (anti-FlhA (top) and anti-FlgN (bottom)).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig1-figsupp5-data2-v2.pdf
Figure 2 with 2 supplements
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).
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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
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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
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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
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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
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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
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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
Figure 2—figure supplement 1
Control blots for Figure 2A-C showing that membrane-embedded FlhA and cytoplasmic FlgN are expressed (cell) but absent from supernatant fractions (sec).
(a) Whole cell (cell) and supernatant (secreted) proteins from late exponential-phase cultures of a Salmonella flgD null strain expressing suppressor mutants isolated from a 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 (-) or wild type FlgD (FlgD) were separated by SDS (15%)-PAGE and analysed by immunoblotting with anti-FlhA and FlgN polyclonal antisera. Apparent molecular weights are in kilodaltons (kDa). (b). Whole cell (cell) and supernatant (secreted) proteins from late exponential-phase cultures of a Salmonella flgD null strain expressing wild type FlgD (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, Δ9–32 1xRpt) were separated by SDS (15%)-PAGE and analysed by immunoblotting with anti-FlhA and anti-FlgN polyclonal antisera. Apparent molecular weights are in kilodaltons (kDa). (c). Whole cell (cell) and supernatant (secreted) proteins from late exponential-phase cultures of a Salmonella flgD null strain expressing 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. Apparent molecular weights are in kilodaltons (kDa). (d). Whole cell (cell) and supernatant (secreted) proteins from late exponential-phase cultures of a Salmonella flgD null strain expressing 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-FlhA and anti-FlgN polyclonal antisera. Apparent molecular weights are in kilodaltons (kDa).
Figure 2—figure supplement 2
Control blots for Figure 3B-C showing that membrane embedded FlhA and cytoplasmic FlgN proteins (cell) are expressed but absent from supernatant fractions (sec).
(a) Whole cell (cell) and supernatant (sec) proteins from late exponential-phase cultures of a Salmonella flgE null strain expressing wild type FlgG (labelled as FlgGwild type), a FlgG variant in which residues 11–35 were deleted (labelled as FlgGshort) 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 FlgGshort + 4 Rpt). All FlgG variants were engineered to contain an internal 3xFLAG tag for immunodetection. Proteins were separated by SDS (15%)-PAGE and analysed by immunoblotting with anti-FlhA or anti-FlgN polyclonal antisera. Apparent molecular weights are in kilodaltons (kDa). (b). Whole cell (cell) and supernatant (secreted) proteins from late exponential-phase cultures of a Salmonella flgD null strain expressing wild type FlgE (labelled as FlgEwild type), a FlgE variant in which residues 9–32 were deleted (labelled as FlgEshort) 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 FlgEshort + 4 Rpt). All FlgE variants were engineered to contain an internal 3xFLAG tag for immunodetection. Proteins were separated by SDS (15%)-PAGE and analysed by immunoblotting with anti-FLAG monoclonal antisera.
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Figure 2—figure supplement 2—source data 1
Full-length protein western blot of cellular and secreted proteins relating to Figure 2—figure supplement 2A (anti-FlhA, bottom).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig2-figsupp2-data1-v2.pdf
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Figure 2—figure supplement 2—source data 2
Full length protein western blot of cellular and secreted proteins relating to Figure 2—figure supplement 2A (anti-FlgN, bottom).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig2-figsupp2-data2-v2.pdf
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Figure 2—figure supplement 2—source data 3
Full-length protein western blot of cellular and secreted proteins relating to Figure 2—figure supplement 2B (anti-FlhA, bottom).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig2-figsupp2-data3-v2.pdf
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Figure 2—figure supplement 2—source data 4
Full-length protein western blot of cellular and secreted proteins relating to Figure 2—figure supplement 2B (anti-FlgN, bottom).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig2-figsupp2-data4-v2.pdf
Figure 3
Effect of the relative position of the N-terminus and GRM on the export of other rod and hook subunits.
(a) Schematic representation of a wild-type subunit (labelled as subunitwild type), a subunit containing a deletion of sequence from between the N-terminus and GRM (labelled as subunitshort) and a subunit in which the deleted sequence was replaced by four repeats of a six-residue sequence Gly-Ser-Thr-Asn-Ala-Ser (yellow, labelled as subunitshort +4 Rpt). (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 FlgGwild type), a FlgG variant in which residues 11–35 were deleted (labelled as FlgGshort) 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 FlgGshort + 4 Rpt). All FlgG variants were engineered to contain an internal 3xFLAG tag for immunodetection. Proteins were separated by SDS (15%)-PAGE and analysed by immunoblotting with anti-FLAG monoclonal 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 FlgEwild type), a FlgE variant in which residues 9–32 were deleted (labelled as FlgEshort) 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 FlgEshort + 4 Rpt). All FlgE variants were engineered to contain an internal 3xFLAG tag for immunodetection. Proteins were separated by SDS (15%)-PAGE and analysed by immunoblotting with anti-FLAG monoclonal antisera.
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Figure 3—source data 1
Full-length protein western blot of cellular and secreted proteins relating to Figure 3B (low exposure).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig3-data1-v2.pdf
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Figure 3—source data 2
Full-length protein western blot of cellular and secreted proteins relating to Figure 3B (high exposure).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig3-data2-v2.pdf
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Figure 3—source data 3
Full-length protein western blot of secreted proteins relating to Figure 3C (low exposure, top).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig3-data3-v2.pdf
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Figure 3—source data 4
Full-length protein western blot of cellular proteins relating to Figure 3C (high exposure, bottom).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig3-data4-v2.pdf
Figure 4
Effect on subunit export of overexpressed FlgDΔ2–5 and variants.
(a) Schematic representation of a FlgD subunit containing a N-terminal hydrophobic signal (orange, labelled as 2–5) and a gate-recognition motif (blue, labelled as GRM). (b) Swimming motility of a Salmonella ΔrecA strain expressing plasmid-encoded wild type FlgD (FlgD), its variants (DΔ2-5ΔGRM, DΔ2–5 or DΔGRM) or empty pTrc99a vector (-). Motility was assessed in 0.25% soft-tryptone agar containing 100 μg/ml ampicillin and 100 μM IPTG and incubated for 4–6 hr at 37 °C. (c) Whole cell (cell) and secreted proteins (secreted) from late-exponential-phase cultures were separated by SDS (15%)-PAGE and analysed by immunoblotting with anti-FliK (hook ruler subunit), anti-FlgK and anti-FlgL (hook-filament junction subunits), anti-FlgD hook cap subunit, anti-FlhA (component of the export machinery) and anti-FlgN (export chaperone for FlgK and FlgL) polyclonal antisera. Apparent molecular weights are in kilodaltons (kDa).(d). A model depicting a FlgDΔ2–5 subunit (left) docked via its gate-recognition motif (GRM, blue) at the subunit binding pocket on FlhBC (PDB: 3B0Z Kuhlen et al., 2020, red), preventing wild type subunits (right) from docking at FlhBC.
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Figure 4—source data 1
Full-length protein western blot of cellular and secreted proteins relating to Figure 4C (anti-FliK, bottom).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig4-data1-v2.pdf
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Figure 4—source data 2
Full-length protein western blot of cellular and secreted proteins relating to Figure 4C (anti-FlgK, top).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig4-data2-v2.pdf
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Figure 4—source data 3
Full-length protein western blot of cellular and secreted proteins relating to Figure 4C (anti-FlgL).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig4-data3-v2.pdf
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Figure 4—source data 4
Full-length protein western blot of cellular proteins relating to Figure 4C (anti-FlgD, top right).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig4-data4-v2.pdf
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Figure 4—source data 5
Full-length protein western blot of secreted proteins relating to Figure 4C (anti-FlgD, bottom).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig4-data5-v2.pdf
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Figure 4—source data 6
Full-length protein western blot of cellular and secreted proteins relating to Figure 4C (anti-FlhA (top) and anti-FlgN (bottom)).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig4-data6-v2.pdf
Figure 5
Effect on subunit export of overexpressed FlgEshort, FlgDshort and variants.
(a) Swimming motility of a Salmonella ΔrecA strain expressing plasmid-encoded wild type FlgE (labelled as FlgE wild type), a FlgE variant in which residues 9–32 were deleted (labelled as FlgEshort), a FlgE variant in which residues 9–32 and residues 39–43 (corresponding to the gate-recognition motif) were deleted (labelled as FlgEshortΔGRM), a FlgE variant in which residues 39–43 were deleted (labelled as FlgEΔGRM) or empty pTrc99a vector (labelled as -). All FlgE variants were engineered to contain an internal 3xFLAG tag for immunodetection. Motility was assessed in 0.25% soft-tryptone agar containing 100 μg/ml ampicillin and 100 μM IPTG and incubated for 4–6 hr at 37 °C (top panel). Whole cell (cell) and secreted proteins (secreted) from late-exponential-phase cultures were separated by SDS (15%)-PAGE and analysed by immunoblotting with anti-FLAG monoclonal antisera (to detect the flag tagged hook subunit FlgE) or anti-FlgD (hook cap subunit), anti-FliD (filament cap subunit), anti-FlgK (hook-filament junction subunit), anti-FliK (hook-ruler subunit), anti-FlhA (component of the export machinery) and anti-FlgN (chaperone for FlgK and FlgL) polyclonal antisera (bottom). Apparent molecular weights are in kilodaltons (kDa).(b) Swimming motility of a Salmonella ΔrecA strain expressing plasmid-encoded wild type FlgD (labelled as FlgD wild type), a FlgD variant in which residues 9–32 were replaced with two repeats of the six amino acid sequence Gly-Ser-Thr-Asn-Ala-Ser (labelled as FlgDshort), a FlgD variant in which residues 9–32 were replaced with two repeats of the six amino acid sequence Gly-Ser-Thr-Asn-Ala-Ser and residues 36–40 were deleted (labelled FlgDshortΔGRM), a FlgD variant in which residues 36–40 were deleted (labelled as FlgDΔGRM) or empty pTrc99a vector (labelled as -). Motility was assessed in 0.25% soft-tryptone agar containing 100 μg/ml ampicillin and 100 μM IPTG and incubated for 4–6 hours at 37 °C (top panel). Whole cell (cell) and secreted proteins (secreted) from late-exponential-phase cultures were separated by SDS (15%)-PAGE and analysed by immunoblotting with anti-FlgD (hook cap subunit), anti-FliD (filament cap subunit), anti-FlgK (hook-filament junction subunit), anti-FliK (hook ruler subunit), anti-FlhA (component of export machinery), and anti-FlgN (chaperone for FlgK and FlgL) polyclonal antisera (bottom). Apparent molecular weights are in kilodaltons (kDa).
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Figure 5—source data 1
Full-length protein western blot of cellular proteins relating to Figure 4A (low contrast, anti-FLAG).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig5-data1-v2.pdf
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Figure 5—source data 2
Full-length protein western blot of secreted proteins relating to Figure 4A (high contrast, anti-FLAG).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig5-data2-v2.pdf
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Figure 5—source data 3
Full-length protein western blot of cellular and secreted proteins relating to Figure 4A (anti-FliD (top) and anti-FlgD (bottom)).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig5-data3-v2.pdf
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Figure 5—source data 4
Full-length protein western blot of cellular and secreted proteins relating to Figure 4A (anti-FlgK).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig5-data4-v2.pdf
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Figure 5—source data 5
Full-length protein western blot of cellular proteins relating to Figure 4A (anti-FliK, left).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig5-data5-v2.pdf
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Figure 5—source data 6
Full-length protein western blot of secreted proteins relating to Figure 4A (anti-FliK, bottom right).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig5-data6-v2.pdf
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Figure 5—source data 7
Full-length protein western blot of cellular and secreted proteins relating to Figure 4A (anti-FlhA, top).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig5-data7-v2.pdf
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Figure 5—source data 8
Full-length protein western blot of cellular and secreted proteins relating to Figure 4A (anti-FlgN, bottom).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig5-data8-v2.pdf
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Figure 5—source data 9
Full-length protein western blot of cellular proteins relating to Figure 4B (anti-FlgD, left).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig5-data9-v2.pdf
-
Figure 5—source data 10
Full-length protein western blot of secreted proteins relating to Figure 4B (anti-FlgD, bottom right).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig5-data10-v2.pdf
-
Figure 5—source data 11
Full-length protein western blot of cellular and secreted proteins relating to Figure 4B (anti-FliD).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig5-data11-v2.pdf
-
Figure 5—source data 12
Full-length protein western blot of cellular and secreted proteins relating to Figure 4B (anti-FlgK, top).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig5-data12-v2.pdf
-
Figure 5—source data 13
Full-length protein western blot of cellular and secreted proteins relating to Figure 4B (anti-FliK).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig5-data13-v2.pdf
-
Figure 5—source data 14
Full-length protein western blot of cellular and secreted proteins relating to Figure 4B (anti-FlhA (top) and anti-FlgN (bottom)).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig5-data14-v2.pdf
Figure 6 with 4 supplements
Suppression of the FlgDshort motility defect by mutations in FliP.
(a) A model depicting subunits docked via their gate-recognition motif (GRM, blue) at the subunit binding pocket on FlhBC (PDB: 3B0Z Meshcheryakov et al., 2013, red) with N-termini of early flagellar subunits adopting either an α-helical conformation separating the N-terminal hydrophobic signal (2–5, orange) and gate-recognition motif (GRM, blue) by ~40–60 ångstrom (where each amino acid is on average separated by ~1.5 Å, left) or an unfolded conformation where the unfolded contour length separating the N-terminal hydrophobic signal (2-5) and gate-recognition motif (GRM) is ~90–150 ångstrom (where each amino acid is on average separated by ~3.5 Å, middle left). Values corresponding to the distance separating the N-terminal hydrophobic signal (2–5, orange) and gate-recognition motif (GRM, blue) of a FlgD subunit variant in which residues 9–32 are replaced with two repeats of the six amino acid sequence Gly-Ser-Thr-Asn-Ala-Ser (FlgDshort) indicate that the N-terminal hydrophobic signal (2–5, orange) and gate-recognition motif (GRM, blue) are separated by ~29 ångstrom (α-helical conformation, middle right) or ~67 ångstrom (unfolded contour length, right). (b) Placement of the crystal structure of FlhBc (PDB:3BOZ Kuhlen et al., 2020; red) and the cryo-EM structure of FliPQR-FlhB (PDB:6S3L Ward et al., 2018) in a tomographic reconstruction of the Salmonella SPI-1 injectisome (EMD-8544 [60]; grey). The minimum distance between the subunit gate-recognition motif binding site on FlhBC (grey) to FlhBN (defined as Salmonella FlhB residue 211 [61]; ~ 78 Å) was estimated by combining: the value corresponding to the distance between the subunit binding pocket on FlhBC (Evans et al., 2013) (grey) and the N-terminal visible residue (D229) in the FlhBC structure (PDB:3BOZ Kuhlen et al., 2020; ~ 52 Å) with the value corresponding to the minimum distance between FlhB residues 211 and 228 (based on a linear α-helical conformation; ~ 26 Å). (c) Swimming motility of recombinant Salmonella flgD null strains producing a chromosomally-encoded FliP-M210A variant (M210A gate, left) or wild type FliP (wild-type gate, right). Wildtype FliP and FliP-M210A were engineered to contain an internal HA tag positioned between residue 21 and 22 to allow immunodetection of FliP. Both strains produced either a pTrc99a plasmid-encoded FlgD subunit variant in which residues 9–32 were replaced with two repeats of the six amino acid sequence Gly-Ser-Thr-Asn-Ala-Ser (FlgDshort; top panel) or a pTrc99a plasmid-encoded wild-type FlgD subunit (FlgDwild type; bottom panel). Motility was assessed in 0.25% soft-tryptone agar containing 100 μg/ml ampicillin and 50 μM IPTG and incubated for 16 hr (top panel) or 4–6 hr at 37 °C (bottom panel). (d). The mean motility halo diameter of recombinant Salmonella flgD null strains producing a chromosomally-encoded FliP-M210A variant (M210A gate, left) or wild-type FliP (wild-type gate, right). Wild-type FliP and FliP-M210A were engineered to contain an internal HA tag positioned between residue 21 and 22 to allow immunodetection of FliP. Both strains produced a pTrc99a plasmid-encoded FlgD subunit variant in which residues 9–32 were replaced with two repeats of the six amino acid sequence Gly-Ser-Thr-Asn-Ala-Ser (FlgDshort). Error bars represent the standard error of the mean calculated from at least three biological replicates. *** indicates a p-value < 0.001.
Figure 6—figure supplement 1
Mutations in FliP suppress the motility defect associated with FlgDshort but do not change the motility of strains producing wild type FlgD.
(a) Whole cell (cell) proteins from late exponential-phase cultures of recombinant Salmonella flgD null strains producing a chromosomally-encoded FliP-M210A variant (M210A gate, left) or wild-type FliP (wild-type gate, right). Wild-type FliP and FliP-M210A were engineered to contain an internal HA tag positioned between residue 21 and 22 to allow immunodetection of FliP (Ward et al., 2018) (bottom panel). Both strains produced either a pTrc99a plasmid-encoded FlgD subunit variant in which residues 9–32 were replaced with two repeats of the six amino acid sequence Gly-Ser-Thr-Asn-Ala-Ser (FlgDshort; top panel) or a pTrc99a plasmid-encoded wild type FlgD subunit (FlgDwild type; middle panel). Proteins were separated by SDS (15%)-PAGE and analysed by immunoblotting with anti-FlgD polyclonal antisera or anti-HA monoclonal antisera. (b). The mean motility halo diameter of recombinant Salmonella flgD null strains producing a chromosomally-encoded FliP-M210A variant (M210A gate, left) or wild-type FliP (wild-type gate, right). Wild-type FliP and FliP-M210A were engineered to contain an internal HA tag positioned between residue 21 and 22 to allow immunodetection of FliP. Both strains produced a pTrc99a plasmid-encoded wild type FlgD. Error bars represent the standard error of the mean calculated from at least three biological replicates. *** indicates a p-value < 0.001.
-
Figure 6—figure supplement 1—source data 1
Full-length protein western blot of cellular proteins relating to Figure 6—figure supplement 1A (anti-HA).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig6-figsupp1-data1-v2.pdf
-
Figure 6—figure supplement 1—source data 2
Full-length protein western blot of cellular proteins relating to Figure 6—figure supplement 1A (anti-FlgD).
- https://cdn.elifesciences.org/articles/66264/elife-66264-fig6-figsupp1-data2-v2.pdf
Figure 6—figure supplement 2
Amino acid sequence alignments of N-terminal regions of Salmonella FlgD cap, FlgE hook and FliK ruler with flagellar subunits from other bacterial species.
(a) The N-terminal sequence of the Salmonella FlgD cap subunit aligned by the conserved gate-recognition motif (GRM, blue) with N-terminal sequences of FlgD from other bacterial species. Small non-polar residues located N-terminal from the minimum distance threshold of 24 residues from the gate-recognition motif are highlighted (yellow). (b). The N-terminal sequence of the Salmonella FlgE hook subunit aligned by the conserved gate-recognition motif (GRM, blue) with N-terminal sequences of FlgE from other bacterial species. Small non-polar residues located N-terminal from the minimum distance threshold of 24 residues from the gate-recognition motif are highlighted (yellow). (c). The N-terminal sequence of the Salmonella FliK ruler subunit aligned by the conserved gate-recognition motif (GRM, blue) with N-terminal sequences of FliK from other bacterial species. Small non-polar residues located N-terminal from the minimum distance threshold of 24 residues from the gate-recognition motif are highlighted (yellow).
Figure 6—figure supplement 3
The substrate binding cytoplasmic domain of FlhB is predicted to be positioned between the plane of the inner membrane and cytoplasmic domain of FlhA.
(a) Cryo-EM structure of the FliPQR-FlhB complex (PDB:6S3L Kuhlen et al., 2020, left) displaying FliPQR (green) and FlhB residues 1–221 (red), the alphafold predicted structure of full-length FlhB (red, middle) and the Cryo-EM structure of the PrgHK-SpaPQR complex (PDB:6PEM) (Hu et al., 2019) displaying PrgHK (magneta) in complex with the FliPQR homologue, SpaPQR (green). (b). Structures from (a) were superimposed in chimera using MatchMaker to generate a predicted model of the PrgHK - FliPQR - full-length FlhB complex (left). A tomographic reconstruction of the Salmonella SPI-1 vT3SS (EMD-20838) (Butan et al., 2019) is shown in grey (right). (c). Placement of the PrgHK – FliPQR – full-length FlhB predicted model in the tomographic reconstruction of the Salmonella SPI-1 vT3SS, which suggests that the substrate binding cytoplasmic domain of FlhB (FlhBC) is positioned below the plane of inner membrane and above the visible nonameric ring formed by the cytoplasmic domain of FlhA (FlhAC). Using the predicted model, the distance between the GRM binding site on FlhBC and the base of the FliPQR-FlhBN complex is estimated to be between 70 and 80 angstrom.
Figure 6—figure supplement 4
A schematic of an early flagellar subunit containing an N-terminal export signal (yellow) and gate-recognition motif (GRM, blue).
The essential gate recognition motif (GRM) of Salmonella FlgD (residues 36–40) was aligned with homologous regions in other early flagellar subunits and with the inner rod and ruler subunits of the injectisome from Salmonella and other bacterial species.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background, (Salmonella enterica serovar Typhimurium) | SJW1103 | doi:10.1099/00221287-130-12-3339 | wildtype | This strain can be obtained from the Fraser lab upon request |
| Strain, strain background, (Salmonella enterica serovar Typhimurium) | recA null | This work | recA gene replaced with kanamycin resistance cassette | This strain can be obtained from the Fraser lab upon request |
| Strain, strain background, (Salmonella enterica serovar Typhimurium) | flgD null | doi:10.1038/nature12682 | flgD gene replaced with kanamycin resistance cassette | This strain can be obtained from the Fraser lab upon request |
| Strain, strain background, (Salmonella enterica serovar Typhimurium) | fliP(M210A)internal HA tag, flgD null | This work | Triple HA tag inserted between residue 21 and 22 of fliP and M210A mutation introduced into the fliP gene, flgD gene replaced with kanamycin resistance cassette. | This strain can be obtained from the Fraser lab upon request |
| Strain, strain background, (Salmonella enterica serovar Typhimurium) | fliPinternal HA tag, flgD null | This work | Triple HA tag inserted between residue 21 and 22 introduced into the fliP gene, flgD gene replaced with kanamycin resistance cassette. | This strain can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD | This work | FlgD residues 1-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆2–5 | This work | FlgD residues 1, 6-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆6–10 | This work | FlgD residues 1–5, 11-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆11–15 | This work | FlgD residues 1–10, 16-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆16–20 | This work | FlgD residues 1–15, 21-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆21–25 | This work | FlgD residues 1–20, 26-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆26–30 | This work | FlgD residues 1–25, 31-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆31–35 | This work | FlgD residues 1–30, 36-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆36–40 | This work | FlgD residues 1–35, 41-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆41–45 | This work | FlgD residues 1–40, 46-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆46–50 | This work | FlgD residues 1–45, 51-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆2–5, ∆GRM | This work | FlgD residues 1, 6–35, 41-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆2–5 19(AGAGAG)20 | This work | FlgD residues 1–19, Ala-Gly-Ala-Gly-Ala-Gly, 20-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆2–5 19(STSTST)20 | This work | FlgD residues 1–19, Ser-Thr-Ser-Thr-Ser-Thr, 20-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆2–5 19(GSGSMT)20 | This work | FlgD residues 1–19, Gly-Ser-Gly-Ser-Met-Thr, 20-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆9–32, 4xRpt | This work | FlgD residues 1–8, 4 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆9–32, 3xRpt | This work | FlgD residues 1–8, 3 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆9–32, 2xRpt (FlgDshort) | This work | FlgD residues 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆9–32, 1xRpt | This work | FlgD residues 1–8, Gly-Ser-Thr-Asn-Ala-Ser, 33-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆9–32 | This work | FlgD residues 1–8, 33-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆9–32, 2xRpt + 1 | This work | FlgD residues 1–8, Gly, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆9–32, 2xRpt + 2 | This work | FlgD residues 1–8, Gly-Ser, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆9–32, 2xRpt + 3 | This work | FlgD residues 1–8, Gly-Ser-Thr, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆9–32, 2xRpt + 4 | This work | FlgD residues 1–8, Gly-Ser-Thr-Asn, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆9–32, 2xRpt + 5 | This work | FlgD residues 1–8, Gly-Ser-Thr-Asn-Ala, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgDshort, ∆GRM | This work | FlgD residues 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33–35, 41-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆2–5 | This work | FlgD residues 1, 6-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆2–5, M7I | This work | FlgD residues 1, 6-232aa, M7I | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆2–5, M7V | This work | FlgD residues 1, 6-232aa, M7V | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆2–5, N8I | This work | FlgD residues 1, 6-232aa, N8I | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆2–5, D9A | This work | FlgD residues 1, 6-232aa, D9A | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆2–5, P10L | This work | FlgD residues 1, 6-232aa, P10L | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆2–5, T11I | This work | FlgD residues 1, 6-232aa, T11I | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆2–5, 23(TTGSGS)24 | This work | FlgD residues 1–23, Thr-Thr-Gly-Ser-Gly-Ser, 24-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆2–5, 23(TTGSGSTTGSGS)24 | This work | FlgD residues 1–23, Thr-Thr-Gly-Ser-Gly-Ser-Thr-Thr-Gly-Ser-Gly-Ser, 24-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆2–5, 27(GSMTGS)28 | This work | FlgD residues 1–27, Gly-Ser-Met-Thr-Gly-Ser, 28-232aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆9–32, 8 (2xGSTNAS)33, V15A | This work | FlgD residues 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa, V15A | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆9–32, 8 (2xGSTNAS)33, V15A, M7I | This work | FlgD residues 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa, M7I | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆9–32, 8 (2xGSTNAS)33, V15A, D9A | This work | FlgD residues 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa, D9A | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆9–32, 8 (2xGSTNAS)33, V15A, T11I | This work | FlgD residues 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa, T11I | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆9–32, 8 (2xGSTNAS)33, V15A, G14V | This work | FlgD residues 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa, G14V | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆9–32, 8 (2xGSTNAS-TNPGSTNAS)33 | This work | FlgD residues 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), (Thr-Asn-Pro-Gly-Ser-Thr-Asn-Ala-Ser) 33-232aa, | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆9–32, 8 (2xGSTNAS-GNASGSTNAS)33 | This work | FlgD residues 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), (Gly-Asn-Ala-Ser-Gly-Ser-Thr-Asn-Ala-Ser) 33-232aa, | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆9–32, 8 (2xGSTNAS-QSSFLTLLVAQLKNQDPTNPLQNNELTTQLA)33 | This work | FlgD residues 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), (Gln-Ser-Ser-Phe-Leu-Thr-Leu-Leu-Val-Ala-Gln-Leu-Lys-Asn-Gln-Asp-Pro-Thr-Asn-Pro-Leu-Asn-Asn-Glu-Leu-Thr-Thr-Gln-Leu-Ala), 33-232aa, | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆9–32, 8 (2xGSTNAS-TNASGSTNAS)33 | This work | FlgD residues 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), (Thr-Asn-Ala-Ser-Gly-Ser-Thr-Asn-Ala-Ser) 33-232aa, | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆9–32, 8 (2xGSTNAS-QSSLGSTNAS)34 | This work | FlgD residues 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), (Gln-Ser-Ser-Leu-Gly-Ser-Thr-Asn-Ala-Ser) 33-232aa, | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆9–32, 8 (2xGSTNAS-QNASGSTNAS)35 | This work | FlgD residues 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), (Gln-Asn-Ala-Ser-Gly-Ser-Thr-Asn-Ala-Ser) 33-232aa, | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgD∆9–32, 8 (2xGSTNAS-TNTFGTLIAS)36 | This work | FlgD residues 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), (Thr-Asn-Thr-Phe-Gly-Thr-Leu-Iso-Ala-Ser) 33-232aa, | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgG | This work | FlgG residues 1–144, FLAGx3, 145-260aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgG∆short | This work | FlgG residues 1–10, 35–144, FLAGx3, 145-260aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgGshort+ linker | This work | FlgG residues 1–10, 4 x(Gly-Ser-Thr-Asn-Ala-Ser) 35–144, FLAGx3, 145-260aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgE | This work | FlgE residues 1–234, FLAGx3, 235-403aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgEshort | This work | FlgE residues 1–8, 33–234, FLAGx3, 235-403aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgEshort+ linker | This work | FlgE residues 1–8, 4 x(Gly-Ser-Thr-Asn-Ala-Ser) 33–234, FLAGx3, 235-403aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgE∆GRM | This work | FlgE residues 1–38, 44–234, FLAGx3, 235-403aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FlgEshort, ∆GRM | This work | FlgE residues 1–8, 33–38, 44–234, FLAGx3, 235-403aa | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FliKmyc | This work | FliK residues 1-405aa, myc | This vector can be obtained from the Fraser lab upon request |
| Recombinant DNA reagent | pTrc99a FliKmyc∆2–8 | This work | FliK residues 1, 9-405aa, myc | This vector can be obtained from the Fraser lab upon request |
| Antibody | anti-FLAG (Mouse monoclonal) | Sigma-Aldrich | Cat# F3165, RRID:AB_259529 | Mouse monoclonal against FLAG tag (1:1000) |
| Antibody | Anti-HA Tag, HRP conjugate(Mouse monoclonal) | Thermo Fisher Scientific | Cat # 26183-HRP, RRID:AB_2533056 | Mouse monoclonal against HA tag (1:1000) |
| Antibody | anti-Myc (9B11), HRP conjugate (Mouse monoclonal) | Cell signalling technology | Cat # 2040, RRID:AB_2148465 | Mouse monoclonal against Myc tag (1:1000) |
| Antibody | anti-FlgD (Rabbit polyclonal) | doi:10.1038/nature12682 | Rabbit polyclonal against Salmonella FlgD (1:1000). This antibody can be obtained from the Fraser lab upon request. | |
| Antibody | anti-FliK (Rabbit polyclonal) | This work | Rabbit polyclonal against Salmonella FliK (1:1000). This antibody can be obtained from the Fraser lab upon request. | |
| Antibody | anti-FlgK (Rabbit polyclonal) | doi: 10.1111/mmi.14731 | Rabbit polyclonal against Salmonella FlgK (1:1000). This antibody can be obtained from the Fraser lab upon request. | |
| Antibody | anti-FlgL (Rabbit polyclonal) | This work | Rabbit polyclonal against Salmonella FlgL (1:1000). This antibody can be obtained from the Fraser lab upon request. | |
| Antibody | anti-FlgN (Rabbit polyclonal) | doi: 10.1111/mmi.14731 | Rabbit polyclonal against Salmonella FlgN (1:1000). This antibody can be obtained from the Fraser lab upon request. | |
| Antibody | anti-FlhA (Rabbit polyclonal) | doi: 10.1111/mmi.14731 | Rabbit polyclonal against Salmonella FlhA (1:1000). This antibody can be obtained from the Fraser lab upon request. |
Table 1
Strains and recombinant plasmids.
| Strains | Description |
|---|---|
| Salmonella typhimurium | |
| SJW1103 | wildtype |
| recA null | ∆recA::kmR |
| flgD null | ∆flgD::kmR |
| fliP(M210A)internalHAtag, flgD null | fliP(M210A) 21 (3xHA tag)22, ∆flgD::kmR |
| fliPinternalHAtag, flgD null | fliP 21 (3xHA tag)22, ∆flgD::kmR |
| Plasmids | |
| pTrc99a FlgD | 1-232aa |
| pTrc99a FlgD∆2–5 | 1, 6-232aa |
| pTrc99a FlgD∆6–10 | 1–5, 11-232aa |
| pTrc99a FlgD∆11–15 | 1–10, 16-232aa |
| pTrc99a FlgD∆16–20 | 1–15, 21-232aa |
| pTrc99a FlgD∆21–25 | 1–20, 26-232aa |
| pTrc99a FlgD∆26–30 | 1–25, 31-232aa |
| pTrc99a FlgD∆31–35 | 1–30, 36-232aa |
| pTrc99a FlgD∆36–40 | 1–35, 41-232aa |
| pTrc99a FlgD∆41–45 | 1–40, 46-232aa |
| pTrc99a FlgD∆46–50 | 1–45, 51-232aa |
| pTrc99a FlgD∆2–5, ∆GRM | 1, 6–35, 41-232aa |
| pTrc99a FlgD∆2–5 19(AGAGAG)20 | 1–19, Ala-Gly-Ala-Gly-Ala-Gly, 20-232aa |
| pTrc99a FlgD∆2–5 19(STSTST)20 | 1–19, Ser-Thr-Ser-Thr-Ser-Thr, 20-232aa |
| pTrc99a FlgD∆2–5 19(GSGSMT)20 | 1–19, Gly-Ser-Gly-Ser-Met-Thr, 20-232aa |
| pTrc99a FlgD∆9–32, 4xRpt | 1–8, 4 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa |
| pTrc99a FlgD∆9–32, 3xRpt | 1–8, 3 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa |
| pTrc99a FlgD∆9–32, 2xRpt (FlgDshort) | 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa |
| pTrc99a FlgD∆9–32, 1xRpt | 1–8, Gly-Ser-Thr-Asn-Ala-Ser, 33-232aa |
| pTrc99a FlgD∆9–32 | 1–8, 33-232aa |
| pTrc99a FlgD∆9–32, 2xRpt + 1 | 1–8, Gly, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa |
| pTrc99a FlgD∆9–32, 2xRpt + 2 | 1–8, Gly-Ser, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa |
| pTrc99a FlgD∆9–32, 2xRpt + 3 | 1–8, Gly-Ser-Thr, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa |
| pTrc99a FlgD∆9–32, 2xRpt + 4 | 1–8, Gly-Ser-Thr-Asn, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa |
| pTrc99a FlgD∆9–32, 2xRpt + 5 | 1–8, Gly-Ser-Thr-Asn-Ala, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa |
| pTrc99a FlgDshort, ∆GRM | 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33–35, 41-232aa |
| pTrc99a FlgD∆2–5 | 1, 6-232aa |
| pTrc99a FlgD∆2–5, M7I | 1, 6-232aa, M7I |
| pTrc99a FlgD∆2–5, M7V | 1, 6-232aa, M7V |
| pTrc99a FlgD∆2–5, N8I | 1, 6-232aa, N8I |
| pTrc99a FlgD∆2–5, D9A | 1, 6-232aa, D9A |
| pTrc99a FlgD∆2–5, P10L | 1, 6-232aa, P10L |
| pTrc99a FlgD∆2–5, T11I | 1, 6-232aa, T11I |
| pTrc99a FlgD∆2–5, 23(TTGSGS)24 | 1–23, Thr-Thr-Gly-Ser-Gly-Ser, 24-232aa |
| pTrc99a FlgD∆2–5, 23(TTGSGSTTGSGS)24 | 1–23, Thr-Thr-Gly-Ser-Gly-Ser-Thr-Thr-Gly-Ser-Gly-Ser, 24-232aa |
| pTrc99a FlgD∆2–5, 27(GSMTGS)28 | 1–27, Gly-Ser-Met-Thr-Gly-Ser, 28-232aa |
| pTrc99a FlgD∆9–32, 8(2xGSTNAS)33, V15A | 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa, V15A |
| pTrc99a FlgD∆9–32, 8(2xGSTNAS)33, V15A, M7I | 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa, M7I |
| pTrc99a FlgD∆9–32, 8(2xGSTNAS)33, V15A, D9A | 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa, D9A |
| pTrc99a FlgD∆9–32, 8(2xGSTNAS)33, V15A, T11I | 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa, T11I |
| pTrc99a FlgD∆9–32, 8(2xGSTNAS)33, V15A, G14V | 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), 33-232aa, G14V |
| pTrc99a FlgD∆9–32, 8(2xGSTNAS-TNPGSTNAS)33 | 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), (Thr-Asn-Pro-Gly-Ser-Thr-Asn-Ala-Ser) 33-232aa, |
| pTrc99a FlgD∆9–32, 8(2xGSTNAS-GNASGSTNAS)33 | 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), (Gly-Asn-Ala-Ser-Gly-Ser-Thr-Asn-Ala-Ser) 33-232aa, |
| pTrc99a FlgD∆9–32, 8(2xGSTNAS-QSSFLTLLVAQLKNQDPTNPLQNNELTTQLA)33 | 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), (Gln-Ser-Ser-Phe-Leu-Thr-Leu-Leu-Val-Ala-Gln-Leu-Lys-Asn-Gln-Asp-Pro-Thr-Asn-Pro-Leu-Asn-Asn-Glu-Leu-Thr-Thr-Gln-Leu-Ala), 33-232aa, |
| pTrc99a FlgD∆9–32, 8(2xGSTNAS-TNASGSTNAS)33 | 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), (Thr-Asn-Ala-Ser-Gly-Ser-Thr-Asn-Ala-Ser) 33-232aa, |
| pTrc99a FlgD∆9–32, 8(2xGSTNAS-QSSLGSTNAS)34 | 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), (Gln-Ser-Ser-Leu-Gly-Ser-Thr-Asn-Ala-Ser) 33-232aa, |
| pTrc99a FlgD∆9–32, 8(2xGSTNAS-QNASGSTNAS)35 | 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), (Gln-Asn-Ala-Ser-Gly-Ser-Thr-Asn-Ala-Ser) 33-232aa, |
| pTrc99a FlgD∆9–32, 8(2xGSTNAS-TNTFGTLIAS)36 | 1–8, 2 x(Gly-Ser-Thr-Asn-Ala-Ser), (Thr-Asn-Thr-Phe-Gly-Thr-Leu-Iso-Ala-Ser) 33-232aa, |
| pTrc99a FlgG | 1–144, FLAGx3, 145-260aa |
| pTrc99a FlgG∆short | 1–10, 35–144, FLAGx3, 145-260aa |
| pTrc99a FlgGshort+ linker | 1–10, 4 x(Gly-Ser-Thr-Asn-Ala-Ser) 35–144, FLAGx3, 145-260aa |
| pTrc99a FlgE | 1–234, FLAGx3, 235-403aa |
| pTrc99a FlgEshort | 1–8, 33–234, FLAGx3, 235-403aa |
| pTrc99a FlgEshort+ linker | 1–8, 4 x(Gly-Ser-Thr-Asn-Ala-Ser) 33–234, FLAGx3, 235-403aa |
| pTrc99a FlgE∆GRM | 1–38, 44–234, FLAGx3, 235-403aa |
| pTrc99a FlgEshort, ∆GRM | 1–8, 33–38, 44–234, FLAGx3, 235-403aa |
| pTrc99a FliKmyc | 1-405aa, myc |
| pTrc99a FliKmyc∆2–8 | 1, 9-405aa, myc |
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Recognition of discrete export signals in early flagellar subunits during bacterial type III secretion
eLife 11:e66264.
https://doi.org/10.7554/eLife.66264
