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. 2011 Sep 20:2:475.
doi: 10.1038/ncomms1488.

An energy transduction mechanism used in bacterial flagellar type III protein export

Tohru Minamino  1 , Yusuke V Morimoto, Noritaka Hara, Keiichi Namba
Affiliations

An energy transduction mechanism used in bacterial flagellar type III protein export

Tohru Minamino et al. Nat Commun. .

Abstract

Flagellar proteins of bacteria are exported by a specific export apparatus. FliI ATPase forms a complex with FliH and FliJ and escorts export substrates from the cytoplasm to the export gate complex, which is made up of six membrane proteins. The export gate complex utilizes proton motive force across the cytoplasmic membrane for protein translocation, but the mechanism remains unknown. Here we show that the export gate complex by itself is a proton-protein antiporter that uses the two components of proton motive force, Δψ and ΔpH, for different steps of the protein export process. However, in the presence of FliH, FliI and FliJ, a specific binding of FliJ with an export gate membrane protein, FlhA, is brought about by the FliH-FliI complex, which turns the export gate into a highly efficient, Δψ-driven protein export apparatus.

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Figures

Figure 1
Figure 1. Effect of Δψ and ΔpH on flagellar protein export.
(a) Effect of valinomycin in the presence of 150 mM KCl. Immunoblotting, using polyclonal anti-FlgD antibody, of whole-cell proteins and culture supernatant fractions prepared from SJW1103 (wild-type, indicated as WT) and MMHI0117 (ΔfliH-fliI flhB(P28T), indicated as ΔfliHI flhB*) grown at 30 °C in a buffer containing 150 mM KCl with or without 20 μM valinomycin at an external pH of 7.0. (b) Effect of potassium benzoate on the secretion level of FlgD (upper panel) and ΔpH change (closed circle) and the quantified secretion level of FlgD (open circle; lower panel) at an external pH of 7.0. Intracellular pH was measured with pHluorin. These data are the average of at least three independent measurements. The experimental errors are less than 10%. (c) Effect of external pH on FlgD secretion over a pH range of 6.0–7.5.
Figure 2
Figure 2. pH dependence of PMF components.
pH dependence of (a) Δψ, (b) intracellular pH, (c) ΔpH and (d) total PMF. Δψ was measured in 10 mM potassium phosphate, 0.1 mM EDTA, and 10 mM sodium lactate at an external pH of 6.0, 6.5, 7.0 or 7.5 with (closed bar) or without (open bar) 20 mM potassium benzoate using TMRM. More than 100 cells were measured. Intracellular pH was measured with pHluorin. At least six independent experiments were conducted. Vertical bars indicate standard deviations.
Figure 3
Figure 3. Solvent-isotope effects on flagellar protein export.
Levels of FlgD secreted by the wild-type (WT) and ΔfliH-fliI flhB(P28T) mutant (ΔfliHI flhB*) cells grown in a buffer containing either H2O or D2O at external pH levels of 6.0 and 7.0. The level of FlgD secretion was analysed by immunoblotting with the polyclonal anti-FlgD antibody.
Figure 4
Figure 4. Effects of FliJ deletion on flagellar protein export.
(a) Immunoblotting, using polyclonal anti-FlgD, of whole-cell proteins and culture supernatant fractions prepared from the wild type (WT), a ΔfliH-fliI double null mutant (ΔfliHI), a fliH-fliI bypass mutant (ΔfliHI flhB*), a fliH-fliI-fliJ triple null mutant (ΔfliHIJ) and a fliH-fliI-fliJ flhB(P28T) (ΔfliHIJ flhB*). (b) Effect of external pH on FlgD secretion by the ΔfliHI flhB*and ΔfliHIJ flhB*mutant strains. (c) Motility assays of MMHIJ001 (ΔfliHIJ) and MMHIJ0117 (ΔfliHIJ flhB*) transformed with pTrc99AFF4 (V) or pMMHI001 (FliH+FliI) in soft agar. The plates were incubated at 30 °C for 24 h. (d) Effect of external pH on FlgD secretion by the ΔfliH-fliI-fliJ flhB(P28T) mutant strain in the presence and absence of FliH and FliI. Immunoblotting, using polyclonal anti-FlgD antibody, of culture supernatant fractions prepared from MMHIJ0117 carrying pTrc99A or pMMHI001 grown at an external pH of 6.0 and 7.0.
Figure 5
Figure 5. Effect of a deletion of residues 328–351 of FlhA on the interaction with the soluble proteins.
(a) Pull-down assays by GST affinity chromatography. The mixture of the soluble fractions (L) prepared from a ΔflhDC-cheW mutant expressing GST (first row), GST-FliH (second row), GST-FliI (third row) or GST-FliJ (forth row) with those from a ΔflhA mutant transformed with pMM108 (N-His-FLAG-FlhA, FlhA) or pMM108-1 (N-His-FLAG-FlhA(Δ328–351), FlhA(Δ328–351)) were loaded onto a GST column. After extensive washing, the proteins were eluted with a buffer containing 10 mM reduced glutathione. The eluted fractions were analysed by CBB staining for GST fusion proteins (upper panels) and immunoblotting with polyclonal anti-FlhA antibody (lower panels). (b) Effect of a ΔfliH-fliI bypass mutation, FlhB(P28T), on the FlhA(Δ328–351) mutants. Motility of NH0004 (ΔflhA ΔfliH-fliI flhB(P28T)) transformed with pTrc99A (V), pMM108 (FlhA) or pMM108-1 (FlhA(Δ328–351)) in soft agar plate. Plates were incubated at 30 °C for 23 h.
Figure 6
Figure 6. Effects of a deletion of residues 13–24 in FliJ on the FliJ–FlhA interaction.
(a) Pull-down assays by GST affinity chromatography. The eluted factions of GST-FliJ or GST-FliJ(Δ13–24) were analysed by CBB staining (upper panels), whereas the eluted FlhA protein was detected by immunoblotting with polyclonal anti-FlhA antibody. (b) Motility of a ΔfliH-fliI-fliJ flhB(P28T) (ΔfliHIJ flhB*) mutant transformed with pTrc99AFF4 (V), pMM404 (FliJ) or pRCJ104 (FliJ(Δ13–24)) in soft agar. The plates were incubated at 30 °C for 7 h. (c) Motility of pseudorevertants from the fliJ(Δ13–24) mutant: wild type (WT), fliJ(Δ13–24) mutant and its pseudorevertants (fliJ(Δ13–24), fliH(Δ96–97) (labelled as fliH*) and fliJ(Δ13–24), fliI(L244R) (labelled as fliI*)). (d) Secretion analysis of FlgD, FlgE and FliC. Immunoblotting, using polyclonal anti-FlgD, anti-FlgE or anti-FliC antibody of whole cell (Cell) and culture supernatant (Sup) fractions prepared from the above strains.
Figure 7
Figure 7. Effect of the FliH and FliI suppressor mutations on the interaction of FliJ(Δ13–24) with FlhA.
(a) Interactions of FliJ with FliH and FliI. Pull-down assays by GST affinity chromatography. The mixture of the soluble fractions (L) prepared from a ΔflhDC-cheW mutant expressing GST (left), GST-FliJ (middle) or GST-FliJ(Δ13–24) (right) with those from the ΔflhDC-cheW mutant producing His-FliH or His-FliI were loaded onto a GST column. After extensive washing, proteins were eluted with a buffer containing 10 mM reduced glutathione. (b) Interaction of GST-FliJ(Δ13–24) with the wild-type FliH–FliI complex (left) and its suppressor mutant variants (middle, FliH(Δ96–97)+FliI; right, FliH+FliI(L244R)). (c) Interactions of GST-FliJ(Δ13–24) with FlhA in the presence of a suppressor mutant variant of the FliH–FliI complex (FliH+FliI(L244R)). (d) Interaction of GST-FlhAC with the suppressor mutant variants of FliH and FliI (FliH*, FliH(Δ96–97); FliI*, FliI(L244R)). The eluted fractions were analysed by CBB staining for GST fusion proteins (upper panels) and immunoblotting with polyclonal anti-FliH, anti-FliI or anti-FlhAC antibody (lower panels).
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References

    1. Lo C. J., Leake M. C., Pilizota T. & Beryy R. M. Nonequivalence of membrane voltage and ion-gradient as driving forces for the bacterial flagellar motor at low load. Biophys. J. 93, 294–302 (2007). - PMC - PubMed
    1. Wiedenmann A., Dimroth P. & von Ballmoos C. Functional asymmetry of the F0 motor in bacterial ATPsynthases. Mol. Microbiol. 72, 479–490 (2009). - PubMed
    1. Macnab R. M. How bacteria assemble flagella. Annu. Rev. Microbiol. 57, 77–100 (2003). - PubMed
    1. Minamino T. & Namba K. Self-assembly and type III protein export of the bacterial flagellum. J. Mol. Microbiol. Biotechnol. 7, 5–17 (2004). - PubMed
    1. Minamino T., Imada K. & Namba K. Mechanisms of type III protein export for bacterial flagellar assembly. Mol. Biosyst. 4, 1105–1115 (2008). - PubMed

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