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Genetic characterization of conserved charged residues in the bacterial flagellar type III export protein FlhA.

Hara N, Namba K, Minamino T - PLoS ONE (2011)

Bottom Line: The addition of FliH and FliI allowed the D45A, R85A, R94K and R270A mutant proteins to work even in the presence of the flhB(P28T) mutation.Suppressor analysis of a flhA(K203W) mutation showed an interaction between FlhA and FliR.Taken all together, we suggest that Asp-208 is directly involved in PMF-driven protein export and that the cooperative interactions of FlhA with FlhB, FliH, FliI, and FliR drive the translocation of export substrate.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan.

ABSTRACT
For assembly of the bacterial flagellum, most of flagellar proteins are transported to the distal end of the flagellum by the flagellar type III protein export apparatus powered by proton motive force (PMF) across the cytoplasmic membrane. FlhA is an integral membrane protein of the export apparatus and is involved in an early stage of the export process along with three soluble proteins, FliH, FliI, and FliJ, but the energy coupling mechanism remains unknown. Here, we carried out site-directed mutagenesis of eight, highly conserved charged residues in putative juxta- and trans-membrane helices of FlhA. Only Asp-208 was an essential acidic residue. Most of the FlhA substitutions were tolerated, but resulted in loss-of-function in the ΔfliH-fliI mutant background, even with the second-site flhB(P28T) mutation that increases the probability of flagellar protein export in the absence of FliH and FliI. The addition of FliH and FliI allowed the D45A, R85A, R94K and R270A mutant proteins to work even in the presence of the flhB(P28T) mutation. Suppressor analysis of a flhA(K203W) mutation showed an interaction between FlhA and FliR. Taken all together, we suggest that Asp-208 is directly involved in PMF-driven protein export and that the cooperative interactions of FlhA with FlhB, FliH, FliI, and FliR drive the translocation of export substrate.

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Effect of alanine substitutions in FlhATM on motility and flagellar protein export.(A) Expression levels of alanine-substituted variants of FlhA in cells. Immunoblotting, using polyclonal anti-FlhAC antibody, of whole cells protein (Cell) prepared from a flhA  strain, NH0001 (ΔflhA), transformed with pUC19-based plasmids encoding various alanine-substituted forms of FlhA. V, pUC19; WT, wild-type FlhA; D45A, FlhA(D45A); R85A, FlhA(R85A); R94A, FlhA(R94A); K203A, FlhA(K203A); R206A, FlhA(R206A); D208A, FlhA(D208A); D249A, FlhA(D249A); R270A, FlhA(R270A). (B) Motility of the flhA  mutant transformed with the above plasmids in soft agar. The plate was incubated at 30°C for 6 hours. (C) Secretion of FlgD. Immunoblotting, using polyclonal anti-FlgD antibody, of culture supernatants (Sup) prepared from the above strains. (D) Dominant negative effect on motility of the wild-type strain SJW1103 in soft agar. The plate was incubated at 30°C for 6 hours.
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pone-0022417-g002: Effect of alanine substitutions in FlhATM on motility and flagellar protein export.(A) Expression levels of alanine-substituted variants of FlhA in cells. Immunoblotting, using polyclonal anti-FlhAC antibody, of whole cells protein (Cell) prepared from a flhA strain, NH0001 (ΔflhA), transformed with pUC19-based plasmids encoding various alanine-substituted forms of FlhA. V, pUC19; WT, wild-type FlhA; D45A, FlhA(D45A); R85A, FlhA(R85A); R94A, FlhA(R94A); K203A, FlhA(K203A); R206A, FlhA(R206A); D208A, FlhA(D208A); D249A, FlhA(D249A); R270A, FlhA(R270A). (B) Motility of the flhA mutant transformed with the above plasmids in soft agar. The plate was incubated at 30°C for 6 hours. (C) Secretion of FlgD. Immunoblotting, using polyclonal anti-FlgD antibody, of culture supernatants (Sup) prepared from the above strains. (D) Dominant negative effect on motility of the wild-type strain SJW1103 in soft agar. The plate was incubated at 30°C for 6 hours.

Mentions: To test the hypothesis that highly conserved charged residues of FlhATM is involved in the energy transduction mechanism, we identified eight highly conserved charged residues, Asp-45, Arg-85, Arg-94, Lys-203, Arg-206, Asp-208, Asp249 and Arg-270, in putative juxta- and trans-membrane helices of FlhA by multiple sequence alignment (Figures 1 and S2) and then replaced each with alanine. Immunoblotting with polyclonal anti-FlhAC antibody detected all the point mutant variants at the wild-type level (Figure 2A), indicating that these substitutions do not affect protein stability.


Genetic characterization of conserved charged residues in the bacterial flagellar type III export protein FlhA.

Hara N, Namba K, Minamino T - PLoS ONE (2011)

Effect of alanine substitutions in FlhATM on motility and flagellar protein export.(A) Expression levels of alanine-substituted variants of FlhA in cells. Immunoblotting, using polyclonal anti-FlhAC antibody, of whole cells protein (Cell) prepared from a flhA  strain, NH0001 (ΔflhA), transformed with pUC19-based plasmids encoding various alanine-substituted forms of FlhA. V, pUC19; WT, wild-type FlhA; D45A, FlhA(D45A); R85A, FlhA(R85A); R94A, FlhA(R94A); K203A, FlhA(K203A); R206A, FlhA(R206A); D208A, FlhA(D208A); D249A, FlhA(D249A); R270A, FlhA(R270A). (B) Motility of the flhA  mutant transformed with the above plasmids in soft agar. The plate was incubated at 30°C for 6 hours. (C) Secretion of FlgD. Immunoblotting, using polyclonal anti-FlgD antibody, of culture supernatants (Sup) prepared from the above strains. (D) Dominant negative effect on motility of the wild-type strain SJW1103 in soft agar. The plate was incubated at 30°C for 6 hours.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139655&req=5

pone-0022417-g002: Effect of alanine substitutions in FlhATM on motility and flagellar protein export.(A) Expression levels of alanine-substituted variants of FlhA in cells. Immunoblotting, using polyclonal anti-FlhAC antibody, of whole cells protein (Cell) prepared from a flhA strain, NH0001 (ΔflhA), transformed with pUC19-based plasmids encoding various alanine-substituted forms of FlhA. V, pUC19; WT, wild-type FlhA; D45A, FlhA(D45A); R85A, FlhA(R85A); R94A, FlhA(R94A); K203A, FlhA(K203A); R206A, FlhA(R206A); D208A, FlhA(D208A); D249A, FlhA(D249A); R270A, FlhA(R270A). (B) Motility of the flhA mutant transformed with the above plasmids in soft agar. The plate was incubated at 30°C for 6 hours. (C) Secretion of FlgD. Immunoblotting, using polyclonal anti-FlgD antibody, of culture supernatants (Sup) prepared from the above strains. (D) Dominant negative effect on motility of the wild-type strain SJW1103 in soft agar. The plate was incubated at 30°C for 6 hours.
Mentions: To test the hypothesis that highly conserved charged residues of FlhATM is involved in the energy transduction mechanism, we identified eight highly conserved charged residues, Asp-45, Arg-85, Arg-94, Lys-203, Arg-206, Asp-208, Asp249 and Arg-270, in putative juxta- and trans-membrane helices of FlhA by multiple sequence alignment (Figures 1 and S2) and then replaced each with alanine. Immunoblotting with polyclonal anti-FlhAC antibody detected all the point mutant variants at the wild-type level (Figure 2A), indicating that these substitutions do not affect protein stability.

Bottom Line: The addition of FliH and FliI allowed the D45A, R85A, R94K and R270A mutant proteins to work even in the presence of the flhB(P28T) mutation.Suppressor analysis of a flhA(K203W) mutation showed an interaction between FlhA and FliR.Taken all together, we suggest that Asp-208 is directly involved in PMF-driven protein export and that the cooperative interactions of FlhA with FlhB, FliH, FliI, and FliR drive the translocation of export substrate.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan.

ABSTRACT
For assembly of the bacterial flagellum, most of flagellar proteins are transported to the distal end of the flagellum by the flagellar type III protein export apparatus powered by proton motive force (PMF) across the cytoplasmic membrane. FlhA is an integral membrane protein of the export apparatus and is involved in an early stage of the export process along with three soluble proteins, FliH, FliI, and FliJ, but the energy coupling mechanism remains unknown. Here, we carried out site-directed mutagenesis of eight, highly conserved charged residues in putative juxta- and trans-membrane helices of FlhA. Only Asp-208 was an essential acidic residue. Most of the FlhA substitutions were tolerated, but resulted in loss-of-function in the ΔfliH-fliI mutant background, even with the second-site flhB(P28T) mutation that increases the probability of flagellar protein export in the absence of FliH and FliI. The addition of FliH and FliI allowed the D45A, R85A, R94K and R270A mutant proteins to work even in the presence of the flhB(P28T) mutation. Suppressor analysis of a flhA(K203W) mutation showed an interaction between FlhA and FliR. Taken all together, we suggest that Asp-208 is directly involved in PMF-driven protein export and that the cooperative interactions of FlhA with FlhB, FliH, FliI, and FliR drive the translocation of export substrate.

Show MeSH
Related in: MedlinePlus