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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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Absolute requirement of a negatively charged residue at position 208 of FlhA.(A) Expression levels of the mutant FlhA proteins. Immunoblotting, with polyclonal anti-FlhAC antibody, of a flhA  strain transformed with pUC19-based plasmids encoding various forms of FlhA. V, pUC19; WT, wild-type FlhA; R94K, FlhA(R94K); R94E, FlhA(R94E); D208E, FlhA(D208E); D208K, FlhA(D208K); D208N, FlhA(D208N); D249E, FlhA(D249E); D249K, FlhA(D249K). (B) Secretion of FlgD. Immunoblotting, using polyclonal anti-FlgD antibody of whole cell (Cell) and culture supernatant (Sup) fractions prepared from the above strains.
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pone-0022417-g003: Absolute requirement of a negatively charged residue at position 208 of FlhA.(A) Expression levels of the mutant FlhA proteins. Immunoblotting, with polyclonal anti-FlhAC antibody, of a flhA strain transformed with pUC19-based plasmids encoding various forms of FlhA. V, pUC19; WT, wild-type FlhA; R94K, FlhA(R94K); R94E, FlhA(R94E); D208E, FlhA(D208E); D208K, FlhA(D208K); D208N, FlhA(D208N); D249E, FlhA(D249E); D249K, FlhA(D249K). (B) Secretion of FlgD. Immunoblotting, using polyclonal anti-FlgD antibody of whole cell (Cell) and culture supernatant (Sup) fractions prepared from the above strains.

Mentions: To probe the role of Arg-94, Asp-208 and Asp-249 in protein export, we mutated these three residues to the following two types: the same charge with a different length of side chain (Arg-to-Lys and Asp-to-Glu); and the oppositely charged residue (Arg-to-Asp and Asp-to-Lys). Immunoblotting with the polyclonal anti-FlhAC antibody revealed that all the mutant variants were as stable as the wild-type (Figure 3A). At positions 94 and 249, neither types of mutations affected the secretion level of FlgD (Figure 3B, lanes 3, 4, 8 and 9), indicating that these residues maintain the function of FlhA regardless of the charge type. While the D208E replacement still permitted the export of FlgD at the wild-type level (Figure 3B, lane 5) the D208K and D208N mutations totally diminished the export (lanes 6 and 7). In agreement with these results, the D208K and D208N mutants as well as the flhA mutant harboring the vector control accumulated much higher amounts of FlgD in the cytoplasm than the wild-type while the others did so more or less at the wild-type levels. FlhA(D208K) and FlhA(D208N) also exerted a dominant negative effect on wild-type motility (data not shown), indicating that they retain the ability to be incorporated into the export apparatus. These results suggest that a negatively charged residue at position 208 of FlhA is essential for the export function.


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

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

Absolute requirement of a negatively charged residue at position 208 of FlhA.(A) Expression levels of the mutant FlhA proteins. Immunoblotting, with polyclonal anti-FlhAC antibody, of a flhA  strain transformed with pUC19-based plasmids encoding various forms of FlhA. V, pUC19; WT, wild-type FlhA; R94K, FlhA(R94K); R94E, FlhA(R94E); D208E, FlhA(D208E); D208K, FlhA(D208K); D208N, FlhA(D208N); D249E, FlhA(D249E); D249K, FlhA(D249K). (B) Secretion of FlgD. Immunoblotting, using polyclonal anti-FlgD antibody of whole cell (Cell) and culture supernatant (Sup) fractions prepared from the above strains.
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Related In: Results  -  Collection

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

pone-0022417-g003: Absolute requirement of a negatively charged residue at position 208 of FlhA.(A) Expression levels of the mutant FlhA proteins. Immunoblotting, with polyclonal anti-FlhAC antibody, of a flhA strain transformed with pUC19-based plasmids encoding various forms of FlhA. V, pUC19; WT, wild-type FlhA; R94K, FlhA(R94K); R94E, FlhA(R94E); D208E, FlhA(D208E); D208K, FlhA(D208K); D208N, FlhA(D208N); D249E, FlhA(D249E); D249K, FlhA(D249K). (B) Secretion of FlgD. Immunoblotting, using polyclonal anti-FlgD antibody of whole cell (Cell) and culture supernatant (Sup) fractions prepared from the above strains.
Mentions: To probe the role of Arg-94, Asp-208 and Asp-249 in protein export, we mutated these three residues to the following two types: the same charge with a different length of side chain (Arg-to-Lys and Asp-to-Glu); and the oppositely charged residue (Arg-to-Asp and Asp-to-Lys). Immunoblotting with the polyclonal anti-FlhAC antibody revealed that all the mutant variants were as stable as the wild-type (Figure 3A). At positions 94 and 249, neither types of mutations affected the secretion level of FlgD (Figure 3B, lanes 3, 4, 8 and 9), indicating that these residues maintain the function of FlhA regardless of the charge type. While the D208E replacement still permitted the export of FlgD at the wild-type level (Figure 3B, lane 5) the D208K and D208N mutations totally diminished the export (lanes 6 and 7). In agreement with these results, the D208K and D208N mutants as well as the flhA mutant harboring the vector control accumulated much higher amounts of FlgD in the cytoplasm than the wild-type while the others did so more or less at the wild-type levels. FlhA(D208K) and FlhA(D208N) also exerted a dominant negative effect on wild-type motility (data not shown), indicating that they retain the ability to be incorporated into the export apparatus. These results suggest that a negatively charged residue at position 208 of FlhA is essential for the export function.

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