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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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FlgD secretion rate of slow motile flhA mutants.(A) Immunoblotting, using polyclonal anti-FlgD antibody, of culture supernatants prepared from NH0005 (ΔflhA PflhDC::T-POP(DEL-25) transformed with pMMHA004 (wild-type FlhA, WT), pNH001(D208E) (FlhA(D208E), D208E) or pNH001(K203A) (FlhA(K203A), K203A). After adding tetracycline, culture supernatants were collected at 0, 15, 30, 45, 60, 75, 90, 120 and 180 min and analyzed with polyclonal anti-FlgD antibody. (B) Relative secretion levels of FlgD, normalized for the level of FlgD in wild-type cells at 180 min. Shown are the mean and standard deviation of three independent experiments.
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pone-0022417-g004: FlgD secretion rate of slow motile flhA mutants.(A) Immunoblotting, using polyclonal anti-FlgD antibody, of culture supernatants prepared from NH0005 (ΔflhA PflhDC::T-POP(DEL-25) transformed with pMMHA004 (wild-type FlhA, WT), pNH001(D208E) (FlhA(D208E), D208E) or pNH001(K203A) (FlhA(K203A), K203A). After adding tetracycline, culture supernatants were collected at 0, 15, 30, 45, 60, 75, 90, 120 and 180 min and analyzed with polyclonal anti-FlgD antibody. (B) Relative secretion levels of FlgD, normalized for the level of FlgD in wild-type cells at 180 min. Shown are the mean and standard deviation of three independent experiments.

Mentions: We found that the motility of the flhA(D208E) (data not shown) and flhA(K203A) (Figure 2B) mutants was worse than that of wild-type cells in soft agar plates whereas the levels of FlgD secretion by these mutants were at the wild-type level when grown overnight in LB (Figure 3B, lane 5, and Figure 2C, lane 6, respectively). These results raise the possibility that these mutants are slow secretors. Secretion rate measurement of the flagellar proteins requires the external onset control of flagellar gene expression. To do this, we inserted a Tn10d (T-POP) transposon upstream of the flagellar master flhDC operon, which is required for the expression of the entire flagellar regulon. As flhDC is transcribed from a tetracycline-inducible promoter PtetA only in the presence of tetracycline, flagellar gene expression can be externally controlled [34]. A flhA mutant containing T-POP was transformed with pUC19-based plasmids encoding wild-type FlhA or each of the FlhA mutants. The transformants were grown at 30°C in LB until OD600 reached ca. 0.5∼0.6. After induction with tetracycline, the culture supernatants were collected at regular time intervals and analyzed by immunoblotting with polyclonal anti-FlgD antibody (Figure 4).


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

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

FlgD secretion rate of slow motile flhA mutants.(A) Immunoblotting, using polyclonal anti-FlgD antibody, of culture supernatants prepared from NH0005 (ΔflhA PflhDC::T-POP(DEL-25) transformed with pMMHA004 (wild-type FlhA, WT), pNH001(D208E) (FlhA(D208E), D208E) or pNH001(K203A) (FlhA(K203A), K203A). After adding tetracycline, culture supernatants were collected at 0, 15, 30, 45, 60, 75, 90, 120 and 180 min and analyzed with polyclonal anti-FlgD antibody. (B) Relative secretion levels of FlgD, normalized for the level of FlgD in wild-type cells at 180 min. Shown are the mean and standard deviation of three independent experiments.
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Related In: Results  -  Collection

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

pone-0022417-g004: FlgD secretion rate of slow motile flhA mutants.(A) Immunoblotting, using polyclonal anti-FlgD antibody, of culture supernatants prepared from NH0005 (ΔflhA PflhDC::T-POP(DEL-25) transformed with pMMHA004 (wild-type FlhA, WT), pNH001(D208E) (FlhA(D208E), D208E) or pNH001(K203A) (FlhA(K203A), K203A). After adding tetracycline, culture supernatants were collected at 0, 15, 30, 45, 60, 75, 90, 120 and 180 min and analyzed with polyclonal anti-FlgD antibody. (B) Relative secretion levels of FlgD, normalized for the level of FlgD in wild-type cells at 180 min. Shown are the mean and standard deviation of three independent experiments.
Mentions: We found that the motility of the flhA(D208E) (data not shown) and flhA(K203A) (Figure 2B) mutants was worse than that of wild-type cells in soft agar plates whereas the levels of FlgD secretion by these mutants were at the wild-type level when grown overnight in LB (Figure 3B, lane 5, and Figure 2C, lane 6, respectively). These results raise the possibility that these mutants are slow secretors. Secretion rate measurement of the flagellar proteins requires the external onset control of flagellar gene expression. To do this, we inserted a Tn10d (T-POP) transposon upstream of the flagellar master flhDC operon, which is required for the expression of the entire flagellar regulon. As flhDC is transcribed from a tetracycline-inducible promoter PtetA only in the presence of tetracycline, flagellar gene expression can be externally controlled [34]. A flhA mutant containing T-POP was transformed with pUC19-based plasmids encoding wild-type FlhA or each of the FlhA mutants. The transformants were grown at 30°C in LB until OD600 reached ca. 0.5∼0.6. After induction with tetracycline, the culture supernatants were collected at regular time intervals and analyzed by immunoblotting with polyclonal anti-FlgD antibody (Figure 4).

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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Related in: MedlinePlus