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The bacterial flagellar protein export apparatus processively transports flagellar proteins even with extremely infrequent ATP hydrolysis.

Minamino T, Morimoto YV, Kinoshita M, Aldridge PD, Namba K - Sci Rep (2014)

Bottom Line: This indicates that the rate of ATP hydrolysis is not at all coupled with the export rate.Deletion of FliI residues 401 to 410 resulted in no flagellar formation although this FliI deletion mutant retained 40% of the ATPase activity, suggesting uncoupling between ATP hydrolysis and activation of the gate.We propose that infrequent ATP hydrolysis by the FliI6FliJ ring is sufficient for gate activation, allowing processive translocation of export substrates for efficient flagellar assembly.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.

ABSTRACT
For self-assembly of the bacterial flagellum, a specific protein export apparatus utilizes ATP and proton motive force (PMF) as the energy source to transport component proteins to the distal growing end. The export apparatus consists of a transmembrane PMF-driven export gate and a cytoplasmic ATPase complex composed of FliH, FliI and FliJ. The FliI(6)FliJ complex is structurally similar to the α(3)β(3)γ complex of F(O)F(1)-ATPase. FliJ allows the gate to efficiently utilize PMF to drive flagellar protein export but it remains unknown how. Here, we report the role of ATP hydrolysis by the FliI(6)FliJ complex. The export apparatus processively transported flagellar proteins to grow flagella even with extremely infrequent or no ATP hydrolysis by FliI mutation (E211D and E211Q, respectively). This indicates that the rate of ATP hydrolysis is not at all coupled with the export rate. Deletion of FliI residues 401 to 410 resulted in no flagellar formation although this FliI deletion mutant retained 40% of the ATPase activity, suggesting uncoupling between ATP hydrolysis and activation of the gate. We propose that infrequent ATP hydrolysis by the FliI6FliJ ring is sufficient for gate activation, allowing processive translocation of export substrates for efficient flagellar assembly.

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Characterization of the fliI(E211Q) and fliI(E211D) mutants.(a) Effect of the E211Q and E211D mutations on the FliI ATPase activity. Measurements were carried out on purified His-FliI (indicated as WT, black filled circle), His-FliI(E211Q) (indicated as E211Q, red filled triangle) or His-FliI(E211D) (indicated as E211D, blue filled square) using malachite green assay. (b) Swarming motility of a ΔfliI mutant transformed with pTrc99A (V), pMM1702 (WT), pKK211 (E211Q) or pMM1702(E211D) (E211D) in soft agar plates. The plate was incubated at 30°C for 6 hours. (c) Effect of the E211Q and E211D mutations on flagellar protein export. Whole cell proteins (Cell) and culture supernatant fractions (Sup) were prepared from the above transformants, subjected to SDS-PAGE, and analyzed by immunoblotting with polyclonal anti-FlgD (1st row), anti-FlgE (2nd row), anti-FliK (3rd row), anti-FlgK (4th row), anti-FlgL (5th row), anti-FliD (6th row) or anti-FliI (7th row) antibody. The positions of molecular mass markers are indicated on the left.
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f2: Characterization of the fliI(E211Q) and fliI(E211D) mutants.(a) Effect of the E211Q and E211D mutations on the FliI ATPase activity. Measurements were carried out on purified His-FliI (indicated as WT, black filled circle), His-FliI(E211Q) (indicated as E211Q, red filled triangle) or His-FliI(E211D) (indicated as E211D, blue filled square) using malachite green assay. (b) Swarming motility of a ΔfliI mutant transformed with pTrc99A (V), pMM1702 (WT), pKK211 (E211Q) or pMM1702(E211D) (E211D) in soft agar plates. The plate was incubated at 30°C for 6 hours. (c) Effect of the E211Q and E211D mutations on flagellar protein export. Whole cell proteins (Cell) and culture supernatant fractions (Sup) were prepared from the above transformants, subjected to SDS-PAGE, and analyzed by immunoblotting with polyclonal anti-FlgD (1st row), anti-FlgE (2nd row), anti-FliK (3rd row), anti-FlgK (4th row), anti-FlgL (5th row), anti-FliD (6th row) or anti-FliI (7th row) antibody. The positions of molecular mass markers are indicated on the left.

Mentions: The carboxyl group of Glu-211 in FliI, which corresponds to Glu-190 in the thermophilic Bacillus F1-ATPase, polarizes a water molecule for the nucleophilic attack to the γ-phosphate of ATP (Fig. 1)111819. To investigate whether ATP hydrolysis by FliI limits the rate of flagellar protein export, we replaced Glu-211 with Asp and measured the ATPase activity. We used FliI(E211Q) as a negative control because the E211Q substitution results in the complete loss of its ATPase activity19. The ATPase activity of FliI(E211D) was measured quantitatively by subtracting the data from FliI(E211Q) as a background, and it was about 100 times lower than that of wild-type FliI (Fig. 2a). The E211Q mutation in FliI considerably reduced both motility (Fig. 2b) and flagellar protein export (Fig. 2c, lane 7). In contrast, more than 80% of the fliI(E211D) mutant cells were motile, and their swimming speed was about 50% of the wild-type level (Fig. 2b and Fig. S1). Consistently, most flagellar proteins were detected in the culture supernatant of this fliI(E211D) mutant (Fig. 2c, lane 8). Interestingly, the levels of FlgD (1st row), FliK (3rd row) and FliD (6th row) secreted by the fliI(E211D) mutant were comparable to the wild-type levels while the secretion levels of FlgE (2nd row), FlgK (4th row) and FlgL (5th row) were four, two, and three-fold lower than wild-type levels, respectively. The E211Q and E211D mutations did not affect the cellular levels of FliI at all (Fig. 2c, 7th row). Therefore, these results indicate that the rate of ATP hydrolysis by FliI does not determine the rate of flagellar protein export.


The bacterial flagellar protein export apparatus processively transports flagellar proteins even with extremely infrequent ATP hydrolysis.

Minamino T, Morimoto YV, Kinoshita M, Aldridge PD, Namba K - Sci Rep (2014)

Characterization of the fliI(E211Q) and fliI(E211D) mutants.(a) Effect of the E211Q and E211D mutations on the FliI ATPase activity. Measurements were carried out on purified His-FliI (indicated as WT, black filled circle), His-FliI(E211Q) (indicated as E211Q, red filled triangle) or His-FliI(E211D) (indicated as E211D, blue filled square) using malachite green assay. (b) Swarming motility of a ΔfliI mutant transformed with pTrc99A (V), pMM1702 (WT), pKK211 (E211Q) or pMM1702(E211D) (E211D) in soft agar plates. The plate was incubated at 30°C for 6 hours. (c) Effect of the E211Q and E211D mutations on flagellar protein export. Whole cell proteins (Cell) and culture supernatant fractions (Sup) were prepared from the above transformants, subjected to SDS-PAGE, and analyzed by immunoblotting with polyclonal anti-FlgD (1st row), anti-FlgE (2nd row), anti-FliK (3rd row), anti-FlgK (4th row), anti-FlgL (5th row), anti-FliD (6th row) or anti-FliI (7th row) antibody. The positions of molecular mass markers are indicated on the left.
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f2: Characterization of the fliI(E211Q) and fliI(E211D) mutants.(a) Effect of the E211Q and E211D mutations on the FliI ATPase activity. Measurements were carried out on purified His-FliI (indicated as WT, black filled circle), His-FliI(E211Q) (indicated as E211Q, red filled triangle) or His-FliI(E211D) (indicated as E211D, blue filled square) using malachite green assay. (b) Swarming motility of a ΔfliI mutant transformed with pTrc99A (V), pMM1702 (WT), pKK211 (E211Q) or pMM1702(E211D) (E211D) in soft agar plates. The plate was incubated at 30°C for 6 hours. (c) Effect of the E211Q and E211D mutations on flagellar protein export. Whole cell proteins (Cell) and culture supernatant fractions (Sup) were prepared from the above transformants, subjected to SDS-PAGE, and analyzed by immunoblotting with polyclonal anti-FlgD (1st row), anti-FlgE (2nd row), anti-FliK (3rd row), anti-FlgK (4th row), anti-FlgL (5th row), anti-FliD (6th row) or anti-FliI (7th row) antibody. The positions of molecular mass markers are indicated on the left.
Mentions: The carboxyl group of Glu-211 in FliI, which corresponds to Glu-190 in the thermophilic Bacillus F1-ATPase, polarizes a water molecule for the nucleophilic attack to the γ-phosphate of ATP (Fig. 1)111819. To investigate whether ATP hydrolysis by FliI limits the rate of flagellar protein export, we replaced Glu-211 with Asp and measured the ATPase activity. We used FliI(E211Q) as a negative control because the E211Q substitution results in the complete loss of its ATPase activity19. The ATPase activity of FliI(E211D) was measured quantitatively by subtracting the data from FliI(E211Q) as a background, and it was about 100 times lower than that of wild-type FliI (Fig. 2a). The E211Q mutation in FliI considerably reduced both motility (Fig. 2b) and flagellar protein export (Fig. 2c, lane 7). In contrast, more than 80% of the fliI(E211D) mutant cells were motile, and their swimming speed was about 50% of the wild-type level (Fig. 2b and Fig. S1). Consistently, most flagellar proteins were detected in the culture supernatant of this fliI(E211D) mutant (Fig. 2c, lane 8). Interestingly, the levels of FlgD (1st row), FliK (3rd row) and FliD (6th row) secreted by the fliI(E211D) mutant were comparable to the wild-type levels while the secretion levels of FlgE (2nd row), FlgK (4th row) and FlgL (5th row) were four, two, and three-fold lower than wild-type levels, respectively. The E211Q and E211D mutations did not affect the cellular levels of FliI at all (Fig. 2c, 7th row). Therefore, these results indicate that the rate of ATP hydrolysis by FliI does not determine the rate of flagellar protein export.

Bottom Line: This indicates that the rate of ATP hydrolysis is not at all coupled with the export rate.Deletion of FliI residues 401 to 410 resulted in no flagellar formation although this FliI deletion mutant retained 40% of the ATPase activity, suggesting uncoupling between ATP hydrolysis and activation of the gate.We propose that infrequent ATP hydrolysis by the FliI6FliJ ring is sufficient for gate activation, allowing processive translocation of export substrates for efficient flagellar assembly.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.

ABSTRACT
For self-assembly of the bacterial flagellum, a specific protein export apparatus utilizes ATP and proton motive force (PMF) as the energy source to transport component proteins to the distal growing end. The export apparatus consists of a transmembrane PMF-driven export gate and a cytoplasmic ATPase complex composed of FliH, FliI and FliJ. The FliI(6)FliJ complex is structurally similar to the α(3)β(3)γ complex of F(O)F(1)-ATPase. FliJ allows the gate to efficiently utilize PMF to drive flagellar protein export but it remains unknown how. Here, we report the role of ATP hydrolysis by the FliI(6)FliJ complex. The export apparatus processively transported flagellar proteins to grow flagella even with extremely infrequent or no ATP hydrolysis by FliI mutation (E211D and E211Q, respectively). This indicates that the rate of ATP hydrolysis is not at all coupled with the export rate. Deletion of FliI residues 401 to 410 resulted in no flagellar formation although this FliI deletion mutant retained 40% of the ATPase activity, suggesting uncoupling between ATP hydrolysis and activation of the gate. We propose that infrequent ATP hydrolysis by the FliI6FliJ ring is sufficient for gate activation, allowing processive translocation of export substrates for efficient flagellar assembly.

Show MeSH
Related in: MedlinePlus