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Comparative study of the ion flux pathway in stator units of proton- and sodium-driven flagellar motors

View Article: PubMed Central - PubMed

ABSTRACT

Flagellar motor proteins, MotA/B and PomA/B, are essential for the motility of Escherichia coli and Vibrio alginolyticus, respectively. Those complexes work as a H+ and a Na+ channel, respectively and play important roles in torque generation as the stators of the flagellar motors. Although Asp32 of MotB and Asp24 of PomB are believed to function as ion binding site(s), the ion flux pathway from the periplasm to the cytoplasm is still unclear. Conserved residues, Ala39 of MotB and Cys31 of PomB, are located on the same sides as Asp32 of MotB and Asp24 of PomB, respectively, in a helical wheel diagram. In this study, a series of mutations were introduced into the Ala39 residue of MotB and the Cys31 residue of PomB. The motility of mutant cells were markedly decreased as the volume of the side chain increased. The loss of function due to the MotB(A39V) and PomB(L28A/C31A) mutations was suppressed by mutations of MotA(M206S) and PomA(L183F), respectively, and the increase in the volume caused by the MotB(A39V) mutation was close to the decrease in the volume caused by the MotA(M206S) mutation. These results demonstrate that Ala39 of MotB and Cys31 of PomB form part of the ion flux pathway and pore with Met206 of MotA and Leu183 of PomA in the MotA/B and PomA/B stator units, respectively.

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(a) Relationship between the volume of the side chain of introduced amino acid residues and the relative swarming size as estimated from Figure 2. Circles colored blue or red show the WT MotA/B and its mutants or WT PomA/B and its mutants, respectively. Three independent experiments were averaged. (b) Relationship between the volume of the side chain of introduced amino acid residues and the relative swimming speed of cells in TG medium for MotA/B expressing E. coli, or in TMN50 medium for PomA/B expressing V. alginolyticus. Circles colored blue or red show the WT MotA/B and its mutants or the WT PomA/B and its mutants, respectively. Three to 5 independent experiments were averaged.
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f3-5_45: (a) Relationship between the volume of the side chain of introduced amino acid residues and the relative swarming size as estimated from Figure 2. Circles colored blue or red show the WT MotA/B and its mutants or WT PomA/B and its mutants, respectively. Three independent experiments were averaged. (b) Relationship between the volume of the side chain of introduced amino acid residues and the relative swimming speed of cells in TG medium for MotA/B expressing E. coli, or in TMN50 medium for PomA/B expressing V. alginolyticus. Circles colored blue or red show the WT MotA/B and its mutants or the WT PomA/B and its mutants, respectively. Three to 5 independent experiments were averaged.

Mentions: An overnight culture of V. alginolyticus in VC medium was incubated in VPG500 medium at a 100-fold dilution and cells were grown at 30°C to exponential growth phase. Cells were centrifuged at 3,500×g for 3 min, and the sedimented cells were resuspended in TMN50 (50 mM Tris-HCl, pH 7.5, 5 mM MgSO4, 5 mM glucose, 50 mM NaCl and 450 mM KCl). Cell suspensions were diluted 50-fold with TMN medium containing various concentrations of NaCl (ionic strength was kept constant to 500 mM by KCl), and the motility of cells were observed immediately using a dark-field microscope. Swimming speeds were determined from at least 200 individual cells as described previously14. Briefly, the speeds were calculated from individual cell tracks measured by a computer-assisted motion analysis program (MetaMorph). It should be noted that the swimming speeds and apparent Km values estimated here are 2~3 fold lower and 7-fold higher, respectively, than the values reported previously10. This resulted from differences in the data collection method. In this study, we employed software (CellTrack 1.2 beta) that could easily and rapidly analyze the motility of large numbers of cells14,15, and the error bars in Figures 3 and 4 are much smaller than those previously reported. Using this analysis, the data contains slow motile and stop-and-go-motion of the cells, and therefore the swimming speeds and apparent Km values were decreased and increased, respectively. It should also be noted that the speeds and Km values become almost identical when highly motile cells showing straight swimming behavior during the measurements were selected and analyzed (data not shown).


Comparative study of the ion flux pathway in stator units of proton- and sodium-driven flagellar motors
(a) Relationship between the volume of the side chain of introduced amino acid residues and the relative swarming size as estimated from Figure 2. Circles colored blue or red show the WT MotA/B and its mutants or WT PomA/B and its mutants, respectively. Three independent experiments were averaged. (b) Relationship between the volume of the side chain of introduced amino acid residues and the relative swimming speed of cells in TG medium for MotA/B expressing E. coli, or in TMN50 medium for PomA/B expressing V. alginolyticus. Circles colored blue or red show the WT MotA/B and its mutants or the WT PomA/B and its mutants, respectively. Three to 5 independent experiments were averaged.
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Related In: Results  -  Collection

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f3-5_45: (a) Relationship between the volume of the side chain of introduced amino acid residues and the relative swarming size as estimated from Figure 2. Circles colored blue or red show the WT MotA/B and its mutants or WT PomA/B and its mutants, respectively. Three independent experiments were averaged. (b) Relationship between the volume of the side chain of introduced amino acid residues and the relative swimming speed of cells in TG medium for MotA/B expressing E. coli, or in TMN50 medium for PomA/B expressing V. alginolyticus. Circles colored blue or red show the WT MotA/B and its mutants or the WT PomA/B and its mutants, respectively. Three to 5 independent experiments were averaged.
Mentions: An overnight culture of V. alginolyticus in VC medium was incubated in VPG500 medium at a 100-fold dilution and cells were grown at 30°C to exponential growth phase. Cells were centrifuged at 3,500×g for 3 min, and the sedimented cells were resuspended in TMN50 (50 mM Tris-HCl, pH 7.5, 5 mM MgSO4, 5 mM glucose, 50 mM NaCl and 450 mM KCl). Cell suspensions were diluted 50-fold with TMN medium containing various concentrations of NaCl (ionic strength was kept constant to 500 mM by KCl), and the motility of cells were observed immediately using a dark-field microscope. Swimming speeds were determined from at least 200 individual cells as described previously14. Briefly, the speeds were calculated from individual cell tracks measured by a computer-assisted motion analysis program (MetaMorph). It should be noted that the swimming speeds and apparent Km values estimated here are 2~3 fold lower and 7-fold higher, respectively, than the values reported previously10. This resulted from differences in the data collection method. In this study, we employed software (CellTrack 1.2 beta) that could easily and rapidly analyze the motility of large numbers of cells14,15, and the error bars in Figures 3 and 4 are much smaller than those previously reported. Using this analysis, the data contains slow motile and stop-and-go-motion of the cells, and therefore the swimming speeds and apparent Km values were decreased and increased, respectively. It should also be noted that the speeds and Km values become almost identical when highly motile cells showing straight swimming behavior during the measurements were selected and analyzed (data not shown).

View Article: PubMed Central - PubMed

ABSTRACT

Flagellar motor proteins, MotA/B and PomA/B, are essential for the motility of Escherichia coli and Vibrio alginolyticus, respectively. Those complexes work as a H+ and a Na+ channel, respectively and play important roles in torque generation as the stators of the flagellar motors. Although Asp32 of MotB and Asp24 of PomB are believed to function as ion binding site(s), the ion flux pathway from the periplasm to the cytoplasm is still unclear. Conserved residues, Ala39 of MotB and Cys31 of PomB, are located on the same sides as Asp32 of MotB and Asp24 of PomB, respectively, in a helical wheel diagram. In this study, a series of mutations were introduced into the Ala39 residue of MotB and the Cys31 residue of PomB. The motility of mutant cells were markedly decreased as the volume of the side chain increased. The loss of function due to the MotB(A39V) and PomB(L28A/C31A) mutations was suppressed by mutations of MotA(M206S) and PomA(L183F), respectively, and the increase in the volume caused by the MotB(A39V) mutation was close to the decrease in the volume caused by the MotA(M206S) mutation. These results demonstrate that Ala39 of MotB and Cys31 of PomB form part of the ion flux pathway and pore with Met206 of MotA and Leu183 of PomA in the MotA/B and PomA/B stator units, respectively.

No MeSH data available.


Related in: MedlinePlus