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

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.

Keywords: bacteria locomotion; ion flux; membrane protein; molecular motor; motility; taxis.

Figure 1

Putative membrane topology of stator proteins, PomA/B, MotA and MotB. Amino acid residues marked by a circle in the transmembrane helix of the B subunit are the sites of attention in this study. Abbreviations: VP, Vibrio parahaemolyticus; VC, Vibrio cholera; SWA, Shewanella oneidensis; RSH, Rhodobacter sphaeroides; Pae, Pseudomonas aeruginosa; EC, Escherichia coli.

Figure 2

Motility of MotB and PomB mutants on soft-agar plates (a)(c), and western blotting analysis of the proteins (b) (d). (a) Swarms of RP6894 (ΔmotAB) cells harboring a plasmid that express WT MotA/B, MotA/B(A39G) (G), MotA/B(A39V) (V), MotA/B(A39C) (C), MotA/B(A39S) (S), MotA/B(A39T) (T), MotA/B(A39Y) (Y) or MotA/B(A39W) (W). Transformants were cultured in liquid medium overnight, and aliquots were spotted onto a plate containing TG and 0.26% agar. Plates were incubated in the presence of 0.04% arabinose at 30°C for 10 hr. Vec. means a pBAD24 plasmid vector. (b) Western blotting analysis of MotA (left panel) and MotB (right panel) in whole cell extracts. Proteins were detected with an anti-MotA266 antibody and an anti-MotB2 antibody. (c) Swarms of NMB191 (ΔpomAB) cells harboring a plasmid that express WT PomA/B, PomA/B(C31G) (G), PomA/B(C31A) (A), PomA/B(C31S) (S), PomA/B(C31V) (V), PomA/B(C31T) (T), PomA/B(C31M) (M), PomA/B(C31I) (I), PomA/B(C31Y) (Y) or PomA/B(C31W) (W). Transformants were cultured in liquid medium overnight, and aliquots were spotted onto a plate containing VPG500 and 0.26% agar. Plates were incubated at 30°C for 6 hr. Vec. means a pSU41 plasmid vector. (b) Western blotting analysis of PomA (left panel) and PomB (right panel) in whole cell extracts. Proteins were detected with an anti-PomA1312 antibody and an anti-PomB93 antibody.

Figure 3

(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.

Figure 4

Estimation of apparent Na⁺ binding affinity with WT PomA/B and PomB mutants. The swimming speeds of cells (WT; wild-type PomA/B, Ala; C31A, Thr; C31T, Gly; C31G, Met; C31M, Val; C31V, Ile; C31I, Ser; C31S) were plotted against NaCl concentrations. Data were fit by the Michaelis-Menten equation.

Figure 5

(a) Swarms of RP6894 (ΔmotAB) cells harboring plasmids that express MotA/B(A39V) (A39V), MotA(M206G)/B(A39V) (A39V/M206G), MotA(M206A)/B(A39V) (A39V/M206A), MotA(M206S)/B(A39V) (A39V/M206S) or MotA(M206T)/B(A39V) (A39V/M206T). Transformants were cultured in liquid medium overnight, and aliquots were spotted onto a plate containing TG and 0.26% agar. Plates were incubated in the presence of 0.04% arabinose at 30°C for 20 hr. Vec. means a pBAD24 plasmid vector. (b) Western blotting analysis of MotA (left panel) and MotB (right panel) in whole cell extracts. Proteins were detected with an anti-MotA266 antibody and an anti-MotB2 antibody. (c) Swarms of NMB191 (ΔpomAB) cells harboring a plasmid that express PomA/B (WT), PomA/B(C31A) (C31A), PomA/B(L28A/C31A) (L28A/C31A) or PomA(L183F)/B(L28A/C31A) (L28A/C31A/L183F). Transformants were cultured in liquid medium overnight, and aliquots were spotted onto a plate containing VPG10 (1% polypeptone, 0.4% K₂HPO₄, 10 mM NaCl, 490 mM KCl, 0.5% glycerol) and 0.25% agar. Plates were incubated in the presence of 0.04% arabinose and 2.5 μg/ml chloramphenicol at 30°C for 12 hr. Vec. means a pBAD33 plasmid vector. (d) Western blotting analysis of PomA (left panel) and PomB (right panel) in whole cell extracts. Proteins were detected with an anti-PomA1312 antibody and an anti-PomB93 antibody.

Figure 6

Model of the ion flux pathway. Asp24 of PomB and Asp32 of MotB form Na⁺ and H⁺ binding sites, respectively, Cys31(PomB)-Leu183(PomA) and Ala39(MotB)-Met206(MotA) serve as a ion channel wall and a regulator for H⁺ and Na⁺ flux, respectively. The ion flux leads to interaction changes and structural change(s) of FliG, and they mediate the flagellar motor rotation.