Article Figures & Data
Figures
- FIG 1
Arrangement of MotA and MotB. (A) Topology of MotA and MotB and location of important amino acid residues. MotA has four transmembrane segments (TM1, TM2, TM3, and TM4). MotB has a single transmembrane segment (TM) and a peptidoglycan-binding motif (PGB). MotB-Asp33 works as a proton pathway. (B) Arrangement of transmembrane segments of MotA and MotB. The MotA/MotB complex is postulated to have two proton flux pathways, shown as light blue circles. (C) The helical wheel projections showing the arrangement of amino acids in segments forming the proton channel (boxed in panel B) is represented. Hydrophilic residues are represented as circles, hydrophobic residues are shown as diamonds, potentially negatively charged residues are shown as triangles, and potentially positively charged residues are shown as pentagons. Hydrophobicity is color coded as well: the most hydrophobic residue is green, and the amount of green decreases proportionally to the hydrophobicity, with zero hydrophobicity indicated in yellow. Hydrophilic residues are shown in red, with pure red being the most hydrophilic (uncharged) residue, and the amount of red decreases proportionally to the hydrophilicity. The potentially charged residues are shown in purple. The plots were generated using a helical wheel projection (D. Armstrong and R. Zidovetzki, unpublished data).
- FIG 2
Effect of MotA(M206I) mutation on proton conductivity. (A) Growth curves of Salmonella cells carrying a plasmid expressing motA motB, motA motB(Δplug), or motA(M206I) motB(Δplug). Protein expression was induced by the addition of 0.2% arabinose at the time indicated with the arrow. Average values of three independent experiments are shown. (B) Immunoblotting using anti-MotB antibody Arabinose (0.2%) was added after a 3-h incubation as shown in panel A, and protein expression was induced for 1 h. (C) Intracellular pH measured using pHluorin in the external pH 5.5. Stator proteins were expressed in E. coli. Results from more than six independent experiments were averaged; error bars represent standard errors.
- FIG 3
Effect of MotA(M206I)/MotB overexpression on motility of wild-type (WT) cells. (A) Fresh colonies of transformants of E. coli RP6894 carrying pBAD24 (Vector), pYC20 (WT), pYC98 (D33N), and pYS1 (M206I) were inoculated in a soft-agar plate with 0.02% arabinose and incubated at 30°C for 11 h. (B) Motility of Salmonella SJW1103 (wild type) carrying the same plasmids in a soft-agar plate with 0.2% arabinose. The plate was incubated at 30°C for 8 h. (C) Immunoblotting of whole-cell proteins prepared from the same strains described for panel B using polyclonal anti-MotA (upper) and anti-MotB (lower) antibodies. The positions of molecular mass markers (in kilodaltons) are shown on the left. Arrowheads indicate the positions of MotA and MotB. (D to F) Arabinose dependence of protein expression, the motile fraction, and swimming speed, as indicated, examined using SJW2241 (ΔmotA motB) carrying pYC20 (white) and pYS1 (gray). Swimming speeds are average values from more than 30 cells; error bars indicate the standard deviations.
- FIG 4
Torque-speed curves of the WT and MotA(M206I) motors. Rotations of the WT (closed symbols) and MotA(M206I) motors (open symbols) were measured using beads with different diameters in motility medium (pH 7.0) with or without Ficoll, indicated as follows: circles, 1.0 μm in 12% Ficoll; triangles, 1.0 μm in medium without Ficoll; squares, 0.5 μm in medium without Ficoll; diamonds, 0.1 μm in medium without Ficoll. Average values and standard deviations are shown.
- FIG 5
Effect of pH changes on motor rotation. Rotation speeds of the WT and MotA(M206I) motors were measured with 1-μm polystyrene beads in the absence (A) or presence (B) of 20 mM potassium benzoate. Average values (circles) and standard deviations (error bars) are shown. No rotation of the M206I motor was observed at pH 5.5 in the presence of benzoate. The right panels show relative values normalized to the rotation speed determined at external pH 7.5.
- FIG 6
Effect of pH changes on the speed per stator unit. (A and B) Typical resurrection traces for Salmonella motors expressing WT (A) or MotA(M206I)/MotB (B) stator units; right panels are speed histograms obtained from speed-versus-time traces. Rotations were measured at external pH 7.0 (left) and 6.0 (right). Horizontal axes in resurrection traces are times after addition of arabinose. Intervals of horizontal gray lines were determined arbitrarily so as to approximately match resurrection steps in each trace as follows: 8.9 Hz for panel A, left; 9.0 Hz for panel A, right; 4.2 Hz for panel B, left; and 4.0 Hz for panel B, right. (C and D) Speed-versus-resurrection step number for WT (C) or MotA(M206I)/MotB (D) stator unit is also shown. The speed for the first step differed between motors due to variance in the number of docked stators. Slopes of regression lines fitted to data points of each motor indicate the speed produced by a single stator unit.
- FIG 7
Effect of pH changes on subcellular localizations of stator units. (A) Epifluorescent images of Salmonella cells expressing MotA/mTurquoise2-MotB, MotA(M206I)/mTurquoise2-MotB, or MotA/mTurquoise2-MotB(D33N) obtained at pH 7.0 and pH 5.5. (B) Effect of benzoate or CCCP on the subcellular localization of WT, MotA(M206I), or MotB(D33N) stator; 20 mM potassium benzoate or 50 μM CCCP was added to medium. The stator localization was quantified from fluorescent images. (C to E) The number of fluorescent spots per cell (C) and fluorescent intensity of each spot (D) were measured; the localization efficiency was evaluated by multiplying the number of spots by the intensity of the spots, representing the total number of docked stators per cell (E). Bars are standard deviations. AU, arbitrary units.
Tables
- TABLE 1
Strains and plasmids used in this study
Strain or plasmid Relevant characteristic(s) Source or reference Salmonella strains SJW1103 Wild type for motility and chemotaxis 47 SJW2241 ΔmotA-motB 48 YSC2123 ΔmotA-motB ΔcheY ΔfimA fliC(Δ204–292) 5 E. coli strains BL21(DE3) Strain for protein expression Novagen RP6894 ΔmotA-motB J. S. Parkinson Plasmids pBAD24 Expression vector 49 pET22b Expression vector Novagen pYC17 pACTrc/pHluorin 50 pYC20 pBAD24/MotA MotB 44 pYC98 pBAD24/MotA MotB(D33N) 42 pYC109 pBAD24/MotA MotB(Δ52–71) 44 pYC112 pBAD24/MotA MotB(Δ52–71, D33N) 42 pYS1 pBAD24/MotA(M206I) MotB This study pYS1(Δplug) pBAD24/MotA(M206I) MotB(Δ52–71) This study pYVM106 pET22b/MotA mTurquoise2-MotB This study pYVM107 pET22b/MotA(M206I) mTurquoise2-MotB This study pYVM108 pET22b/MotA mTurquoise2-MotB(D33N) This study pYVM109 E. coli MotA mTurquoise2-MotB/pET22b This study pYVM110 E. coli MotA mTurquoise2-MotB(D32N)/pET22b This study
Supplemental material
- Supplemental file 1 -
Fig. S1 [Resurrection traces obtained by induction of MotA/MotB or MotA(M206I)/MotB proteins], S2 [Effect of external pH on expression of MotA/MotB or MotA(M206I)/MotB proteins], S3 (Effect of pH changes on fluorescent intensity of mTuquoise2-MotB), S4 (Effect of CCCP on subcellular localizations of stator units in E. coli), and S5 [Effect of changes in pH on steady-state rotation of fully induced WT and MotA(M206I) motors]
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- Supplemental file 1 -
