Distinct roles of highly conserved charged residues at the MotA-FliG interface in bacterial flagellar motor rotation

Abstract

Electrostatic interactions between the stator protein MotA and the rotor protein FliG are important for bacterial flagellar motor rotation. Arg90 and Glu98 of MotA are required not only for torque generation but also for stator assembly around the rotor, but their actual roles remain unknown. Here we analyzed the roles of functionally important charged residues at the MotA-FliG interface in motor performance. About 75% of the motA(R90E) cells and 45% of the motA(E98K) cells showed no fluorescent spots of green fluorescent protein (GFP)-MotB, indicating reduced efficiency of stator assembly around the rotor. The FliG(D289K) and FliG(R281V) mutations, which restore the motility of the motA(R90E) and motA(E98K) mutants, respectively, showed reduced numbers and intensity of GFP-MotB spots as well. The FliG(D289K) mutation significantly recovered the localization of GFP-MotB to the motor in the motA(R90E) mutant, whereas the FliG(R281V) mutation did not recover the GFP-MotB localization in the motA(E98K) mutant. These results suggest that the MotA-Arg90-FliG-Asp289 interaction is critical for the proper positioning of the stators around the rotor, whereas the MotA-Glu98-FliG-Arg281 interaction is more important for torque generation.

Fig 1

Schematic diagram of possible electrostatic interactions at the rotor-stator interface. The stator complex consists of four copies of MotA and two copies of MotB. FliG is a rotor protein and is postulated to occupy the upper part of the C ring. The interactions between MotA-Arg90 and FliG-Asp289 and between MotA-Glu98 and FliG-Arg281 are important for motor function (17).

Fig 2

Effect of the MotA(R90E) mutation on torque generation by the flagellar motor. (A) Torque-speed curve of fully induced wild-type and MotA(R90E) mutant motor. The Salmonella YSC2122 strain [ΔmotA-motB, fliC(Δ204-292)] was transformed with pYC20 (wild-type MotA/B [WT]) or pYC20(R90E) [MotA(R90E)/B, abbreviated as R90E], and the resulting transformants were grown in LB containing 100 μg of ampicillin/ml and 100 μM arabinose for 5 h at 30°C. Rotation measurements of individual flagellar motors were carried out by tracking the position of 1.5-μm (circle), 1.0-μm (triangle), 0.8-μm (square), or 0.5-μm (diamond) beads attached to the sticky flagellar filament. All of the measurements were made at around 23°C. (B) Speed histogram of fully induced wild-type (left) or MotA(R90E) mutant motor (right). Rotation rate of each flagellar motor labeled with 1.0-μm bead was determined using 1-s data windows at an interval of 0.1 s. The number labels above the data represent the units corresponding to multiples of 7 Hz.

Fig 3

Effect of MotA(R90E) and MotA(E98K) mutations on the proton conductivity of the unplugged, activated MotA/B(Δ52-71) proton channel. Intracellular pH of YSC2123 cells (ΔmotA-motB ΔfliC::pHluorin) was measured using pHluorin expressed in the cytoplasm after the induction of MotA/B (WT), MotA/B(Δ52-71) (Δ52-71), MotA(R90E)/B(Δ52-71) (R90E/Δ52-71), or MotA(E98K)/B(Δ52-71) (E98K/Δ52-71) at an external pH of 5.5. The data that exhibited a statistically significant differences (***, P < 0.001) compared to the wild-type MotA/B (#) are highlighted with asterisks.

Fig 4

Effect of FliG and MotA mutations on free-swimming motility. The fraction and speed of free-swimming cells are indicated for the following strains: YVM003 (WT/WT), YVM033 (WT/D289K), YVM034 (WT/R281V), YVM037 (WT/R281D), YVM031 (R90E/WT), YVM032 (E98K/WT), YVM035 (R90E/D289K), YVM036 (E98K/R281V), and YVM039 (E98K/R281D). The swimming fraction is the fraction of swimming cells. The swimming speed is the average speed of >40 cells, and vertical lines indicate the standard deviations. If the fraction of motile cells was <5% of the total cells, the swimming speed is presented as zero. Measurements were made at around 23°C.

Fig 5

Effect of FliG and MotA mutations on the subcellular localization of GFP-MotB. (A) Bright-field (BF) and epifluorescence (EPI) images of YVM003 (WT/WT), YVM033 (WT/D289K), YVM034 (WT/R281V), YVM037 (WT/R281D), YVM031 (R90E/WT), YVM032 (E98K/WT), YVM035 (R90E/D289K), YVM036 (E98K/R281V), and YVM039 (E98K/R281D). The cells were incubated at 30°C for 16 h in LB and then observed by fluorescence microscopy. Measurements were made at around 23°C. (B) Population fraction of the cells with different numbers of GFP-MotB spots in each strain described above. More than 400 cells were counted.

Fig 6

Multicopy effect of the wild-type MotA/B complex on free-swimming motility of fliG mutants. The fraction and speed of free-swimming cells of YVM003 (WT) transformed with pYC20 (MotAB), YVM033 (D289K) harboring pYC20, and YVM034 (R281V) carrying pYC20. The swimming fraction is the fraction of swimming cells. The cells were incubated at 30°C for 5 h in LB with 0.2, 0.02, or 0.002% arabinose. Measurements were made at around 23°C.