Abstract
The bacterial flagellar motor converts the energy of proton flow through the MotA/MotB complex into mechanical works required for motor rotation. The rotational force is generated by electrostatic interactions between the stator protein MotA and the rotor protein FliG. The Arg-90 and Glu-98 from MotA interact with Asp-289 and Arg-281 of FliG, respectively. An increase in the expression level of the wild-type MotA/MotB complex inhibits motility of the gfp-motB fliG(R281V) mutant but not the fliG(R281V) mutant, suggesting that the MotA/GFP-MotB complex cannot work together with wild-type MotA/MotB in the presence of the fliG(R281V) mutation. However, it remains unknown why. Here, we investigated the effect of the GFP fusion to MotB at its N-terminus on the MotA/MotB function. Over-expression of wild-type MotA/MotB significantly reduced the growth rate of the gfp-motB fliG(R281V) mutant. The over-expression of the MotA/GFP-MotB complex caused an excessive proton leakage through its proton channel, thereby inhibiting cell growth. These results suggest that the GFP tag on the MotB N-terminus affects well-regulated proton translocation through the MotA/MotB proton channel. Therefore, we propose that the N-terminal cytoplasmic tail of MotB couples the gating of the proton channel with the MotA–FliG interaction responsible for torque generation.
Highlights
Many bacteria are propelled by rotating flagella to swim in liquid environments
When the intracellular pH decreases, the dissociation rate of protons from the cytoplasmic entrance of the MotA/MotB proton channel into the cytoplasm is reduced significantly, resulting in a slower torque generation cycle of the motor to cause severely impaired motility [47]. These observations lead to a plausible hypothesis that the motility inhibition of the gfp-motB fliG(R281V) strain caused by over-expression of the MotA/MotB complex [36] may be a consequence of reduction in intracellular pH caused by undesirable proton
Because the green fluorescent protein (GFP) tag to MotB increases the proton channel activity of the MotA/MotB complex (Figure 3b), we propose that the interaction between Glu-98 of MotA and Arg-281 of FliG play an important role in the activation mechanism of the MotA/MotB proton channel and that physical communications between MotAC and MotBNCT promote the opening of the cytoplasmic side of the proton channel of the MotA/MotB complex
Summary
The basal body is located at the base of the flagellar filament acting as a helical propeller and works as a rotary motor powered by the electrochemical potential difference of cations, such as proton and sodium ion, across the membrane that translocate those cations through the transmembrane channel of the stator complex associated around the rotor [1,2]. The proton-driven flagellar motor of Salmonella enterica generates the rotational force through processive interactions between the rotor and multiple stator units [3,4,5,6,7]. The flagellar motor autonomously controls the number of functional stator units around the rotor in response to changes in the environment [15]
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