Sodium-powered stators of the bacterial flagellar motor can generate torque in the presence of phenamil with mutations near the peptidoglycan-binding region.

The bacterial flagellar motor (BFM) is a molecular complex which powers the rotation of the filament that propels swimming bacteria. Rotational torque is generated by harnessing the flow of ions through ion channels known as stators which couple the energy from the ion gradient across the inner membrane to rotation of the rotor. Here we used error-prone PCR to introduce single point mutations into the sodium-powered Vibrio alginolyticus/Eschrichia Coli chimeric stator PotB. We then selected for motors that exhibited resistance to the sodium-channel inhibitor phenamil. We found that single mutations that inferred resistance to phenamil occurred at two sites: 1) the transmembrane domain of PotB, corresponding to the TM region of the PomB stator from V. alginolyticus, and 2) near the peptidoglycan (PG) binding region that corresponds to the C-terminal region of the MotB stator from E. coli. We corroborated our swim plate observations with single cell rotation assays to confirm that individual cells could drive rotation of flagellar motors in the presence of up to 100 M phenamil. Our results demonstrate that it is not only the pore region of the stator that moderates the effect of motility in the presence of ion-channel blockers. We hypothesise that mutations in the PG region can allow motors to function in the presence of phenamil by allowing multiple semi-functioning stators to persist on the motor and drive flagellar rotation.