Structural Basis Of Assembly And Torque Transmission Of The Bacterial Flagellar Motor

The bacterial flagellar motor drives the rotation of flagellar filaments at speeds of300 to 2400 revolutions per second to facilitate bacterial mobility,which is essential for bacterial survival and pathogenicity.The flagellar motor is composed of over 25 different proteins.It is one of nature’s most remarkable nanoscale molecular engines with nearly 100% efficiency in energy conversion.However,the detailed structure,assembly and torque mechanisms of the bacterial flagellar motor remain elusive.To investigate the structure of the bacterial flagellar motor,we generated a mutant strain of the Salmonella enterica Typhimurium LT2 strain with double deletions of the flagellin-related genes fliC and fliD to avoid the effects of flagellins on purification of the flagellar motor.The Salmonella LT2-Δfli CD strain produced the flagellar motor and hook normally but did not assemble the filament.We optimized the purification procedure of bacterial flagellar motor with a mild condition based on previous studies.The purified flagellar motor-hook complex was highly stable in the negative staining and cryo-EM analyses.Reconstruction of the cryo-EM imaging data generated the local density maps of the flagellar motor-hook complex in the assembled state,including a 3.9-(?) density map of the global complex,a 2.8-(?) density map of the LP ring,a 3.2-(?) density map of the rod,a 3.6-(?) density map of the export apparatus,a 3.6-(?) density map of the MS ring and a 3.4-(?) density map of the hook.The overall structure of the flagellar motor-hook complex was modeled with 175 subunits from 12 different proteins,and with a molecular mass of approximately 6.3MDa.The export apparatus consists of 5 FliP,4 FliQ,and 1 FliR subunits and adopts a right-hand helical symmetry.6 FliE and 5 FlgB subunits trigger the remodeling of the export apparatus from a closed to an open conformation to initiate the rod assembly,thereby 6 FlgC,5 FlgF,and 24 FlgG are sequentially assembled upon FliE and FlgB.The MS ring consists of 34 FliF subunits.10 peptides protruding from the MS ring with the FlgB and FliE subunits mediate torque transmission from the MS ring to the rod and overcome the symmetry mismatch between the rotational and helical structures in the motor.FlgH and FlgI interact with each other in a 1-versus-4 manner.The LP ring applies electrostatic forces and hydrogen bonds to enable the high-speed rotation of the rod without significant structural obstacles or energy consumption.The rod connects the hook via a tight tubular junction structure to transmit the torque.In summary,our work provides detailed molecular insights into the components,structural assembly,and torque transmission mechanisms of the bacterial flagellar motor,and provides valuable information for antibiotics development,synthetic biology,and nanomachines design in the future.