Structure-function studies of the bacterial flagellar motor protein FliG

Previous studies have shown that the FliG, FliM, and FliN proteins form a complex on the rotor portion of the bacterial flagellar motor. In addition, genetic analyses of nonmotile (Mot-) mutants have suggested that FliG, FliM, and FliN may be directly involved in torque generation. Subsequently, it was shown that overexpression of the mutant proteins restores motility to all of the Mot- fliM and fliN mutants. These results suggest that FliM and FliN are unlikely to be directly involved in torque generation. Here, previously reported, randomly isolated Mot- mutations in fliG are examined. Overexpression of the FliG Mot-mutant proteins does not restore motility to any of the mutants, which.suggests that FliG directly participates in torque generation. The locations of nonflagellate and Motmutations in the fliG coding sequence suggest that FliG consists of two functional domains: an N-terminal domain required for flagellar assembly and a C-terminal domain required for motility. Next, site-directed mutagenesis was used to identify amino acid residues in the C-terminal domain of Escherichia coli FliG that are critical for torque generation. The results indicate that five charged residues in FhG, Lys264, Arg281, Asp288, Asp289, and Arg297, form the rotor portion of the active site in the bacterial flagellar motor, as several mutations at these positions completely abolish motility. Double mutants with charged residue substitutions in both the rotor protein FliG and the stator protein MotA also were examined. A pattern of synergistic motility defects, which indicates that the active site residues of FliG interact electrostatically with charged residues in MotA, was observed. Lastly, the C-terminal FliG motility domain from the hyperthermophilic eubacterium Thermotoga maritima was crystallized and its three-dimensional structure determined. The active site residues critical for torque generation are shown to form a highly charged ridge on the surface of the protein. This ridge likely delineates the pathway of motor rotation. Nonchernotactic mutations identify residues in another region of the protein that are critical for controlling the direction of motor rotation. A model for the arrangement and stoichiometry of the FliG motility domain in the bacterial flagellar motor is presented.