The FliG protein of Escherichia coli is essential for assembly and function of the flagellar motor. Certain mutations in FliG give a non-motile, or Mot-, phenotype, in which flagella are assembled but do not rotate. Mutations with this property are clustered in a C-terminal segment of FliG that is stable when expressed alone, and thus probably constitutes an independently folded domain. Previously, we suggested that this domain forms the rotor portion of the active site for torque generation in the motor. In this work, we have used a mutational approach to identify the amino acid residues in the C-terminal domain of FliG that are most important for motor function. Site-directed mutagenesis was used to replace each of the conserved residues in this domain with alanine, and the effects on motor function were measured. Because charged residues have often been suggested to have important roles in torque generation, conserved charged residues were changed individually and in all pairwise combinations. The results show that three charged residues of FliG, Arg279, Asp286 and Asp287, are directly involved in torque generation. Mutations in these residues cause motility defects that suggest that they function jointly, in an active site whose most important property is a specific arrangement of charges. Two other charged residues, Lys262 and Arg295, may also be involved in torque generation, but are less critical than Arg279, Asp286 or Asp287. Uncharged residues of the FliG motility domain do not appear to have direct roles in torque generation, although some are needed for the stability of the protein or for normal clockwise/counter-clockwise switching. The Mot-mutations of fliG isolated previously by random mutagenesis do not alter the putative active-site residues, but render the proteins abnormally susceptible to proteolysis, suggesting significantly altered conformations or reduced stabilities.
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