RESIDUES OF THE CYTOPLASMIC DOMAIN OF MOTA ESSENTIAL FOR TORQUE GENERATION IN THE BACTERIAL FLAGELLAR MOTOR

The MotA protein of Escherichia coli is a component of the flagellum t hat functions, together with MotB, in transmembrane proton conduction. MotA and MotB are believed to form the stator of the flagellar motor. They are integral membrane proteins; MotA has a large (ca 22 kDa) dom ain in the cytoplasm, and MotB a much smaller one. (ca 3 kDa). Recent work suggests that cytoplasmically located parts of MotA and/or MotB m ight be present at the active site for torque generation in the motor. To test the proposal that the cytoplasmic domain of MotA functions in torque generation, and to identify the amino acid residues most impor tant for function, we have carried out a mutational analysis of this d omain. Using random mutagenesis, many mutations of cytoplasmic residue s of MotA were isolated, which either abolish or impair torque generat ion. hn most cases the residues affected are not conserved, and many o f the replacements involve loss or gain of a proline residue, which su ggests that these mutations disrupt function by altering the protein c onformation rather than by directly affecting residues of an active si te. Using site-directed mutagenesis, the conserved residues in the cyt oplasmic domain of MotA were replaced, either singly or, in the case b f charged residues, in various combinations. The results identify four residues of MotA that are import-ant for motor function. These are Ar g90 and Glu98, located in the cytoplasmic domain, and Pro173 and Pro22 2, located at the interface between the cytoplasmic domain and the mem brane-spanning domain. Possible roles for these residues in torque gen eration are discussed. (C) 1997 Academic Press Limited.