VIBRIO-ALGINOLYTICUS MUTANTS RESISTANT TO PHENAMIL, A SPECIFIC INHIBITOR OF THE SODIUM-DRIVEN FLAGELLAR MOTOR

The polar flagella of Vibrio alginolyticus are driven by sodium motive forte and those motors are specifically and strongly inhibited by phe namil, an amiloride analog that is thought to interact with a sodium c hannel of the flagellar motor. To study the sodium ion coupling site, we isolated motility mutants resistant to phenamil and named the pheno type Mpa(r) for motility resistant to phenamil. The motility of the wi ld-type (Mpa(s)) was inhibited by 50 mu M phenamil, whereas Mpa(r) str ains were still motile in the presence of 200 mu M phenamil. The K-i v alue for phenamil in the Mpa(r) strain was estimated to be five times larger than that in the Mpa(s) strain. However, the sensitivities to a miloride or benzamil, another amiloride analog, were not distinctly ch anged in the Mpa(r) strain. The rotation rate of the wild-type Na+-dri ven motor fluctuates greatly in the presence of phenamil, which can be explained in terms of a relatively slow dissociation rate of phenamil from the motor. We therefore studied the stability of the rotation of the Mpa(r) and Mpa(s) motors by phenamil. The speed fluctuations of t he Mpa(r) motors were distinctly reduced relative to the Mpa(s) motors . The steadier rotation of the Mpa(r) motors can be explained by an in crease in the phenamil dissociation rate from a sodium channel of the motor, which suggests that a phenamil-specific binding site of the mot or is mutated in the Mpa(r) strain. (C) 1997 Academic Press Limited