Random mutagenesis of the pomA gene encoding a putative channel component of the Na+-driven polar flagellar motor of Vibrio alginolyticus

摘要

PomA and PomB are integral membrane proteins and are essential for the rotation of the Na+-driven polar flagellar motor of Vibrio alginolyticus. On the basis of their similarity to MotA and MotB, which are the proton-conducting components of the H+-driven motor, they are thought to form the Na+-channel complex and to be essential for mechanochemical coupling in the motor. To investigate PomA function, random mutagenesis of the pomA gene by using hydroxylamine was carried out. We isolated 37 non-motile mutants (26 independent mutations) and most of the mutations were dominant; these mutant alleles are able to inhibit the motility of wild-type cells when greatly overexpressed. The mutant PomA proteins could be detected by immunoblotting, except for those with deletions or truncations. Many of the dominant mutations were mapped to the putative third or fourth transmembrane segments, which are the most conserved regions. Some mutations that showed strong dominance were in highly conserved residues. T186I is the mutation of a polar residue located in a transmembrane segment that might be involved in ion translocation. P199L occurred in a residue that is thought to mediate conformational changes essential for torque generation in MotA. These results suggest that PomA and MotA have very similar structures and roles, and the basic mechanism for torque generation will be similar in the proton and sodium motors.