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

PomA and PomB are integral membrane proteins and are essential for the rota tion of the Na+-driven polar flagellar motor of Vibrio alginolyticus. On th e basis of their similarity to MotA and MotB, which are the proton-conducti ng components of the H+-driven motor they are thought to form the Na+-chann el complex and to be essential for mechanochemical coupling in the motor. T o investigate PomA function, random mutagenesis of the pomA gene by using h ydroxylamine was carried out. We Isolated 37 non-motile mutants (26 indepen dent mutations) and most of the mutations were dominant; these mutant allel es are able to inhibit the motility of wild-type cells when greatly overexp ressed. The mutant PomA proteins could be detected by immunoblotting. excep t for those with deletions or truncations. Many of the dominant mutations w ere mapped to the putative third or fourth transmembrane segments, which ar e the most conserved regions. Some mutations that showed strong dominance w ere in highly conserved residues. T1861 is the mutation of a polar residue located in a transmembrane segment that might be involved in ion translocat ion. 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 mechan ism for torque generation will be similar in the proton and sodium motors.