Intragenic suppressor of a plug deletion nonmotility mutation in potB, a chimeric stator protein of sodium-driven flagella

Abstract

The torque of bacterial flagellar motors is generated by interactions between the rotor and the stator and is coupled to the influx of H+ or Na+ through the stator. A chimeric protein, PotB, in which the N-terminal region of Vibrio alginolyticus PomB was fused to the C-terminal region of Escherichia coli MotB, can function with PomA as a Na+ -driven stator in E. coli. Here, we constructed a deletion variant of PotB (with a deletion of residues 41 to 91 [δ41-91], called PotBδL), which lacks the periplasmic linker region including the segment that works as a "plug" to inhibit premature ion influx. This variant did not confer motile ability, but we isolated a Na+-driven, spontaneous suppressor mutant, which has a point mutation (R109P) in the MotB/PomB-specific α-helix that connects the transmembrane and peptidoglycan binding domains of PotBδL in the region of MotB. Overproduction of the PomA/PotBδL(R109P) stator inhibited the growth of E. coli cells, suggesting that this stator has high Na+-conducting activity. Mutational analyses of Arg109 and nearby residues suggest that the structural alteration in this α-helix optimizes PotBδL conformation and restores the proper arrangement of transmembrane helices to form a functional channel pore. We speculate that this α-helix plays a key role in assembly-coupled stator activation. © 2012, American Society for Microbiology.