title: 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.
authors.affiliation: Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, Japan.
authors.forename: Shiwei
authors.initials: S
authors.lastname: Zhu
authors.forename: Michio
authors.initials: M
authors.lastname: Homma
authors.forename: Seiji
authors.initials: S
authors.lastname: Kojima
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D005407:Flagella / Q000235:genetics / Q000378:metabolism*; D020409:Molecular Motor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D044149:Vibrio alginolyticus / Q000235:genetics / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D020409:Molecular Motor Proteins; C064779:MotB protein, Bacteria; C103352:PomA protein, bacteria; C107541:PomB protein, Vibrio; D011993:Recombinant Fusion Proteins; D015222:Sodium Channels; D012964:Sodium