Characterization of the Periplasmic Domain of MotB and Implications for Its Role in the Stator Assembly of the Bacterial Flagellar Motor▿

摘要

MotA and MotB are integral membrane proteins that form the stator complex of the proton-driven bacterial flagellar motor. The stator complex functions as a proton channel and couples proton flow with torque generation. The stator must be anchored to an appropriate place on the motor, and this is believed to occur through a putative peptidoglycan-binding (PGB) motif within the C-terminal periplasmic domain of MotB. In this study, we constructed and characterized an N-terminally truncated variant of Salmonella enterica serovar Typhimurium MotB consisting of residues 78 through 309 (MotBC). MotBC significantly inhibited the motility of wild-type cells when exported into the periplasm. Some point mutations in the PGB motif enhanced the motility inhibition, while an in-frame deletion variant, MotBC(Δ197-210), showed a significantly reduced inhibitory effect. Wild-type MotBC and its point mutant variants formed a stable homodimer, while the deletion variant was monomeric. A small amount of MotB was coisolated only with the secreted form of MotBC-His6 by Ni-nitrilotriacetic acid affinity chromatography, suggesting that the motility inhibition results from MotB-MotBC heterodimer formation in the periplasm. However, the monomeric mutant variant MotBC(Δ197-210) did not bind to MotB, suggesting that MotBC is directly involved in stator assembly. We propose that the MotBC dimer domain plays an important role in targeting and stable anchoring of the MotA/MotB complex to putative stator-binding sites of the motor.