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

Dynamic NanoMachine Project, ICORP, JST, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan,¹ Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan²

Received 25 October 2007/ Accepted 19 February 2008

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 (MotB_C). MotB_C 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, MotB_C(197-210), showed a significantly reduced inhibitory effect. Wild-type MotB_C 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 MotB_C-His₆ by Ni-nitrilotriacetic acid affinity chromatography, suggesting that the motility inhibition results from MotB-MotB_C heterodimer formation in the periplasm. However, the monomeric mutant variant MotB_C(197-210) did not bind to MotB, suggesting that MotB_C is directly involved in stator assembly. We propose that the MotB_C dimer domain plays an important role in targeting and stable anchoring of the MotA/MotB complex to putative stator-binding sites of the motor.

Published ahead of print on 29 February 2008.