scholarly journals The C-terminal periplasmic domain of MotB is responsible for load-dependent control of the number of stators of the bacterial flagellar motor

BIOPHYSICS ◽  
2013 ◽  
Vol 9 (0) ◽  
pp. 173-181 ◽  
Author(s):  
David J. Castillo ◽  
Shuichi Nakamura ◽  
Yusuke V. Morimoto ◽  
Yong-Suk Che ◽  
Nobunori Kami-ike ◽  
...  
Keyword(s):  
Flagellar Motor ◽  
Periplasmic Domain ◽  
Bacterial Flagellar Motor

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

2008 ◽  
Vol 190 (9) ◽  
pp. 3314-3322 ◽  
Author(s):  
Seiji Kojima ◽  
Yukio Furukawa ◽  
Hideyuki Matsunami ◽  
Tohru Minamino ◽  
Keiichi Namba
Keyword(s):  
Point Mutations ◽  
Nitrilotriacetic Acid ◽  
Inhibitory Effect ◽  
Proton Channel ◽  
Flagellar Motor ◽  
Wild Type ◽  
Deletion Variant ◽  
Periplasmic Domain ◽  
Bacterial Flagellar Motor ◽  
Motility Inhibition

ABSTRACT 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.

scholarly journals Site-Directed Cross-Linking Identifies the Stator-Rotor Interaction Surfaces in a Hybrid Bacterial Flagellar Motor

2021 ◽  
Vol 203 (9) ◽  
Author(s):  
Hiroyuki Terashima ◽  
Seiji Kojima ◽  
Michio Homma
Keyword(s):  
Escherichia Coli ◽  
Cross Linking ◽  
Physical Interaction ◽  
Flagellar Motor ◽  
Intact Cells ◽  
Content Type ◽  
Bacterial Flagellum ◽  
Cross Links ◽  
Bacterial Flagellar Motor ◽  
Rotary Motor

ABSTRACT The bacterial flagellum is the motility organelle powered by a rotary motor. The rotor and stator elements of the motor are located in the cytoplasmic membrane and cytoplasm. The stator units assemble around the rotor, and an ion flux (typically H+ or Na+) conducted through a channel of the stator induces conformational changes that generate rotor torque. Electrostatic interactions between the stator protein PomA in Vibrio (MotA in Escherichia coli) and the rotor protein FliG have been shown by genetic analyses but have not been demonstrated biochemically. Here, we used site-directed photo-cross-linking and disulfide cross-linking to provide direct evidence for the interaction. We introduced a UV-reactive amino acid, p-benzoyl-l-phenylalanine (pBPA), into the cytoplasmic region of PomA or the C-terminal region of FliG in intact cells. After UV irradiation, pBPA inserted at a number of positions in PomA and formed a cross-link with FliG. PomA residue K89 gave the highest yield of cross-links, suggesting that it is the PomA residue nearest to FliG. UV-induced cross-linking stopped motor rotation, and the isolated hook-basal body contained the cross-linked products. pBPA inserted to replace residue R281 or D288 in FliG formed cross-links with the Escherichia coli stator protein, MotA. A cysteine residue introduced in place of PomA K89 formed disulfide cross-links with cysteine inserted in place of FliG residues R281 and D288 and some other flanking positions. These results provide the first demonstration of direct physical interaction between specific residues in FliG and PomA/MotA. IMPORTANCE The bacterial flagellum is a unique organelle that functions as a rotary motor. The interaction between the stator and rotor is indispensable for stator assembly into the motor and the generation of motor torque. However, the interface of the stator-rotor interaction has only been defined by mutational analysis. Here, we detected the stator-rotor interaction using site-directed photo-cross-linking and disulfide cross-linking approaches. We identified several residues in the PomA stator, especially K89, that are in close proximity to the rotor. Moreover, we identified several pairs of stator and rotor residues that interact. This study directly demonstrates the nature of the stator-rotor interaction and suggests how stator units assemble around the rotor and generate torque in the bacterial flagellar motor.

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