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 GFP Fusion to the N-Terminus of MotB Affects the Proton Channel Activity of the Bacterial Flagellar Motor in Salmonella

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1255
Author(s):  
Yusuke V. Morimoto ◽  
Keiichi Namba ◽  
Tohru Minamino
Keyword(s):  
Electrostatic Interactions ◽  
Proton Translocation ◽  
Proton Channel ◽  
Flagellar Motor ◽  
Wild Type ◽  
Over Expression ◽  
N Terminus ◽  
Flow Through ◽  
Proton Leakage ◽  
Bacterial Flagellar Motor

The bacterial flagellar motor converts the energy of proton flow through the MotA/MotB complex into mechanical works required for motor rotation. The rotational force is generated by electrostatic interactions between the stator protein MotA and the rotor protein FliG. The Arg-90 and Glu-98 from MotA interact with Asp-289 and Arg-281 of FliG, respectively. An increase in the expression level of the wild-type MotA/MotB complex inhibits motility of the gfp-motBfliG(R281V) mutant but not the fliG(R281V) mutant, suggesting that the MotA/GFP-MotB complex cannot work together with wild-type MotA/MotB in the presence of the fliG(R281V) mutation. However, it remains unknown why. Here, we investigated the effect of the GFP fusion to MotB at its N-terminus on the MotA/MotB function. Over-expression of wild-type MotA/MotB significantly reduced the growth rate of the gfp-motBfliG(R281V) mutant. The over-expression of the MotA/GFP-MotB complex caused an excessive proton leakage through its proton channel, thereby inhibiting cell growth. These results suggest that the GFP tag on the MotB N-terminus affects well-regulated proton translocation through the MotA/MotB proton channel. Therefore, we propose that the N-terminal cytoplasmic tail of MotB couples the gating of the proton channel with the MotA–FliG interaction responsible for torque generation.

scholarly journals Effect of the MotA(M206I) Mutation on Torque Generation and Stator Assembly in theSalmonellaH+-Driven Flagellar Motor

2019 ◽  
Vol 201 (6) ◽  
Author(s):  
Yuya Suzuki ◽  
Yusuke V. Morimoto ◽  
Kodai Oono ◽  
Fumio Hayashi ◽  
Kenji Oosawa ◽  
...  
Keyword(s):  
Ion Flux ◽  
Motive Force ◽  
Flagellar Motor ◽  
Wild Type ◽  
Torque Generation ◽  
Unit Speed ◽  
Bacterial Flagellar Motor ◽  
Ph Dependent ◽  
Type Motor ◽  
External Ph

ABSTRACTThe bacterial flagellar motor is composed of a rotor and a dozen stators and converts the ion flux through the stator into torque. Each stator unit alternates in its attachment to and detachment from the rotor even during rotation. In some species, stator assembly depends on the input energy, but it remains unclear how an electrochemical potential across the membrane (e.g., proton motive force [PMF]) or ion flux is involved in stator assembly dynamics. Here, we focused on pH dependence of a slow motile MotA(M206I) mutant ofSalmonella. The MotA(M206I) motor produces torque comparable to that of the wild-type motor near stall, but its rotation rate is considerably decreased as the external load is reduced. Rotation assays of flagella labeled with 1-μm beads showed that the rotation rate of the MotA(M206I) motor is increased by lowering the external pH whereas that of the wild-type motor is not. Measurements of the speed produced by a single stator unit using 1-μm beads showed that the unit speed of the MotA(M206I) is about 60% of that of the wild-type and that a decrease in external pH did not affect the MotA(M206I) unit speed. Analysis of the subcellular stator localization revealed that the number of functional stators is restored by lowering the external pH. The pH-dependent improvement of stator assembly was observed even when the PMF was collapsed and proton transfer was inhibited. These results suggest that MotA-Met206 is responsible for not only load-dependent energy coupling between the proton influx and rotation but also pH-dependent stator assembly.IMPORTANCEThe bacterial flagellar motor is a rotary nanomachine driven by the electrochemical transmembrane potential (ion motive force). About 10 stators (MotA/MotB complexes) are docked around a rotor, and the stator recruitment depends on the load, ion motive force, and coupling ion flux. The MotA(M206I) mutation slows motor rotation and decreases the number of docked stators inSalmonella. We show that lowering the external pH improves the assembly of the mutant stators. Neither the collapse of the ion motive force nor a mutation mimicking the proton-binding state inhibited stator localization to the motor. These results suggest that MotA-Met206 is involved in torque generation and proton translocation and that stator assembly is stabilized by protonation of the stator.

scholarly journals Suppressor Analysis of the MotB(D33E) Mutation To Probe Bacterial Flagellar Motor Dynamics Coupled with Proton Translocation

2008 ◽  
Vol 190 (20) ◽  
pp. 6660-6667 ◽  
Author(s):  
Yong-Suk Che ◽  
Shuichi Nakamura ◽  
Seiji Kojima ◽  
Nobunori Kami-ike ◽  
Keiichi Namba ◽  
...  
Keyword(s):  
High Speed ◽  
Proton Translocation ◽  
Energy Coupling ◽  
Flagellar Motor ◽  
Transmembrane Helices ◽  
Wild Type ◽  
Torque Generation ◽  
Serovar Typhimurium ◽  
Bacterial Flagellar Motor ◽  
Protein Instability

ABSTRACT MotA and MotB form the stator of the proton-driven bacterial flagellar motor, which conducts protons and couples proton flow with motor rotation. Asp-33 of Salmonella enterica serovar Typhimurium MotB, which is a putative proton-binding site, is critical for torque generation. However, the mechanism of energy coupling remains unknown. Here, we carried out genetic and motility analysis of a slowly motile motB(D33E) mutant and its pseudorevertants. We first confirmed that the poor motility of the motB(D33E) mutant is due to neither protein instability, mislocalization, nor impaired interaction with MotA. We isolated 17 pseudorevertants and identified the suppressor mutations in the transmembrane helices TM2 and TM3 of MotA and in TM and the periplasmic domain of MotB. The stall torque produced by the motB(D33E) mutant motor was about half of the wild-type level, while those for the pseudorevertants were recovered nearly to the wild-type levels. However, the high-speed rotations of the motors under low-load conditions were still significantly impaired, suggesting that the rate of proton translocation is still severely limited at high speed. These results suggest that the second-site mutations recover a torque generation step involving stator-rotor interactions coupled with protonation/deprotonation of Glu-33 but not maximum proton conductivity.

scholarly journals Sodium-powered stators of the bacterial flagellar motor can generate torque in the presence of phenamil with mutations near the peptidoglycan-binding region

2018 ◽  
Author(s):  
Tsubasa Ishida ◽  
Rie Ito ◽  
Jessica Clark ◽  
Nicholas J Matzke ◽  
Yoshiyuki Sowa ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Single Point ◽  
Point Mutations ◽  
Ion Source ◽  
Channel Blockers ◽  
Flagellar Motor ◽  
Binding Region ◽  
Ion Channel Blockers ◽  
Flagellar Motors ◽  
Bacterial Flagellar Motor

SummaryThe bacterial flagellar motor (BFM) powers the rotation that propels swimming bacteria. Rotational torque is generated by harnessing the flow of ions through ion channels known as stators which couple the energy from the ion gradient across the inner membrane to rotation of the rotor. Here we used error-prone PCR to introduce single point mutations into the sodium-powered Vibrio alginolyticus / Escherichia coli chimeric stator PotB and selected for motors that exhibited motility in the presence of the sodium-channel inhibitor phenamil. We found single mutations that enable motility under phenamil occurred at two sites: 1) the transmembrane domain of PotB, corresponding to the TM region of the PomB stator from V. alginolyticus, and 2) near the peptidoglycan (PG) binding region that corresponds to the C-terminal region of the MotB stator from E. coli. Single cell rotation assays confirmed that individual flagellar motors could rotate in up to 100 µM phenamil. Using phylogenetic logistic regression, we found correlation between natural residue variation and ion source at positions corresponding to PotB F22Y, but not at other sites. Our results demonstrate that it is not only the pore region of the stator that moderates motility in the presence of ion-channel blockers.

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

ALL-Associated JAK1 Mutants Activate the JAK/STAT Pathway Via IL-9Rα Homodimers

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2848-2848
Author(s):  
Tekla Hornakova ◽  
Judith Staerk ◽  
Yohan Royer ◽  
Elisabetta Flex ◽  
Marco Tartaglia ◽  
...  
Keyword(s):  
Ectopic Expression ◽  
Point Mutations ◽  
Inhibitory Effect ◽  
Dominant Negative ◽  
Hek293 Cells ◽  
Cytokine Receptors ◽  
Receptor Complex ◽  
Wild Type ◽  
Constitutive Activation ◽  
Stat Pathway

Abstract Point mutations in the JAK2 tyrosine kinase play a major role in the development of myeloproliferative syndromes by activating preformed homodimers of cytokine receptors such as EpoR, TpoR and GM-CSFR. More recently, a series of activating mutations in JAK1 have also been shown to be associated with B and T cell acute lymphoblastic leukemias, but little is known about the mechanisms involved in the constitutive activation of these mutants and about their mode of interaction with cytokine receptors. Here, we studied the ability of several ALL-associated JAK1 mutants (V658F, A634D, R724H, R879C) to activate the JAK/STAT pathway upon ectopic expression in HEK293 cells, alone or together with the other components of the IL-9 receptor complex (IL-9Rα, γc and JAK3). Expression of the JAK1 mutants alone failed to trigger STAT activation, but coexpression of the IL-9Rα chain promoted JAK1 mutant phosphorylation and STATs activation, even without γc and JAK3 which are required for IL-9 responsiveness. Mutation of the FERM domain of JAK1, which is critical for cytokine receptor association, or of the single tyrosine of IL-9Rα involved in STAT recruitment abolished this activity, indicating that JAK1 mutants need to associate with IL-9R and phosphorylate this tyrosine residue to activate STAT factors. Several lines of evidence indicated that IL-9Rα homodimerization was involved in this process. IL-9Rα variants with deletion or mutation of the JAK-interacting BOX1 region not only failed to promote JAK1 activation, but acted as dominant negative forms reverting the effect of wild-type IL-9Rα. Coimmunoprecipitation experiments also showed the formation of IL-9Rα homodimers. Interestingly, this process was partially inhibited by the expression of γc, suggesting that overlapping residues are involved in IL-9Rα homodimerization and IL-9Rα/γc heterodimerization. Coexpression of wild-type JAK3 partially reverted the inhibitory effect of γc, indicating that JAK3 can cooperate with JAK1 mutants within the IL-9 receptor complex, even in the absence of the cytokine. Similar results were also observed with IL-2Rβ, which binds JAK1, but not for JAK2-associated homodimeric receptors such as TpoR or EpoR. Taken together, our results show that IL-9Rα and IL-2Rβ homodimers can efficiently mediate constitutive activation of ALL-associated JAK1 mutants, even in cells lacking the expression of γc, which is required for the activation of wild-type JAK1 to their respective physiological ligands, IL-9 and IL-2.

scholarly journals Novel Insights into Conformational Rearrangements of the Bacterial Flagellar Switch Complex

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Tomofumi Sakai ◽  
Tomoko Miyata ◽  
Naoya Terahara ◽  
Koichiro Mori ◽  
Yumi Inoue ◽  
...  
Keyword(s):  
Proton Translocation ◽  
Coupling Efficiency ◽  
Structure And Function ◽  
Proton Channel ◽  
Flagellar Motor ◽  
Wild Type ◽  
Suppressor Mutations ◽  
Type Motor ◽  
And Function ◽  
Intersubunit Interactions

ABSTRACTThe flagellar motor can spin in both counterclockwise (CCW) and clockwise (CW) directions. The flagellar motor consists of a rotor and multiple stator units, which act as a proton channel. The rotor is composed of the transmembrane MS ring made of FliF and the cytoplasmic C ring consisting of FliG, FliM, and FliN. The C ring is directly involved in rotation and directional switching. TheSalmonellaFliF-FliG deletion fusion motor missing 56 residues from the C terminus of FliF and 94 residues from the N terminus of FliG keeps a domain responsible for the interaction with the stator intact, but its motor function is reduced significantly. Here, we report the structure and function of the FliF-FliG deletion fusion motor. The FliF-FliG deletion fusion not only resulted in a strong CW switch bias but also affected rotor-stator interactions coupled with proton translocation through the proton channel of the stator unit. The energy coupling efficiency of the deletion fusion motor was the same as that of the wild-type motor. Extragenic suppressor mutations in FliG, FliM, or FliN not only relieved the strong CW switch bias but also increased the motor speed at low load. The FliF-FliG deletion fusion made intersubunit interactions between C ring proteins tighter compared to the wild-type motor, whereas the suppressor mutations affect such tighter intersubunit interactions. We propose that a change of intersubunit interactions between the C ring proteins may be required for high-speed motor rotation as well as direction switching.IMPORTANCEThe bacterial flagellar motor is a bidirectional rotary motor for motility and chemotaxis, which often plays an important role in infection. The motor is a large transmembrane protein complex composed of a rotor and multiple stator units, which also act as a proton channel. Motor torque is generated through their cyclic association and dissociation coupled with proton translocation through the proton channel. A large cytoplasmic ring of the motor, called C ring, is responsible for rotation and switching by interacting with the stator, but the mechanism remains unknown. By analyzing the structure and function of the wild-type motor and a mutant motor missing part of the C ring connecting itself with the transmembrane rotor ring while keeping a stator-interacting domain for bidirectional torque generation intact, we found interesting clues to the change in the C ring conformation for the switching and rotation involving loose and tight intersubunit interactions.

scholarly journals Regulation of Switching Frequency and Bias of the Bacterial Flagellar Motor by CheY and Fumarate

1998 ◽  
Vol 180 (13) ◽  
pp. 3375-3380 ◽  
Author(s):  
Marco Montrone ◽  
Michael Eisenbach ◽  
Dieter Oesterhelt ◽  
Wolfgang Marwan
Keyword(s):  
Response Curve ◽  
Conformational Transition ◽  
Switching Frequency ◽  
Flagellar Motor ◽  
Wild Type ◽  
Clockwise Rotation ◽  
Transient Change ◽  
Computer Aided ◽  
Bacterial Flagellar Motor

ABSTRACT The effect of CheY and fumarate on switching frequency and rotational bias of the bacterial flagellar motor was analyzed by computer-aided tracking of tethered Escherichia coli. Plots of cells overexpressing CheY in a gutted background showed a bell-shaped correlation curve of switching frequency and bias centering at about 50% clockwise rotation. Gutted cells (i.e., withcheA to cheZ deleted) with a low CheY level but a high cytoplasmic fumarate concentration displayed the same correlation of switching frequency and bias as cells overexpressing CheY at the wild-type fumarate level. Hence, a high fumarate level can phenotypically mimic CheY overexpression by simultaneously changing the switching frequency and the bias. A linear correlation of cytoplasmic fumarate concentration and clockwise rotation bias was found and predicts exclusively counterclockwise rotation without switching when fumarate is absent. This suggests that (i) fumarate is essential for clockwise rotation in vivo and (ii) any metabolically induced fluctuation of its cytoplasmic concentration will result in a transient change in bias and switching probability. A high fumarate level resulted in a dose-response curve linking bias and cytoplasmic CheY concentration that was offset but with a slope similar to that for a low fumarate level. It is concluded that fumarate and CheY act additively presumably at different reaction steps in the conformational transition of the switch complex from counterclockwise to clockwise motor rotation.

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