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 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 Yersinia enterocolitica Type III Secretion Depends on the Proton Motive Force but Not on the Flagellar Motor Components MotA and MotB

2004 ◽  
Vol 72 (7) ◽  
pp. 4004-4009 ◽  
Author(s):  
Gottfried Wilharm ◽  
Verena Lehmann ◽  
Kristina Krauss ◽  
Beatrix Lehnert ◽  
Susanna Richter ◽  
...  
Keyword(s):  
Yersinia Enterocolitica ◽  
Type Iii Secretion ◽  
Proton Motive Force ◽  
Motive Force ◽  
Host Cells ◽  
Infection Model ◽  
Flagellar Motor ◽  
Wild Type ◽  
Secretion Systems ◽  
Type Iii

ABSTRACT The flagellum is believed to be the common ancestor of all type III secretion systems (TTSSs). In Yersinia enterocolitica, expression of the flagellar TTSS and the Ysc (Yop secretion) TTSS are inversely regulated. We therefore hypothesized that the Ysc TTSS may adopt flagellar motor components in order to use the pathogenicity-related translocon in a drill-like manner. As a prerequisite for this hypothesis, we first tested a requirement for the proton motive force by both systems using the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Motility as well as type III-dependent secretion of Yop proteins was inhibited by CCCP. We deleted motAB, which resulted in an immotile phenotype. This mutant, however, secreted amounts of Yops to the supernatant comparable to those of the wild type. Translocation of Yops into host cells was also not affected by the motAB deletion. Virulence of the mutant was comparable to that of the wild type in the mouse oral infection model. Thus, the hypothesis that the Ysc TTSS might adopt flagellar motor components was not confirmed. The finding that, in addition to consumption of ATP, Ysc TTSS requires the proton motive force is discussed.

scholarly journals Speed of the bacterial flagellar motor near zero load depends on the number of stator units

2017 ◽  
Vol 114 (44) ◽  
pp. 11603-11608 ◽  
Author(s):  
Ashley L. Nord ◽  
Yoshiyuki Sowa ◽  
Bradley C. Steel ◽  
Chien-Jung Lo ◽  
Richard M. Berry
Keyword(s):  
Escherichia Coli ◽  
Motive Force ◽  
Flagellar Motor ◽  
Single Curve ◽  
Ion Gradient ◽  
Membrane Bound ◽  
Bacterial Flagellar Motor ◽  
Ion Conducting ◽  
Relationship Of

The bacterial flagellar motor (BFM) rotates hundreds of times per second to propel bacteria driven by an electrochemical ion gradient. The motor consists of a rotor 50 nm in diameter surrounded by up to 11 ion-conducting stator units, which exchange between motors and a membrane-bound pool. Measurements of the torque–speed relationship guide the development of models of the motor mechanism. In contrast to previous reports that speed near zero torque is independent of the number of stator units, we observe multiple speeds that we attribute to different numbers of units near zero torque in both Na+- and H+-driven motors. We measure the full torque–speed relationship of one and two H+units inEscherichia coliby selecting the number of H+units and controlling the number of Na+units in hybrid motors. These experiments confirm that speed near zero torque in H+-driven motors increases with the stator number. We also measured 75 torque–speed curves for Na+-driven chimeric motors at different ion-motive force and stator number. Torque and speed were proportional to ion-motive force and number of stator units at all loads, allowing all 77 measured torque–speed curves to be collapsed onto a single curve by simple rescaling.

scholarly journals Bacterial flagellar motor

2008 ◽  
Vol 41 (2) ◽  
pp. 103-132 ◽  
Author(s):  
Yoshiyuki Sowa ◽  
Richard M. Berry
Keyword(s):  
Single Molecule ◽  
Cell Envelope ◽  
Molecular Motor ◽  
Motive Force ◽  
Flagellar Motor ◽  
Large Size ◽  
Structural Details ◽  
Bacterial Flagellar Motor ◽  
And Function ◽  
Rotary Motor

AbstractThe bacterial flagellar motor is a reversible rotary nano-machine, about 45 nm in diameter, embedded in the bacterial cell envelope. It is powered by the flux of H+or Na+ions across the cytoplasmic membrane driven by an electrochemical gradient, the proton-motive force or the sodium-motive force. Each motor rotates a helical filament at several hundreds of revolutions per second (hertz). In many species, the motor switches direction stochastically, with the switching rates controlled by a network of sensory and signalling proteins. The bacterial flagellar motor was confirmed as a rotary motor in the early 1970s, the first direct observation of the function of a single molecular motor. However, because of the large size and complexity of the motor, much remains to be discovered, in particular, the structural details of the torque-generating mechanism. This review outlines what has been learned about the structure and function of the motor using a combination of genetics, single-molecule and biophysical techniques, with a focus on recent results and single-molecule techniques.

scholarly journals Temperature Dependences of Torque Generation and Membrane Voltage in the Bacterial Flagellar Motor

2013 ◽  
Vol 105 (12) ◽  
pp. 2801-2810 ◽  
Author(s):  
Yuichi Inoue ◽  
Matthew A.B. Baker ◽  
Hajime Fukuoka ◽  
Hiroto Takahashi ◽  
Richard M. Berry ◽  
...  
Keyword(s):  
Membrane Voltage ◽  
Flagellar Motor ◽  
Torque Generation ◽  
Temperature Dependences ◽  
Bacterial Flagellar Motor

scholarly journals Mechanics of torque generation in the bacterial flagellar motor

2015 ◽  
Vol 112 (32) ◽  
pp. E4381-E4389 ◽  
Author(s):  
Kranthi K. Mandadapu ◽  
Jasmine A. Nirody ◽  
Richard M. Berry ◽  
George Oster
Keyword(s):  
Conformational Changes ◽  
Specific Model ◽  
Power Stroke ◽  
Flagellar Motor ◽  
Mechanical Explanation ◽  
Proposed Model ◽  
Torque Generation ◽  
Bacterial Flagellar Motor ◽  
Α Helix ◽  
Fundamental Forces

The bacterial flagellar motor (BFM) is responsible for driving bacterial locomotion and chemotaxis, fundamental processes in pathogenesis and biofilm formation. In the BFM, torque is generated at the interface between transmembrane proteins (stators) and a rotor. It is well established that the passage of ions down a transmembrane gradient through the stator complex provides the energy for torque generation. However, the physics involved in this energy conversion remain poorly understood. Here we propose a mechanically specific model for torque generation in the BFM. In particular, we identify roles for two fundamental forces involved in torque generation: electrostatic and steric. We propose that electrostatic forces serve to position the stator, whereas steric forces comprise the actual “power stroke.” Specifically, we propose that ion-induced conformational changes about a proline “hinge” residue in a stator α-helix are directly responsible for generating the power stroke. Our model predictions fit well with recent experiments on a single-stator motor. The proposed model provides a mechanical explanation for several fundamental properties of the flagellar motor, including torque–speed and speed–ion motive force relationships, backstepping, variation in step sizes, and the effects of key mutations in the stator.

scholarly journals Evidence for symmetry in the elementary process of bidirectional torque generation by the bacterial flagellar motor

2010 ◽  
Vol 107 (41) ◽  
pp. 17616-17620 ◽  
Author(s):  
Shuichi Nakamura ◽  
Nobunori Kami-ike ◽  
Jun-ichi P. Yokota ◽  
Tohru Minamino ◽  
Keiichi Namba
Keyword(s):  
Elementary Process ◽  
Flagellar Motor ◽  
Torque Generation ◽  
Bacterial Flagellar Motor
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