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.

scholarly journals Impact of fluorescent protein fusions on the bacterial flagellar motor

2017 ◽  
Author(s):  
M Heo ◽  
AL Nord ◽  
D Chamousset ◽  
E van Rijn ◽  
HJE Beaumont ◽  
...  
Keyword(s):  
Side Effects ◽  
Protein Function ◽  
Fusion Proteins ◽  
Fluorescent Proteins ◽  
Protein Complexes ◽  
Switching Frequency ◽  
Flagellar Motor ◽  
Internal Dynamics ◽  
Biologically Relevant ◽  
Bacterial Flagellar Motor

AbstractFluorescent fusion proteins open a direct and unique window onto protein function. However, they also introduce the risk of perturbation of the function of the native protein. Successful applications of fluorescent fusions therefore rely on a careful assessment and minimization of the side effects. Such insight, however, is still lacking for many applications of fluorescent fusions. This is particularly relevant in the study of the internal dynamics of motor protein complexes, where both the chemical and mechanical reaction coordinates can be affected. Fluorescent proteins fused to thestatorof the bacterial flagellar motor (BFM) complex have previously been used to successfully unveil the internal subunit dynamics of the motor. Here we report the effects of three different fluorescent proteins fused to the stator, all of which altered BFM behavior. The torque generated by individual stators was reduced while their stoichiometry in the complex remained unaffected. MotB fusions decreased the rotation-direction switching frequency of single motors and induced a novel BFM behavior: a bias-dependent asymmetry in the speed attained in the two rotation directions. All these effects could be mitigated by the insertion of a linker at the fusion point. These findings provide a quantitative account of the effects of fluorescent fusions on BFM dynamics and their alleviation—new insights that advance the use of fluorescent fusions to probe the dynamics of protein complexes.Author summaryMuch of what is known about the biology of proteins was discovered by fusing them to fluorescent proteins that allow detection of their location. But the label comes at a cost: the presence of the tag can alter the behavior of the protein of interest in unforeseen, yet biologically relevant ways. These side effects limit the depth to which fluorescent proteins can be used to probe protein function. One of the systems that has been successfully studied with fluorescent fusions for which these effects have not been addressed are dynamic protein complexes that carry out mechanical work. We examined how fluorescent proteins fused to a component of the bacterial flagellar motor complex impacts its function. Our findings show that the fusion proteins altered biologically relevant dynamical properties of the motor, including induction of a novel mechanical behavior, and demonstrate an approach to alleviate this. These results advance our ability to dissect the bacterial flagellar motor, and the internal dynamics of protein complexes in general, with fluorescent fusion proteins while causing minimal perturbation.

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.

Assembly and Dynamics of the Bacterial Flagellum

2020 ◽  
Vol 74 (1) ◽  
pp. 181-200 ◽  
Author(s):  
Judith P. Armitage ◽  
Richard M. Berry
Keyword(s):  
Single Molecule ◽  
Protein Complexes ◽  
Complex Structure ◽  
Live Cells ◽  
Flagellar Motor ◽  
Interacting Protein ◽  
Bacterial Flagellum ◽  
Bacterial Flagellar Motor ◽  
The Stability

The bacterial flagellar motor is the most complex structure in the bacterial cell, driving the ion-driven rotation of the helical flagellum. The ordered expression of the regulon and the assembly of the series of interacting protein rings, spanning the inner and outer membranes to form the ∼45–50-nm protein complex, have made investigation of the structure and mechanism a major challenge since its recognition as a rotating nanomachine about 40 years ago. Painstaking molecular genetics, biochemistry, and electron microscopy revealed a tiny electric motor spinning in the bacterial membrane. Over the last decade, new single-molecule and in vivo biophysical methods have allowed investigation of the stability of this and other large protein complexes, working in their natural environment inside live cells. This has revealed that in the bacterial flagellar motor, protein molecules in both the rotor and stator exchange with freely circulating pools of spares on a timescale of minutes, even while motors are continuously rotating. This constant exchange has allowed the evolution of modified components allowing bacteria to keep swimming as the viscosity or the ion composition of the outside environment changes.

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 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 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 Asymmetric random walks reveal that the chemotaxis network modulates flagellar rotational bias in Helicobacter pylori

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jyot D Antani ◽  
Anita X Sumali ◽  
Tanmay P Lele ◽  
Pushkar P Lele
Keyword(s):  
Helicobacter Pylori ◽  
Random Walks ◽  
Hydrodynamic Interactions ◽  
Flagellar Motor ◽  
Clockwise Rotation ◽  
Reversal Frequency ◽  
Bacterial Flagellar Motor ◽  
H Pylori ◽  
Bounding Surfaces ◽  
The Relationship

The canonical chemotaxis network modulates the bias for a particular direction of rotation in the bacterial flagellar motor to help the cell migrate toward favorable chemical environments. How the chemotaxis network in Helicobacter pylori modulates flagellar functions is unknown, which limits our understanding of chemotaxis in this species. Here, we determined that H. pylori swim faster (slower) whenever their flagella rotate counterclockwise (clockwise) by analyzing their hydrodynamic interactions with bounding surfaces. This asymmetry in swimming helped quantify the rotational bias. Upon exposure to a chemo-attractant, the bias decreased and the cells tended to swim exclusively in the faster mode. In the absence of a key chemotaxis protein, CheY, the bias was zero. The relationship between the reversal frequency and the rotational bias was unimodal. Thus, H. pylori’s chemotaxis network appears to modulate the probability of clockwise rotation in otherwise counterclockwise-rotating flagella, similar to the canonical network.

Three-Dimensional Subcellular Organization of Hepatocytes in Intact Liver: Simultaneous Visualization of Cytoskeletal and Membrane Markers by Confocal Microscopy

1996 ◽  
Vol 54 ◽  
pp. 288-289
Author(s):  
Greg V. Martin ◽  
Ann L. Hubbard
Keyword(s):  
Three Dimensional ◽  
Integral Membrane Proteins ◽  
Polarized Secretion ◽  
Microscope Images ◽  
Computer Aided ◽  
Intracellular Organelles ◽  
Cytoskeletal Elements ◽  
Simultaneous Visualization

The microtubule (MT) cytoskeleton is necessary for many of the polarized functions of hepatocytes. Among the functions dependent on the MT-based cytoskeleton are polarized secretion of proteins, delivery of endocytosed material to lysosomes, and transcytosis of integral plasma membrane (PM) proteins. Although microtubules have been shown to be crucial to the establishment and maintenance of functional and structural polarization in the hepatocyte, little is known about the architecture of the hepatocyte MT cytoskeleton in vivo, particularly with regard to its relationship to PM domains and membranous organelles. Using an in situ extraction technique that preserves both microtubules and cellular membranes, we have developed a protocol for immunofluorescent co-localization of cytoskeletal elements and integral membrane proteins within 20 µm cryosections of fixed rat liver. Computer-aided 3D reconstruction of multi-spectral confocal microscope images was used to visualize the spatial relationships among the MT cytoskeleton, PM domains and intracellular organelles.

[18F]-florbetaben PET/CT Imaging in the Alzheimer’s Disease Mouse Model APPswe/PS1dE9

2018 ◽  
Vol 16 (1) ◽  
pp. 49-55 ◽  
Author(s):  
J. Stenzel ◽  
C. Rühlmann ◽  
T. Lindner ◽  
S. Polei ◽  
S. Teipel ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Mouse Model ◽  
New Drugs ◽  
Imaging Data ◽  
Wild Type ◽  
Preclinical Research ◽  
Standardized Uptake Values ◽  
Pet Ct

Background: Positron-emission-tomography (PET) using 18F labeled florbetaben allows noninvasive in vivo-assessment of amyloid-beta (Aβ), a pathological hallmark of Alzheimer’s disease (AD). In preclinical research, [<sup>18</sup>F]-florbetaben-PET has already been used to test the amyloid-lowering potential of new drugs, both in humans and in transgenic models of cerebral amyloidosis. The aim of this study was to characterize the spatial pattern of cerebral uptake of [<sup>18</sup>F]-florbetaben in the APPswe/ PS1dE9 mouse model of AD in comparison to histologically determined number and size of cerebral Aβ plaques. Methods: Both, APPswe/PS1dE9 and wild type mice at an age of 12 months were investigated by smallanimal PET/CT after intravenous injection of [<sup>18</sup>F]-florbetaben. High-resolution magnetic resonance imaging data were used for quantification of the PET data by volume of interest analysis. The standardized uptake values (SUVs) of [<sup>18</sup>F]-florbetaben in vivo as well as post mortem cerebral Aβ plaque load in cortex, hippocampus and cerebellum were analyzed. Results: Visual inspection and SUVs revealed an increased cerebral uptake of [<sup>18</sup>F]-florbetaben in APPswe/ PS1dE9 mice compared with wild type mice especially in the cortex, the hippocampus and the cerebellum. However, SUV ratios (SUVRs) relative to cerebellum revealed only significant differences in the hippocampus between the APPswe/PS1dE9 and wild type mice but not in cortex; this differential effect may reflect the lower plaque area in the cortex than in the hippocampus as found in the histological analysis. Conclusion: The findings suggest that histopathological characteristics of Aβ plaque size and spatial distribution can be depicted in vivo using [<sup>18</sup>F]-florbetaben in the APPswe/PS1dE9 mouse model.

scholarly journals Stu2p binds tubulin and undergoes an open-to-closed conformational change

2006 ◽  
Vol 172 (7) ◽  
pp. 1009-1022 ◽  
Author(s):  
Jawdat Al-Bassam ◽  
Mark van Breugel ◽  
Stephen C. Harrison ◽  
Anthony Hyman
Keyword(s):  
Conformational Change ◽  
Conformational Transition ◽  
Budding Yeast ◽  
Microtubule Dynamics ◽  
Open Structure ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Common

Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat–containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.

Sign in / Sign up

Export Citation Format

Share Document