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1.
J Bacteriol ; 204(12): e0027822, 2022 12 20.
Article in English | MEDLINE | ID: mdl-36448786

ABSTRACT

In isotropic environments, an Escherichia coli cell exhibits coordinated rotational switching of its flagellar motors, produced by fluctuations in the intracellular concentration of phosphorylated CheY (CheY-P) emanating from chemoreceptor signaling arrays. In this study, we show that these CheY-P fluctuations arise through modifications of chemoreceptors by two sensory adaptation enzymes: the methyltransferase CheR and the methylesterase CheB. A cell containing CheR, CheB, and the serine chemoreceptor Tsr exhibited motor synchrony, whereas a cell lacking CheR and CheB or containing enzymatically inactive forms did not. Tsr variants with different combinations of methylation-mimicking Q residues at the adaptation sites also failed to show coordinated motor switching in cells lacking CheR and CheB. Cells containing CheR, CheB, and Tsr [NDND], a variant in which the adaptation site residues are not substrates for CheR or CheB modifications, also lacked motor synchrony. TsrΔNWETF, which lacks a C-terminal pentapeptide-binding site for CheR and CheB, and the ribose-galactose receptor Trg, which natively lacks this motif, failed to produce coordinated motor switching, despite the presence of CheR and CheB. However, addition of the NWETF sequence to Trg enabled Trg-NWETF to produce motor synchrony, as the sole receptor type in cells containing CheR and CheB. Finally, CheBc, the catalytic domain of CheB, supported motor coordination in combination with CheR and Tsr. These results indicate that the coordination of motor switching requires CheR/CheB-mediated changes in receptor modification state. We conclude that the opposing receptor substrate-site preferences of CheR and CheB produce spontaneous blinking of the chemoreceptor array's output activity. IMPORTANCE Under steady-state conditions with no external stimuli, an Escherichia coli cell coordinately switches the rotational direction of its flagellar motors. Here, we demonstrate that the CheR and CheB enzymes of the chemoreceptor sensory adaptation system mediate this coordination. Stochastic fluctuations in receptor adaptation states trigger changes in signal output from the receptor array, and this array blinking generates fluctuations in CheY-P concentration that coordinate directional switching of the flagellar motors. Thus, in the absence of chemoeffector gradients, the sensory adaptation system controls run-tumble swimming of the cell, its optimal foraging strategy.


Subject(s)
Escherichia coli Proteins , Escherichia coli , Escherichia coli/genetics , Escherichia coli/metabolism , Chemotaxis , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , Chemoreceptor Cells , Escherichia coli Proteins/metabolism , Methyl-Accepting Chemotaxis Proteins/metabolism
2.
Front Microbiol ; 12: 765739, 2021.
Article in English | MEDLINE | ID: mdl-34899649

ABSTRACT

Bacterial flagellar motor (BFM) is a large membrane-spanning molecular rotary machine for swimming motility. Torque is generated by the interaction between the rotor and multiple stator units powered by ion-motive force (IMF). The number of bound stator units is dynamically changed in response to the external load and the IMF. However, the detailed dynamics of stator unit exchange process remains unclear. Here, we directly measured the speed changes of sodium-driven chimeric BFMs under fast perfusion of different sodium concentration conditions using computer-controlled, high-throughput microfluidic devices. We found the sodium-driven chimeric BFMs maintained constant speed over a wide range of sodium concentrations by adjusting stator units in compensation to the sodium-motive force (SMF) changes. The BFM has the maximum number of stator units and is most stable at 5 mM sodium concentration rather than higher sodium concentration. Upon rapid exchange from high to low sodium concentration, the number of functional stator units shows a rapidly excessive reduction and then resurrection that is different from predictions of simple absorption model. This may imply the existence of a metastable hidden state of the stator unit during the sudden loss of sodium ions.

3.
Biomolecules ; 10(11)2020 11 12.
Article in English | MEDLINE | ID: mdl-33198296

ABSTRACT

Signal transduction utilizing membrane-spanning receptors and cytoplasmic regulator proteins is a fundamental process for all living organisms, but quantitative studies of the behavior of signaling proteins, such as their diffusion within a cell, are limited. In this study, we show that fluctuations in the concentration of the signaling molecule, phosphorylated CheY, constitute the basis of chemotaxis signaling. To analyze the propagation of the CheY-P signal quantitatively, we measured the coordination of directional switching between flagellar motors on the same cell. We analyzed the time lags of the switching of two motors in both CCW-to-CW and CW-to-CCW switching (∆tCCW-CW and ∆tCW-CCW). In wild-type cells, both time lags increased as a function of the relative distance of two motors from the polar receptor array. The apparent diffusion coefficient estimated for ∆t values was ~9 µm2/s. The distance-dependency of ∆tCW-CCW disappeared upon loss of polar localization of the CheY-P phosphatase, CheZ. The distance-dependency of the response time for an instantaneously applied serine attractant signal also disappeared with the loss of polar localization of CheZ. These results were modeled by calculating the diffusion of CheY and CheY-P in cells in which phosphorylation and dephosphorylation occur in different subcellular regions. We conclude that diffusion of signaling molecules and their production and destruction through spontaneous activity of the receptor array generates fluctuations in CheY-P concentration over timescales of several hundred milliseconds. Signal fluctuation coordinates rotation among flagella and regulates steady-state run-and-tumble swimming of cells to facilitate efficient responses to environmental chemical signals.


Subject(s)
Escherichia coli/metabolism , Flagella/metabolism , Methyl-Accepting Chemotaxis Proteins/metabolism , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli Proteins , Flagella/chemistry , Flagella/genetics , Methyl-Accepting Chemotaxis Proteins/genetics , Phosphorylation , Rotation , Signal Transduction
4.
Methods Mol Biol ; 1593: 215-226, 2017.
Article in English | MEDLINE | ID: mdl-28389957

ABSTRACT

To elucidate the mechanisms by which cells respond to extracellular stimuli, the behavior of intracellular signaling proteins in a single cell should be directly examined, while simultaneously recording the cellular response. In Escherichia coli, an extracellular chemotactic stimulus is thought to induce a switch in the rotational direction of the flagellar motor, elicited by the binding and dissociation of the phosphorylated form of CheY (CheY-P) to and from the motor. We recently provided direct evidence for the binding of CheY-P to a functioning flagellar motor in live cells. Here, we describe the method for simultaneously measuring the fluorescent signal of the CheY-enhanced green fluorescent protein fusion protein (CheY-EGFP) and the rotational switching of the flagellar motor. By performing fluorescence and bright-field microscopy simultaneously, the rotational switch of the flagellar motor was shown to be induced by the binding and dissociation of CheY-P, and the number of CheY-P molecules bound to the motor was estimated.


Subject(s)
Chemotaxis/physiology , Escherichia coli/metabolism , Escherichia coli/physiology , Signal Transduction/physiology , Cytoplasm/metabolism , Cytoplasm/physiology , Escherichia coli Proteins/metabolism , Flagella/metabolism , Green Fluorescent Proteins/metabolism , Membrane Proteins/metabolism , Phosphorylation/physiology , Protein Binding/physiology
5.
Biophys J ; 107(3): 730-739, 2014 Aug 05.
Article in English | MEDLINE | ID: mdl-25099812

ABSTRACT

In response to an attractant or repellant, an Escherichia coli cell controls the rotational direction of its flagellar motor by a chemotaxis system. When an E. coli cell senses an attractant, a reduction in the intracellular concentration of a chemotaxis protein, phosphorylated CheY (CheY-P), induces counterclockwise (CCW) rotation of the flagellar motor, and this cellular response is thought to occur in several hundred milliseconds. Here, to measure the signaling process occurring inside a single E. coli cell, including the recognition of an attractant by a receptor cluster, the inactivation of histidine kinase CheA, and the diffusion of CheY and CheY-P molecules, we applied a serine stimulus by instantaneous photorelease from a caged compound and examined the cellular response at a temporal resolution of several hundred microseconds. We quantified the clockwise (CW) and CCW durations immediately after the photorelease of serine as the response time and the duration of the response, respectively. The results showed that the response time depended on the distance between the receptor and motor, indicating that the decreased CheY-P concentration induced by serine propagates through the cytoplasm from the receptor-kinase cluster toward the motor with a timing that is explained by the diffusion of CheY and CheY-P molecules. The response time included 240 ms for enzymatic reactions in addition to the time required for diffusion of the signaling molecule. The measured response time and duration of the response also revealed that the E. coli cell senses a similar serine concentration regardless of whether the serine concentration is increasing or decreasing. These detailed quantitative findings increase our understanding of the signal transduction process that occurs inside cells during bacterial chemotaxis.


Subject(s)
Bacterial Proteins/metabolism , Chemotaxis , Escherichia coli/metabolism , Membrane Proteins/metabolism , Escherichia coli/drug effects , Escherichia coli/physiology , Escherichia coli Proteins , Histidine Kinase , Methyl-Accepting Chemotaxis Proteins , Reaction Time , Serine/pharmacology , Single-Cell Analysis
6.
Langmuir ; 30(25): 7289-95, 2014 Jul 01.
Article in English | MEDLINE | ID: mdl-24898450

ABSTRACT

Vesicle formation is a fundamental kinetic process related to the vesicle budding and endocytosis in a cell. In the vesicle formation by artificial means, transformation of lamellar lipid aggregates into spherical architectures is a key process and known to be prompted by e.g. heat, infrared irradiation, and alternating electric field induction. Here we report UV-light-driven formation of vesicles from particles consisting of crumpled phospholipid multilayer membranes involving a photoactive amphiphilic compound composed of 1,4-bis(4-phenylethynyl)benzene (BPEB) units. The particles can readily be prepared from a mixture of these components, which is casted on the glass surface followed by addition of water under ultrasonic radiation. Interestingly, upon irradiation with UV light, micrometer-size vesicles were generated from the particles. Neither infrared light irradiation nor heating prompted the vesicle formation. Taking advantage of the benefits of light, we successfully demonstrated micrometer-scale spatiotemporal control of single vesicle formation. It is also revealed that the BPEB units in the amphiphile are essential for this phenomenon.


Subject(s)
Membranes, Artificial , Ultraviolet Rays , Phospholipids/chemistry
7.
Sci Signal ; 7(319): ra32, 2014 Apr 01.
Article in English | MEDLINE | ID: mdl-24692593

ABSTRACT

The bacterial chemotaxis system regulates the rotational direction of flagellar motors through an intracellular signaling molecule, the phosphorylated form of CheY (CheY-P). The binding of CheY-P to a motor is believed to switch the motor's rotational direction from counterclockwise to clockwise. We demonstrated that the rotational switch of a motor was directly regulated by the binding and dissociation of CheY-P by simultaneously visualizing CheY tagged with green fluorescent protein and the rotational switching of a motor in live cells. The binding of 13 ± 7 CheY-P molecules was sufficient to induce clockwise rotation, and CheY-P molecules bound to and dissociated from a motor within ~100 ms during switching. Thus, we have directly measured the regulation of the output from a signal transduction pathway by intracellular signaling proteins.


Subject(s)
Bacterial Proteins/metabolism , Chemotaxis , Green Fluorescent Proteins/metabolism , Microscopy, Fluorescence/methods , Signal Transduction , Bacterial Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli/physiology , Escherichia coli Proteins , Flagella/metabolism , Flagella/physiology , Green Fluorescent Proteins/genetics , Immunoblotting , Intracellular Space/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Methyl-Accepting Chemotaxis Proteins , Molecular Dynamics Simulation , Molecular Motor Proteins/metabolism , Phosphorylation , Protein Binding , Rotation , Time-Lapse Imaging
8.
J Biol Chem ; 289(4): 2205-16, 2014 Jan 24.
Article in English | MEDLINE | ID: mdl-24302735

ABSTRACT

In pancreatic islets, insulin secretion occurs via synchronous elevation of Ca(2+) levels throughout the islets during high glucose conditions. This Ca(2+) elevation has two phases: a quick increase, observed after the glucose stimulus, followed by prolonged oscillations. In these processes, the elevation of intracellular ATP levels generated from glucose is assumed to inhibit ATP-sensitive K(+) channels, leading to the depolarization of membranes, which in turn induces Ca(2+) elevation in the islets. However, little is known about the dynamics of intracellular ATP levels and their correlation with Ca(2+) levels in the islets in response to changing glucose levels. In this study, a genetically encoded fluorescent biosensor for ATP and a fluorescent Ca(2+) dye were employed to simultaneously monitor the dynamics of intracellular ATP and Ca(2+) levels, respectively, inside single isolated islets. We observed rapid increases in cytosolic and mitochondrial ATP levels after stimulation with glucose, as well as with methyl pyruvate or leucine/glutamine. High ATP levels were sustained as long as high glucose levels persisted. Inhibition of ATP production suppressed the initial Ca(2+) increase, suggesting that enhanced energy metabolism triggers the initial phase of Ca(2+) influx. On the other hand, cytosolic ATP levels did not fluctuate significantly with the Ca(2+) level in the subsequent oscillation phases. Importantly, Ca(2+) oscillations stopped immediately before ATP levels decreased significantly. These results might explain how food or glucose intake evokes insulin secretion and how the resulting decrease in plasma glucose levels leads to cessation of secretion.


Subject(s)
Adenosine Triphosphate/metabolism , Calcium Signaling/physiology , Calcium/metabolism , Glucose/metabolism , Islets of Langerhans/metabolism , Membrane Potentials/physiology , Adenosine Triphosphate/genetics , Animals , Calcium Signaling/drug effects , Cell Line, Tumor , Cytosol/metabolism , Glucose/pharmacology , Glutamine/genetics , Glutamine/metabolism , Islets of Langerhans/cytology , Leucine/genetics , Leucine/metabolism , Membrane Potentials/drug effects , Mice , Mitochondria/genetics , Mitochondria/metabolism , Pyruvic Acid/metabolism , Sweetening Agents/metabolism , Sweetening Agents/pharmacology
9.
Biophys J ; 105(12): 2801-10, 2013 Dec 17.
Article in English | MEDLINE | ID: mdl-24359752

ABSTRACT

In their natural habitats bacteria are frequently exposed to sudden changes in temperature that have been shown to affect their swimming. With our believed to be new methods of rapid temperature control for single-molecule microscopy, we measured here the thermal response of the Na(+)-driven chimeric motor expressed in Escherichia coli cells. Motor torque at low load (0.35 µm bead) increased linearly with temperature, twofold between 15°C and 40°C, and torque at high load (1.0 µm bead) was independent of temperature, as reported for the H(+)-driven motor. Single cell membrane voltages were measured by fluorescence imaging and these were almost constant (∼120 mV) over the same temperature range. When the motor was heated above 40°C for 1-2 min the torque at high load dropped reversibly, recovering upon cooling below 40°C. This response was repeatable over as many as 10 heating cycles. Both increases and decreases in torque showed stepwise torque changes with unitary size ∼150 pN nm, close to the torque of a single stator at room temperature (∼180 pN nm), indicating that dynamic stator dissociation occurs at high temperature, with rebinding upon cooling. Our results suggest that the temperature-dependent assembly of stators is a general feature of flagellar motors.


Subject(s)
Bacterial Proteins/chemistry , Escherichia coli/chemistry , Membrane Potentials , Temperature , Torque , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins/genetics , Escherichia coli/physiology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Sodium/chemistry , Vibrio alginolyticus/chemistry
10.
Biophysics (Nagoya-shi) ; 8: 59-66, 2012.
Article in English | MEDLINE | ID: mdl-27857608

ABSTRACT

Escherichia coli cells swim toward a favorable environment by chemotaxis. The chemotaxis system regulates the swimming behavior of the bacteria by controlling the rotational direction of their flagellar motors. Extracellular stimuli sensed by chemoreceptors are transduced to an intracellular signal molecule, phosphorylated CheY (CheY-P), that switches the rotational direction of the flagellar motors from counterclockwise (CCW) to clockwise (CW) or from CW to CCW. Many studies have focused on identifying the proteins involved in the chemotaxis system, and findings on the structures and intracellular localizations of these proteins have largely elucidated the molecular pathway. On the other hand, quantitative evaluations of the chemotaxis system, including the process of intracellular signaling by the propagation of CheY-P and the rotational switching of flagellar motor by binding of CheY-P molecules, are still uncertain. For instance, scientific consensus has held that the flagellar motors of an E. coli cell switch rotational direction asynchronously. However, recent work shows that the rotational switching of any two different motors on a single E. coli cell is highly coordinated; a sub-second switching delay between motors is clearly correlated with the relative distance of each motor from the chemoreceptor patch located at one pole of the cell. In this review of previous studies and our recent findings, we discuss the regulatory mechanism of the multiple flagellar motors on an individual E. coli cell and the intracellular signaling process that can be inferred from this coordinated switching.

11.
J Mol Biol ; 414(1): 62-74, 2011 Nov 18.
Article in English | MEDLINE | ID: mdl-21986199

ABSTRACT

The torque of the bacterial flagellar motor is generated by the rotor-stator interaction coupled with specific ion translocation through the stator channel. To produce a fully functional motor, multiple stator units must be properly incorporated around the rotor by an as yet unknown mechanism to engage the rotor-stator interactions. Here, we investigated stator assembly using a mutational approach of the Na(+)-driven polar flagellar motor of Vibrio alginolyticus, whose stator is localized at the flagellated cell pole. We mutated a rotor protein, FliG, which is located at the C ring of the basal body and closely participates in torque generation, and found that point mutation L259Q, L270R or L271P completely abolishes both motility and polar localization of the stator without affecting flagellation. Likewise, mutations V274E and L279P severely affected motility and stator assembly. Those residues are localized at the core of the globular C-terminal domain of FliG when mapped onto the crystal structure of FliG from Thermotoga maritima, which suggests that those mutations induce quite large structural alterations at the interface responsible for the rotor-stator interaction. These results show that the C-terminal domain of FliG is critical for the proper assembly of PomA/PomB stator complexes around the rotor and probably functions as the target of the stator at the rotor side.


Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Movement/physiology , Flagella/physiology , Mutation/genetics , Sodium Channels/metabolism , Sodium/metabolism , Vibrio alginolyticus/physiology , Mutagenesis, Site-Directed , Vibrio Infections/metabolism , Vibrio Infections/microbiology
12.
Biophys J ; 100(9): 2193-200, 2011 May 04.
Article in English | MEDLINE | ID: mdl-21539787

ABSTRACT

An Escherichia coli cell transduces extracellular stimuli sensed by chemoreceptors to the state of an intracellular signal molecule, which regulates the switching of the rotational direction of the flagellar motors from counterclockwise (CCW) to clockwise (CW) and from CW back to CCW. Here, we performed high-speed imaging of flagellar motor rotation and show that the switching of two different motors on a cell is controlled coordinatedly by an intracellular signal protein, phosphorylated CheY (CheY-P). The switching is highly coordinated with a subsecond delay between motors in clear correlation with the distance of each motor from the chemoreceptor patch localized at a cell pole, which would be explained by the diffusive motion of CheY-P molecules in the cell. The coordinated switching becomes disordered by the expression of a constitutively active CheY mutant that mimics the CW-rotation stimulating function. The coordinated switching requires CheZ, which is the phosphatase for CheY-P. Our results suggest that a transient increase and decrease in the concentration of CheY-P caused by a spontaneous burst of its production by the chemoreceptor patch followed by its dephosphorylation by CheZ, which is probably a wavelike propagation in a subsecond timescale, triggers and regulates the coordinated switching of flagellar motors.


Subject(s)
Escherichia coli/cytology , Escherichia coli/metabolism , Flagella/metabolism , Molecular Motor Proteins/metabolism , Artificial Cells/cytology , Artificial Cells/metabolism , Bacterial Proteins/metabolism , Chemoreceptor Cells/cytology , Chemoreceptor Cells/metabolism , Escherichia coli Proteins/metabolism , Gene Deletion , Green Fluorescent Proteins/metabolism , Intracellular Space/metabolism , Membrane Proteins/metabolism , Methyl-Accepting Chemotaxis Proteins , Models, Biological , Mutant Proteins/metabolism , Rotation
13.
Biochem Biophys Res Commun ; 394(1): 130-5, 2010 Mar 26.
Article in English | MEDLINE | ID: mdl-20184859

ABSTRACT

The bacterial flagellar motor is a rotary motor driven by the electrochemical potential of a coupling ion. The interaction between a rotor and stator units is thought to generate torque. The overall structure of flagellar motor has been thought to be static, however, it was recently proved that stators are exchanged in a rotating motor. Understanding the dynamics of rotor components in functioning motor is important for the clarifying of working mechanism of bacterial flagellar motor. In this study, we focused on the dynamics and the turnover of rotor components in a functioning flagellar motor. Expression systems for GFP-FliN, FliM-GFP, and GFP-FliG were constructed, and each GFP-fusion was functionally incorporated into the flagellar motor. To investigate whether the rotor components are exchanged in a rotating motor, we performed fluorescence recovery after photobleaching experiments using total internal reflection fluorescence microscopy. After photobleaching, in a tethered cell producing GFP-FliN or FliM-GFP, the recovery of fluorescence at the rotational center was observed. However, in a cell producing GFP-FliG, no recovery of fluorescence was observed. The transition phase of fluorescence intensity after full or partially photobleaching allowed the turnover of FliN subunits to be calculated as 0.0007s(-1), meaning that FliN would be exchanged in tens of minutes. These novel findings indicate that a bacterial flagellar motor is not a static structure even in functioning state. This is the first report for the exchange of rotor components in a functioning bacterial flagellar motor.


Subject(s)
Escherichia coli/physiology , Flagella/physiology , Bacterial Proteins/metabolism , Bacterial Proteins/physiology , Escherichia coli/metabolism , Flagella/metabolism , Green Fluorescent Proteins/metabolism , Microscopy, Fluorescence , Photobleaching , Rotation
14.
J Bacteriol ; 192(6): 1740-3, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20097863

ABSTRACT

Escherichia coli chemoreceptors can sense changes in temperature for thermotaxis. Here we found that the aerotaxis transducer Aer, a homolog of chemoreceptors lacking a periplasmic domain, mediates thermoresponses. We propose that thermosensing by the chemoreceptors is a general attribute of their highly conserved cytoplasmic domain (or their less conserved transmembrane domain).


Subject(s)
Carrier Proteins/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Temperature , Carrier Proteins/genetics , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial/physiology , Intercellular Signaling Peptides and Proteins , Signal Transduction
15.
Mol Microbiol ; 71(4): 825-35, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19183284

ABSTRACT

The bacterial flagellar motor is driven by the electrochemical potential of specific ions, H(+) or Na(+). The motor consists of a rotor and stator, and their interaction generates rotation. The stator, which is composed of PomA and PomB in the Na(+) motor of Vibrio alginolyticus, is thought to be a torque generator converting the energy of ion flux into mechanical power. We found that specific mutations in PomB, including D24N, F33C and S248F, which caused motility defects, affected the assembly of stator complexes into the polar flagellar motor using green fluorescent protein-fused stator proteins. D24 of PomB is the predicted Na(+)-binding site. Furthermore, we demonstrated that the coupling ion, Na(+), is required for stator assembly and that phenamil (an inhibitor of the Na(+)-driven motor) inhibited the assembly. Carbonyl cyanide m-chlorophenylhydrazone, which is a proton ionophore that collapses the sodium motive force in this organism at neutral pH, also inhibited the assembly. Thus we conclude that the process of Na(+) influx through the channel, including Na(+) binding, is essential for the assembly of the stator complex to the flagellar motor as well as for torque generation.


Subject(s)
Bacterial Proteins/metabolism , Flagella/metabolism , Molecular Motor Proteins/metabolism , Sodium Channels/metabolism , Sodium/metabolism , Vibrio alginolyticus/metabolism , Bacterial Proteins/genetics , Binding Sites , Flagella/genetics , Hydrazones/pharmacology , Molecular Motor Proteins/genetics , Mutation , Protein Structure, Tertiary , Sodium Channels/genetics , Torque , Vibrio alginolyticus/genetics
16.
J Mol Biol ; 376(5): 1251-9, 2008 Mar 07.
Article in English | MEDLINE | ID: mdl-18207160

ABSTRACT

The bacterial flagellar motor is a rotary motor in the cell envelope of bacteria that couples ion flow across the cytoplasmic membrane to torque generation by independent stators anchored to the cell wall. The recent observation of stepwise rotation of a Na(+)-driven chimeric motor in Escherichia coli promises to reveal the mechanism of the motor in unprecedented detail. We measured torque-speed relationships of this chimeric motor using back focal plane interferometry of polystyrene beads attached to flagellar filaments in the presence of high sodium-motive force (85 mM Na(+)). With full expression of stator proteins the torque-speed curve had the same shape as those of wild-type E. coli and Vibrio alginolyticus motors: the torque is approximately constant (at approximately 2200 pN nm) from stall up to a "knee" speed of approximately 420 Hz, and then falls linearly with speed, extrapolating to zero torque at approximately 910 Hz. Motors containing one to five stators generated approximately 200 pN nm per stator at speeds up to approximately 100 Hz/stator; the knee speed in 4- and 5-stator motors is not significantly slower than in the fully induced motor. This is consistent with the hypothesis that the absolute torque depends on stator number, but the speed dependence does not. In motors with point mutations in either of two critical conserved charged residues in the cytoplasmic domain of PomA, R88A and R232E, the zero-torque speed was reduced to approximately 400 Hz. The torque at low speed was unchanged by mutation R88A but was reduced to approximately 1500 pN nm by R232E. These results, interpreted using a simple kinetic model, indicate that the basic mechanism of torque generation is the same regardless of stator type and coupling ion and that the electrostatic interaction between stator and rotor proteins is related to the torque-speed relationship.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Bacterial Outer Membrane Proteins/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Flagella/metabolism , Molecular Motor Proteins/metabolism , Sodium/metabolism , Torque , Bacterial Outer Membrane Proteins/genetics , Escherichia coli/chemistry , Vibrio alginolyticus/chemistry , Vibrio alginolyticus/metabolism
18.
J Mol Biol ; 367(3): 692-701, 2007 Mar 30.
Article in English | MEDLINE | ID: mdl-17289075

ABSTRACT

The bacterial flagellar motor is a rotary motor driven by the electrochemical potentials of specific ions across the cell membrane. Direct interactions between the rotor protein FliG and the stator protein MotA are thought to generate the rotational torque. Here, we used total internal reflection fluorescent microscopy to observe the localization of green fluorescent protein (GFP)-fused FliG in Escherichia coli cells. We identified three types of fluorescent punctate signals: immobile dots, mobile dots that exhibited simple diffusion, and mobile dots that exhibited restricted diffusion. When GFP-FliG was expressed in a DeltafliG background, most of the cells were not mobile. When the cells were tethered to a glass side, however, rotating cells were commonly observed and a single fluorescent dot was always observed at the rotational center of the tethered cell. These fluorescent dots were likely positions at which functional GFP-FliG had been incorporated into a flagellar motor. Our results suggest that flagellar basal bodies diffuse in the cytoplasmic membrane until the axial structure and/or other structures assemble.


Subject(s)
Bacterial Proteins/metabolism , Cell Membrane/metabolism , Flagella/metabolism , Molecular Motor Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Molecular Motor Proteins/chemistry , Molecular Motor Proteins/genetics , Movement , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
19.
Mol Microbiol ; 62(4): 1170-80, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17038120

ABSTRACT

The four motor proteins PomA, PomB, MotX and MotY, which are believed to be stator proteins, are essential for motility by the Na(+)-driven flagella of Vibrio alginolyticus. When we purified the flagellar basal bodies, MotX and MotY were detected in the basal body, which is the supramolecular complex comprised of the rotor and the bushing, but PomA and PomB were not. By antibody labelling, MotX and MotY were detected around the LP ring. These results indicate that MotX and MotY associate with the basal body. The basal body had a new ring structure beneath the LP ring, which was named the T ring. This structure was changed or lost in the basal body from a DeltamotX or DeltamotY strain. The T ring probably comprises MotX and MotY. In the absence of MotX or MotY, we demonstrated that PomA and PomB were not localized to a cell pole. From the above results, we suggest that MotX and MotY of the T ring are involved in the incorporation and/or stabilization of the PomA/PomB complex in the motor.


Subject(s)
Bacterial Outer Membrane Proteins/analysis , Bacterial Proteins/analysis , Flagella/chemistry , Membrane Proteins/analysis , Molecular Motor Proteins/analysis , Vibrio alginolyticus/chemistry , Bacterial Outer Membrane Proteins/physiology , Bacterial Proteins/physiology , Flagella/physiology , Flagella/ultrastructure , Membrane Proteins/physiology , Molecular Motor Proteins/physiology , Sodium/metabolism , Sodium Channels/metabolism , Sodium Channels/physiology , Vibrio alginolyticus/cytology , Vibrio alginolyticus/physiology
20.
J Bacteriol ; 188(4): 1466-72, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16452430

ABSTRACT

In Escherichia coli, rotation of the flagellar motor has been shown to depend upon electrostatic interactions between charged residues of the stator protein MotA and the rotor protein FliG. These charged residues are conserved in the Na+-driven polar flagellum of Vibrio alginolyticus, but mutational studies in V. alginolyticus suggested that they are relatively unimportant for motor rotation. The electrostatic interactions detected in E. coli therefore might not be a general feature of flagellar motors, or, alternatively, the V. alginolyticus motor might rely on similar interactions but incorporate additional features that make it more robust against mutation. Here, we have carried out a comparative study of chimeric motors that were resident in E. coli but engineered to use V. alginolyticus stator components, rotor components, or both. Charged residues in the V. alginolyticus rotor and stator proteins were found to be essential for motor rotation when the proteins functioned in the setting of the E. coli motor. Patterns of synergism and suppression in rotor/stator double mutants indicate that the V. alginolyticus proteins interact in essentially the same way as their counterparts in E. coli. The robustness of the rotor-stator interface in V. alginolyticus is in part due to the presence of additional charged residues in PomA but appears mainly due to other factors, because an E. coli motor using both rotor and stator components from V. alginolyticus remained sensitive to mutation. Motor function in V. alginolyticus may be enhanced by the proteins MotX and MotY.


Subject(s)
Escherichia coli/physiology , Hydrogen , Molecular Motor Proteins , Protons , Sodium , Bacterial Proteins/biosynthesis , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Escherichia coli/metabolism , Flagella/chemistry , Locomotion , Membrane Proteins/biosynthesis , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mutation , Vibrio alginolyticus/chemistry
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