Counterclockwise States Research Articles

Structural basis of the bacterial flagellar motor rotational switching.

The bacterial flagellar motor is a huge bidirectional rotary nanomachine that drives rotation of the flagellum for bacterial motility. The cytoplasmic C ring of the flagellar motor functions as the switch complex for the rotational direction switching from counterclockwise to clockwise. However, the structural basis of the rotational switching and how the C ring is assembled have long remained elusive. Here, we present two high-resolution cryo-electron microscopy structures of the C ring-containing flagellar basal body-hook complex from Salmonella Typhimurium,which are in the default counterclockwise state and in a constitutively active CheY mutant-induced clockwise state, respectively. In both complexes, the C ring consists of four subrings, but is in two different conformations. The CheY proteins are bound into an open groove between two adjacent protomers on the surface of the middle subring of the C ring and interact with the FliG and FliM subunits. The binding of the CheY protein induces a significant upward shift of the C ring towards the MS ring and inward movements of its protomers towards the motor center, which eventually remodels the structures of the FliG subunits and reverses the orientations and surface electrostatic potential of the αtorque helices to trigger the counterclockwise-to-clockwise rotational switching. The conformational changes of the FliG subunits reveal that the stator units on the motor require a relocation process in the inner membrane during the rotational switching. This study provides unprecedented molecular insights into the rotational switching mechanism and a detailed overall structural view of the bacterial flagellar motors.

Frequency-adjustable OAM antenna with co-divergent beams for IoT applications

A frequency-tunable Orbital Angular Momentum (OAM)-generating antenna array system is introduced for the first time to provide high-data-rate and secure communication for Internet of Things (IoT) infrastructures and devices. Propagation of simultaneous OAM modes brings about the high-data-rate and frequency tunability leads to interference suppression for secure communication. Regarding the design evolution of the proposed structure, the novel Multiple-Input-Multiple-Output (MIMO) antenna consisting of two input ports creates a 2 × 2 UCA, resulting in two 2 × 2 sub-UCA. Feeding each 2 × 2 sub-array with [0°, 90°, 180°, 270°] phase gradients in clockwise and counterclockwise states gives rise to dual-mode OAM-carrying waves with simultaneous excitation capability. It should be mentioned that each MIMO element owns frequency tunability, resulting in a frequency-adjustable dual-mode OAM antenna array. A prototypes of the suggested MIMO antenna as well as the OAM array are fabricated and measured. Changing the applied DC-voltage to the varactor diodes embedded in capacitive stubs of the antenna elements shifts the operating frequency in the MIMO antenna and consequently in the OAM array. Concerning the unique radiation characteristics of the OAM antenna, vortex beams having helical phase fronts are observed, confirming the generation of expected OAM waves. Therefore, it is asserted that the introduced structure, for the first time, proposes new features of frequency-tunability, and co-divergent simultaneous OAM propagation using the MIMO-UCA technique to provide high-data-rate and secure communication in IoT applications.

Unidirectional mode selection in bistable quantum cascade ring lasers.

Ideal ring resonators are characterized by travelling-wave counter-propagating modes, but in practice travelling waves can only be realized under unidirectional operation, which has proved elusive. Here, we have designed and fabricated a monolithic quantum cascade ring laser coupled to an active waveguide that allows for robust, deterministic and controllable unidirectional operation. Spontaneous emission injection through the active waveguide enables dynamical switching between the clockwise and counterclockwise states of the ring laser with as little as 1.6% modulation of the electrical input. We show that this behavior stems from a perturbation in the bistable dynamics of the ring laser. In addition to switching and bistability, our novel coupler design for quantum cascade ring lasers offers an efficient mechanism for outcoupling and light detection.

A Mechanism of Modulating the Direction of Flagellar Rotation in Bacteria by Fumarate and Fumarate Reductase

Fumarate, an electron acceptor in anaerobic respiration of Escherichia coli, has an additional function of assisting the flagellar motor to shift from counterclockwise to clockwise rotation, with a consequent modulation of the bacterial swimming behavior. Fumarate transmits its effect to the motor via the fumarate reductase complex (FrdABCD), shown to bind to FliG—one of the motor’s switch proteins. How binding of the FrdABCD respiratory enzyme to FliG enhances clockwise rotation and how fumarate is involved in this activity have remained puzzling. Here we show that the FrdA subunit in the presence of fumarate is sufficient for binding to FliG and for clockwise enhancement. We further demonstrate by in vitro binding assays and super-resolution microscopy in vivo that the mechanism by which fumarate-occupied FrdA enhances clockwise rotation involves its preferential binding to the clockwise state of FliG (FliGcw). Continuum electrostatics combined with docking analysis and conformational sampling endorsed the experimental conclusions and suggested that the FrdA–FliGcw interaction is driven by the positive electrostatic potential generated by FrdA and the negatively charged areas of FliG. They further demonstrated that fumarate changes FrdA’s conformation to one that can bind to FliGcw. These findings also show that the reason for the failure of the succinate dehydrogenase flavoprotein SdhA (an almost-identical analog of FrdA shown to bind to FliG equally well) to enhance clockwise rotation is that it has no binding preference for FliGcw. We suggest that this mechanism is physiologically important as it can modulate the magnitude of ΔG0 between the clockwise and counterclockwise states of the motor to tune the motor to the growth conditions of the bacteria.

Switching dynamics of the bacterial flagellar motor near zero load.

Switching dynamics of flagellar motors of Escherichia coli is commonly observed through markers attached to the flagellar filaments. To eliminate possible complications resulting from the conformational transitions of these filaments and to look at the output of motors more directly, we monitored motor rotation by attaching nanogold spheres to the hooks of cells lacking filaments. We observed exponentially distributed counterclockwise (CCW) and clockwise (CW) intervals and Lorentzian power spectra of the switching time series consistent with models that treat motor switching as a two-state Poisson process.

Influence of vortex domain switching on the electrocaloric property of a ferroelectric nanoparticle

The influence of vortex domain switching on the electrocaloric (EC) property of a PbTiO3 nanoparticle is studied using the phase field method combined with thermodynamics analysis. The calculation results reveal that both a very large negative adiabatic temperature change (ΔT = −6.34 K) and a positive adiabatic temperature change (ΔT = 4.16 K) appear in the PbTiO3 nanoparticle when the vortex domain switches from the clockwise state to the counterclockwise state under a curled electric field. The obtained results will be of help in understanding the EC property in ferroelectrics with multidomain structure.

Dynamics of mechanosensing in the bacterial flagellar motor

Mechanosensing by flagella is thought to trigger bacterial swarmer-cell differentiation, an important step in pathogenesis. How flagellar motors sense mechanical stimuli is not known. To study this problem, we suddenly increased the viscous drag on motors by a large factor, from very low loads experienced by motors driving hooks or hooks with short filament stubs, to high loads, experienced by motors driving tethered cells or 1-μm latex beads. From the initial speed (after the load change), we inferred that motors running at very low loads are driven by one or at most two force-generating units. Following the load change, motors gradually adapted by increasing their speeds in a stepwise manner (over a period of a few minutes). Motors initially spun exclusively counterclockwise, but then increased the fraction of time that they spun clockwise over a time span similar to that observed for adaptation in speed. Single-motor total internal reflection fluorescence imaging of YFP-MotB (part of a stator force-generating unit) confirmed that the response to sudden increments in load occurred by the addition of new force-generating units. We estimate that 6-11 force-generating units drive motors at high loads. Wild-type motors and motors locked in the clockwise or counterclockwise state behaved in a similar manner, as did motors in cells deleted for the motor protein gene fliL or for genes in the chemotaxis signaling pathway. Thus, it appears that stators themselves act as dynamic mechanosensors. They change their structure in response to changes in external load. How such changes might impact cellular functions other than motility remains an interesting question.

Symmetry theory of the flexomagnetoelectric interaction in the magnetic vortices and skyrmions

Symmetry classification of the magnetic vortices and skyrmions has been suggested. Relation between symmetry based predictions and direct calculation has been shown. It was shown that electric dipole moment of the vortex is located inside the small vortex core. The antivortices and antiskyrmions do not carry the total core electric dipole induced by the flexomagnetoelectric interaction in the hexoctahedral cubic crystal. The volumetric bound electric charge is distributed around the core. Switching of the core electric dipole direction produces the switching of the core magnetization or vortex chirality and vice versa. The vortices and skyrmions with time-invariant enantiomorphism have two degenerative states: clockwise and counterclockwise state.

Optical Characterization of All-Magnetic NOT Gate Operation in Vortex Rings

We have demonstrated deterministic operation of inverters (NOT gates) using vortex magnetization states in pairs of linked 5 mum diameter permalloy rings with a small region of overlap. The input ring is clipped so that the state can be controlled by global fields, and is designed to switch at lower fields than the unclipped output ring, which then inverts the logical state of the input. Optical coatings over half of each ring provide distinct magnetooptic Kerr effect (MOKE) signatures for saturation, clockwise, and counterclockwise states in our focused beam system. We demonstrate that excessive overlap between the rings leads to overall shape anisotropy dominating the behavior of the structure.

Fumarate modulates bacterial flagellar rotation by lowering the free energy difference between the clockwise and counterclockwise states of the motor

Switching flagellar rotation from one direction to another is an essential part of bacterial chemotaxis. Fumarate has been shown to possess the capacity to restore to flagella of cytoplasm-free, CheY-containing bacterial envelopes the ability to switch directions and to increase the probability of reversal in intact cells. Neither the target of fumarate action nor the mechanism of function is known. To distinguish between the two potential targets of fumarate, the response regulator CheY and the flagellar switch-motor complex, we compared flagellar rotation between isogenic strains that lacked CheY and had either low or high levels of fumarate. The difference in the fumarate levels was due to a deletion of the genes encoding the enzymes that synthesize and metabolize fumarate; succinate dehydrogenase and fumarase, respectively. The strains were in a gutted background (i.e. a background deleted for the cytoplasmic chemotaxis proteins and some of the receptors), and switching was achieved by carrying out the measurements at 2.5°C, where it has been demonstrated that gutted cells switch spontaneously. The flagellar rotation of the strain with the highest level of fumarate was the most clockwise-biased and had the highest reversal frequency, indicating that fumarate is effective even in the absence of CheY. Fumarate reduced the free energy difference of the counterclockwise-to-clockwise transition and had no appreciable effect on the activation energy of this transition. Similar observations were made at room temperature, provided that intracellular CheY was present. In a wild-type background, both mutants made rings on semi-solid agar typical of normal chemotaxis. Taken together, the results suggest that the target of fumarate is the switch-motor complex, that fumarate acts by increasing the probability of the clockwise state, and that a fumarate level as low as that found in succinate dehydrogenase mutants is sufficient for normal chemotaxis.

Control of direction of flagellar rotation in bacterial chemotaxis.

The motile behavior of the bacterium Escherichia coli depends on the direction of rotation of its flagellar motors. Binding of the phosphorylated signaling molecule CheY to a motor component FliM is known to enhance clockwise rotation. It is difficult to study this interaction in vivo, because the dynamics of phosphorylation of CheY by its kinase CheA and the hydrolysis of CheY (accelerated by CheZ) are not under direct experimental control. Here, we examine instead the interaction with the flagellar motor of a double mutant CheY13DK106YW that is active without phosphorylation. The behavioral assays were carried out on tethered cells lacking CheA and CheZ. The effects of variation in intracellular concentration of the mutant protein were highly nonlinear. However, they can be explained by a thermal isomerization model in which the free energies of clockwise and counterclockwise states depend linearly on the amount of CheY bound.

Multiple kinetic states for the flagellar motor switch.

By means of a computerized video processing system, the flagellar motors of Escherichia coli were shown to have multiple kinetic states for each rotational direction. High-resolution analysis of flagellar motors revealed new kinetic states both in wild-type cells and in a strain deleted of other signal-transducing genes to which CheY had been introduced. This strain, RP1091, retained residual kinase activity that could phosphorylate CheY, complicating the biochemical identification of certain kinetic states. The behavioral effect of CheY on single flagellar motors was ultrasensitive, with an apparent Hill coefficient of 5.5 +/- 1.9 (SD) and a half-maximal effect at 10.1 +/- 0.5 (SD) microM CheY. Based on the CheY concentration dependence, a two-state model is clearly excluded, even for the simpler system of CheY-induced rotational reversals in the deletion strain. The data are best described by a four-state model, with two clockwise and two counterclockwise states.