Bidirectional Optical Control of Proton Motive Force in Escherichia coli Using Microbial Rhodopsins.

Proton (H+) motive force (PMF) serves as the energy source for the flagellar motor rotation, crucial for microbial motility. Here, to control PMF using light, we introduced light-driven inward and outward proton pump rhodopsins, RmXeR and AR3, into Escherichia coli. The motility of E. coli cells expressing RmXeR and AR3 significantly decreased and increased upon illumination, respectively. Tethered cell experiments revealed that, upon illumination, the torque of the flagellar motor decreased to nearly zero (28 pN nm) with RmXeR, while it increased to 1170 pN nm with AR3. These alterations in PMF correspond to +146 mV (RmXeR) and -140 mV (AR3), respectively. Thus, bidirectional optical control of PMF in E. coli was successfully achieved by using proton pump rhodopsins. This system holds a potential for enhancing our understanding of the roles of PMF in various biological functions.

Flagellar polymorphism-dependent bacterial swimming motility in a structured environment.

Most motile bacteria use supramolecular motility machinery called bacterial flagellum, which converts the chemical energy gained from ion flux into mechanical rotation. Bacterial cells sense their external environment through a two-component regulatory system consisting of a histidine kinase and response regulator. Combining these systems allows the cells to move toward favorable environments and away from their repellents. A representative example of flagellar motility is run-and-tumble swimming in Escherichia coli, where the counter-clockwise (CCW) rotation of a flagellar bundle propels the cell forward, and the clockwise (CW) rotation undergoes cell re-orientation (tumbling) upon switching the direction of flagellar motor rotation from CCW to CW. In this mini review, we focus on several types of chemotactic behaviors that respond to changes in flagellar shape and direction of rotation. Moreover, our single-cell analysis demonstrated back-and-forth swimming motility of an original E. coli strain. We propose that polymorphic flagellar changes are required to enhance bacterial movement in a structured environment as a colony spread on an agar plate.

The rapid evolution of flagellar ion selectivity in experimental populations of E. coli.

Determining which cellular processes facilitate adaptation requires a tractable experimental model where an environmental cue can generate variants that rescue function. The bacterial flagellar motor (BFM) is an excellent candidate-an ancient and highly conserved molecular complex for bacterial propulsion toward favorable environments. Motor rotation is often powered by H+ or Na+ ion transit through the torque-generating stator subunit of the motor complex, and ion selectivity has adapted over evolutionary time scales. Here, we used CRISPR engineering to replace the native Escherichia coli H+-powered stator with Na+-powered stator genes and report the spontaneous reversion of our edit in a low-sodium environment. We followed the evolution of the stators during their reversion to H+-powered motility and used both whole-genome and RNA sequencing to identify genes involved in the cell's adaptation. Our transplant of an unfit protein and the cells' rapid response to this edit demonstrate the adaptability of the stator subunit and highlight the hierarchical modularity of the flagellar motor.

Bayesian-based decipherment of in-depth information in bacterial chemical sensing beyond pleasant/unpleasant responses.

Chemical sensing is vital to the survival of all organisms. Bacterial chemotaxis is conducted by multiple receptors that sense chemicals to regulate a single signalling system controlling the transition between the direction (clockwise vs. counterclockwise) of flagellar rotation. Such an integrated system seems better suited to judge chemicals as either favourable or unfavourable, but not for identification purposes though differences in their affinities to the receptors may cause difference in response strength. Here, an experimental setup was developed to monitor behaviours of multiple cells stimulated simultaneously as well as a statistical framework based on Bayesian inferences. Although responses of individual cells varied substantially, ensemble averaging of the time courses seemed characteristic to attractant species, indicating we can extract information of input chemical species from responses of the bacterium. Furthermore, two similar, but distinct, beverages elicited attractant responses of cells with profiles distinguishable with the Bayesian procedure. These results provide a basis for novel bio-inspired sensors that could be used with other cell types to sense wider ranges of chemicals.

Distinct chemotactic behavior in the original Escherichia coli K-12 depending on forward-and-backward swimming, not on run-tumble movements.

Most motile bacteria are propelled by rigid, helical, flagellar filaments and display distinct swimming patterns to explore their favorable environments. Escherichia coli cells have a reversible rotary motor at the base of each filament. They exhibit a run-tumble swimming pattern, driven by switching of the rotational direction, which causes polymorphic flagellar transformation. Here we report a novel swimming mode in E. coli ATCC10798, which is one of the original K-12 clones. High-speed tracking of single ATCC10798 cells showed forward and backward swimming with an average turning angle of 150°. The flagellar helicity remained right-handed with a 1.3 µm pitch and 0.14 µm helix radius, which is consistent with the feature of a curly type, regardless of motor switching; the flagella of ATCC10798 did not show polymorphic transformation. The torque and rotational switching of the motor was almost identical to the E. coli W3110 strain, which is a derivative of K-12 and a wild-type for chemotaxis. The single point mutation of N87K in FliC, one of the filament subunits, is critical to the change in flagellar morphology and swimming pattern, and lack of flagellar polymorphism. E. coli cells expressing FliC(N87K) sensed ascending a chemotactic gradient in liquid but did not spread on a semi-solid surface. Based on these results, we concluded that a flagellar polymorphism is essential for spreading in structured environments.

Coupling Ion Specificity of the Flagellar Stator Proteins MotA1/MotB1 of Paenibacillus sp. TCA20.

The bacterial flagellar motor is a reversible rotary molecular nanomachine, which couples ion flux across the cytoplasmic membrane to torque generation. It comprises a rotor and multiple stator complexes, and each stator complex functions as an ion channel and determines the ion specificity of the motor. Although coupling ions for the motor rotation were presumed to be only monovalent cations, such as H+ and Na+, the stator complex MotA1/MotB1 of Paenibacillus sp. TCA20 (MotA1TCA/MotB1TCA) was reported to use divalent cations as coupling ions, such as Ca2+ and Mg2+. In this study, we initially aimed to measure the motor torque generated by MotA1TCA/MotB1TCA under the control of divalent cation motive force; however, we identified that the coupling ion of MotA1TCAMotB1TCA is very likely to be a monovalent ion. We engineered a series of functional chimeric stator proteins between MotB1TCA and Escherichia coli MotB. E. coli ΔmotAB cells expressing MotA1TCA and the chimeric MotB presented significant motility in the absence of divalent cations. Moreover, we confirmed that MotA1TCA/MotB1TCA in Bacillus subtilis ΔmotABΔmotPS cells generates torque without divalent cations. Based on two independent experimental results, we conclude that the MotA1TCA/MotB1TCA complex directly converts the energy released from monovalent cation flux to motor rotation.

High pressure inhibits signaling protein binding to the flagellar motor and bacterial chemotaxis through enhanced hydration.

High pressure below 100 MPa interferes inter-molecular interactions without causing pressure denaturation of proteins. In Escherichia coli, the binding of the chemotaxis signaling protein CheY to the flagellar motor protein FliM induces reversal of the motor rotation. Using molecular dynamics (MD) simulations and parallel cascade selection MD (PaCS-MD), we show that high pressure increases the water density in the first hydration shell of CheY and considerably induces water penetration into the CheY-FliM interface. PaCS-MD enabled us to observe pressure-induced dissociation of the CheY-FliM complex at atomic resolution. Pressure dependence of binding free energy indicates that the increase of pressure from 0.1 to 100 MPa significantly weakens the binding. Using high-pressure microscopy, we observed that high hydrostatic pressure fixes the motor rotation to the counter-clockwise direction. In conclusion, the application of pressure enhances hydration of the proteins and weakens the binding of CheY to FliM, preventing reversal of the flagellar motor.

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

The bacterial flagellar motor powers the rotation that propels the 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: (i) the transmembrane domain of PotB, corresponding to the TM region of the PomB stator from V. alginolyticus and (ii) near the peptidoglycan 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.

Dimerization site 2 of the bacterial DNA-binding protein H-NS is required for gene silencing and stiffened nucleoprotein filament formation.

The bacterial nucleoid-associated protein H-NS is a DNA-binding protein, playing a major role in gene regulation. To regulate transcription, H-NS silences genes, including horizontally acquired foreign genes. Escherichia coli H-NS is 137 residues long and consists of two discrete and independent structural domains: an N-terminal oligomerization domain and a C-terminal DNA-binding domain, joined by a flexible linker. The N-terminal oligomerization domain is composed of two dimerization sites, dimerization sites 1 and 2, which are both required for H-NS oligomerization, but the exact role of dimerization site 2 in gene silencing is unclear. To this end, we constructed a whole set of single amino acid substitution variants spanning residues 2 to 137. Using a well-characterized H-NS target, the slp promoter of the glutamic acid-dependent acid resistance (GAD) cluster promoters, we screened for any variants defective in gene silencing. Focusing on the function of dimerization site 2, we analyzed four variants, I70C/I70A and L75C/L75A, which all could actively bind DNA but are defective in gene silencing. Atomic force microscopy analysis of DNA-H-NS complexes revealed that all of these four variants formed condensed complexes on DNA, whereas WT H-NS formed rigid and extended nucleoprotein filaments, a conformation required for gene silencing. Single-molecule stretching experiments confirmed that the four variants had lost the ability to form stiffened filaments. We conclude that dimerization site 2 of H-NS plays a key role in the formation of rigid H-NS nucleoprotein filament structures required for gene silencing.

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

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 in Escherichia coli by 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.

Measurements of the Rotation of the Flagellar Motor by Bead Assay.

The bacterial flagellar motor is a reversible rotary nano-machine powered by the ion flux across the cytoplasmic membrane. Each motor rotates a long helical filament that extends from the cell body at several hundreds revolutions per second. The output of the motor is characterized by its generated torque and rotational speed. The torque can be calculated as the rotational frictional drag coefficient multiplied by the angular velocity. Varieties of methods, including a bead assay, have been developed to measure the flagellar rotation rate under various load conditions on the motor. In this chapter, we describe a method to monitor the motor rotation through a position of a 1 µm bead attached to a truncated flagellar filament.

Bacterial Flagellar Motor Switch in Response to CheY-P Regulation and Motor Structural Alterations.

The bacterial flagellar motor (BFM) is a molecular machine that rotates the helical filaments and propels the bacteria swimming toward favorable conditions. In our previous works, we built a stochastic conformational spread model to explain the dynamic and cooperative behavior of BFM switching. Here, we extended this model to test whether it can explain the latest experimental observations regarding CheY-P regulation and motor structural adaptivity. We show that our model predicts a strong correlation between rotational direction and the number of CheY-Ps bound to the switch complex, in agreement with the latest finding from Fukuoka et al. It also predicts that the switching sensitivity of the BFM can be fine-tuned by incorporating additional units into the switch complex, as recently demonstrated by Yuan et al., who showed that stoichiometry of FliM undergoes dynamic change to maintain ultrasensitivity in the motor switching response. In addition, by locking some rotor switching units on the switch complex into the stable clockwise-only conformation, our model has accurately simulated recent experiments expressing clockwise-locked FliG(ΔPAA) into the switch complex and reproduced the increased switching rate of the motor.

Substrate-dependent dynamics of the multidrug efflux transporter AcrB of Escherichia coli.

The resistance-nodulation-cell division (RND)-type xenobiotic efflux system plays a major role in the multidrug resistance of gram-negative bacteria. The only constitutively expressed RND system of Escherichia coli consists of the inner membrane transporter AcrB, the membrane fusion protein AcrA, and the outer membrane channel TolC. The latter two components are shared with another RND-type transporter AcrD, whose expression is induced by environmental stimuli. Here, we demonstrate how RND-type ternary complexes, which span two membranes and the cell wall, form in vivo. Total internal reflection fluorescence (TIRF) microscopy revealed that most fluorescent foci formed by AcrB fused to green fluorescent protein (GFP) were stationary in the presence of TolC but showed lateral displacements when tolC was deleted. The fraction of stationary AcrB-GFP foci decreased with increasing levels of AcrD. We propose that the AcrB-containing complex becomes unstable upon the induction of AcrD, which presumably replaces AcrB, a process we call "transporter exchange." This instability is suppressed by AcrB-specific substrates, suggesting that the ternary complex is stabilised when it is in action. These results suggest that the assembly of the RND-type efflux system is dynamically regulated in response to external stimuli, shedding new light on the adaptive antibiotic resistance of bacteria.

Liquid-based iterative recombineering method tolerant to counter-selection escapes.

Selection-based recombineering is a flexible and proven technology to precisely modify bacterial genomes at single base resolution. It consists of two steps of homologous recombination followed by selection/counter-selection. However, the shortage of efficient counter-selectable markers limits the throughput of this method. Additionally, the emergence of 'selection escapees' can affect recombinant pools generated through this method, and they must be manually removed at each step of selection-based recombineering. Here, we report a series of efforts to improve the throughput and robustness of selection-based recombineering and to achieve seamless and automatable genome engineering. Using the nucleoside kinase activity of herpes simplex virus thymidine kinase (hsvTK) on the non-natural nucleoside dP, a highly efficient, rapid, and liquid-based counter-selection system was established. By duplicating hsvtk gene, combined with careful control of the population size for the subsequent round, we effectively eliminated selection escapes, enabling seamless and multiple insertions/replacement of gene-size fragments in the chromosome. Four rounds of recombineering could thus be completed in 10 days, requiring only liquid handling and without any need for colony isolation or genotype confirmation. The simplicity and robustness of our method make it broadly accessible for multi-locus chromosomal modifications.

Hybrid-fuel bacterial flagellar motors in Escherichia coli.

The bacterial flagellar motor rotates driven by an electrochemical ion gradient across the cytoplasmic membrane, either H(+) or Na(+) ions. The motor consists of a rotor ∼50 nm in diameter surrounded by multiple torque-generating ion-conducting stator units. Stator units exchange spontaneously between the motor and a pool in the cytoplasmic membrane on a timescale of minutes, and their stability in the motor is dependent upon the ion gradient. We report a genetically engineered hybrid-fuel flagellar motor in Escherichia coli that contains both H(+)- and Na(+)-driven stator components and runs on both types of ion gradient. We controlled the number of each type of stator unit in the motor by protein expression levels and Na(+) concentration ([Na(+)]), using speed changes of single motors driving 1-µm polystyrene beads to determine stator unit numbers. De-energized motors changed from locked to freely rotating on a timescale similar to that of spontaneous stator unit exchange. Hybrid motor speed is simply the sum of speeds attributable to individual stator units of each type. With Na(+) and H(+) stator components expressed at high and medium levels, respectively, Na(+) stator units dominate at high [Na(+)] and are replaced by H(+) units when Na(+) is removed. Thus, competition between stator units for spaces in a motor and sensitivity of each type to its own ion gradient combine to allow hybrid motors to adapt to the prevailing ion gradient. We speculate that a similar process may occur in species that naturally express both H(+) and Na(+) stator components sharing a common rotor.

Populational heterogeneity vs. temporal fluctuation in Escherichia coli flagellar motor switching.

The dynamic switching of the bacterial flagellar motor regulates cell motility in bacterial chemotaxis. It has been reported under physiological conditions that the switching bias of the flagellar motor undergoes large temporal fluctuations, which reflects noise propagating in the chemotactic signaling network. On the other hand, nongenetic heterogeneity is also observed in flagellar motor switching, as a large group of switching motors show different switching bias and frequency under the same physiological condition. In this work, we present simultaneous measurement of groups of Escherichia coli flagellar motor switching and compare them to long time recording of single switching motors. Consistent with previous studies, we observed temporal fluctuations in switching bias in long time recording experiments. However, the variability in switching bias at the populational level showed much higher volatility than its temporal fluctuation. These results suggested stable individuality in E. coli motor switching. We speculate that uneven expression of key regulatory proteins with amplification by the ultrasensitive response of the motor can account for the observed populational heterogeneity and temporal fluctuations.

Mechanism and kinetics of a sodium-driven bacterial flagellar motor.

The bacterial flagellar motor is a large rotary molecular machine that propels swimming bacteria, powered by a transmembrane electrochemical potential difference. It consists of an ∼50-nm rotor and up to ∼10 independent stators anchored to the cell wall. We measured torque-speed relationships of single-stator motors under 25 different combinations of electrical and chemical potential. All 25 torque-speed curves had the same concave-down shape as fully energized wild-type motors, and each stator passes at least 37 ± 2 ions per revolution. We used the results to explore the 25-dimensional parameter space of generalized kinetic models for the motor mechanism, finding 830 parameter sets consistent with the data. Analysis of these sets showed that the motor mechanism has a "powerstroke" in either ion binding or transit; ion transit is channel-like rather than carrier-like; and the rate-limiting step in the motor cycle is ion binding at low concentration, ion transit, or release at high concentration.

High hydrostatic pressure induces counterclockwise to clockwise reversals of the Escherichia coli flagellar motor.

The bacterial flagellar motor is a reversible rotary machine that rotates a left-handed helical filament, allowing bacteria to swim toward a more favorable environment. The direction of rotation reverses from counterclockwise (CCW) to clockwise (CW), and vice versa, in response to input from the chemotaxis signaling circuit. CW rotation is normally caused by binding of the phosphorylated response regulator CheY (CheY-P), and strains lacking CheY are typically locked in CCW rotation. The detailed mechanism of switching remains unresolved because it is technically difficult to regulate the level of CheY-P within the concentration range that produces flagellar reversals. Here, we demonstrate that high hydrostatic pressure can induce CW rotation even in the absence of CheY-P. The rotation of single flagellar motors in Escherichia coli cells with the cheY gene deleted was monitored at various pressures and temperatures. Application of >120 MPa pressure induced a reversal from CCW to CW at 20°C, although at that temperature, no motor rotated CW at ambient pressure (0.1 MPa). At lower temperatures, pressure-induced changes in direction were observed at pressures of <120 MPa. CW rotation increased with pressure in a sigmoidal fashion, as it does in response to increasing concentrations of CheY-P. Application of pressure generally promotes the formation of clusters of ordered water molecules on the surfaces of proteins. It is possible that hydration of the switch complex at high pressure induces structural changes similar to those caused by the binding of CheY-P.

Microscopic analysis of bacterial motility at high pressure.

The bacterial flagellar motor is a molecular machine that converts an ion flux to the rotation of a helical flagellar filament. Counterclockwise rotation of the filaments allows them to join in a bundle and propel the cell forward. Loss of motility can be caused by environmental factors such as temperature, pH, and solvation. Hydrostatic pressure is also a physical inhibitor of bacterial motility, but the detailed mechanism of this inhibition is still unknown. Here, we developed a high-pressure microscope that enables us to acquire high-resolution microscopic images, regardless of applied pressures. We also characterized the pressure dependence of the motility of swimming Escherichia coli cells and the rotation of single flagellar motors. The fraction and speed of swimming cells decreased with increased pressure. At 80 MPa, all cells stopped swimming and simply diffused in solution. After the release of pressure, most cells immediately recovered their initial motility. Direct observation of the motility of single flagellar motors revealed that at 80 MPa, the motors generate torque that should be sufficient to join rotating filaments in a bundle. The discrepancy in the behavior of free swimming cells and individual motors could be due to the applied pressure inhibiting the formation of rotating filament bundles that can propel the cell body in an aqueous environment.

Steps and bumps: precision extraction of discrete states of molecular machines.

We report statistical time-series analysis tools providing improvements in the rapid, precision extraction of discrete state dynamics from time traces of experimental observations of molecular machines. By building physical knowledge and statistical innovations into analysis tools, we provide techniques for estimating discrete state transitions buried in highly correlated molecular noise. We demonstrate the effectiveness of our approach on simulated and real examples of steplike rotation of the bacterial flagellar motor and the F1-ATPase enzyme. We show that our method can clearly identify molecular steps, periodicities and cascaded processes that are too weak for existing algorithms to detect, and can do so much faster than existing algorithms. Our techniques represent a step in the direction toward automated analysis of high-sample-rate, molecular-machine dynamics. Modular, open-source software that implements these techniques is provided.