Structure and Function of Stator Units of the Bacterial Flagellar Motor.

Many bacteria use the flagellum for locomotion and chemotaxis. Its bidirectional rotation is driven by a membrane-embedded motor, which uses energy from the transmembrane ion gradient to generate torque at the interface between stator units and rotor. The structural organization of the stator unit (MotAB), its conformational changes upon ion transport, and how these changes power rotation of the flagellum remain unknown. Here, we present ~3 Å-resolution cryoelectron microscopy reconstructions of the stator unit in different functional states. We show that the stator unit consists of a dimer of MotB surrounded by a pentamer of MotA. Combining structural data with mutagenesis and functional studies, we identify key residues involved in torque generation and present a detailed mechanistic model for motor function and switching of rotational direction.

Structural basis of torque generation in the bi-directional bacterial flagellar motor.

The flagellar stator unit is an oligomeric complex of two membrane proteins (MotA5B2) that powers bi-directional rotation of the bacterial flagellum. Harnessing the ion motive force across the cytoplasmic membrane, the stator unit operates as a miniature rotary motor itself to provide torque for rotation of the flagellum. Recent cryo-electron microscopic (cryo-EM) structures of the stator unit provided novel insights into its assembly, function, and subunit stoichiometry, revealing the ion flux pathway and the torque generation mechanism. Furthermore, in situ cryo-electron tomography (cryo-ET) studies revealed unprecedented details of the interactions between stator unit and rotor. In this review, we summarize recent advances in our understanding of the structure and function of the flagellar stator unit, torque generation, and directional switching of the motor.

Signaling events that occur when cells of Escherichia coli encounter a glass surface.

Bacterial cells interact with solid surfaces and change their lifestyle from single free-swimming cells to sessile communal structures (biofilms). Cyclic di-guanosine monophosphate (c-di-GMP) is central to this process, yet we lack tools for direct dynamic visualization of c-di-GMP in single cells. Here, we developed a fluorescent protein-based c-di-GMP-sensing system for Escherichia coli that allowed us to visualize initial signaling events and assess the role played by the flagellar motor. The sensor was pH sensitive, and the events that appeared on a seconds' timescale were alkaline spikes in the intracellular pH. These spikes were not apparent when signals from different cells were averaged. Instead, a signal appeared on a minutes' timescale that proved to be due to an increase in intracellular c-di-GMP. This increase, but not the alkaline spikes, depended upon a functional flagellar motor. The kinetics and the amplitude of both the pH and c-di-GMP responses displayed cell-to-cell variability indicative of the distinct ways the cells approached and interacted with the surface. The energetic status of a cell can modulate these events. In particular, the alkaline spikes displayed an oscillatory behavior and the c-di-GMP increase was modest in the presence of glucose.

Mechanosensitive remodeling of the bacterial flagellar motor is independent of direction of rotation.

Motility is important for the survival and dispersal of many bacteria, and it often plays a role during infections. Regulation of bacterial motility by chemical stimuli is well studied, but recent work has added a new dimension to the problem of motility control. The bidirectional flagellar motor of the bacterium Escherichia coli recruits or releases torque-generating units (stator units) in response to changes in load. Here, we show that this mechanosensitive remodeling of the flagellar motor is independent of direction of rotation. Remodeling rate constants in clockwise rotating motors and in counterclockwise rotating motors, measured previously, fall on the same curve if plotted against torque. Increased torque decreases the off rate of stator units from the motor, thereby increasing the number of active stator units at steady state. A simple mathematical model based on observed dynamics provides quantitative insight into the underlying molecular interactions. The torque-dependent remodeling mechanism represents a robust strategy to quickly regulate output (torque) in response to changes in demand (load).

Synchronized strobed phase contrast and fluorescence microscopy: the interlaced standard reimagined.

We propose a simple, cost-effective method for synchronized phase contrast and fluorescence video acquisition in live samples. Counter-phased pulses of phase contrast illumination and fluorescence excitation light are synchronized with the exposure of the two fields of an interlaced camera sensor. This results in a video sequence in which each frame contains both exposure modes, each in half of its pixels. The method allows real-time acquisition and display of synchronized and spatially aligned phase contrast and fluorescence image sequences that can be separated by de-interlacing in two independent videos. The method can be implemented on any fluorescence microscope with a camera port without needing to modify the optical path.

Torque-dependent remodeling of the bacterial flagellar motor.

Multisubunit protein complexes are ubiquitous in biology and perform a plethora of essential functions. Most of the scientific literature treats such assemblies as static: their function is assumed to be independent of their manner of assembly, and their structure is assumed to remain intact until they are degraded. Recent observations of the bacterial flagellar motor, among others, bring these notions into question. The torque-generating stator units of the motor assemble and disassemble in response to changes in load. Here, we used electrorotation to drive tethered cells forward, which decreases motor load, and measured the resulting stator dynamics. No disassembly occurred while the torque remained high, but all of the stator units were released when the motor was spun near the zero-torque speed. When the electrorotation was turned off, so that the load was again high, stator units were recruited, increasing motor speed in a stepwise fashion. A model in which speed affects the binding rate and torque affects the free energy of bound stator units captures the observed torque-dependent stator assembly dynamics, providing a quantitative framework for the environmentally regulated self-assembly of a major macromolecular machine.

Cargo transport shapes the spatial organization of a microbial community.

The human microbiome is an assemblage of diverse bacteria that interact with one another to form communities. Bacteria in a given community are arranged in a 3D matrix with many degrees of freedom. Snapshots of the community display well-defined structures, but the steps required for their assembly are not understood. Here, we show that this construction is carried out with the help of gliding bacteria. Gliding is defined as the motion of cells over a solid or semisolid surface without the necessity of growth or the aid of pili or flagella. Genomic analysis suggests that gliding bacteria are present in human microbial communities. We focus on Capnocytophaga gingivalis, which is present in abundance in the human oral microbiome. Tracking of fluorescently labeled single cells and of gas bubbles carried by fluid flow shows that swarms of C. gingivalis are layered, with cells in the upper layers moving more rapidly than those in the lower layers. Thus, cells also glide on top of one another. Cells of nonmotile bacterial species attach to the surface of C. gingivalis and are propelled as cargo. The cargo cell moves along the length of a C. gingivalis cell, looping from one pole to the other. Multicolor fluorescent spectral imaging of cells of different live but nonmotile bacterial species reveals their long-range transport in a polymicrobial community. A swarm of C. gingivalis transports some nonmotile bacterial species more efficiently than others and helps to shape the spatial organization of a polymicrobial community.

Internal and external components of the bacterial flagellar motor rotate as a unit.

Most bacteria that swim, including Escherichia coli, are propelled by helical filaments, each driven at its base by a rotary motor powered by a proton or a sodium ion electrochemical gradient. Each motor contains a number of stator complexes, comprising 4MotA 2MotB or 4PomA 2PomB, proteins anchored to the rigid peptidoglycan layer of the cell wall. These proteins exert torque on a rotor that spans the inner membrane. A shaft connected to the rotor passes through the peptidoglycan and the outer membrane through bushings, the P and L rings, connecting to the filament by a flexible coupling known as the hook. Although the external components, the hook and the filament, are known to rotate, having been tethered to glass or marked by latex beads, the rotation of the internal components has remained only a reasonable assumption. Here, by using polarized light to bleach and probe an internal YFP-FliN fusion, we show that the innermost components of the cytoplasmic ring rotate at a rate similar to that of the hook.

CW and CCW Conformations of the E. coli Flagellar Motor C-Ring Evaluated by Fluorescence Anisotropy.

The molecular cascade that controls switching of the direction of rotation of Escherichia coli flagellar motors is well known, but the conformational changes that allow the rotor to switch are still unclear. The signaling molecule CheY, when phosphorylated, binds to the C-ring at the base of the rotor, raising the probability that the motor spins clockwise. When the concentration of CheY-P is so low that the motor rotates exclusively counterclockwise (CCW), the C-ring recruits more monomers of FliM and tetramers of FliN, the proteins to which CheY-P binds, thus increasing the motor's sensitivity to CheY-P and allowing it to switch once again. Motors that rotate exclusively CCW have more FliM and FliN subunits in their C-rings than motors that rotate exclusively clockwise. How are the new subunits accommodated? Does the diameter of the C-ring increase, or do FliM and FliN get packed in a different pattern, keeping the overall diameter of the C-ring constant? Here, by measuring fluorescence anisotropy of yellow fluorescent protein-labeled motors, we show that the CCW C-rings accommodate more FliM monomers without changing the spacing between them, and more FliN monomers at the same time as increasing their effective spacing and/or changing their orientation within the tetrameric structure.

Adaptation at the output of the chemotaxis signalling pathway.

In the bacterial chemotaxis network, receptor clusters process input, and flagellar motors generate output. Receptor and motor complexes are coupled by the diffusible protein CheY-P. Receptor output (the steady-state concentration of CheY-P) varies from cell to cell. However, the motor is ultrasensitive, with a narrow operating range of CheY-P concentrations. How the match between receptor output and motor input might be optimized is unclear. Here we show that the motor can shift its operating range by changing its composition. The number of FliM subunits in the C-ring increases in response to a decrement in the concentration of CheY-P, increasing motor sensitivity. This shift in sensitivity explains the slow partial adaptation observed in mutants that lack the receptor methyltransferase and methylesterase and why motors show signal-dependent FliM turnover. Adaptive remodelling is likely to be a common feature in the operation of many molecular machines.

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.

The Screw-Like Movement of a Gliding Bacterium Is Powered by Spiral Motion of Cell-Surface Adhesins.

Flavobacterium johnsoniae, a rod-shaped bacterium, glides over surfaces at speeds of ∼2 µm/s. The propulsion of a cell-surface adhesin, SprB, is known to enable gliding. We used cephalexin to generate elongated cells with irregular shapes and followed their displacement in three dimensions. These cells rolled about their long axes as they moved forward, following a right-handed trajectory. We coated gold nanoparticles with an SprB antibody and tracked them in three dimensions in an evanescent field where the nanoparticles appeared brighter when they were closer to the glass. The nanoparticles followed a right-handed spiral trajectory on the surface of the cell. Thus, if SprB were to adhere to the glass rather than to a nanoparticle, the cell would move forward along a right-handed trajectory, as observed, but in a direction opposite to that of the nanoparticle.

Visualizing Flagella while Tracking Bacteria.

A complete description of the swimming behavior of a bacterium requires measurement of the displacement and orientation of the cell body together with a description of the movement of the flagella. We rebuilt a tracking microscope so that we could visualize flagellar filaments of tracked cells by fluorescence. We studied Escherichia coli (cells of various lengths, including swarm cells), Bacillus subtilis (wild-type and a mutant with fewer flagella), and a motile Streptococcus (now Enterococcus). The run-and-tumble statistics were nearly the same regardless of cell shape, length, and flagellation; however, swarm cells rarely tumbled, and cells of Enterococcus tended to swim in loops when moving slowly. There were events in which filaments underwent polymorphic transformations but remained in bundles, leading to small deflections in direction of travel. Tumble speeds were ∼2/3 as large as run speeds, and the rates of change of swimming direction while running or tumbling were smaller when cells swam more rapidly. If a smaller fraction of filaments were involved in tumbles, the tumble intervals were shorter and the angles between runs were smaller.

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.

Switching of bacterial flagellar motors [corrected] triggered by mutant FliG.

Binding of the chemotaxis response regulator CheY-P promotes switching between rotational states in flagellar motors of the bacterium Escherichia coli. Here, we induced switching in the absence of CheY-P by introducing copies of a mutant FliG locked in the clockwise (CW) conformation (FliG(CW)). The composition of the mixed FliG ring was estimated via fluorescence imaging, and the probability of CW rotation (CWbias) was determined from the rotation of tethered cells. The results were interpreted in the framework of a 1D Ising model. The data could be fit by assuming that mutant subunits are more stable in the CW conformation than in the counterclockwise conformation. We found that CWbias varies depending on the spatial arrangement of the assembled subunits in the FliG ring. This offers a possible explanation for a previous observation of hysteresis in the switch function in analogous mixed FliM motors-in motors containing identical fractions of mutant FliM(CW) in otherwise wild-type motors, the CWbias differed depending on whether mutant subunits were expressed in strains with native motors or native subunits were expressed in strains with mutant motors.

Mutations That Stimulate flhDC Expression in Escherichia coli K-12.

UNLABELLED: Motility is a beneficial attribute that enables cells to access and explore new environments and to escape detrimental ones. The organelle of motility in Escherichia coli is the flagellum, and its production is initiated by the activating transcription factors FlhD and FlhC. The expression of these factors by the flhDC operon is highly regulated and influenced by environmental conditions. The flhDC promoter is recognized by σ(70) and is dependent on the transcriptional activator cyclic AMP (cAMP)-cAMP receptor protein complex (cAMP-CRP). A number of K-12 strains exhibit limited motility due to low expression levels of flhDC. We report here a large number of mutations that stimulate flhDC expression in such strains. They include single nucleotide changes in the -10 element of the promoter, in the promoter spacer, and in the cAMP-CRP binding region. In addition, we show that insertion sequence (IS) elements or a kanamycin gene located hundreds of base pairs upstream of the promoter can effectively enhance transcription, suggesting that the topology of a large upstream region plays a significant role in the regulation of flhDC expression. None of the mutations eliminated the requirement for cAMP-CRP for activation. However, several mutations allowed expression in the absence of the nucleoid organizing protein, H-NS, which is normally required for flhDC expression. IMPORTANCE: The flhDC operon of Escherichia coli encodes transcription factors that initiate flagellar synthesis, an energetically costly process that is highly regulated. Few deregulating mutations have been reported thus far. This paper describes new single nucleotide mutations that stimulate flhDC expression, including a number that map to the promoter spacer region. In addition, this work shows that insertion sequence elements or a kanamycin gene located far upstream from the promoter or repressor binding sites also stimulate transcription, indicating a role of regional topology in the regulation of flhDC expression.

Water reservoir maintained by cell growth fuels the spreading of a bacterial swarm.

Flagellated bacteria can swim across moist surfaces within a thin layer of fluid, a means for surface colonization known as swarming. This fluid spreads with the swarm, but how it does so is unclear. We used micron-sized air bubbles to study the motion of this fluid within swarms of Escherichia coli. The bubbles moved diffusively, with drift. Bubbles starting at the swarm edge drifted inward for the first 5 s and then moved outward. Bubbles starting 30 µm from the swarm edge moved inward for the first 20 s, wandered around in place for the next 40 s, and then moved outward. Bubbles starting at 200 or 300 µm from the edge moved outward or wandered around in place, respectively. So the general trend was inward near the outer edge of the swarm and outward farther inside, with flows converging on a region about 100 µm from the swarm edge. We measured cellular metabolic activities with cells expressing a short-lived GFP and cell densities with cells labeled with a membrane fluorescent dye. The fluorescence plots were similar, with peaks about 80 µm from the swarm edge and slopes that mimicked the particle drift rates. These plots suggest that net fluid flow is driven by cell growth. Fluid depth is largest in the multilayered region between approximately 30 and 200 µm from the swarm edge, where fluid agitation is more vigorous. This water reservoir travels with the swarm, fueling its spreading. Intercellular communication is not required; cells need only grow.

Mechanism for adaptive remodeling of the bacterial flagellar switch.

The bacterial flagellar motor has been shown in previous work to adapt to changes in the steady-state concentration of the chemotaxis signaling molecule, CheY-P, by changing the FliM content. We show here that the number of FliM molecules in the motor and the fraction of FliM molecules that exchange depend on the direction of flagellar rotation, not on CheY-P binding per se. Our results are consistent with a model in which the structural differences associated with the direction of rotation modulate the strength of FliM binding. When the motor spins counterclockwise, FliM binding strengthens, the fraction of FliM molecules that exchanges decreases, and the ring content increases. The larger number of CheY-P binding sites enhances the motor's sensitivity, i.e., the motor adapts. An interesting unresolved question is how additional copies of FliM might be accommodated.

Osmotic pressure in a bacterial swarm.

Using Escherichia coli as a model organism, we studied how water is recruited by a bacterial swarm. A previous analysis of trajectories of small air bubbles revealed a stream of fluid flowing in a clockwise direction ahead of the swarm. A companion study suggested that water moves out of the agar into the swarm in a narrow region centered ∼ 30 µm from the leading edge of the swarm and then back into the agar (at a smaller rate) in a region centered ∼ 120 µm back from the leading edge. Presumably, these flows are driven by changes in osmolarity. Here, we utilized green/red fluorescent liposomes as reporters of osmolarity to verify this hypothesis. The stream of fluid that flows in front of the swarm contains osmolytes. Two distinct regions are observed inside the swarm near its leading edge: an outer high-osmolarity band (∼ 30 mOsm higher than the agar baseline) and an inner low-osmolarity band (isotonic or slightly hypotonic to the agar baseline). This profile supports the fluid-flow model derived from the drift of air bubbles and provides new (to our knowledge) insights into water maintenance in bacterial swarms. High osmotic pressure at the leading edge of the swarm extracts water from the underlying agar and promotes motility. The osmolyte is of high molecular weight and probably is lipopolysaccharide.

Microbubbles reveal chiral fluid flows in bacterial swarms.

Flagellated bacteria can swim within a thin film of fluid that coats a solid surface, such as agar; this is a means for colony expansion known as swarming. We found that micrometer-sized bubbles make excellent tracers for the motion of this fluid. The microbubbles form explosively when small aliquots of an aqueous suspension of droplets of a water-insoluble surfactant (Span 83) are placed on the agar ahead of a swarm, as the water is absorbed by the agar and the droplets are exposed to air. Using these bubbles, we discovered an extensive stream (or river) of swarm fluid flowing clockwise along the leading edge of an Escherichia coli swarm, at speeds of order 10 µm/s, about three times faster than the swarm expansion. The flow is generated by the action of counterclockwise rotating flagella of cells stuck to the substratum, which drives fluid clockwise around isolated cells (when viewed from above), counterclockwise between cells in dilute arrays, and clockwise in front of cells at the swarm edge. The river provides an avenue for long-range communication in the swarming colony, ideally suited for secretory vesicles that diffuse poorly. These findings broaden our understanding of swarming dynamics and have implications for the engineering of bacterial-driven microfluidic devices.