The ancient E-ring in bacterial flagellar motors

The bacterial flagellum is a locomotive organelle that propels the bacterial cell body in liquid environments. The flagellum is a supramolecular complex composed of about 30 different proteins and consists of at least three parts: a rotary motor, a universal joint, and a helical filament. The flagellar motor of Escherichia coli and Salmonella enterica is powered by an inward-directed electrochemical potential difference of protons across the cytoplasmic membrane. The flagellar motor consists of a rotor made of FliF, FliG, FliM and FliN and a dozen stators consisting of MotA and MotB. FliG, FliM and FliN also act as a molecular switch, enabling the motor to spin in both counterclockwise and clockwise directions. Each stator is anchored to the peptidoglycan layer through the C-terminal periplasmic domain of MotB and acts as a proton channel to couple the proton flow through the channel with torque generation. Highly conserved charged residues at the rotor–stator interface are required not only for torque generation but also for stator assembly around the rotor. In this review, we will summarize our current understanding of the structure and function of the proton-driven bacterial flagellar motor.

Bacterial flagellar motility is controlled by the binding of CheY proteins to the cytoplasmic switch complex of the flagellar motor, resulting in changes in swimming speed or direction. Despite its importance for motor function, structural information about the interaction between effector proteins and the motor are scarce. To address this gap in knowledge, we used electron cryotomography and subtomogram averaging to visualize such interactions inside Caulobacter crescentus cells. In C. crescentus, several CheY homologs regulate motor function for different aspects of the bacterial lifestyle. We used subtomogram averaging to image binding of the CheY family protein CleD to the cytoplasmic Cring switch complex, the control center of the flagellar motor. This unambiguously confirmed the orientation of the motor switch protein FliM and the binding of a member of the CheY protein family to the outside rim of the C ring. We also uncovered previously unknown structural elaborations of the alphaproteobacterial flagellar motor, including two novel periplasmic ring structures, and the stator ring harboring eleven stator units, adding to our growing catalog of bacterial flagellar diversity.

Bacterial flagellar motor (BFM) is a protein complex used for bacterial motility and chemotaxis that involves in energy transformation, torque generation and switching. FliL is a single-transmembrane protein associated with flagellar motor function. We performed biochemical and biophysical approaches to investigate the functional roles of FliL associated with stator-units. Firstly, we found the periplasmic region of FliL is crucial for its polar localization. Also, the plug mutation in stator-unit affected the polar localization of FliL implying the activation of stator-unit is important for FliL recruitment. Secondly, we applied single-molecule fluorescent microscopy to study the role of FliL in stator-unit assembly. Using molecular counting by photobleaching, we found the stoichiometry of stator-unit and FliL protein would be 1:1 in a functional motor. Moreover, the turnover time of stator-units are slightly increased in the absence of FliL. By further investigation of protein dynamics on membrane, we found the diffusions of stator-units and FliL are independent. Surprisingly, the FliL diffusion rate without stator-units is unexpectedly slow indicating a protein-complex forming event. Our results suggest that FliL plays a supporting role to the stator in the BFM.

The bacterial flagellar motor (BFM) is responsible for driving bacterial locomotion and chemotaxis, fundamental processes in pathogenesis and biofilm formation. In the BFM, torque is generated at the interface between transmembrane proteins (stators) and a rotor. It is well established that the passage of ions down a transmembrane gradient through the stator complex provides the energy for torque generation. However, the physics involved in this energy conversion remain poorly understood. Here we propose a mechanically specific model for torque generation in the BFM. In particular, we identify roles for two fundamental forces involved in torque generation: electrostatic and steric. We propose that electrostatic forces serve to position the stator, whereas steric forces comprise the actual "power stroke." Specifically, we propose that ion-induced conformational changes about a proline "hinge" residue in a stator α-helix are directly responsible for generating the power stroke. Our model predictions fit well with recent experiments on a single-stator motor. The proposed model provides a mechanical explanation for several fundamental properties of the flagellar motor, including torque-speed and speed-ion motive force relationships, backstepping, variation in step sizes, and the effects of key mutations in the stator.

Rotation of the bacterial flagellar motor is powered by a transmembrane gradient of protons or, in some species, sodium ions. The molecular mechanism of coupling between ion flow and motor rotation is not understood. The proteins most closely involved in motor rotation are MotA, MotB, and FliG. MotA and MotB are transmembrane proteins that function in transmembrane proton conduction and that are believed to form the stator. FliG is a soluble protein located on the cytoplasmic face of the rotor. Two other proteins, FliM and FliN, are known to bind to FliG and have also been suggested to be involved to some extent in torque generation. Proton (or sodium)-binding sites in the motor are likely to be important to its function and might be formed from the side chains of acidic residues. To investigate the role of acidic residues in the function of the flagellar motor, we mutated each of the conserved acidic residues in the five proteins that have been suggested to be involved in torque generation and measured the effects on motility. None of the conserved acidic residues of MotA, FliG, FliM, or FliN proved essential for torque generation. An acidic residue at position 32 of MotB did prove essential. Of 15 different substitutions studied at this position, only the conservative-replacement D32E mutant retained any function. Previous studies, together with additional data presented here, indicate that the proteins involved in motor rotation do not contain any conserved basic residues that are critical for motor rotation per se. We propose that Asp 32 of MotB functions as a proton-binding site in the bacterial flagellar motor and that no other conserved, protonatable residues function in this capacity.

Microfluidic actuators are extremely important components of microfluidic systems used in biological, chemical and biochemical analyses. We are in the process of developing hybrid microsystem devices that utilize flagellar motors in tethered Escherichia coli (E.coli) cells as actuators. In this study we describe the robustness of the flagellar motor actuators. To examine the robustness of the actuators we observe the behavior of the cells when exposed to a hydrodynamic load. With this method we are able to determine the torque generated by the flagellar motor and the adhesion force of the flagellar filament to the substrate. Using hydrodynamic loading we are also able to synchronize the rotation of multiple cells. Finally, we present data which represents the lifetime of the flagellar motor actuators.

Bacterial swimming is mostly powered by the bacterial flagellar motor and the number of proteins involved in the flagellar motor can vary. Quantifying the proteins present in flagellar motors from a range of species delivers insight into how motility has changed throughout history and provides a platform for estimating from its genome whether a species is likely to be motile. We conducted sequence and structural homology searches for 54 flagellar pathway proteins across 11 365 bacterial genomes and developed a classifier with up to 95% accuracy that could predict whether a strain was motile or not. We then mapped the evolution of flagellar motility across the Genome Taxonomy Database bacterial tree of life. We confirmed that the last common bacterial ancestor had flagellar motility and that the rate of loss of this motility was four-fold higher than the rate of gain. We showed that the presence of filament protein homologues was highly phylogenetically correlated with motility and that all species classified as motile contained at least one filament homologue. We calculated the rate of gain and loss for each flagellar protein and that the filament protein FliC was highly correlated with motility across the tree of life. We then measured the correlation of each flagellar motor protein with FliC and showed that the filament, rotor, and rod and hook proteins were all highly correlated with FliC, and thus with motility. We calculated the differential rates of gain and loss for each flagellar protein and quantified which genomes encoded for partial sets of flagellar proteins, indicating potential pathways by which motility could be lost. Overall, we show that filament, rod and hook and rotor proteins are conserved when flagellar motility is preserved and that the presence or absence of a FliC homologue is a good, simple predictor of whether or not a species has flagellar motility.

The bacterial flagellar motor is a molecular nanomachine, the assembly and regulation of which requires many accessory proteins. Their identity, structure and function are often discovered through characterisation of mutants with impaired motility. Here, we demonstrate the functional association of the Helicobacter pylori peptidoglycan-associated lipoprotein (HpPal) with the flagellar motor by analysing the motility phenotype of the ∆pal mutant, and present the results of the preliminary X-ray crystallographic analysis of its globular C-terminal domain HpPal-C. Purified HpPal-C behaved as a dimer in solution. Crystals of HpPal-C were grown by the hanging drop vapour diffusion method using medium molecular weight polyethylene glycol (PEG) Smear as the precipitating agent. The crystals belong to the primitive orthorhombic space group P1 with unit cell parameters a = 50.7, b = 63.0, c = 75.1 Å. X-ray diffraction data were collected to 1.8 Å resolution on the Australian Synchrotron beamline MX2. Calculation of the Matthews coefficient (VM=2.24 Å3/Da) and molecular replacement showed that the asymmetric unit contains two protein subunits. This study is an important step towards elucidation of the non-canonical role of H. pylori Pal in the regulation, or function of, the flagellar motor.

The bacterial flagellar motor is a complex biological rotary molecular motor which is situated in the cell envelopes of bacteria. Whereas most biological motors use adenosine triphosphate (ATP) as their energy source, the rotation of the flagellar motor is driven by a flow of charged ions across the bacterial plasma membrane. The motor powers the rotation of helical flagellar filaments at speeds of up to several hundred hertz. These rotating filaments act like propellers, pushing the cells through their environment. The motors are regulated by one of the best‐characterized biological signalling pathways, the chemotaxis pathway. This pathway changes the swimming pattern of the bacteria in response to changes in the concentration of external chemicals so that they move into environments which are optimal for their growth. Key concepts: Bacteria swim using a small biological rotary motor which is powered by the movement of charged ions across the plasma membrane. The flagellar motor consists of a rotor which rotates against stator units that are anchored to the peptidoglycan cell wall. Torque is generated by the interaction of the stator units, MotA and MotB, with FliG in the rotor. Despite the fact that the driving ions always flow in one direction through the stator units, many flagellar motors can switch between clockwise and counterclockwise rotation. A complex signalling pathway regulates the motor output in response to environmental signals ensuring that bacteria swim towards nutrient rich environments.

Most biological processes are performed by multiprotein complexes. Traditionally described as static entities, evidence is now emerging that their components can be highly dynamic, exchanging constantly with cellular pools. The bacterial flagellar motor contains approximately 13 different proteins and provides an ideal system to study functional molecular complexes. It is powered by transmembrane ion flux through a ring of stator complexes that push on a central rotor. The Escherichia coli motor switches direction stochastically in response to binding of the response regulator CheY to the rotor switch component FliM. Much is known of the static motor structure, but we are just beginning to understand the dynamics of its individual components. Here we measure the stoichiometry and turnover of FliM in functioning flagellar motors, by using high-resolution fluorescence microscopy of E. coli expressing genomically encoded YPet derivatives of FliM at physiological levels. We show that the approximately 30 FliM molecules per motor exist in two discrete populations, one tightly associated with the motor and the other undergoing stochastic turnover. This turnover of FliM molecules depends on the presence of active CheY, suggesting a potential role in the process of motor switching. In many ways the bacterial flagellar motor is as an archetype macromolecular assembly, and our results may have further implications for the functional relevance of protein turnover in other large molecular complexes.

The flagellar motor rotates bi-directionally in counter-clockwise (CCW) and clockwise (CW) directions. The motor consists of a stator and a rotor. Recent structural studies have revealed that the stator is composed of a pentameric ring of A subunits and a dimer axis of B subunits. Highly conserved charged and neighboring residues of the A subunit interacts with the rotor, generating torque through a gear-like mechanism. The rotational direction is controlled by chemotaxis signaling transmitted to the rotor, with less evidence for the stator being involved. In this study, we report novel mutations that affect the switching of the rotational direction at the putative interaction site of the stator to generate rotational force. Our results highlight an aspect of flagellar motor function that appropriate switching of the interaction states between the stator and rotor is critical for controlling the rotational direction.

A number of extracellular helical protein polymers are crucial for supporting bacterial motility. The bacterial flagellum is a polymeric appendage used to support cellular motility. Historically, structural studies of flagellar and other filaments were limited to those present as or locked into straightened states. Here, we present a robust workflow that produces biologically relevant high-resolution cryo-electron microscopy (cryo-EM) structures of bacterial flagellar filaments. We highlight how a simple purification method, centered around several centrifugation steps, exploits the process of filament ejection in Caulobacter crescentus and results in isolated filaments amenable to transmission electron microscopy (TEM) studies. The quality of the sample is validated by SDS-PAGE and negative stain TEM analysis before a sample is vitrified for cryogenic electron microscopy (cryo-EM) data collection. We provide a detailed protocol for reconstructing either straight or curved flagellar filaments by cryo-EM helical reconstruction methods, followed by an overview of model building and validation. In our hands, this workflow resulted in several flagellar structures below 3 Å resolution, with one data set reaching a global resolution of 2.1 Å. The application of this workflow supports structure-function studies to better understand the molecular interactions that regulate filament architecture in biologically relevant states. Future work will not only examine interactions that regulate bacterial flagellar and other filament organization but also provide a foundation for developing new helical biopolymers for biotech applications.Key features• Rapid high-quality purification of bacterial flagella via simple bacterial culturing, centrifugation, and resuspension methods.• High-throughput cryo-EM data collection of filamentous objects.• Use of cryoSPARC implementations of helical reconstruction algorithms to generate high-resolution 3D structures of bacterial flagella or other helical polymers.

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.

Helicobacter pylori encodes homologues of PilM, PilN and PilO from bacteria with Type IV pili, where these proteins form a pilus alignment complex. Inactivation of pilO changes H. pylori motility in semi-solid media, suggesting a link to the chemosensory pathways or flagellar motor. Here, we showed that mutation of the pilO or pilN gene in H. pylori strain SS1 reduced the mean linear swimming speed in liquid media, implicating PilO and PilN in the function, or regulation of, the flagellar motor. We also demonstrated that the soluble variants of H. pylori PilN and PilO share common biochemical properties with their Type IV pili counterparts which suggests their adapted function in the bacterial flagellar motor may be similar to that in the Type IV pili.

The bacterial flagellar motor, one of the few rotary motors in nature, produces torque to drive the flagellar filament by ion translocation through membrane-bound stator complexes. We used the light-driven proton pump proteorhodopsin (pR) to control the proton-motive force (PMF) in vivo by illumination. pR excitation was shown to be sufficient to replace native PMF generation, and when excited in cells with intact native PMF generation systems increased motor speed beyond the physiological norm. We characterized the effects of rapid in vivo PMF changes on the flagellar motor. Transient PMF disruption events from loss of illumination caused motors to stop, with rapid recovery of their previous rotation rate after return of illumination. However, extended periods of PMF loss led to stepwise increases in rotation rate upon PMF return as stators returned to the motor. The rate constant for stator binding to a putative single binding site on the motor was calculated to be 0.06 s(-1). Using GFP-tagged MotB stator proteins, we found that transient PMF disruption leads to reversible stator diffusion away from the flagellar motor, showing that PMF presence is necessary for continued motor integrity, and calculated a stator dissociation rate of 0.038 s(-1).