Effect of fluid elasticity on the emergence of oscillations in an active elastic filament.

Many microorganisms propel themselves through complex media by deforming their flagella. The beat is thought to emerge from interactions between forces of the surrounding fluid, the passive elastic response from deformations of the flagellum and active forces from internal molecular motors. The beat varies in response to changes in the fluid rheology, including elasticity, but there are limited data on how systematic changes in elasticity alter the beat. This work analyses a related problem with fixed-strength driving force: the emergence of beating of an elastic planar filament driven by a follower force at the tip of a viscoelastic fluid. This analysis examines how the onset of oscillations depends on the strength of the force and viscoelastic parameters. Compared to a Newtonian fluid, it takes more force to induce the instability in viscoelastic fluids, and the frequency of the oscillation is higher. The linear analysis predicts that the frequency increases with the fluid relaxation time. Using numerical simulations, the model predictions are compared with experimental data on frequency changes in the bi-flagellated alga Chlamydomonas reinhardtii. The model shows the same trends in response to changes in both fluid viscosity and Deborah number and thus provides a possible mechanistic understanding of the experimental observations.

The younger flagellum sets the beat for Chlamydomonas reinhardtii.

Eukaryotes swim with coordinated flagellar (ciliary) beating and steer by fine-tuning the coordination. The model organism for studying flagellate motility, Chlamydomonas reinhardtii, employs synchronous, breaststroke-like flagellar beating to swim, and it modulates the beating amplitudes differentially to steer. This strategy hinges on both inherent flagellar asymmetries (e.g. different response to chemical messengers) and such asymmetries being effectively coordinated in the synchronous beating. In C. reinhardtii, the synchrony of beating is known to be supported by a mechanical connection between flagella; however, how flagellar asymmetries persist in the synchrony remains elusive. For example, it has been speculated for decades that one flagellum leads the beating, as its dynamic properties (i.e. frequency, waveform, etc.) appear to be copied by the other one. In this study, we combine experiments, computations, and modeling efforts to elucidate the roles played by each flagellum in synchronous beating. With a non-invasive technique to selectively load each flagellum, we show that the coordinated beating essentially only responds to load exerted on the cis flagellum; and that such asymmetry in response derives from a unilateral coupling between the two flagella. Our results highlight a distinct role for each flagellum in coordination and have implication for biflagellates' tactic behaviors.

Many single-cell organisms use tiny hair-like structures called flagella to move around. To direct this movement, the flagella must work together and beat in a synchronous manner. In some organisms, coordination is achieved by each flagellum reacting to the flow generated by neighbouring flagella. In others, flagella are joined together by fiber connections between their bases, which allow movement to be coordinated through mechanical signals sent between flagella. One such organism is Chlamydomonas reinhardtii, a type of algae frequently used to study flagellar coordination. Its two flagella ­ named trans and cis because of their positions relative to the cell's eyespot ­ propel the cell through water using breaststroke-like movements. To steer, C. reinhardtii adjusts the strength of the strokes made by each flagellum. Despite this asymmetry, the flagella must continue to beat in synchrony to move efficiently. To understand how the cell manages these differences, Wei et al. exposed each flagellum to carefully generated oscillations in water so that each was exposed to different forces and their separate responses could be measured. A combination of experiments, modelling and computer simulations were then used to work out how the two flagella coordinate to steer the cell. Wei et al. found that only the cis flagellum coordinates the beating, with the trans flagellum simply copying the motion of the cis. A direct consequence of such one-way coupling is that only forces on the cis flagellum influence the coordinated beating dynamics of both flagella. These findings shed light on the unique roles of each flagellum in the coordinated movement in C. reinhardtii and have implications for how other organisms with mechanically-connected flagella navigate their environments.

CryoEM structures reveal how the bacterial flagellum rotates and switches direction.

Bacterial chemotaxis requires bidirectional flagellar rotation at different rates. Rotation is driven by a flagellar motor, which is a supercomplex containing multiple rings. Architectural uncertainty regarding the cytoplasmic C-ring, or 'switch', limits our understanding of how the motor transmits torque and direction to the flagellar rod. Here we report cryogenic electron microscopy structures for Salmonella enterica serovar typhimurium inner membrane MS-ring and C-ring in a counterclockwise pose (4.0 Å) and isolated C-ring in a clockwise pose alone (4.6 Å) and bound to a regulator (5.9 Å). Conformational differences between rotational poses include a 180° shift in FliF/FliG domains that rotates the outward-facing MotA/B binding site to inward facing. The regulator has specificity for the clockwise pose by bridging elements unique to this conformation. We used these structures to propose how the switch reverses rotation and transmits torque to the flagellum, which advances the understanding of bacterial chemotaxis and bidirectional motor rotation.

Ecological relevance of flagellar motility in soil bacterial communities.

Flagellar motility is a key bacterial trait as it allows bacteria to navigate their immediate surroundings. Not all bacteria are capable of flagellar motility, and the distribution of this trait, its ecological associations, and the life history strategies of flagellated taxa remain poorly characterized. We developed and validated a genome-based approach to infer the potential for flagellar motility across 12 bacterial phyla (26 192 unique genomes). The capacity for flagellar motility was associated with a higher prevalence of genes for carbohydrate metabolism and higher maximum potential growth rates, suggesting that flagellar motility is more prevalent in environments with higher carbon availability. To test this hypothesis, we applied a method to infer the prevalence of flagellar motility in whole bacterial communities from metagenomic data and quantified the prevalence of flagellar motility across four independent field studies that each captured putative gradients in soil carbon availability (148 metagenomes). We observed a positive relationship between the prevalence of bacterial flagellar motility and soil carbon availability in all datasets. Since soil carbon availability is often correlated with other factors that could influence the prevalence of flagellar motility, we validated these observations using metagenomic data from a soil incubation experiment where carbon availability was directly manipulated with glucose amendments. This confirmed that the prevalence of bacterial flagellar motility is consistently associated with soil carbon availability over other potential confounding factors. This work highlights the value of combining predictive genomic and metagenomic approaches to expand our understanding of microbial phenotypic traits and reveal their general environmental associations.

It's not all about flagella - sticky invasion by pathogenic spirochetes.

Pathogenic spirochetes cause a range of serious human diseases such as Lyme disease (LD), syphilis, leptospirosis, relapsing fever (RF), and periodontal disease. Motility is a critical virulence factor for spirochetes. From the mechanical perspective of the infection, it has been widely believed that flagella are the sole key players governing the migration and dissemination of these pathogens in the host. Here, we highlight the important contribution of spirochetal surface-exposed adhesive molecules and their dynamic interactions with host molecules in the process of infection, specifically in spirochetal swimming and crawling migration. We believe that these recent findings overturn the prevailing view depicting the spirochetal body to be just an inert elastic bag, which does not affect spirochetal cell locomotion.

Bacterial flagella hijack type IV pili proteins to control motility.

Bacterial flagella and type IV pili (TFP) are surface appendages that enable motility and mechanosensing through distinct mechanisms. These structures were previously thought to have no components in common. Here, we report that TFP and some flagella share proteins PilO, PilN, and PilM, which we identified as part of the Helicobacter pylori flagellar motor. H. pylori mutants lacking PilO or PilN migrated better than wild type in semisolid agar because they continued swimming rather than aggregated into microcolonies, mimicking the TFP-regulated surface response. Like their TFP homologs, flagellar PilO/PilN heterodimers formed a peripheral cage that encircled the flagellar motor. These results indicate that PilO and PilN act similarly in flagella and TFP by differentially regulating motility and microcolony formation when bacteria encounter surfaces.

Viscosity-dependent determinants of Campylobacter jejuni impacting the velocity of flagellar motility.

IMPORTANCE: Bacteria can adapt flagellar motor output in response to the load that the extracellular milieu imparts on the flagellar filament to enable propulsion. Bacteria can adapt flagellar motor output in response to the load that the extracellular milieu imparts on the flagellar filament to enable propulsion through diverse environments. These changes may involve increasing power and torque in high-viscosity environments or reducing power and flagellar rotation upon contact with a surface. C. jejuni swimming velocity in low-viscosity environments is comparable to other bacterial flagellates and increases significantly as external viscosity increases. In this work, we provide evidence that the mechanics of the C. jejuni flagellar motor has evolved to naturally promote high swimming velocity in high-viscosity environments. We found that C. jejuni produces VidA and VidB as auxiliary proteins to specifically affect flagellar motor activity in low viscosity to reduce swimming velocity. Our findings provide some of the first insights into different mechanisms that exist in bacteria to alter the mechanics of a flagellar motor, depending on the viscosity of extracellular environments.

Flagellar dynamics reveal fluctuations and kinetic limit in the Escherichia coli chemotaxis network.

The Escherichia coli chemotaxis network, by which bacteria modulate their random run/tumble swimming pattern to navigate their environment, must cope with unavoidable number fluctuations ("noise") in its molecular constituents like other signaling networks. The probability of clockwise (CW) flagellar rotation, or CW bias, is a measure of the chemotaxis network's output, and its temporal fluctuations provide a proxy for network noise. Here we quantify fluctuations in the chemotaxis signaling network from the switching statistics of flagella, observed using time-resolved fluorescence microscopy of individual optically trapped E. coli cells. This approach allows noise to be quantified across the dynamic range of the network. Large CW bias fluctuations are revealed at steady state, which may play a critical role in driving flagellar switching and cell tumbling. When the network is stimulated chemically to higher activity, fluctuations dramatically decrease. A stochastic theoretical model, inspired by work on gene expression noise, points to CheY activation occurring in bursts, driving CW bias fluctuations. This model also shows that an intrinsic kinetic ceiling on network activity places an upper limit on activated CheY and CW bias, which when encountered suppresses network fluctuations. This limit may also prevent cells from tumbling unproductively in steep gradients.

Multiflagellarity leads to the size-independent swimming speed of peritrichous bacteria.

To swim through a viscous fluid, a flagellated bacterium must overcome the fluid drag on its body by rotating a flagellum or a bundle of multiple flagella. Because the drag increases with the size of bacteria, it is expected theoretically that the swimming speed of a bacterium inversely correlates with its body length. Nevertheless, despite extensive research, the fundamental size-speed relation of flagellated bacteria remains unclear with different experiments reporting conflicting results. Here, by critically reviewing the existing evidence and synergizing our own experiments of large sample sizes, hydrodynamic modeling, and simulations, we demonstrate that the average swimming speed of Escherichia coli, a premier model of peritrichous bacteria, is independent of their body length. Our quantitative analysis shows that such a counterintuitive relation is the consequence of the collective flagellar dynamics dictated by the linear correlation between the body length and the number of flagella of bacteria. Notably, our study reveals how bacteria utilize the increasing number of flagella to regulate the flagellar motor load. The collective load sharing among multiple flagella results in a lower load on each flagellar motor and therefore faster flagellar rotation, which compensates for the higher fluid drag on the longer bodies of bacteria. Without this balancing mechanism, the swimming speed of monotrichous bacteria generically decreases with increasing body length, a feature limiting the size variation of the bacteria. Altogether, our study resolves a long-standing controversy over the size-speed relation of flagellated bacteria and provides insights into the functional benefit of multiflagellarity in bacteria.

Flagellar motors of swimming bacteria contain an incomplete set of stator units to ensure robust motility.

Flagellated bacteria, like Escherichia coli, swim by rotating helical flagellar filaments powered by rotary flagellar motors at their base. Motor dynamics are sensitive to the load it drives. It was previously thought that motor load was high when driving filament rotation in free liquid environments. However, torque measurements from swimming bacteria revealed substantially lower values compared to single-motor studies. We addressed this inconsistency through motor resurrection experiments, abruptly attaching a 1-micrometer-diameter bead to the filament to ensure high load. Unexpectedly, we found that the motor works with only half the complement of stator units when driving filament rotation. This suggests that the motor is not under high load during bacterial swimming, which we confirmed by measuring the torque-speed relationship by varying media viscosity. Therefore, the motor operates in an intermediate-load region, adaptively regulating its stator number on the basis of external load conditions. This ensures the robustness of bacterial motility when swimming in diverse load conditions and varying flagella numbers.

Regulation of major bacterial survival strategies by transcripts sequestration in a membraneless organelle.

TmaR, the only known pole-localizer protein in Escherichia coli, was shown to cluster at the cell poles and control localization and activity of the major sugar regulator in a tyrosine phosphorylation-dependent manner. Here, we show that TmaR assembles by phase separation (PS) via heterotypic interactions with RNA in vivo and in vitro. An unbiased automated mutant screen combined with directed mutagenesis and genetic manipulations uncovered the importance of a predicted nucleic-acid-binding domain, a disordered region, and charged patches, one containing the phosphorylated tyrosine, for TmaR condensation. We demonstrate that, by protecting flagella-related transcripts, TmaR controls flagella production and, thus, cell motility and biofilm formation. These results connect PS in bacteria to survival and provide an explanation for the linkage between metabolism and motility. Intriguingly, a point mutation or increase in its cellular concentration induces irreversible liquid-to-solid transition of TmaR, similar to human disease-causing proteins, which affects cell morphology and division.

Precise Measurement of the Stoichiometry of the Adaptive Bacterial Flagellar Switch.

The cytoplasmic ring (C-ring) of the bacterial flagellar motor controls the motor rotation direction, thereby controlling bacterial run-and-tumble behavior. The C-ring has been shown to undergo adaptive remodeling in response to changes in motor directional bias. However, the stoichiometry and arrangement of the C-ring is still unclear due to contradiction between the results from fluorescence studies and cryo-electron microscopy (cryo-EM) structural analysis. Here, by using the copy number of FliG molecules (34) in the C-ring as a reference, we precisely measured the copy numbers of FliM molecules in motors rotating exclusively counterclockwise (CCW) and clockwise (CW). We surprisingly found that there are on average 45 and 58 FliM molecules in CW and CCW rotating motors, respectively, which are much higher than previous estimates. Our results suggested a new mechanism of C-ring adaptation, that is, extra FliM molecules could be bound to the primary C-ring with probability depending on the motor rotational direction. We further confirmed that all of the FliM molecules in the C-ring function in chemotaxis signaling transduction because all of them could be bound by the chemotactic response regulator CheY-P. Our measurements provided new insights into the structure and arrangement of the flagellar switch. IMPORTANCE The bacterial flagellar switch can undergo adaptive remodeling in response to changes in motor rotation direction, thereby shifting its operating point to match the output of the chemotaxis signaling pathway. However, it remains unclear how the flagellar switch accomplishes this adaptive remodeling. Here, via precise fluorescence studies, we measured the absolute copy numbers of the critical component in the switch for motors rotating counterclockwise and clockwise, obtaining much larger numbers than previous relative estimates. Our results suggested a new mechanism of adaptive remodeling of the flagellar switch and provided new insights for updating the conformation spread model of the switch.

FliL Functions in Diverse Microbes to Negatively Modulate Motor Output via Its N-Terminal Region.

The flagellar motor protein FliL is conserved across many microbes, but its exact role has been obscured by varying fliL mutant phenotypes. We reanalyzed results from fliL studies and found they utilized alleles that differed in the amount of N- and C-terminal regions that were retained. Alleles that retain the N-terminal cytoplasmic and transmembrane helix (TM) regions in the absence of the C-terminal periplasmic domain result in loss of motility, while alleles that completely lack the N-terminal region, independent of the periplasmic domain, retain motility. We then tested this prediction in Helicobacter pylori fliL and found support for the idea. This analysis suggests that FliL function may be more conserved across bacteria than previously thought, that it is not essential for motility, and that the N-terminal region has the negative ability to regulate motor function. IMPORTANCE FliL is a protein found in the flagellar motor of bacteria, but what it does was not clear. To study FliL function, scientists often remove it and see what happens. Loss of FliL was thought to have different effects depending on the microbe. We uncovered, however, that part of the confusion arose because scientists inadvertently removed different parts of the protein. Our analysis and data suggest that leaving the N-terminal regions blocks motility, while fully removing FliL allows normal motility. This finding will help scientists understand FliL because it clarifies what needs to be removed to fully eliminate the protein, and also that the N-terminal region can block motility.

Absence of CEP78 causes photoreceptor and sperm flagella impairments in mice and a human individual.

Cone-rod dystrophy (CRD) is a genetically inherited retinal disease that can be associated with male infertility, while the specific genetic mechanisms are not well known. Here, we report CEP78 as a causative gene of a particular syndrome including CRD and male infertility with multiple morphological abnormalities of sperm flagella (MMAF) both in human and mouse. Cep78 knockout mice exhibited impaired function and morphology of photoreceptors, typified by reduced ERG amplitudes, disrupted translocation of cone arrestin, attenuated and disorganized photoreceptor outer segments (OS) disks and widen OS bases, as well as interrupted connecting cilia elongation and abnormal structures. Cep78 deletion also caused male infertility and MMAF, with disordered '9+2' structure and triplet microtubules in sperm flagella. Intraflagellar transport (IFT) proteins IFT20 and TTC21A are identified as interacting proteins of CEP78. Furthermore, CEP78 regulated the interaction, stability, and centriolar localization of its interacting protein. Insufficiency of CEP78 or its interacting protein causes abnormal centriole elongation and cilia shortening. Absence of CEP78 protein in human caused similar phenotypes in vision and MMAF as Cep78-/- mice. Collectively, our study supports the important roles of CEP78 defects in centriole and ciliary dysfunctions and molecular pathogenesis of such multi-system syndrome.

Swarming Motility Assays in Salmonella.

Salmonella enterica has six subspecies, of which the subspecies enterica is the most important for human health. The dispersal and infectivity of this species are dependent upon flagella-driven motility. Two kinds of flagella-mediated movements have been described-swimming individually in bulk liquid and swarming collectively over a surface substrate. During swarming, the bacteria acquire a distinct physiology, the most significant consequence of which is acquisition of adaptive resistance to antibiotics. Described here are protocols to cultivate, verify, and study swimming and swarming motility in S. enterica, and an additional "border-crossing" assay, where cells "primed" to swarm are presented with an environmental challenge such as antibiotics to assess their propensity to handle the challenge.

Testing the ion-current model for flagellar length sensing and IFT regulation.

Eukaryotic cilia and flagella are microtubule-based organelles whose relatively simple shape makes them ideal for investigating the fundamental question of organelle size regulation. Most of the flagellar materials are transported from the cell body via an active transport process called intraflagellar transport (IFT). The rate of IFT entry into flagella, known as IFT injection, has been shown to negatively correlate with flagellar length. However, it remains unknown how the cell measures the length of its flagella and controls IFT injection. One of the most-discussed theoretical models for length sensing to control IFT is the ion-current model, which posits that there is a uniform distribution of Ca2+ channels along the flagellum and that the Ca2+ current from the flagellum into the cell body increases linearly with flagellar length. In this model, the cell uses the Ca2+ current to negatively regulate IFT injection. The recent discovery that IFT entry into flagella is regulated by the phosphorylation of kinesin through a calcium-dependent protein kinase has provided further impetus for the ion-current model. To test this model, we measured and manipulated the levels of Ca2+ inside of Chlamydomonas flagella and quantified IFT injection. Although the concentration of Ca2+ inside of flagella was weakly correlated with the length of flagella, we found that IFT injection was reduced in calcium-deficient flagella, rather than increased as the model predicted, and that variation in IFT injection was uncorrelated with the occurrence of flagellar Ca2+ spikes. Thus, Ca2+ does not appear to function as a negative regulator of IFT injection, hence it cannot form the basis of a stable length control system.

Steady-state running rate sets the speed and accuracy of accumulation of swimming bacteria.

We study the chemotaxis of a population of genetically identical swimming bacteria undergoing run and tumble dynamics driven by stochastic switching between clockwise and counterclockwise rotation of the flagellar rotary system, where the steady-state rate of the switching changes in different environments. Understanding chemotaxis quantitatively requires that one links the measured steady-state switching rates of the rotary system, as well as the directional changes of individual swimming bacteria in a gradient of chemoattractant/repellant, to the efficiency of a population of bacteria in moving up/down the gradient. Here we achieve this by using a probabilistic model, parametrized with our experimental data, and show that the response of a population to the gradient is complex. We find the changes to the steady-state switching rate in the absence of gradients affect the average speed of the swimming bacterial population response as well as the width of the distribution. Both must be taken into account when optimizing the overall response of the population in complex environments.

Conversion of anterograde into retrograde trains is an intrinsic property of intraflagellar transport.

Cilia or eukaryotic flagella are microtubule-based organelles found across the eukaryotic tree of life. Their very high aspect ratio and crowded interior are unfavorable to diffusive transport of most components required for their assembly and maintenance. Instead, a system of intraflagellar transport (IFT) trains moves cargo rapidly up and down the cilium (Figure 1A).1-3 Anterograde IFT, from the cell body to the ciliary tip, is driven by kinesin-II motors, whereas retrograde IFT is powered by cytoplasmic dynein-1b motors.4 Both motors are associated with long chains of IFT protein complexes, known as IFT trains, and their cargoes.5-8 The conversion from anterograde to retrograde motility at the ciliary tip involves (1) the dissociation of kinesin motors from trains,9 (2) a fundamental restructuring of the train from the anterograde to the retrograde architecture,8,10,11 (3) the unloading and reloading of cargo,2 and (4) the activation of the dynein motors.8,12 A prominent hypothesis is that there is dedicated calcium-dependent protein-based machinery at the ciliary tip to mediate these processes.4,13 However, the mechanisms of IFT turnaround have remained elusive. In this study, we use mechanical and chemical methods to block IFT at intermediate positions along the cilia of the green algae Chlamydomonas reinhardtii, in normal and calcium-depleted conditions. We show that IFT turnaround, kinesin dissociation, and dynein-1b activation can consistently be induced at arbitrary distances from the ciliary tip, with no stationary tip machinery being required. Instead, we demonstrate that the anterograde-to-retrograde conversion is a calcium-independent intrinsic ability of IFT.

Encapsulated bacteria deform lipid vesicles into flagellated swimmers.

We study a synthetic system of motile Escherichia coli bacteria encapsulated inside giant lipid vesicles. Forces exerted by the bacteria on the inner side of the membrane are sufficient to extrude membrane tubes filled with one or several bacteria. We show that a physical coupling between the membrane tube and the flagella of the enclosed cells transforms the tube into an effective helical flagellum propelling the vesicle. We develop a simple theoretical model to estimate the propulsive force from the speed of the vesicles and demonstrate the good efficiency of this coupling mechanism. Together, these results point to design principles for conferring motility to synthetic cells.

Structural and biochemical analyses of the flagellar expression regulator DegU from Listeria monocytogenes.

Listeria monocytogenes is a pathogenic bacterium that produces flagella, the locomotory organelles, in a temperature-dependent manner. At 37 °C inside humans, L. monocytogenes employs MogR to repress the expression of flagellar proteins, thereby preventing the production of flagella. However, in the low-temperature environment outside of the host, the antirepressor GmaR inactivates MogR, allowing flagellar formation. Additionally, DegU is necessary for flagellar expression at low temperatures. DegU transcriptionally activates the expression of GmaR and flagellar proteins by binding the operator DNA in the fliN-gmaR promoter as a response regulator of a two-component regulatory system. To determine the DegU-mediated regulation mechanism, we performed structural and biochemical analyses on the recognition of operator DNA by DegU. The DegU-DNA interaction is primarily mediated by a C-terminal DNA-binding domain (DBD) and can be fortified by an N-terminal receiver domain (RD). The DegU DBD adopts a tetrahelical helix-turn-helix structure and assembles into a dimer. The DegU DBD dimer recognizes the operator DNA using a positive patch. Unexpectedly, unlike typical response regulators, DegU interacts with operator DNA in both unphosphorylated and phosphorylated states with similar binding affinities. Therefore, we conclude that DegU is a noncanonical response regulator that is constitutively active irrespective of phosphorylation.