Swimming ability and flagellar motility of sperm packets of the volvocine green alga Pleodorina starrii.

Eukaryotic flagella collectively form metachronal waves that facilitate the ability to cause flow or swim. Among such flagellated and planktonic swimmers, large volvocine genera such as Eudorina, Pleodorina and Volvox form bundles of small male gametes (sperm) called "sperm packets" for sexual reproduction. Although these sperm packets reportedly have flagella and the ability to swim, previous studies on volvocine motility have focused on asexual forms and the swimming characteristics of sperm packets remain unknown. However, it is important to quantify the motility of sperm packets and sperm in order to gain insights into the significance of motility in the sexual reproduction of planktonic algae. In this study, we quantitatively described the behavior of three flagellated forms of a male strain of Pleodorina starrii-asexual colonies, sperm packets, and single dissociated sperm-with emphasis on comparison of the two multicellular forms. Despite being smaller, sperm packets swam approximately 1.4 times faster than the asexual colonies of the same male strain. Body length was approximately 0.5 times smaller in the sperm packets than in asexual colonies. The flagella from sperm packets and asexual colonies showed asymmetric waveforms, whereas those from dissociated single sperm showed symmetric waveforms, suggesting the presence of a switching mechanism between sperm packets and dissociated sperm. Flagella from sperm packets were approximately 0.5 times shorter and had a beat period approximately twice as long as those from asexual colonies. The flagella of sperm packets were densely distributed over the anterior part of the body, whereas the flagella of asexual colonies were sparse and evenly distributed. The distribution of flagella, but not the number of flagella, appear to illustrate a significant difference in the speeds of sperm packets and asexual colonies. Our findings reveal novel aspects of the regulation of eukaryotic flagella and shed light on the role of flagellar motility in sexual reproduction of planktonic algae.

Development of functional spermatozoa in mammalian spermiogenesis.

Infertility is a global health problem affecting one in six couples, with 50% of cases attributed to male infertility. Spermatozoa are male gametes, specialized cells that can be divided into two parts: the head and the flagellum. The head contains a vesicle called the acrosome that undergoes exocytosis and the flagellum is a motility apparatus that propels the spermatozoa forward and can be divided into two components, axonemes and accessory structures. For spermatozoa to fertilize oocytes, the acrosome and flagellum must be formed correctly. In this Review, we describe comprehensively how functional spermatozoa develop in mammals during spermiogenesis, including the formation of acrosomes, axonemes and accessory structures by focusing on analyses of mouse models.

Calaxin is a key factor for calcium-dependent waveform control in zebrafish sperm.

Calcium is critical for regulating the waveform of motile cilia and flagella. Calaxin is currently the only known molecule involved in the calcium-dependent regulation in ascidians. We have recently shown that Calaxin stabilizes outer arm dynein (OAD), and the knockout of Calaxin results in primary ciliary dyskinesia phenotypes in vertebrates. However, from the knockout experiments, it was not clear which functions depend on calcium and how Calaxin regulates the waveform. To address this question, here, we generated transgenic zebrafish expressing a mutant E130A-Calaxin deficient in calcium binding. E130A-Calaxin restored the OAD reduction of calaxin -/- sperm and the abnormal movement of calaxin -/- left-right organizer cilia, showing that Calaxin's stabilization of OADs is calcium-independent. In contrast, our quantitative analysis of E130A-Calaxin sperms showed that the calcium-induced asymmetric beating was not restored, linking Calaxin's calcium-binding ability with an asymmetric flagellar beating for the first time. Our data show that Calaxin is a calcium-dependent regulator of the ciliary beating and a calcium-independent OAD stabilizer.

Influence of bacterial swimming and hydrodynamics on attachment of phages.

Bacteriophages ("phages") are viruses that infect bacteria. Since they do not actively self-propel, phages rely on thermal diffusion to find target cells-but can also be advected by fluid flows, such as those generated by motile bacteria themselves in bulk fluids. How does the flow field generated by a swimming bacterium influence how it encounters phages? Here, we address this question using coupled molecular dynamics and lattice Boltzmann simulations of flagellated bacteria swimming through a bulk fluid containing uniformly-dispersed phages. We find that while swimming increases the rate at which phages attach to both the cell body and flagellar propeller, hydrodynamic interactions strongly suppress this increase at the cell body, but conversely enhance this increase at the flagellar bundle. Our results highlight the pivotal influence of hydrodynamics on the interactions between bacteria and phages, as well as other diffusible species, in microbial environments.

Multiflagellate Swimming Controlled by Hydrodynamic Interactions.

Many eukaryotic microorganisms propelled by multiple flagella can swim very rapidly with distinct gaits. Here, we model a three-dimensional mutiflagellate swimmer, resembling the microalgae. When the flagella are actuated synchronously, the swimming efficiency can be enhanced or reduced by interflagella hydrodynamic interactions (HIs), determined by the intrinsic tilting angle of the flagella. The asynchronous gait with a phase difference between neighboring flagella can reduce oscillatory motion via the basal mechanical coupling. In the presence of a spherical body, simulations taking into account the flagella-body interactions reveal the advantage of anterior configuration compared with posterior configuration, where in the latter case an optimal flagella number arises. Apart from understanding the role of HIs in the multiflagellate microorganisms, this work could also guide laboratory fabrications of novel microswimmers.

Foraging mechanisms in excavate flagellates shed light on the functional ecology of early eukaryotes.

The phagotrophic flagellates described as "typical excavates" have been hypothesized to be morphologically similar to the Last Eukaryotic Common Ancestor and understanding the functional ecology of excavates may therefore help shed light on the ecology of these early eukaryotes. Typical excavates are characterized by a posterior flagellum equipped with a vane that beats in a ventral groove. Here, we combined flow visualization and observations of prey capture in representatives of the three clades of excavates with computational fluid dynamic modeling, to understand the functional significance of this cell architecture. We record substantial differences amongst species in the orientation of the vane and the beat plane of the posterior flagellum. Clearance rate magnitudes estimated from flow visualization and modeling are both like that of other similarly sized flagellates. The interaction between a vaned flagellum beating in a confinement is modeled to produce a very efficient feeding current at low energy costs, irrespective of the beat plane and vane orientation and of all other morphological variations. Given this predicted uniformity of function, we suggest that the foraging systems of typical excavates studied here may be good proxies to understand those potentially used by our distant ancestors more than 1 billion years ago.

The Ugd, a capsular polysaccharide synthesis protein, regulates the bacterial motility in Vibrio alginolyticus.

Vibrio alginolyticus is one of the most common opportunistic pathogens in marine animals and humans. In this study, A transposon mutation library of the V. alginolyticus E110 was used to identify motility-related genes, and we found three flagellar and one capsular polysaccharide (CPS) synthesis-related genes were linked to swarming motility. Then, gene deletion and complementation further confirmed that CPS synthesis-related gene ugd is involved in the swarming motility of V. alginolyticus. Phenotype assays showed that the Δugd mutant reduced CPS production, decreased biofilm formation, impaired swimming ability, and increased cytotoxicity compared to the wild-type strain. Transcriptome analysis showed that 655 genes (15%) were upregulated and 914 genes (21%) were downregulated in the Δugd strain. KEGG pathway and heatmap analysis revealed that genes involved in two-component systems (TCSs), chemotaxis, and flagella assembly pathways were downregulated in the Δugd mutant. On the other hand, genes involved in pathways of human diseases, biosynthesis ABC transporters, and metabolism were upregulated in the Δugd mutant. The RT-qPCR further validated that ugd-regulated genes are associated with motility, biofilm formation, virulence, and TCSs. These findings imply that ugd may be an important player in the control of some physiological processes in V. alginolyticus, highlighting its potential as a target for future research and potential therapeutic interventions.

Counterclockwise rotation of the flagellum promotes biofilm initiation in Helicobacter pylori.

Motility promotes biofilm initiation during the early steps of this process: microbial surface association and attachment. Motility is controlled in part by chemotaxis signaling, so it seems reasonable that chemotaxis may also affect biofilm formation. There is a gap, however, in our understanding of the interactions between chemotaxis and biofilm formation, partly because most studies analyzed the phenotype of only a single chemotaxis signaling mutant, e.g., cheA. Here, we addressed the role of chemotaxis in biofilm formation using a full set of chemotaxis signaling mutants in Helicobacter pylori, a class I carcinogen that infects more than half the world's population and forms biofilms. Using mutants that lack each chemotaxis signaling protein, we found that chemotaxis signaling affected the biofilm initiation stage, but not mature biofilm formation. Surprisingly, some chemotaxis mutants elevated biofilm initiation, while others inhibited it in a manner that was not tied to chemotaxis ability or ligand input. Instead, the biofilm phenotype correlated with flagellar rotational bias. Specifically, mutants with a counterclockwise bias promoted biofilm initiation, e.g., ∆cheA, ∆cheW, or ∆cheV1; in contrast, those with a clockwise bias inhibited it, e.g., ∆cheZ, ∆chePep, or ∆cheV3. We tested this correlation using a counterclockwise bias-locked flagellum, which induced biofilm formation independent of the chemotaxis system. These CCW flagella, however, were not sufficient to induce biofilm formation, suggesting there are downstream players. Overall, our work highlights the new finding that flagellar rotational direction promotes biofilm initiation, with the chemotaxis signaling system operating as one mechanism to control flagellar rotation. IMPORTANCE: Chemotaxis signaling systems have been reported to contribute to biofilm formation in many bacteria; however, how they regulate biofilm formation remains largely unknown. Chemotaxis systems are composed of many distinct kinds of proteins, but most previous work analyzed the biofilm effect of loss of only a few. Here, we explored chemotaxis' role during biofilm formation in the human-associated pathogenic bacterium Helicobacter pylori. We found that chemotaxis proteins are involved in biofilm initiation in a manner that correlated with how they affected flagellar rotation. Biofilm initiation was high in mutants with counterclockwise (CCW) flagellar bias and low in those with clockwise bias. We supported the idea that a major driver of biofilm formation is flagellar rotational direction using a CCW-locked flagellar mutant, which stays CCW independent of chemotaxis input and showed elevated biofilm initiation. Our data suggest that CCW-rotating flagella, independent of chemotaxis inputs, are a biofilm-promoting signal.

Spatiotemporal dynamics of the proton motive force on single bacterial cells.

Electrochemical gradients across biological membranes are vital for cellular bioenergetics. In bacteria, the proton motive force (PMF) drives essential processes like adenosine triphosphate production and motility. Traditionally viewed as temporally and spatially stable, recent research reveals a dynamic PMF behavior at both single-cell and community levels. Moreover, the observed lateral segregation of respiratory complexes could suggest a spatial heterogeneity of the PMF. Using a light-activated proton pump and detecting the activity of the bacterial flagellar motor, we perturb and probe the PMF of single cells. Spatially homogeneous PMF perturbations reveal millisecond-scale temporal dynamics and an asymmetrical capacitive response. Localized perturbations show a rapid lateral PMF homogenization, faster than proton diffusion, akin to the electrotonic potential spread observed in passive neurons, explained by cable theory. These observations imply a global coupling between PMF sources and consumers along the membrane, precluding sustained PMF spatial heterogeneity but allowing for rapid temporal changes.

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.

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.

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.

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.

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.

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.