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 lack of Tex44 causes severe subfertility with flagellar abnormalities in male mice.

By analyzing a mouse Interspecific Recombinant Congenic Strain (IRCS), we previously identified a quantitative trait locus (QTL), called Mafq1 on mouse chromosome 1, that is associated with male hypofertility and ultrastructural sperm abnormalities. Within this locus, we identified a new candidate gene that could be implicated in a reproductive phenotype: Tex44 (Testis-expressed protein 44). We thus performed a CRISPR/Cas9-mediated complete deletion of this gene in mice in order to study its function. Tex44-KO males were severely hypofertile in vivo and in vitro due to a drastic reduction of sperm motility which itself resulted from important morphological sperm abnormalities. Namely, Tex44-KO sperm showed a disorganized junction between the midpiece and the principal piece of the flagellum, leading to a 180° flagellar bending in this region. In addition, the loss of some axonemal microtubule doublets and outer dense fibers in the flagellum's principal piece has been observed. Our results suggest that, in mice, TEX44 is implicated in the correct set-up of the sperm flagellum during spermiogenesis and its absence leads to flagellar abnormalities and consequently to severe male hypofertility.

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.

IFT cargo and motors associate sequentially with IFT trains to enter cilia of C. elegans.

Intraflagellar transport (IFT) orchestrates entry of proteins into primary cilia. At the ciliary base, assembled IFT trains, driven by kinesin-2 motors, can transport cargo proteins into the cilium, across the crowded transition zone. How trains assemble at the base and how proteins associate with them is far from understood. Here, we use single-molecule imaging in the cilia of C. elegans chemosensory neurons to directly visualize the entry of kinesin-2 motors, kinesin-II and OSM-3, as well as anterograde cargo proteins, IFT dynein and tubulin. Single-particle tracking shows that IFT components associate with trains sequentially, both in time and space. Super-resolution maps of IFT components in wild-type and mutant worms reveal ciliary ultrastructure and show that kinesin-II is essential for axonemal organization. Finally, imaging cilia lacking kinesin-II and/or transition zone function uncovers the interplay of kinesin-II and OSM-3 in driving efficient transport of IFT trains across the transition zone.

Overexpression of the flagellar motor protein MotB sensitizes Bacillus subtilis to aminoglycosides in a motility-independent manner.

The flagellar motor proteins, MotA and MotB, form a complex that rotates the flagella by utilizing the proton motive force (PMF) at the bacterial cell membrane. Although PMF affects the susceptibility to aminoglycosides, the effect of flagellar motor proteins on the susceptibility to aminoglycosides has not been investigated. Here, we found that MotB overexpression increased susceptibility to aminoglycosides, such as kanamycin and gentamicin, in Bacillus subtilis without affecting swimming motility. MotB overexpression did not affect susceptibility to ribosome-targeting antibiotics other than aminoglycosides, cell wall-targeting antibiotics, DNA synthesis-inhibiting antibiotics, or antibiotics inhibiting RNA synthesis. Meanwhile, MotB overexpression increased the susceptibility to aminoglycosides even in the motA-deletion mutant, which lacks swimming motility. Overexpression of the MotB mutant protein carrying an amino acid substitution at the proton-binding site (D24A) resulted in the loss of the enhanced aminoglycoside-sensitive phenotype. These results suggested that MotB overexpression sensitizes B. subtilis to aminoglycosides in a motility-independent manner. Notably, the aminoglycoside-sensitive phenotype induced by MotB requires the proton-binding site but not the MotA/MotB complex formation.

Trapping and scattering of a multiflagellated bacterium by a hard surface.

Thiovulum majus, which is one of the fastest known bacteria, swims using hundreds of flagella. Unlike typical pusher cells, which swim in circular paths over hard surfaces, T. majus localize near hard boundaries by turning their flagella to exert a net force normal to the surface. To probe the torques that stabilize this hydrodynamically bound state, the trajectories of several thousand collisions between a T. majus cell and a wall of a quasi-two-dimensional microfluidic chamber are analyzed. Measuring the fraction of cells escaping the wall either to the left or to the right of the point of contact-and how this probability varies with incident angle and time spent in contact with the surface-maps the scattering dynamics onto a first passage problem. These measurements are compared to the prediction of a Fokker-Planck equation to fit the angular velocity of a cell in contact with a hard surface. This analysis reveals a bound state with a narrow basin of attraction in which cells orient their flagella normal to the surface. The escape angle predicted by matching these near field dynamics with the far-field hydrodynamics is consistent with observation. We discuss the significance of these results for the ecology of T. majus and their self-organization into active chiral crystals.

The MBO2/FAP58 heterodimer stabilizes assembly of inner arm dynein b and reveals axoneme asymmetries involved in ciliary waveform.

Cilia generate three-dimensional waveforms required for cell motility and transport of fluid, mucus, and particles over the cell surface. This movement is driven by multiple dynein motors attached to nine outer doublet microtubules that form the axoneme. The outer and inner arm dyneins are organized into 96-nm repeats tandemly arrayed along the length of the doublets. Motility is regulated in part by projections from the two central pair microtubules that contact radial spokes located near the base of the inner dynein arms in each repeat. Although much is known about the structures and protein complexes within the axoneme, many questions remain about the regulatory mechanisms that allow the cilia to modify their waveforms in response to internal or external stimuli. Here, we used Chlamydomonas mbo (move backwards only) mutants with altered waveforms to identify at least two conserved proteins, MBO2/CCDC146 and FAP58/CCDC147, that form part of a L-shaped structure that varies between doublet microtubules. Comparative proteomics identified additional missing proteins that are altered in other motility mutants, revealing overlapping protein defects. Cryo-electron tomography and epitope tagging revealed that the L-shaped, MBO2/FAP58 structure interconnects inner dynein arms with multiple regulatory complexes, consistent with its function in modifying the ciliary waveform.

Structure and tethering mechanism of dynein-2 intermediate chains in intraflagellar transport.

Dynein-2 is a large multiprotein complex that powers retrograde intraflagellar transport (IFT) of cargoes within cilia/flagella, but the molecular mechanism underlying this function is still emerging. Distinctively, dynein-2 contains two identical force-generating heavy chains that interact with two different intermediate chains (WDR34 and WDR60). Here, we dissect regulation of dynein-2 function by WDR34 and WDR60 using an integrative approach including cryo-electron microscopy and CRISPR/Cas9-enabled cell biology. A 3.9 Å resolution structure shows how WDR34 and WDR60 use surprisingly different interactions to engage equivalent sites of the two heavy chains. We show that cilia can assemble in the absence of either WDR34 or WDR60 individually, but not both subunits. Dynein-2-dependent distribution of cargoes depends more strongly on WDR60, because the unique N-terminal extension of WDR60 facilitates dynein-2 targeting to cilia. Strikingly, this N-terminal extension can be transplanted onto WDR34 and retain function, suggesting it acts as a flexible tether to the IFT "trains" that assemble at the ciliary base. We discuss how use of unstructured tethers represents an emerging theme in IFT train interactions.

Identifying the suite of genes central to swimming in the biocontrol bacterium Pseudomonas protegens Pf-5.

Swimming motility is a key bacterial trait, important to success in many niches. Biocontrol bacteria, such as Pseudomonas protegens Pf-5, are increasingly used in agriculture to control crop diseases, where motility is important for colonization of the plant rhizosphere. Swimming motility typically involves a suite of flagella and chemotaxis genes, but the specific gene set employed for both regulation and biogenesis can differ substantially between organisms. Here we used transposon-directed insertion site sequencing (TraDIS), a genome-wide approach, to identify 249 genes involved in P. protegens Pf-5 swimming motility. In addition to the expected flagella and chemotaxis, we also identified a suite of additional genes important for swimming, including genes related to peptidoglycan turnover, O-antigen biosynthesis, cell division, signal transduction, c-di-GMP turnover and phosphate transport, and 27 conserved hypothetical proteins. Gene knockout mutants and TraDIS data suggest that defects in the Pst phosphate transport system lead to enhanced swimming motility. Overall, this study expands our knowledge of pseudomonad motility and highlights the utility of a TraDIS-based approach for analysing the functions of thousands of genes. This work sets a foundation for understanding how swimming motility may be related to the inconsistency in biocontrol bacteria performance in the field.

How P. aeruginosa cells with diverse stator composition collectively swarm.

Swarming is a macroscopic phenomenon in which surface bacteria organize into a motile population. The flagellar motor that drives swarming in Pseudomonas aeruginosa is powered by stators MotAB and MotCD. Deletion of the MotCD stator eliminates swarming, whereas deletion of the MotAB stator enhances swarming. Interestingly, we measured a strongly asymmetric stator availability in the wild-type (WT) strain, with MotAB stators produced at an approximately 40-fold higher level than MotCD stators. However, utilization of MotCD stators in free swimming cells requires higher liquid viscosities, while MotAB stators are readily utilized at low viscosities. Importantly, we find that cells with MotCD stators are ~10× more likely to have an active motor compared to cells uses the MotAB stators. The spectrum of motility intermittency can either cooperatively shut down or promote flagellum motility in WT populations. In P. aeruginosa, transition from a static solid-like biofilm to a dynamic liquid-like swarm is not achieved at a single critical value of flagellum torque or stator fraction but is collectively controlled by diverse combinations of flagellum activities and motor intermittencies via dynamic stator utilization. Experimental and computational results indicate that the initiation or arrest of flagellum-driven swarming motility does not occur from individual fitness or motility performance but rather related to concepts from the "jamming transition" in active granular matter.IMPORTANCEIt is now known that there exist multifactorial influences on swarming motility for P. aeruginosa, but it is not clear precisely why stator selection in the flagellum motor is so important. We show differential production and utilization of the stators. Moreover, we find the unanticipated result that the two motor configurations have significantly different motor intermittencies: the fraction of flagellum-active cells in a population on average with MotCD is active ~10× more often than with MotAB. What emerges from this complex landscape of stator utilization and resultant motor output is an intrinsically heterogeneous population of motile cells. We show how consequences of stator recruitment led to swarming motility and how the stators potentially relate to surface sensing circuitry.

Structural basis of directional switching by the bacterial flagellum.

The bacterial flagellum is a macromolecular protein complex that harvests energy from uni-directional ion flow across the inner membrane to power bacterial swimming via rotation of the flagellar filament. Rotation is bi-directional, with binding of a cytoplasmic chemotactic response regulator controlling reversal, though the structural and mechanistic bases for rotational switching are not well understood. Here we present cryoelectron microscopy structures of intact Salmonella flagellar basal bodies (3.2-5.5 Å), including the cytoplasmic C-ring complexes required for power transmission, in both counter-clockwise and clockwise rotational conformations. These reveal 180° movements of both the N- and C-terminal domains of the FliG protein, which, when combined with a high-resolution cryoelectron microscopy structure of the MotA5B2 stator, show that the stator shifts from the outside to the inside of the C-ring. This enables rotational switching and reveals how uni-directional ion flow across the inner membrane is used to accomplish bi-directional rotation of the flagellum.

The swimming defect caused by the absence of the transcriptional regulator LdtR in Sinorhizobium meliloti is restored by mutations in the motility genes motA and motS.

The flagellar motor is a powerful macromolecular machine used to propel bacteria through various environments. We determined that flagellar motility of the alpha-proteobacterium Sinorhizobium meliloti is nearly abolished in the absence of the transcriptional regulator LdtR, known to influence peptidoglycan remodeling and stress response. LdtR does not regulate motility gene transcription. Remarkably, the motility defects of the ΔldtR mutant can be restored by secondary mutations in the motility gene motA or a previously uncharacterized gene in the flagellar regulon, which we named motS. MotS is not essential for S. meliloti motility and may serve an accessory role in flagellar motor function. Structural modeling predicts that MotS comprised an N-terminal transmembrane segment, a long-disordered region, and a conserved ß-sandwich domain. The C terminus of MotS is localized in the periplasm. Genetics based substitution of MotA with MotAG12S also restored the ΔldtR motility defect. The MotAG12S variant protein features a local polarity shift at the periphery of the MotAB stator units. We propose that MotS may be required for optimal alignment of stators in wild-type flagellar motors but becomes detrimental in cells with altered peptidoglycan. Similarly, the polarity shift in stator units composed of MotB/MotAG12S might stabilize its interaction with altered peptidoglycan.

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.

Roles for CEP170 in cilia function and dynein-2 assembly.

Primary cilia are essential eukaryotic organelles required for signalling and secretion. Dynein-2 is a microtubule-motor protein complex and is required for ciliogenesis via its role in facilitating retrograde intraflagellar transport (IFT) from the cilia tip to the cell body. Dynein-2 must be assembled and loaded onto IFT trains for entry into cilia for this process to occur, but how dynein-2 is assembled and how it is recycled back into a cilium remain poorly understood. Here, we identify centrosomal protein of 170 kDa (CEP170) as a dynein-2-interacting protein in mammalian cells. We show that loss of CEP170 perturbs intraflagellar transport and hedgehog signalling, and alters the stability of dynein-2 holoenzyme complex. Together, our data indicate a role for CEP170 in supporting cilia function and dynein-2 assembly.

Oceanimonas pelagia sp. nov., a novel biosurfactant-producing and plastic-degrading potential bacterium isolated from marine coastal sediment.

A marine bacterial strain, named NTOU-MSR1T, was isolated from marine sediment of northern coast of Taiwan. This bacterium was Gram-stain-negative, aerobic, and motile, with a single flagellum. Its rod-shaped cells measured approximately 0.5-0.6 µm in width and 1.8-2.0 µm in length. NTOU-MSR1T grew at temperatures ranging from 10 to 45 °C, optimally at 30 °C. The pH range for growth was 7.0-10.0, with optimal growth at pH 7.0-8.0. It tolerated NaCl concentrations up to 12%. The cell membrane predominantly contained fatty acids such C16:1ω7c, C18:1ω7c, and C16:0. The overall genome relatedness indices indicated that strain NTOU-MSR1T had an average nucleotide identity (ANI) of 87.88% and a digital DNA-DNA hybridization (dDDH) value of 35.40% compared to its closest related species, O. marisflavi 102-Na3T. These values fell below the 95% and 70% threshold for species delineation, respectively. These findings suggested that the strain NTOU-MSR1T was a new member of the Oceanimonas genus. Its genomic DNA had a G + C content of 61.0 mol%. Genomic analysis revealed genes associated with the catechol branch of ß- ketoadipate pathway for degrading polycyclic aromatic hydrocarbons, resistance to heavy metal, biosynthesis of polyhydroxybutyrate and the production of glycoside hydrolases (GH19, GH23, and GH103) for chitin and glycan digestion. Additionally, NTOU-MSR1T was capable of synthesizing biosurfactants and potentially degrading plastic. The proposed name for this new species is Oceanimonas pelagia, with the type strain designated as NTOU-MSR1T (= BCRC 81403T = JCM 36023T).

Tektin bundle interacting protein, TEKTIP1, functions to stabilize the tektin bundle and axoneme in mouse sperm flagella.

Tektins are microtubule inner proteins (MIPs) and localize at the inside lumen of doublet microtubules (DMTs) of cilia/flagella. TEKTIP1, a newly identified protein by cryo-electron microscopy (cryo-EM), is proposed to be localized at the center of the tektin bundle and hypothesized to recruit tektins or stabilize the bundle. However, the physiological role of TEKTIP1 is unknown. In this study, we generated Tektip1-knockout (Tektip1-/-) mice and showed that they were male subfertile primarily due to reduced sperm motility. A high percentage of sperm from Tektip1-/- mice showed moderately disorganized axoneme structures and abnormal flagellar waveforms. TEKTIP1 predominately interacted with TEKT3 among tektins. Loss of TEKTIP1 partially disturbed the organization of tektin bundle by mainly affecting the native status of TEKT3 and its interaction with other tektins. Collectively, our study reveals the physiological role and potential molecular mechanism of TEKTIP1 in axonemal structure and sperm motility, highlights the importance of MIPs in stabilizing DMTs, and suggests a potential relevance of TEKTIP1 deficiency to human asthenospermia. Tektip1-/- mice will be an excellent animal model to study the DMT organization of sperm flagella using cryo-EM in future.

Rotation of the Fla2 flagella of Cereibacter sphaeroides requires the periplasmic proteins MotK and MotE that interact with the flagellar stator protein MotB2.

The bacterial flagellum is a complex structure formed by more than 25 different proteins, this appendage comprises three conserved structures: the basal body, the hook and filament. The basal body, embedded in the cell envelope, is the most complex structure and houses the export apparatus and the motor. In situ images of the flagellar motor in different species have revealed a huge diversity of structures that surround the well-conserved periplasmic components of the basal body. The identity of the proteins that form these novel structures in many cases has been elucidated genetically and biochemically, but in others they remain to be identified or characterized. In this work, we report that in the alpha proteobacteria Cereibacter sphaeroides the novel protein MotK along with MotE are essential for flagellar rotation. We show evidence that these periplasmic proteins interact with each other and with MotB2. Moreover, these proteins localize to the flagellated pole and MotK localization is dependent on MotB2 and MotA2. These results together suggest that the role of MotK and MotE is to activate or recruit the flagellar stators to the flagellar structure.