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.

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.

Protein subcellular relocalization and function of duplicated flagellar calcium binding protein genes in honey bee trypanosomatid parasite.

The honey bee trypanosomatid parasite, Lotmaria passim, contains two genes that encode the flagellar calcium binding protein (FCaBP) through tandem duplication in its genome. FCaBPs localize in the flagellum and entire body membrane of L. passim through specific N-terminal sorting sequences. This finding suggests that this is an example of protein subcellular relocalization resulting from gene duplication, altering the intracellular localization of FCaBP. However, this phenomenon may not have occurred in Leishmania, as one or both of the duplicated genes have become pseudogenes. Multiple copies of the FCaBP gene are present in several Trypanosoma species and Leptomonas pyrrhocoris, indicating rapid evolution of this gene in trypanosomatid parasites. The N-terminal flagellar sorting sequence of L. passim FCaBP1 is in close proximity to the BBSome complex, while that of Trypanosoma brucei FCaBP does not direct GFP to the flagellum in L. passim. Deletion of the two FCaBP genes in L. passim affected growth and impaired flagellar morphogenesis and motility, but it did not impact host infection. Therefore, FCaBP represents a duplicated gene with a rapid evolutionary history that is essential for flagellar structure and function in a trypanosomatid parasite.

Structure-guided discovery of protein and glycan components in native mastigonemes.

Mastigonemes, the hair-like lateral appendages lining cilia or flagella, participate in mechanosensation and cellular motion, but their constituents and structure have remained unclear. Here, we report the cryo-EM structure of native mastigonemes isolated from Chlamydomonas at 3.0 Å resolution. The long stem assembles as a super spiral, with each helical turn comprising four pairs of anti-parallel mastigoneme-like protein 1 (Mst1). A large array of arabinoglycans, which represents a common class of glycosylation in plants and algae, is resolved surrounding the type II poly-hydroxyproline (Hyp) helix in Mst1. The EM map unveils a mastigoneme axial protein (Mstax) that is rich in heavily glycosylated Hyp and contains a PKD2-like transmembrane domain (TMD). Mstax, with nearly 8,000 residues spanning from the intracellular region to the distal end of the mastigoneme, provides the framework for Mst1 assembly. Our study provides insights into the complexity of protein and glycan interactions in native bio-architectures.

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.

Flagellotropic phages: common yet diverse host interaction strategies.

Many bacteriophages (phages) interact with flagella and rely on bacterial motility for successful infection of their hosts. Yet, limited information is available on how phages have evolved to recognize and bind both flagella and subsequent surface receptors for phage DNA injection. Here, we present an update on the current knowledge of flagellotropic phages using a few well-studied phages as examples to unravel the molecular details of bacterial host recognition. We discuss the recent advances in the role of globular exposed flagellin domains and flagella glycosylation in phage binding to the flagella. In addition, we present diverse types of surface receptors and phage components responsible for the interaction with the host. Finally, we point to questions remaining to be answered and new approaches to study this unique group of phages.

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.

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.

The O-glycan is essential for the induction of protective antibodies against lethal infection by flagella A-bearing Pseudomonas aeruginosa.

To address the problem of increased antimicrobial resistance, we developed a glycoconjugate vaccine comprised of O-polysaccharides (OPS) of the four most prevalent serotypes of Klebsiella pneumoniae (KP) linked to recombinant flagellin types A and B (rFlaA and rFlaB) of Pseudomonas aeruginosa (PA). Flagellin is the major subunit of the flagellar filament. Flagella A and B, essential virulence factors for PA, are glycosylated with different glycans. We previously reported that while both rFlaA and rFlaB were highly immunogenic, only the rFlaB antisera reduced PA motility and protected mice from lethal PA infection in a mouse model of thermal injury. Since recombinant flagellin is not glycosylated, we examined the possibility that the glycan on native FlaA (nFlaA) might be critical to functional immune responses. We compared the ability of nFlaA to that of native, deglycosylated FlaA (dnFlaA) to induce functionally active antisera. O glycan was removed from nFlaA with trifluoromethanesulfonic acid. Despite the similar high-titered anti-FlaA antibody levels elicited by nFlaA, rFlaA, and dnFlaA, only the nFlaA antisera inhibited PA motility and protected mice following lethal intraperitoneal bacterial challenge. Both the protective efficacy and carrier protein function of nFlaA were retained when conjugated to KP O1 OPS. We conclude that unlike the case with FlaB O glycan, the FlaA glycan is an important epitope for the induction of functionally active anti-FlaA antibodies.

Identification of a new export signal that targets early subunits to the flagellar type III secretion export machinery.

Type III secretion systems (T3SSs) are essential for motility and virulence in many bacterial pathogens. Proteins destined for the flagellar T3SS contain at least two export signals in their N-terminal D0 domain. Here, we describe a third carboxy (C)-terminal signal in early flagellar subunits that facilitates subunit targeting to the export machinery. Mutational analysis identified critical residues within the flagellar hook subunit C-terminal export signal. The flagellar ATPase and cytoplasmic ring components were not required for this targeting, indicating that core export machinery components facilitate substrate targeting via the C-terminal export signal. More broadly, these results demonstrate that multiple distinct export signals within type III secretion substrates facilitate distinct export events at the T3SS export machinery. Our data establish key events in the export mechanism of type III secretion systems: targeting of subunits to and their sequential interactions with key components of the export machinery. IMPORTANCE: Many bacterial pathogens utilize T3SS to inject virulence proteins (effectors) into host cells or to assemble flagella on the bacterial cell surface. Bacterial flagella present a paradigm for how cells build and operate complex cell-surface "nanomachines." Efficient subunit targeting from the bacterial cytosol to type III secretion systems is essential for rapid assembly and secretion by T3SSs. Subunits are thought to dock at the export machinery before being unfolded and translocated into the export channel. However, little is known about how subunits dock at the export machinery and the events that occur post docking. Here, we identified a new export signal within the C-termini of subunits that is essential for targeting of subunits to the type III export machinery. We show that this new export signal and previously identified export signals are recognized separately and sequentially, revealing a pathway for subunit transit through the type III export machinery in which sequential recognition events carry out different roles at major steps in the export pathway.

Clostridioides difficile Flagella.

Clostridioides difficile is an important pathogen for humans with a lead in nosocomial infection, but it is also more and more common in communities. Our knowledge of the pathology has historically been focused on the toxins produced by the bacteria that remain its major virulence factors. But the dysbiosis of the intestinal microbiota creating the conditions for the colonization appears to be fundamental for our understanding of the disease. Colonization implies several steps for the bacteria that do or do not use their capacity of motility with the synthesis of flagella. In this review, we focus on the current understanding of different topics on the C. difficile flagellum, ranging from its genetic organization to the vaccinal interest in it.

Centriolar appendages evolve into the inner sheath of mammalian flagella.

The annulus, a septin-based structure in vertebrate sperm connecting the MP and PP, has unclear migration mechanics. In this issue, Hoque et al. (https://doi.org/10.1083/jcb.202307147) report that the CBY3/CIBAR1 complex ensures its precise positioning by regulating membrane properties.

Roles of linker region flanked by transmembrane and peptidoglycan binding region of PomB in energy conversion of the Vibrio flagellar motor.

The flagellar components of Vibrio spp., PomA and PomB, form a complex that transduces sodium ion and contributes to rotate flagella. The transmembrane protein PomB is attached to the basal body T-ring by its periplasmic region and has a plug segment following the transmembrane helix to prevent ion flux. Previously we showed that PomB deleted from E41 to R120 (Δ41-120) was functionally comparable to the full-length PomB. In this study, three deletions after the plug region, PomB (Δ61-120), PomB (Δ61-140), and PomB (Δ71-150), were generated. PomB (Δ61-120) conferred motility, whereas the other two mutants showed almost no motility in soft agar plate; however, we observed some swimming cells with speed comparable for the wild-type cells. When the two PomB mutants were introduced into a wild-type strain, the swimming ability was not affected by the mutant PomBs. Then, we purified the mutant PomAB complexes to confirm the stator formation. When plug mutations were introduced into the PomB mutants, the reduced motility by the deletion was rescued, suggesting that the stator was activated. Our results indicate that the deletions prevent the stator activation and the linker and plug regions, from E41 to S150, are not essential for the motor function of PomB but are important for its regulation.

Twists and turns: 40 years of investigating how and why bacteria swim.

Fifty years of research has transformed our understanding of bacterial movement from one of description, based on a limited number of electron micrographs and some low-magnification studies of cells moving towards or away from chemical effectors, to probably the best understood behavioural system in biology. We have a molecular understanding of how bacteria sense and respond to changes in their environment and detailed structural insights into the workings of one of the most complex motor structures we know of. Thanks to advances in genomics we also understand how, through evolution, different species have tuned and adapted a core shared system to optimize behaviour in their specific environment. In this review, I will highlight some of the unexpected findings we made during my over 40-year career, how those findings changed some of our understanding of bacterial behaviour and biochemistry and some of the battles to have those observations accepted.

Wave-like oscillations of clamped microtubules driven by collective dynein transport.

Microtubules (MTs) are observed to move and buckle driven by ATP-dependent molecular motors in both mitotic and interphasic eukaryotic cells as well as in specialized structures such as flagella and cilia with a stereotypical geometry. In previous work, clamped MTs driven by a few kinesin motors were seen to buckle and occasionally flap in what was referred to as flagella-like motion. Theoretical models of active-filament dynamics and a following force have predicted that, with sufficient force and binding-unbinding, such clamped filaments should spontaneously undergo periodic buckling oscillations. However, a systematic experimental test of the theory and reconciliation to a model was lacking. Here, we have engineered a minimal system of MTs clamped at their plus ends and transported by a sheet of dynein motors that demonstrate the emergence of spontaneous traveling-wave oscillations along single filaments. The frequencies of tip oscillations are in the millihertz range and are statistically indistinguishable in the onset and recovery phases. We develop a 2D computational model of clamped MTs binding and unbinding stochastically to motors in a "gliding-assay" geometry. The simulated MTs oscillate with a frequency comparable to experiment. The model predicts the effect of MT length and motor density on qualitative transitions between distinct phases of flapping, regular oscillations, and looping. We develop an effective "order parameter" based on the relative deflection along the filament and orthogonal to it. The transitions predicted in simulations are validated by experimental data. These results demonstrate a role for geometry, MT buckling, and collective molecular motor activity in the emergence of oscillatory dynamics.

Label-free proteomic comparison reveals ciliary and nonciliary phenotypes of IFT-A mutants.

DIFFRAC is a powerful method for systematically comparing proteome content and organization between samples in a high-throughput manner. By subjecting control and experimental protein extracts to native chromatography and quantifying the contents of each fraction using mass spectrometry, it enables the quantitative detection of alterations to protein complexes and abundances. Here, we applied DIFFRAC to investigate the consequences of genetic loss of Ift122, a subunit of the intraflagellar transport-A (IFT-A) protein complex that plays a vital role in the formation and function of cilia and flagella, on the proteome of Tetrahymena thermophila. A single DIFFRAC experiment was sufficient to detect changes in protein behavior that mirrored known effects of IFT-A loss and revealed new biology. We uncovered several novel IFT-A-regulated proteins, which we validated through live imaging in Xenopus multiciliated cells, shedding new light on both the ciliary and non-ciliary functions of IFT-A. Our findings underscore the robustness of DIFFRAC for revealing proteomic changes in response to genetic or biochemical perturbation.

Microbial Primer: The bacterial flagellum - how bacteria swim.

Bacteria swim using membrane-spanning, electrochemical gradient-powered motors that rotate semi-rigid helical filaments. This primer provides a brief overview of the basic synthesis, structure and operation of these nanomachines. Details and variations on the basic system can be found in suggested further reading.