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The Helicobacter pylori flagellar motor contains several accessory structures that are not found in the archetypal Escherichia coli and Salmonella enterica motors. H . pylori hp0838 encodes a previously uncharacterized lipoprotein and is in an operon with flgP , which encodes a motor accessory protein. Deletion analysis of hp0838 in H . pylori B128 showed that the gene is not required for motility in soft agar medium, but the mutant displayed a reduced growth rate and an increased sensitivity to bacitracin, which is an antibiotic that is normally excluded by the outer membrane. Introducing a plasmid-borne copy of hp0838 into the H . pylori Δ hp0838 mutant suppressed the fitness defect and antibiotic sensitivity of the strain. A variant of the Δ hp0838 mutant containing a frameshift mutation in pflA , which resulted in paralyzed flagella, displayed wild-type growth rate and resistance to bacitracin, suggesting the fitness defect and antibiotic sensitivity of the Δ hp0838 mutant are dependent on flagellar rotation. Comparative analysis of in-situ structures of the wild type and Δ hp0838 mutant motors revealed the Δ hp0838 mutant motor lacked a previously undescribed ring structure with 18-fold symmetry located near the outer membrane. Given its role in formation of the motor outer ring, HP0838 was designated FapH ( f lagellar a ccessory p rotein in H elicobacter pylori ) and the motor accessory formed the protein was named the FapH ring. Our data suggest that the FapH ring helps to preserve outer membrane barrier function during flagellar rotation. Given that FapH homologs are present in many members of the phylum Campylobacterota, they may have similar roles in protecting the outer membrane from damage due to flagellar rotation in these bacteria.

Spirochetes are a widespread group of bacteria with a distinct morphology. Some spirochetes are important human pathogens that utilize periplasmic flagella to achieve motility and host infection. The motors that drive the rotation of periplasmic flagella have a unique spirochete-specific feature, termed the collar, crucial for the flat-wave morphology and motility of the Lyme disease spirochete Borrelia burgdorferi . Here, we deploy cryo-electron tomography and subtomogram averaging to determine high-resolution in-situ structures of the B . burgdorferi flagellar motor. Comparative analysis and molecular modeling of in-situ flagellar motor structures from B . burgdorferi mutants lacking each of the known collar proteins (FlcA, FlcB, FlcC, FlbB, and Bb0236/FlcD) uncover a complex protein network at the base of the collar. Importantly, our data suggest that FlbB forms a novel periplasmic ring around the rotor but also acts as a scaffold supporting collar assembly and subsequent recruitment of stator complexes. The complex protein network based on the FlbB ring effectively bridges the rotor and 16 torque-generating stator complexes in each flagellar motor, thus contributing to the specialized motility and lifestyle of spirochetes in complex environments.

Bacteria switch only intermittently to motile planktonic lifestyles under favorable conditions. Under chronic nutrient deprivation, however, bacteria orchestrate a switch to stationary phase, conserving energy by altering metabolism and stopping motility. About two-thirds of bacteria use flagella to swim, but how bacteria deactivate this large molecular machine remains unclear. Here, we describe the previously unreported ejection of polar motors by [gamma]-proteobacteria. We show that these bacteria eject their flagella at the base of the flagellar hook when nutrients are depleted, leaving a relic of a former flagellar motor in the outer membrane. Subtomogram averages of the full motor and relic reveal that this is an active process, as a plug protein appears in the relic, likely to prevent leakage across their outer membrane; furthermore, we show that ejection is triggered only under nutritional depletion and is independent of the filament as a possible mechanosensor. We show that filament ejection is a widespread phenomenon demonstrated by the appearance of relic structures in diverse [gamma]-proteobacteria including Plesiomonas shigelloides, Vibrio cholerae, Vibrio fischeri, Shewanella putrefaciens, and Pseudomonas aeruginosa. While the molecular details remain to be determined, our results demonstrate a novel mechanism for bacteria to halt costly motility when nutrients become scarce.

The assembly and physiological function of cilia and flagella depend on the stable association of A- and B-tubules, which form axonemal doublet microtubules (DMTs). However, the mechanisms underlying the connection of B-tubules to A-tubules to form DMTs in mammalian cilia/flagella are unclear. CFAP77 encodes an outer junction (OJ) protein within DMTs that is conserved across many species and cell types. In this study, Cfap77 -KO mice were generated to reveal that CFAP77 is essential for sperm progressive motility and male fertility. Loss of CFAP77 led to opened B-tubules specifically at the OJ regions of axonemal DMTs as revealed by conventional transmission electron microscopy. Cryo-electron tomography was used to further resolve the in situ structure of sperm axonemal DMTs directly from Cfap77 -KO mice, which exhibited a loss of large filamentous density corresponding to the CFAP77-CCDC105-TEX43 ternary subcomplex at the OJ regions. Additionally, sperm proteomic analysis confirmed that CFAP77 knockout led to the complete loss of this ternary complex. Our work not only explores the physiological role of the OJ protein CFAP77 in axonemal A- and B-tubule connections in mammals but also combines in situ structural biology and knockout mice to reveal the underlying structural/molecular mechanism involved.

In its simplest form, bacterial flagellar filaments are composed of flagellin proteins with just two helical inner domains, which together comprise the filament core. Although this minimal filament is sufficient to provide motility in many flagellated bacteria, most bacteria produce flagella composed of flagellin proteins with one or more outer domains arranged in a variety of supramolecular architectures radiating from the inner core. Flagellin outer domains are known to be involved in adhesion, proteolysis and immune evasion but have not been thought to be required for motility. Here we show that in the Pseudomonas aeruginosa PAO1 strain, a bacterium that forms a ridged filament with a dimerization of its flagellin outer domains, motility is categorically dependent on these flagellin outer domains. Moreover, a comprehensive network of intermolecular interactions connecting the inner domains to the outer domains, the outer domains to one another, and the outer domains back to the inner domain filament core, is required for motility. This inter-domain connectivity confers PAO1 flagella with increased stability, essential for its motility in viscous environments. Additionally, we find that such ridged flagellar filaments are not unique to Pseudomonas but are, instead, present throughout diverse bacterial phyla.

Bacterial locomotion by rotating flagella is achieved through the hook, which transmits torque from the motor to the filament. The hook is a tubular structure composed of a single type of protein, yet it adopts a curved shape. To perform its function, it must be simultaneously flexible and torsionally rigid. The molecular mechanism by which chemically identical subunits form such a dynamic structure is unknown. Here, we show the complete structure of the hook from Salmonella enterica in its supercoiled ‘curved’ state, at 2.9 Å resolution. Subunits in the curved hook are grouped into 11 distinctive conformations, each shared along 11 protofilaments. The domains of the elongated hook subunit behave as rigid bodies connected by two hinge regions. The reconstituted model demonstrates how identical subunits can dynamically change conformation by physical interactions while bending. These multiple subunit states contradict the two-state model, which is a key feature of flagellar polymorphism.

MotY homologs are present in a variety of monotrichous bacterial strains and are thought to form an additional structural T ring in flagellar motors. While MotY potentially plays an important role in motor torque generation, its impact on motor output dynamics remains poorly understood. In this study, we investigate the role of MotY in P. aeruginosa , elucidating its interactions with the two sets of stator units (MotAB and MotCD) using Förster resonance energy transfer (FRET) assays. Employing a newly developed bead assay, we characterize the dynamic behavior of flagellar motors in motY mutants, identifying MotY as the key functional protein to affect the clockwise bias of naturally unbiased motors in P. aeruginosa . Our findings reveal that MotY enhances stator assembly efficiency without affecting the overall assembly of the flagellar structure. Additionally, we demonstrate that MotY is essential for maintaining motor torque and regulating switching rates. Our study highlights the physiological significance of MotY in fine-tuning flagellar motor function in complex environments.

Key Points The African trypanosome Trypanosoma brucei is a unicellular pathogen that causes lethal sleeping sickness in humans, which is a devastating and neglected tropical disease that is endemic to vast regions of Africa. T. brucei also infects wild and domestic livestock, which limits sustainable development, and it is thus considered to be both a cause and consequence of poverty. T. brucei has a single flagellum that is present throughout the parasite and its life cycle. The flagellum has conserved and unique features. It emerges from a membrane invagination at the posterior end of the cell and remains attached to the cell body for most of its length. The flagellum contains cytoskeletal structures, which are ensheathed by a specialized flagellar membrane that interfaces with the external environment and that has a protein and lipid composition that is distinct from the rest of the cell surface. The T. brucei flagellum has multiple functions and is essential for parasite motility, viability, transmission and pathogenesis. Flagellum-mediated motility is powered by the axoneme, which is a biological machine that converts dynein motor structural changes into flagellum beating and parasite propulsion. T. brucei motility is crucial for movement through host tissues and provides a surprising immune-evasion mechanism. In addition to motility, the T. brucei flagellum is an important morphogenetic hub that controls cell shape and size, directs organelle segregation and governs cell division. These functions are modulated during developmental transitions of the parasite and are achieved by the direct or indirect physical connections of the flagellum to other cellular elements. The flagellum is a crucial host–pathogen interface that has important roles in parasite transmission and virulence. Flagellar proteins mediate attachment to host tissues, carry out uptake of host growth factors and promote parasite survival by inhibiting host immunity. T. brucei is an excellent model system to study the biology of the highly conserved eukaryotic flagellum and offers valuable insights into how flagella assemble, move and sense the environment. Continued studies of the T.brucei flagellum hold the promise of having a great impact on human health, as human flagella are paramount in human development and physiology. In addition, the flagella of many human pathogens are salient but unexplained structures that await further study. The protozoan parasite Trypanosoma brucei has a single flagellum that is present in all of its different developmental stages. In this Review, Langousis and Hill discuss the structural and functional features of the flagellum and highlight its central role in the virulence and transmission of this important human pathogen. Trypanosoma brucei is a pathogenic unicellular eukaryote that infects humans and other mammals in sub-Saharan Africa. A central feature of trypanosome biology is the single flagellum of the parasite, which is an essential and multifunctional organelle that facilitates cell propulsion, controls cell morphogenesis and directs cytokinesis. Moreover, the flagellar membrane is a specialized subdomain of the cell surface that mediates attachment to host tissues and harbours multiple virulence factors. In this Review, we discuss the structure, assembly and function of the trypanosome flagellum, including canonical roles in cell motility as well as novel and emerging roles in cell morphogenesis and host–parasite interactions.

Type 3 secretion systems (T3SSs) are essential components of two complex bacterial machineries: the flagellum, which drives cell motility, and the non-flagellar T3SS (NF-T3SS), which delivers effectors into eukaryotic cells. Yet the origin, specialization, and diversification of these machineries remained unclear. We developed computational tools to identify homologous components of the two systems and to discriminate between them. Our analysis of >1,000 genomes identified 921 T3SSs, including 222 NF-T3SSs. Phylogenomic and comparative analyses of these systems argue that the NF-T3SS arose from an exaptation of the flagellum, i.e. the recruitment of part of the flagellum structure for the evolution of the new protein delivery function. This reconstructed chronology of the exaptation process proceeded in at least two steps. An intermediate ancestral form of NF-T3SS, whose descendants still exist in Myxococcales, lacked elements that are essential for motility and included a subset of NF-T3SS features. We argue that this ancestral version was involved in protein translocation. A second major step in the evolution of NF-T3SSs occurred via recruitment of secretins to the NF-T3SS, an event that occurred at least three times from different systems. In rhizobiales, a partial homologous gene replacement of the secretin resulted in two genes of complementary function. Acquisition of a secretin was followed by the rapid adaptation of the resulting NF-T3SSs to multiple, distinct eukaryotic cell envelopes where they became key in parasitic and mutualistic associations between prokaryotes and eukaryotes. Our work elucidates major steps of the evolutionary scenario leading to extant NF-T3SSs. It demonstrates how molecular evolution can convert one complex molecular machine into a second, equally complex machine by successive deletions, innovations, and recruitment from other molecular systems.

Two studies now provide structural insights into the stator–rotor interaction, stator assembly and stator maintenance of the bacterial flagellum.