Helicobacter pylori FlgN binds its substrate FlgK and the flagellum ATPase FliI in a similar manner observed for the FliT chaperone.

In bacterial flagellum biogenesis, secretion of the hook-filament junction proteins FlgK and FlgL and completion of the flagellum requires the FlgN chaperone. Similarly, the related FliT chaperone is necessary for the secretion of the filament cap protein FliD and binds the flagellar export gate protein FlhA and the flagellum ATPase FliI. FlgN and FliT require FliJ for effective substrate secretion. In Helicobacter pylori, neither FlgN, FliT, nor FliJ have been annotated. We demonstrate that the genome location of HP1120 is identical to that of flgN in other flagellated bacteria and that HP1120 is the homolog of Campylobacter jejuni FlgN. A modeled HP1120 structure contains three α-helices and resembles the FliT chaperone, sharing a similar substrate-binding pocket. Using pulldowns and thermophoresis, we show that both HP1120 and a HP1120Δ126-144 deletion mutant bind to FlgK with nanomolar affinity, but not to the filament cap protein FliD, confirming that HP1120 is FlgN. Based on size-exclusion chromatography and multi-angle light scattering, H. pylori FlgN binds to FlgK with 1:1 stoichiometry. Overall structural similarities between FlgN and FliT suggest that substrate recognition on FlgN primarily involves an antiparallel coiled-coil interface between the third helix of FlgN and the C-terminal helix of the substrate. A FlgNΔ126-144 N100A, Y103A, S111I triple mutant targeting this interface significantly impairs the binding of FlgK. Finally, we demonstrate that FlgNΔ126-144 , like FliT, binds with sub-micromolar affinity to the flagellum ATPase FliI or its N-terminal domain. Hence FlgN and FliT likely couple delivery of low-abundance export substrates to the flagellum ATPase FliI.

Tuning the stator subunit of the flagellar motor with coiled-coil engineering.

Many bacteria swim driven by an extracellular filament rotated by the bacterial flagellar motor. This motor is powered by the stator complex, MotA5 MotB2 , an heptameric complex which forms an ion channel which couples energy from the ion motive force to torque generation. Recent structural work revealed that stator complex consists of a ring of five MotA subunits which rotate around a central dimer of MotB subunits. Transmembrane (TM) domains TM3 and TM4 from MotA combine with the single TM domain from MotB to form two separate ion channels within this complex. Much is known about the ion binding site and ion specificity; however, to date, no modeling has been undertaken to explore the MotB-MotB dimer stability and the role of MotB conformational dynamics during rotation. Here, we modeled the central MotB dimer using coiled-coil engineering and modeling principles and calculated free energies to identify stable states in the operating cycle of the stator. We found three stable coiled-coil states with dimer interface angles of 28°, 56°, and 64°. We tested the effect of strategic mutagenesis on the comparative energy of the states and correlated motility with a specific hierarchy of stability between the three states. In general, our results indicate agreement with existing models describing a 36° rotation step of the MotA pentameric ring during the power stroke and provide an energetic basis for the coordinated rotation of the central MotB dimer based on coiled-coil modeling.

Structure and electromechanical coupling of a voltage-gated Na+/H+ exchanger.

Voltage-sensing domains control the activation of voltage-gated ion channels, with a few exceptions1. One such exception is the sperm-specific Na+/H+ exchanger SLC9C1, which is the only known transporter to be regulated by voltage-sensing domains2-5. After hyperpolarization of sperm flagella, SLC9C1 becomes active, causing pH alkalinization and CatSper Ca2+ channel activation, which drives chemotaxis2,6. SLC9C1 activation is further regulated by cAMP2,7, which is produced by soluble adenyl cyclase (sAC). SLC9C1 is therefore an essential component of the pH-sAC-cAMP signalling pathway in metazoa8,9, required for sperm motility and fertilization4. Despite its importance, the molecular basis of SLC9C1 voltage activation is unclear. Here we report cryo-electron microscopy (cryo-EM) structures of sea urchin SLC9C1 in detergent and nanodiscs. We show that the voltage-sensing domains are positioned in an unusual configuration, sandwiching each side of the SLC9C1 homodimer. The S4 segment is very long, 90 Å in length, and connects the voltage-sensing domains to the cytoplasmic cyclic-nucleotide-binding domains. The S4 segment is in the up configuration-the inactive state of SLC9C1. Consistently, although a negatively charged cavity is accessible for Na+ to bind to the ion-transporting domains of SLC9C1, an intracellular helix connected to S4 restricts their movement. On the basis of the differences in the cryo-EM structure of SLC9C1 in the presence of cAMP, we propose that, upon hyperpolarization, the S4 segment moves down, removing this constriction and enabling Na+/H+ exchange.

Structure, Assembly, and Function of Flagella Responsible for Bacterial Locomotion.

Many motile bacteria use flagella for locomotion under a variety of environmental conditions. Because bacterial flagella are under the control of sensory signal transduction pathways, each cell is able to autonomously control its flagellum-driven locomotion and move to an environment favorable for survival. The flagellum of Salmonella enterica serovar Typhimurium is a supramolecular assembly consisting of at least three distinct functional parts: a basal body that acts as a bidirectional rotary motor together with multiple force generators, each of which serves as a transmembrane proton channel to couple the proton flow through the channel with torque generation; a filament that functions as a helical propeller that produces propulsion; and a hook that works as a universal joint that transmits the torque produced by the rotary motor to the helical propeller. At the base of the flagellum is a type III secretion system that transports flagellar structural subunits from the cytoplasm to the distal end of the growing flagellar structure, where assembly takes place. In recent years, high-resolution cryo-electron microscopy (cryoEM) image analysis has revealed the overall structure of the flagellum, and this structural information has made it possible to discuss flagellar assembly and function at the atomic level. In this article, we describe what is known about the structure, assembly, and function of Salmonella flagella.

Axonemal structures reveal mechanoregulatory and disease mechanisms.

Motile cilia and flagella beat rhythmically on the surface of cells to power the flow of fluid and to enable spermatozoa and unicellular eukaryotes to swim. In humans, defective ciliary motility can lead to male infertility and a congenital disorder called primary ciliary dyskinesia (PCD), in which impaired clearance of mucus by the cilia causes chronic respiratory infections1. Ciliary movement is generated by the axoneme, a molecular machine consisting of microtubules, ATP-powered dynein motors and regulatory complexes2. The size and complexity of the axoneme has so far prevented the development of an atomic model, hindering efforts to understand how it functions. Here we capitalize on recent developments in artificial intelligence-enabled structure prediction and cryo-electron microscopy (cryo-EM) to determine the structure of the 96-nm modular repeats of axonemes from the flagella of the alga Chlamydomonas reinhardtii and human respiratory cilia. Our atomic models provide insights into the conservation and specialization of axonemes, the interconnectivity between dyneins and their regulators, and the mechanisms that maintain axonemal periodicity. Correlated conformational changes in mechanoregulatory complexes with their associated axonemal dynein motors provide a mechanism for the long-hypothesized mechanotransduction pathway to regulate ciliary motility. Structures of respiratory-cilia doublet microtubules from four individuals with PCD reveal how the loss of individual docking factors can selectively eradicate periodically repeating structures.

Structure and Assembly of the Proteus mirabilis Flagellar Motor by Cryo-Electron Tomography.

Proteus mirabilis is a Gram-negative Gammaproteobacterium and a major causative agent of urinary tract infections in humans. It is characterized by its ability to switch between swimming motility in liquid media and swarming on solid surfaces. Here, we used cryo-electron tomography and subtomogram averaging to reveal the structure of the flagellar motor of P. mirabilis at nanometer resolution in intact cells. We found that P. mirabilis has a motor that is structurally similar to those of Escherichia coli and Salmonella enterica, lacking the periplasmic elaborations that characterize other more specialized gammaproteobacterial motors. In addition, no density corresponding to stators was present in the subtomogram average suggesting that the stators are dynamic. Finally, several assembly intermediates of the motor were seen that support the inside-out assembly pathway.

Questioning rotary functionality in the bacterial flagellar system and proposing a murburn model for motility.

Bacterial flagellar system (BFS) was the primary example of a purported 'rotary-motor' functionality in a natural assembly. This mandates the translation of a circular motion of components inside into a linear displacement of the cell body outside, which is supposedly orchestrated with the following features of the BFS: (i) A chemical/electrical differential generates proton motive force (pmf, including a trans-membrane potential, TMP), which is electro-mechanically transduced by inward movement of protons via BFS. (ii) Membrane-bound proteins of BFS serve as stators and the slender filament acts as an external propeller, culminating into a hook-rod that pierces the membrane to connect to a 'broader assembly of deterministically movable rotor'. We had disclaimed the purported pmf/TMP-based respiratory/photosynthetic physiology involving Complex V, which was also perceived as a 'rotary machine' earlier. We pointed out that the murburn redox logic was operative therein. We pursue the following similar perspectives in BFS-context: (i) Low probability for the evolutionary attainment of an ordered/synchronized teaming of about two dozen types of proteins (assembled across five-seven distinct phases) towards the singular agendum of rotary motility. (ii) Vital redox activity (not the gambit of pmf/TMP!) powers the molecular and macroscopic activities of cells, including flagella. (iii) Flagellar movement is noted even in ambiances lacking/countering the directionality mandates sought by pmf/TMP. (iv) Structural features of BFS lack component(s) capable of harnessing/achieving pmf/TMP and functional rotation. A viable murburn model for conversion of molecular/biochemical activity into macroscopic/mechanical outcomes is proposed herein for understanding BFS-assisted motility. HIGHLIGHTSThe motor-like functionalism of bacterial flagellar system (BFS) is analyzedProton/Ion-differential based powering of BFS is unviable in bacteriaUncouplers-sponsored effects were misinterpreted, resulting in a detour in BFS researchThese findings mandate new explanation for nano-bio-mechanical movements in BFSA minimalist murburn model for the bacterial flagella-aided movement is proposedCommunicated by Ramaswamy H. Sarma.

RABL4/IFT27 in a nucleotide-independent manner promotes phospholipase D ciliary retrieval via facilitating BBSome reassembly at the ciliary tip.

Certain ciliary transmembrane and membrane-associated signaling proteins export from cilia as intraflagellar transport (IFT) cargoes in a BBSome-dependent manner. Upon reaching the ciliary tip via anterograde IFT, the BBSome disassembles before being reassembled to form an intact entity for cargo phospholipase D (PLD) coupling. During this BBSome remodeling process, Chlamydomonas Rab-like 4 GTPase IFT27, by binding its partner IFT25 to form the heterodimeric IFT25/27, is indispensable for BBSome reassembly. Here, we show that IFT27 binds IFT25 in an IFT27 nucleotide-independent manner. IFT25/27 and the IFT subcomplexes IFT-A and -B are irrelevant for maintaining the stability of one another. GTP-loading onto IFT27 enhances the IFT25/27 affinity for binding to the IFT-B subcomplex core IFT-B1 entity in cytoplasm, while GDP-bound IFT27 does not prevent IFT25/27 from entering and cycling through cilia by integrating into IFT-B1. Upon at the ciliary tip, IFT25/27 cycles on and off IFT-B1 and this process is irrelevant with the nucleotide state of IFT27. During BBSome remodeling at the ciliary tip, IFT25/27 promotes BBSome reassembly independent of IFT27 nucleotide state, making postremodeled BBSomes available for PLD to interact with. Thus, IFT25/27 facilitates BBSome-dependent PLD export from cilia via controlling availability of intact BBSomes at the ciliary tip, while IFT27 nucleotide state does not participate in this regulatory event.

Live-Cell Imaging of the Assembly and Ejection Processes of the Bacterial Flagella by Fluorescence Microscopy.

Bacterial flagella are molecular machines used for motility and chemotaxis. The flagellum consists of a thin extracellular helical filament as a propeller, a short hook as a universal joint, and a basal body as a rotary motor. The filament is made up of more than 20,000 flagellin molecules and can grow to several micrometers long but only 20 nanometers thick. The regulation of flagellar assembly and ejection is important for bacterial environmental adaptation. However, due to the technical difficulty to observe these nanostructures in live cells, our understanding of the flagellar growth and loss is limited. In the last three decades, the development of fluorescence microscopy and fluorescence labeling of specific cellular structure has made it possible to perform the real-time observation of bacterial flagellar assembly and ejection processes. Furthermore, flagella are not only critical for bacterial motility but also important antigens stimulating host immune responses. The complete understanding of bacterial flagellar production and ejection is valuable for understanding macromolecular self-assembly, cell adaptation, and pathogen-host interactions.

Purification of Na+-Driven MotPS Stator Complexes and Single-Molecule Imaging by High-Speed Atomic Force Microscopy.

The stator unit of the bacterial flagellar motor coordinates the number of active stators in the motor by sensing changes in external load and ion motive force across the cytoplasmic membrane. The structural dynamics of the stator unit at the single-molecule level is key to understanding the sensing mechanism and motor assembly. High-speed atomic force microscopy (HS-AFM) is a powerful tool for directly observing dynamically acting biological molecules with high spatiotemporal resolution without interfering with their function. Here, we describe protocols for single-molecule imaging of the Na+-driven MotPS stator complex by HS-AFM.

IFT52 plays an essential role in sensory cilia formation and neuronal sensory function in Drosophila.

Cilia are microtubule-based, hair-like organelles involved in sensory function or motility, playing critical roles in many physiological processes such as reproduction, organ development, and sensory perception. In insects, cilia are restricted to certain sensory neurons and sperms, being important for chemical and mechanical sensing, and fertility. Although great progress has been made regarding the mechanism of cilia assembly, the formation of insect cilia remains poorly understand, even in the insect model organism Drosophila. Intraflagellar transport (IFT) is a cilia-specific complex that traffics protein cargos bidirectionally along the ciliary axoneme and is essential for most cilia. Here we investigated the role of IFT52, a core component of IFT-B, in cilia/flagellar formation in Drosophila. We show that Drosophila IFT52 is distributed along the sensory neuronal cilia, and is essential for sensory cilia formation. Deletion of Ift52 results in severe defects in cilia-related sensory behaviors. It should be noted that IFT52 is not detected in spermatocyte cilia or sperm flagella of Drosophila. Accordingly, ift52 mutants can produce sperms with normal motility, supporting a dispensable role of IFT in Drosophila sperm flagella formation. Altogether, IFT52 is a conserved protein essential for sensory cilia formation and sensory neuronal function in insects.

Beyond deep-sea mining sublethal effects: Delayed mortality from acute Cu exposure of the cold-water octocoral Viminella flagellum.

The potential release of metals, especially copper (Cu) during mining of seafloor massive sulphides (SMS), represents a potential toxicological threat to cold-water coral (CWC) habitats. Herein, we evaluated for the first time the response of the whip coral Viminella flagellum to short-term acute Cu exposure. Nubbins of V. flagellum were exposed to Cu concentrations of 0 (control); 60; 150; 250; 450 and 600 µg/L for 96 h. After exposure, V. flagellum nubbins were transferred to a continuous flow-through aquarium and feed once a day for 3 weeks. No immediate mortality was detected during the short-term Cu exposure. However, a delayed mortality, which was concentration dependent was observed. The first signs of tissue loss occurred after 1 week of recovery in non-contaminated conditions in V. flagellum nubbins previously exposed to Cu concentrations of 60 and 150 µg/L followed by nubbins exposed to Cu concentrations of 250, 450 µg/L after 2 weeks and 600 µg/L after 3 weeks. A delayed mortality impact should be considered in future Cu tolerance experiments and scenarios of deep-sea mining exploitation.

Structure of MotA, a flagellar stator protein, from hyperthermophile.

Many motile bacteria swim and swarm toward favorable environments using the flagellum, which is rotated by a motor embedded in the inner membrane. The motor is composed of the rotor and the stator, and the motor torque is generated by the change of the interaction between the rotor and the stator induced by the ion flow through the stator. A stator unit consists of two types of membrane proteins termed A and B. Recent cryo-EM studies on the stators from mesophiles revealed that the stator consists of five A and two B subunits, whereas the low-resolution EM analysis showed that purified hyperthermophilic MotA forms a tetramer. To clarify the assembly formation and factors enhancing thermostability of the hyperthermophilic stator, we determined the cryo-EM structure of MotA from Aquifex aeolicus (Aa-MotA), a hyperthermophilic bacterium, at 3.42 Å resolution. Aa-MotA forms a pentamer with pseudo C5 symmetry. A simulated model of the Aa-MotA5MotB2 stator complex resembles the structures of mesophilic stator complexes, suggesting that Aa-MotA can assemble into a pentamer equivalent to the stator complex without MotB. The distribution of hydrophobic residues of MotA pentamers suggests that the extremely hydrophobic nature in the subunit boundary and the transmembrane region is a key factor to stabilize hyperthermophilic Aa-MotA.

Structure and Assembly of the Bacterial Flagellum.

The bacterial flagellum is a large macromolecular assembly that acts as propeller, providing motility through the rotation of a long extracellular filament. It is composed of over 20 different proteins, many of them highly oligomeric. Accordingly, it has attracted a huge amount of interest amongst researchers and the wider public alike. Nonetheless, most of its molecular details had long remained elusive.This however has changed recently, with the emergence of cryo-EM to determine the structure of protein assemblies at near-atomic resolution. Within a few years, the atomic details of most of the flagellar components have been elucidated, revealing not only its overall architecture but also the molecular details of its rotation mechanism. However, many questions remained unaddressed, notably on the complexity of the assembly of such an intricate machinery.In this chapter, we review the current state of our understanding of the bacterial flagellum structure, focusing on the recent development from cryo-EM. We also highlight the various elements that still remain to be fully characterized. Finally, we summarize the existing model for flagellum assembly and discuss some of the outstanding questions that are still pending in our understanding of the diversity of assembly pathways.

Single p197 molecules of the mitochondrial genome segregation system of Trypanosoma brucei determine the distance between basal body and outer membrane.

The tripartite attachment complex (TAC) couples the segregation of the single unit mitochondrial DNA of trypanosomes with the basal body (BB) of the flagellum. Here, we studied the architecture of the exclusion zone filament (EZF) of the TAC, the only known component of which is p197, that connects the BB with the mitochondrial outer membrane (OM). We show that p197 has three domains that are all essential for mitochondrial DNA inheritance. The C terminus of p197 interacts with the mature and probasal body (pro-BB), whereas its N terminus binds to the peripheral OM protein TAC65. The large central region of p197 has a high α-helical content and likely acts as a flexible spacer. Ultrastructure expansion microscopy (U-ExM) of cell lines exclusively expressing p197 versions of different lengths that contain both N- and C-terminal epitope tags demonstrates that full-length p197 alone can bridge the ∼270-nm distance between the BB and the cytosolic face of the OM. Thus U-ExM allows the localization of distinct domains within the same molecules and suggests that p197 is the TAC subunit most proximal to the BB. In addition, U-ExM revealed that p197 acts as a spacer molecule, as two shorter versions of p197, with the repeat domain either removed or replaced by the central domain of the Trypanosoma cruzi p197 ortholog reduced the distance between the BB and the OM in proportion to their predicted molecular weight.

Self-assembled flagella protein nanofibers induce enhanced mucosal immunity.

Nanofibers are potential vaccines or adjuvants for vaccination at the mucosal interface. However, how their lengths affect the mucosal immunity is not well understood. Using length-tunable flagella (self-assembled from a protein termed flagellin) as model protein nanofibers, we studied the mechanisms of their interaction with mucosal interface to induce immune responses length-dependently. Briefly, through tuning flagellin assembly, length-controlled protein nanofibers were prepared. The shorter nanofibers exhibited more pronounced toll-like receptor 5 (TLR5) and inflammasomes activation accompanied by pyroptosis, as a result of cellular uptake, lysosomal damage, and mitochondrial reactive oxygen species generation. Accordingly, the shorter nanofibers elevated the IgA level in mucosal secretions and enhanced the serum IgG level in ovalbumin-based intranasal vaccinations. These mucosal and systematic antibody responses were correlated with the mucus penetration capacity of the nanofibers. Intranasal administration of vaccines (human papillomavirus type 16 peptides) adjuvanted with shorter nanofibers significantly elicited cytotoxic T lymphocyte responses, strongly inhibiting tumor growth and improving survival rates in a TC-1 cervical cancer model. This work suggests that length-dependent immune responses of nanofibers can be elucidated for designing nanofibrous vaccines and adjuvants for both infectious diseases and cancer.

Direct Measurement of the Stall Torque of the Flagellar Motor in Escherichia coli with Magnetic Tweezers.

The flagellar motor drives the rotation of flagellar filaments, propelling the swimming of flagellated bacteria. The maximum torque the motor generates, the stall torque, is a key characteristic of the motor function. Direct measurements of the stall torque carried out 3 decades ago suffered from large experimental uncertainties, and subsequently there were only indirect measurements. Here, we applied magnetic tweezers to directly measure the stall torque in E. coli. We precisely calibrated the torsional stiffness of the magnetic tweezers and performed motor resurrection experiments at stall, accomplishing a precise determination of the stall torque per torque-generating unit (stator unit). From our measurements, each stator passes 2 protons per step, indicating a tight coupling between motor rotation and proton flux. IMPORTANCE The maximum torque the bacterial flagellar motor generates, the stall torque, is a critical parameter that describes the motor energetics. As the motor operates in equilibrium near stall, from the stall torque one can determine how many protons each torque-generating unit (stator) of the motor passes per revolution and then test whether motor rotation and proton flux are tightly or loosely coupled, which has been controversial in recent years. Direct measurements performed 3 decades ago suffered from large uncertainties, and subsequently, only indirect measurements were attempted, obtaining a range of values inconsistent with the previous direct measurements. Here, we developed a method that used magnetic tweezers to perform motor resurrection experiments at stall, resulting in a direct precise measurement of the stall torque per stator. Our study resolved the previous inconsistencies and provided direct experimental support for the tight coupling mechanism between motor rotation and proton flux.

Flagellin outer domain dimerization modulates motility in pathogenic and soil bacteria from viscous environments.

Flagellar filaments function as the propellers of the bacterial flagellum and their supercoiling is key to motility. The outer domains on the surface of the filament are non-critical for motility in many bacteria and their structures and functions are not conserved. Here, we show the atomic cryo-electron microscopy structures for flagellar filaments from enterohemorrhagic Escherichia coli O157:H7, enteropathogenic E. coli O127:H6, Achromobacter, and Sinorhizobium meliloti, where the outer domains dimerize or tetramerize to form either a sheath or a screw-like surface. These dimers are formed by 180° rotations of half of the outer domains. The outer domain sheath (ODS) plays a role in bacterial motility by stabilizing an intermediate waveform and prolonging the tumbling of E. coli cells. Bacteria with these ODS and screw-like flagellar filaments are commonly found in soil and human intestinal environments of relatively high viscosity suggesting a role for the dimerization in these environments.

Biosynthesis of Linear Protein Nanoarrays Using the Flagellar Axoneme.

Applications in biotechnology and synthetic biology often make use of soluble proteins, but there are many potential advantages of anchoring enzymes to a stable substrate, including stability and the possibility for substrate channeling. To avoid the necessity of protein purification and chemical immobilization, there has been growing interest in bio-assembly of protein-containing nanoparticles, exploiting the self-assembly of viral capsid proteins or other proteins that form polyhedral structures. However, these nanoparticles are limited in size, which constrains the packaging and the accessibility of the proteins. An axoneme, the insoluble protein core of the eukaryotic flagellum or cilium, is a highly ordered protein structure that can be several microns in length, orders of magnitude larger than other types of nanoparticles. We show that when proteins of interest are fused to specific axonemal proteins and expressed in living Chlamydomonas reinhardtii cells, they become incorporated into linear arrays, which have the advantages of high protein loading capacity and single-step purification with retention of biomass. The arrays can be isolated as membrane-enclosed vesicles or as exposed protein arrays. The approach is demonstrated for both a fluorescent protein and an enzyme (beta-lactamase), showing that incorporation into axonemes retains protein function in a stable, easily isolated array form.

Cryo-electron Tomography Reveals the Roles of FliY in Helicobacter pylori Flagellar Motor Assembly.

Helicobacter pylori plays a causative role in gastric diseases. The pathogenicity of H. pylori depends on its ability to colonize the stomach guided by motility. FliY is a unique flagellar motor switch component coexisting with the classical FliG, FliM, and FliN switch proteins in some bacteria and has been shown to be essential for flagellation. However, the functional importance of FliY in H. pylori flagellar motor assembly is not well understood. Here, we applied cryo-electron tomography and subtomogram averaging to analyze the in situ structures of flagellar motors from wild-type strain, fliY-null mutant and complementation mutants expressing the N-terminal or C-terminal domain of FliY. Loss of full-length FliY or its C-terminal domain interrupted the formation of an intact C ring and soluble export apparatus, as well as the hook and flagellar filaments. Complementation with FliY C-terminal domain restored all these missing components of flagellar motor. Taken together, these results provide structural insights into the roles of FliY, especially its C-terminal domain in flagellar motor assembly in H. pylori. IMPORTANCE Helicobacter pylori is the major risk factor related with gastric diseases. Flagellar motor is one of the most important virulence factors in H. pylori. However, the assembly mechanism of H. pylori flagellar motor is not fully understood yet. Previous report mainly described the overall structures of flagellum but had not focused on its specific components. Here, we focus on H. pylori flagellar C-ring protein FliY. We directly visualize the flagellar structures of H. pylori wild-type and FliY N-/C-terminal complementary strains by cryo-electron tomography and subtomogram averaging. Our results show that deletion of FliY or its C-terminal domain causes the loss of C ring, whereas deletion of FliY N-terminal does not affect C-ring assembly and flagellar structures. Our results provide direct evidence that C-ring protein FliY, especially its C-terminal domain, plays an indispensable role in H. pylori motor assembly and flagellar formation. This study will deepen our understanding about H. pylori pathogenesis.