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A number of extracellular helical protein polymers are crucial for supporting bacterial motility. The bacterial flagellum is a polymeric appendage used to support cellular motility. Historically, structural studies of flagellar and other filaments were limited to those present as or locked into straightened states. Here, we present a robust workflow that produces biologically relevant high-resolution cryo-electron microscopy (cryo-EM) structures of bacterial flagellar filaments. We highlight how a simple purification method, centered around several centrifugation steps, exploits the process of filament ejection in Caulobacter crescentus and results in isolated filaments amenable to transmission electron microscopy (TEM) studies. The quality of the sample is validated by SDS-PAGE and negative stain TEM analysis before a sample is vitrified for cryogenic electron microscopy (cryo-EM) data collection. We provide a detailed protocol for reconstructing either straight or curved flagellar filaments by cryo-EM helical reconstruction methods, followed by an overview of model building and validation. In our hands, this workflow resulted in several flagellar structures below 3 Å resolution, with one data set reaching a global resolution of 2.1 Å. The application of this workflow supports structure-function studies to better understand the molecular interactions that regulate filament architecture in biologically relevant states. Future work will not only examine interactions that regulate bacterial flagellar and other filament organization but also provide a foundation for developing new helical biopolymers for biotech applications. Key features • Rapid high-quality purification of bacterial flagella via simple bacterial culturing, centrifugation, and resuspension methods. • High-throughput cryo-EM data collection of filamentous objects. • Use of cryoSPARC implementations of helical reconstruction algorithms to generate high-resolution 3D structures of bacterial flagella or other helical polymers.

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Swarming motility is a type of movement used by pathogenic flagellated bacteria as virulence factor to colonize surfaces and cause damage to the host. Vibrio parahaemolyticus is a pathogenic flagellated bacterium that increases its virulence by switching from swimmer to swarming cells. The hosts of pathogenic V. parahaemolyticus include farmed shrimp. Therefore, methods to detect and quantify this movement are important to control shrimp diseases caused by pathogenic V. parahaemolyticus strains. We developed an optimized swarming motility assay by identifying the most optimal type of agar, and drying time of the culture medium, agar concentration and volume of the bacterial culture to achieve the fastest swarming motility during the migration of V. parahaemolyticus on Petri dishes during a 24-hour incubation period. The method includes data analysis that could be used as a tool to identify potential anti-virulence products by comparing the slopes of the linearized diameters of the swarming halos of bacteria treated with the products, as they migrate on Petri dishes over a 24-hour incubation period. Here we report:•A simple method for detection and quantification of swarming motility halos of V. parahaemolyticus bacteria.•A method that could be used as a tool to identify potential anti-virulence products.

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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.

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The combination of immunotherapy and chemotherapy to ablate tumors has attracted substantial attention due to the ability to simultaneously elicit antitumor immune responses and trigger direct tumor cell death. However, conventional combinational strategies mainly focus on the employment of drug carriers to deliver immunomodulators, chemotherapeutics, or their combinations, always suffering from complicated preparation and carrier-relevant side effects. Here, the fabrication of bacterial flagellum-drug nanoconjugates (FDNCs) for carrier-free immunochemotherapy is described. FDNCs are simply prepared by attaching chemotherapeutics to amine residues of flagellin through an acid-sensitive and traceless cis-aconityl linker. By virtue of native nanofibrous structure and immunogenicity, bacterial flagella not only show long-term tumor retention and highly efficient cell internalization, but also provoke robust systemic antitumor immune responses. Meanwhile, conjugated chemotherapeutics exhibit an acid-mediated release profile and durable intratumoral exposure, which can induce potent tumor cell inhibition via direct killing. More importantly, this combination is able to augment immunoactivation effects associated with chemotherapy-enabled immunogenic tumor cell death to further enhance antitumor efficacy. By leveraging the innate response of the immune system to pathogens, the conjugation of therapeutic agents with self-adjuvant bacterial flagella provides an alternative approach to develop carrier-free nanotherapeutics for tumor immunochemotherapy.

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Most motile bacteria use supramolecular motility machinery called bacterial flagellum, which converts the chemical energy gained from ion flux into mechanical rotation. Bacterial cells sense their external environment through a two-component regulatory system consisting of a histidine kinase and response regulator. Combining these systems allows the cells to move toward favorable environments and away from their repellents. A representative example of flagellar motility is run-and-tumble swimming in Escherichia coli, where the counter-clockwise (CCW) rotation of a flagellar bundle propels the cell forward, and the clockwise (CW) rotation undergoes cell re-orientation (tumbling) upon switching the direction of flagellar motor rotation from CCW to CW. In this mini review, we focus on several types of chemotactic behaviors that respond to changes in flagellar shape and direction of rotation. Moreover, our single-cell analysis demonstrated back-and-forth swimming motility of an original E. coli strain. We propose that polymorphic flagellar changes are required to enhance bacterial movement in a structured environment as a colony spread on an agar plate.

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Two of the most fascinating bacterial nanomachines-the broadly disseminated rotary flagellum at the heart of cellular motility and the eukaryotic cell-puncturing injectisome essential to specific pathogenic species-utilize at their core a conserved export machinery called the type III secretion system (T3SS). The T3SS not only secretes the components that self-assemble into their extracellular appendages but also, in the case of the injectisome, subsequently directly translocates modulating effector proteins from the bacterial cell into the infected host. The injectisome is thought to have evolved from the flagellum as a minimal secretory system lacking motility, with the subsequent acquisition of additional components tailored to its specialized role in manipulating eukaryotic hosts for pathogenic advantage. Both nanomachines have long been the focus of intense interest, but advances in structural and functional understanding have taken a significant step forward since 2015, facilitated by the revolutionary advances in cryo-electron microscopy technologies. With several seminal structures of each nanomachine now captured, we review here the molecular similarities and differences that underlie their diverse functions.

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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.

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Many motile bacteria employ the flagellar type III secretion system (fT3SS) to build the flagellum on the cell surface. The fT3SS consists of a transmembrane export gate complex, which acts as a proton/protein antiporter that couples proton flow with flagellar protein export, and a cytoplasmic ATPase ring complex, which works as an activator of the export gate complex. Three transmembrane proteins, FliP, FliQ, and FliR, form a core structure of the export gate complex, and this core complex serves as a polypeptide channel that allows flagellar structural subunits to be translocated across the cytoplasmic membrane. Here, we describe the methods for overproduction, solubilization, and purification of the Salmonella FliP/FliQ/FliR complex.

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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.

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The bacterial flagellum is a large assembly of about 30 different proteins and is divided into three parts: the filament that acts as a screw propeller, the hook as a universal joint, and the basal body as a rotary motor. In the case of Salmonella, the filament length is 10-15 µm, which is more than ten times longer than the size of the cell. The filament is composed of only one component protein, flagellin, and is made of 11 protofilaments. The filament can form 12 different supercoiled structures as polymorphic forms. Each protofilament can take either the L (left-handed) or R (right-handed) state, and the number ratio of the protofilaments in these two states determines the shape of the supercoil. Some point mutations in flagellin make the filament straight by making all the protofilaments in one of the two states. The straight filaments enable us to use their helical symmetries for structural analysis by electron cryomicroscopy (cryoEM) and single particle image analysis. Here, we describe the methods for the purification of the flagellar filament and cryoEM data collection and image analysis.

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The bacterial flagellum employs a rotary motor embedded on the cell surface. The motor consists of the stator and rotor elements and is driven by ion influx (typically H+ or Na+) through an ion channel of the stator. Ion influx induces conformational changes in the stator, followed by changes in the interactions between the stator and rotor. The driving force to rotate the flagellum is thought to be generated by changing the stator-rotor interactions. In this chapter, we describe two methods for investigating the interactions between the stator and rotor: site-directed in vivo photo-crosslinking and site-directed in vivo cysteine disulfide crosslinking.

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The flagellar motor of marine Vibrio is driven by the sodium-motive force across the inner membrane. The stator complex, consisting of two membrane proteins PomA and PomB, is responsible for energy conversion in the motor. To understand the coupling of the Na+ flux with torque generation, it is essential to clearly identify the Na+-binding sites and the Na+ flux pathway through the stator channel. Although residues essential for Na+ flux have been identified by using mutational analysis, it has been difficult to observe Na+ binding to the PomAB stator complex. Here we describe a method to monitor the binding of Na+ to purified PomAB stator complex using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. This method demonstrates that Na+-binding sites are formed by critical aspartic acid and threonine residues located in the transmembrane segments of PomAB.

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Kin discrimination in nature is an effective way for bacteria to stabilize population cooperation and maintain progeny benefits. However, so far, the research on kin discrimination for Bacillus still has concentrated on "attack and defense" between cells and diffusion-dependent molecular signals of quorum sensing, kin recognition in Bacillus, however, has not been reported. To determine whether flagellar is involve in the kin recognition of Bacillus, we constructed Bacillus velezensis SQR9 assembled with flagellin of its kin and non-kin strains, and performed a swarm boundary assay with SQR9, then analyzed sequence variation of flagellin and other flagellar structural proteins in B. velezensis genus. Our results showed that SQR9 assembled with flagellin of non-kin strains was more likely to form a border phenotype with wild-type strain SQR9 in swarm assay than that of kin strains, and that non-kin strains had greater variation in flagellin than kin strains. In B. velezensis, these variations in flagellin were prevalent and had evolved significantly faster than other flagellar structural proteins. Therefore, we proposed that flagellin is an effective tool partly involved in the kin recognition of B. velezensis strains. IMPORTANCE Kin selection plays an important role in stabilizing population cooperation and maintaining the progeny benefits for bacteria in nature. However, to date, the role of flagellin in kin recognition in Bacillus has not been reported. By using rhizospheric Bacillus velezensis SQR9, we accomplished flagellin region interchange among its related strains, and show that flagellin acts as a mediator to distinguish kin from non-kin in B. velezensis. We demonstrated the polymorphism of flagellin in B. velezensis through alignment analysis of flagellin protein sequences. Therefore, it was proposed that flagellin was likely to be an effective tool for mediating kin recognition in B. velezensis.

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Flagellar structural subunits are transported via the flagellar type III secretion system (fT3SS) and assemble at the distal end of the growing flagellar structure. The C-terminal cytoplasmic domain of FlhA (FlhAC) serves as a docking platform for export substrates and flagellar chaperones and plays an important role in hierarchical protein targeting and export. FlhAC consists of domains D1, D2, D3, and D4 and adopts open and closed conformations. Gly-368 of Salmonella FlhA is located within the highly conserved GYXLI motif and is critical for the dynamic domain motions of FlhAC. However, it remains unclear how it works. Here, we report that periodic conformational changes of the GYXLI motif induce a remodeling of hydrophobic side chain interaction networks in FlhAC and promote the cyclic open-close domain motions of FlhAC. The temperature-sensitive flhA(G368C) mutation stabilized a completely closed conformation at 42°C through strong hydrophobic interactions between Gln-498 of domain D1 and Pro-667 of domain D4 and between Phe-459 of domain D2 and Pro-646 of domain D4, thereby inhibiting flagellar protein export by the fT3SS. Its intragenic suppressor mutations reorganized the hydrophobic interaction networks in the closed FlhAC structure, restoring the protein export activity of the fT3SS to a significant degree. Furthermore, the conformational flexibility of the GYXLI motif was critical for flagellar protein export. We propose that the conserved GYXLI motif acts as a structural switch to induce the dynamic domain motions of FlhAC required for efficient and rapid protein export by the fT3SS. IMPORTANCE Many motile bacteria employ the flagellar type III secretion system (fT3SS) to construct flagella beyond the cytoplasmic membrane. The C-terminal cytoplasmic domain of FlhA (FlhAC), a transmembrane subunit of the fT3SS, provides binding sites for export substrates and flagellar export chaperones to coordinate flagellar protein export with assembly. FlhAC undergoes cyclic open-close domain motions. The highly conserved Gly-368 residue of FlhA is postulated to be critical for dynamic domain motions of FlhAC. However, it remains unknown how it works. Here, we carried out mutational analysis of FlhAC combined with molecular dynamics simulation and provide evidence that the conformational flexibility of FlhAC by Gly-368 is important for remodeling hydrophobic side chain interaction networks in FlhAC to facilitate its cyclic open-close domain motions, allowing the fT3SS to transport flagellar structural subunits for efficient and rapid flagellar assembly.

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Marine bacterium Vibrio alginolyticus forms a single flagellum at a cell pole. In Vibrio, two proteins (GTPase FlhF and ATPase FlhG) regulate the number of flagella. We previously isolated the NMB155 mutant that forms multiple flagella despite the absence of mutations in flhF and flhG. Whole-genome sequencing of NMB155 identified an E9K mutation in FliM that is a component of C-ring in the flagellar rotor. Mutations in FliM result in defects in flagellar formation (fla) and flagellar rotation (che or mot); however, there are a few reports indicating that FliM mutations increase the number of flagella. Here, we determined that the E9K mutation confers the multi-flagellar phenotype and also the che phenotype. The co-expression of wild-type FliM and FliM-E9K indicated that they were competitive in regard to determining the flagellar number. The ATPase activity of FlhG has been correlated with the number of flagella. We observed that the ATPase activity of FlhG was increased by the addition of FliM but not by the addition of FliM-E9K in vitro. This indicates that FliM interacts with FlhG to increase its ATPase activity, and the E9K mutation may inhibit this interaction. FliM may control the ATPase activity of FlhG to properly regulate the number of the polar flagellum at the cell pole.

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Vibrio has a polar flagellum driven by sodium ions for swimming. The force-generating stator unit consists of PomA and PomB. PomA contains four transmembrane regions and a cytoplasmic domain of approximately 100 residues, which interacts with the rotor protein, FliG, to be important for the force generation of rotation. The 3D structure of the stator shows that the cytosolic interface (CI) helix of PomA is located parallel to the inner membrane. In this study, we investigated the function of CI helix and its role as stator. Systematic proline mutagenesis showed that residues K64, F66 and M67 were important for this function. The mutant stators did not assemble around the rotor. Moreover, the growth defect caused by PomB plug deletion was suppressed by these mutations. We speculate that the mutations affect the structure of the helices extending from TM3 and TM4 and reduce the structural stability of the stator complex. This study suggests that the helices parallel to the inner membrane play important roles in various processes, such as the hoop-like function in securing the stability of the stator complex and the ion conduction pathway, which may lead to the elucidation of the ion permeation and assembly mechanism of the stator.

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Typical second messengers include cyclic AMP (cAMP), cyclic GMP (cGMP), and inositol phosphate. In bacteria, cyclic diguanylate (c-di-GMP), which is not used in animals, is widely used as a second messenger for environmental responses. Initially found as a regulator of cellulose synthesis, this small molecule is known to be widely present in bacteria. A wide variety of synthesis and degradation enzymes for c-di-GMP exist, and the activities of effector proteins are regulated by changing the cellular c-di-GMP concentration in response to the environment. It has been shown well that c-di-GMP plays an essential role in pathogenic cycle and is involved in flagellar motility in Vibrio cholerae. In this review, we aim to explain the direct or indirect regulatory mechanisms of c-di-GMP in bacteria, focusing on the study of c-di-GMP in Vibrio spp. and in flagella, which are our research subjects.

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In 1980s, the most genes involved in the bacterial flagellar function and formation had been isolated, although many of their functions or roles were not clarified. Bacterial flagella are the primary locomotive organ and are not necessary for growing in vitro but are probably essential for living in natural condition and are involved in the pathogenicity. In vitro, the flagella-deficient strains can grow at rates similar to wild-type strains. More than 50 genes are responsible for flagellar function, and the flagellum is constructed by more than 20 structural proteins. The maintenance cost of flagellum is high as several genes are required for its development. The fact that it evolved as a motor organ even with such high cost shows that the motility is indispensable to survive under the harsh environment of Earth. In this review, we focus on flagella-related research conducted by the authors for about 40 years and flagellar research focused on Vibrio spp.

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Bacterial flagella are the best-known rotational organelles in the biological world. The spiral-shaped flagellar filaments that extend from the cell surface rotate like a screw to create a propulsive force. At the base of the flagellar filament lies a protein motor that consists of a stator and a rotor embedded in the membrane. The stator is composed of two types of membrane subunits, PomA (similar to MotA in Escherichia coli) and PomB (similar to MotB in E. coli), which are energy converters that assemble around the rotor to couple rotation with the ion flow. Recently, stator structures, where two MotB molecules are inserted into the center of a ring made of five MotA molecules, were reported. This structure inspired a model in which the MotA ring rotates around the MotB dimer in response to ion influx. Here, we focus on the Vibrio PomB plug region, which is involved in flagellar motor activation. We investigated the plug region using site-directed photo-cross-linking and disulfide cross-linking experiments. Our results demonstrated that the plug interacts with the extracellular short loop region of PomA, which is located between transmembrane helices 3 and 4. Although the motor stopped rotating after cross-linking, its function recovered after treatment with a reducing reagent that disrupted the disulfide bond. Our results support the hypothesis, which has been inferred from the stator structure, that the plug region terminates the ion influx by blocking the rotation of the rotor as a spanner. IMPORTANCE The biological flagellar motor resembles a mechanical motor. It is composed of a stator and a rotor. The force is transmitted to the rotor by the gear-like stator movements. It has been proposed that the pentamer of MotA subunits revolves around the axis of the B subunit dimer in response to ion flow. The plug region of the B subunit regulates the ion flow. Here, we demonstrated that the ion flow was terminated by cross-linking the plug region of PomB with PomA. These findings support the rotation hypothesis and explain the role of the plug region in blocking the rotation of the stator unit.

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We report evidence further supporting homology between proteins in the F1 FO -ATP synthetase and the bacterial flagellar motor (BFM). BFM proteins FliH, FliI, and FliJ have been hypothesized to be homologous to FO -b + F1 -δ, F1 -α/ß, and F1 -γ, with similar structure and interactions. We conduct a further test by constructing a gene order dataset, examining the order of fliH, fliI, and fliJ genes across the phylogenetic breadth of flagellar and nonflagellar type 3 secretion systems, and comparing this to published surveys of gene order in the F1 FO -ATP synthetase, its N-ATPase relatives, and the bacterial/archaeal V- and A-type ATPases. Strikingly, the fliHIJ gene order was deeply conserved, with the few exceptions appearing derived, and exactly matching the widely conserved F-ATPase gene order atpFHAG, coding for subunits b-δ-α-γ. The V/A-type ATPases have a similar conserved gene order. Our results confirm homology between these systems, and suggest a rare case of synteny conserved over billions of years, predating the Last Universal Common Ancestor (LUCA).

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