Structural Insights into Type III Secretion Systems of the Bacterial Flagellum and Injectisome.

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.

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.

Bacterial Flagellum-Drug Nanoconjugates for Carrier-Free Immunochemotherapy.

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.

Membrane voltage-dependent activation mechanism of the bacterial flagellar protein export apparatus.

The proton motive force (PMF) consists of the electric potential difference (Δψ), which is measured as membrane voltage, and the proton concentration difference (ΔpH) across the cytoplasmic membrane. The flagellar protein export machinery is composed of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase ring complex consisting of FliH, FliI, and FliJ. ATP hydrolysis by the FliI ATPase activates the export gate complex to become an active protein transporter utilizing Δψ to drive proton-coupled protein export. An interaction between FliJ and a transmembrane ion channel protein, FlhA, is a critical step for Δψ-driven protein export. To clarify how Δψ is utilized for flagellar protein export, we analyzed the export properties of the export gate complex in the absence of FliH and FliI. The protein transport activity of the export gate complex was very low at external pH 7.0 but increased significantly with an increase in Δψ by an upward shift of external pH from 7.0 to 8.5. This observation suggests that the export gate complex is equipped with a voltage-gated mechanism. An increase in the cytoplasmic level of FliJ and a gain-of-function mutation in FlhA significantly reduced the Δψ dependency of flagellar protein export by the export gate complex. However, deletion of FliJ decreased Δψ-dependent protein export significantly. We propose that Δψ is required for efficient interaction between FliJ and FlhA to open the FlhA ion channel to conduct protons to drive flagellar protein export in a Δψ-dependent manner.

Formation of multiple flagella caused by a mutation of the flagellar rotor protein FliM in Vibrio alginolyticus.

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.

Roles of the second messenger c-di-GMP in bacteria: Focusing on the topics of flagellar regulation and Vibrio spp.

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.

Flagellar export apparatus and ATP synthetase: Homology evidenced by synteny predating the Last Universal Common Ancestor.

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).

Achievements in bacterial flagellar research with focus on Vibrio species.

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.

Architecture and Assembly of the Bacterial Flagellar Motor Complex.

One of the central systems responsible for bacterial motility is the flagellum. The bacterial flagellum is a macromolecular protein complex that is more than five times the cell length. Flagella-driven motility is coordinated via a chemosensory signal transduction pathway, and so bacterial cells sense changes in the environment and migrate towards more desirable locations. The flagellum of Salmonella enterica serovar Typhimurium is composed of a bi-directional rotary motor, a universal joint and a helical propeller. The flagellar motor, which structurally resembles an artificial motor, is embedded within the cell envelop and spins at several hundred revolutions per second. In contrast to an artificial motor, the energy utilized for high-speed flagellar motor rotation is the inward-directed proton flow through a transmembrane proton channel of the stator unit of the flagellar motor. The flagellar motor realizes efficient chemotaxis while performing high-speed movement by an ingenious directional switching mechanism of the motor rotation. To build the universal joint and helical propeller structures outside the cell body, the flagellar motor contains its own protein transporter called a type III protein export apparatus. In this chapter we summarize the structure and assembly of the Salmonella flagellar motor complex.

Putative Spanner Function of the Vibrio PomB Plug Region in the Stator Rotation Model for Flagellar Motor.

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.

Characterization of the MinD/ParA-type ATPase FlhG in Vibrio alginolyticus and implications for function of its monomeric form.

FlhG is a MinD/ParA-type ATPase that works as a negative regulator for flagellar biogenesis. In Vibrio alginolyticus, FlhG functions antagonistically with the positive regulator FlhF to generate a single polar flagellum. Here, we examined the effects of ADP and ATP on the aggregation and dimerization of Vibrio FlhG. Purified FlhG aggregated after exposure to low NaCl conditions, and its aggregation was suppressed in the presence of ADP or ATP. FlhG mutants at putative ATP-binding (K31A) or catalytic (D60A) residues showed similar aggregation profiles to the wild type, but ATP caused strong aggregation of the ATPase-stimulated D171A mutant although ADP significantly suppressed the aggregation. Results of size exclusion chromatography of purified FlhG or Vibrio cell lysates suggested that FlhG exists as a monomer in solution, and ATP does not induce FlhG dimerization. The K31A and D60A mutants eluted at monomer fractions regardless of nucleotides, but ATP shifted the elution peak of the D171A mutant to slightly earlier, presumably because of a subtle conformational change. Our results suggest that monomeric FlhG can function in vivo, whose active conformation aggregates easily.

Molecular Organization and Assembly of the Export Apparatus of Flagellar Type III Secretion Systems.

The bacterial flagellum is a supramolecular motility machine consisting of the basal body, the hook, and the filament. For construction of the flagellum beyond the cellular membranes, a type III protein export apparatus uses ATP and proton-motive force (PMF) across the cytoplasmic membrane as the energy sources to transport flagellar component proteins from the cytoplasm to the distal end of the growing flagellar structure. The protein export apparatus consists of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase complex. In addition, the basal body C ring acts as a sorting platform for the cytoplasmic ATPase complex that efficiently brings export substrates and type III export chaperone-substrate complexes from the cytoplasm to the export gate complex. In this book chapter, we will summarize our current understanding of molecular organization and assembly of the flagellar type III protein export apparatus.

Characterization of FlgP, an Essential Protein for Flagellar Assembly in Rhodobacter sphaeroides.

The flagellar lipoprotein FlgP has been identified in several species of bacteria, and its absence provokes different phenotypes. In this study, we show that in the alphaproteobacterium Rhodobacter sphaeroides, a ΔflgP mutant is unable to assemble the hook and the filament. In contrast, the membrane/supramembrane (MS) ring and the flagellar rod appear to be assembled. In the absence of FlgP a severe defect in the transition from rod to hook polymerization occurs. In agreement with this idea, we noticed a reduction in the amount of intracellular flagellin and the chemotactic protein CheY4, both encoded by genes dependent on σ28 This suggests that in the absence of flgP the switch to export the anti-sigma factor, FlgM, does not occur. The presence of FlgP was detected by Western blot in samples of isolated wild-type filament basal bodies, indicating that FlgP is an integral part of the flagellar structure. In this regard, we show that FlgP interacts with FlgH and FlgT, indicating that FlgP should be localized closely to the L and H rings. We propose that FlgP could affect the architecture of the L ring, which has been recently identified to be responsible for the rod-hook transition.IMPORTANCE Flagellar based motility confers a selective advantage on bacteria by allowing migration to favorable environments or in pathogenic species to reach the optimal niche for colonization. The flagellar structure has been well established in Salmonella However, other accessory components have been identified in other species. Many of these have been implied in adapting the flagellar function to enable faster rotation, or higher torque. FlgP has been proposed to be the main component of the basal disk located underlying the outer membrane in Campylobacter jejuni and Vibrio fischeri Its role is still unclear, and its absence impacts motility differently in different species. The study of these new components will bring a better understanding of the evolution of this complex organelle.

Flagellin glycosylation with pseudaminic acid in Campylobacter and Helicobacter: prospects for development of novel therapeutics.

Many pathogenic bacteria require flagella-mediated motility to colonise and persist in their hosts. Helicobacter pylori and Campylobacter jejuni are flagellated epsilonproteobacteria associated with several human pathologies, including gastritis, acute diarrhea, gastric carcinoma and neurological disorders. In both species, glycosylation of flagellin with an unusual sugar pseudaminic acid (Pse) plays a crucial role in the biosynthesis of functional flagella, and thereby in bacterial motility and pathogenesis. Pse is found only in pathogenic bacteria. Its biosynthesis via six consecutive enzymatic steps has been extensively studied in H. pylori and C. jejuni. This review highlights the importance of flagella glycosylation and details structural insights into the enzymes in the Pse pathway obtained via a combination of biochemical, crystallographic, and mutagenesis studies of the enzyme-substrate and -inhibitor complexes. It is anticipated that understanding the underlying structural and molecular basis of the catalytic mechanisms of the Pse-synthesising enzymes will pave the way for the development of novel antimicrobials.

Insight into the flagella type III export revealed by the complex structure of the type III ATPase and its regulator.

FliI and FliJ form the FliI6FliJ ATPase complex of the bacterial flagellar export apparatus, a member of the type III secretion system. The FliI6FliJ complex is structurally similar to the α3ß3γ complex of F1-ATPase. The FliH homodimer binds to FliI to connect the ATPase complex to the flagellar base, but the details are unknown. Here we report the structure of the homodimer of a C-terminal fragment of FliH (FliHC2) in complex with FliI. FliHC2 shows an unusually asymmetric homodimeric structure that markedly resembles the peripheral stalk of the A/V-type ATPases. The FliHC2-FliI hexamer model reveals that the C-terminal domains of the FliI ATPase face the cell membrane in a way similar to the F/A/V-type ATPases. We discuss the mechanism of flagellar ATPase complex formation and a common origin shared by the type III secretion system and the F/A/V-type ATPases.

Biochemical and Phylogenetic Study of SltF, a Flagellar Lytic Transglycosylase from Rhodobacter sphaeroides.

In this work, we have characterized the soluble lytic transglycosylase (SltF) from Rhodobacter sphaeroides that interacts with the scaffolding protein FlgJ in the periplasm to open space at the cell wall peptidoglycan heteropolymer for the emerging rod. The characterization of the genetic context of flgJ and sltF in alphaproteobacteria shows that these two separate genes coexist frequently in a flagellar gene cluster. Two domains of unknown function in SltF were studied, and the results show that the deletion of a 17-amino-acid segment near the N terminus does not show a recognizable phenotype, whereas the deletion of 47 and 95 amino acids of the C terminus of SltF disrupts the interaction with FlgJ without affecting the transglycosylase catalytic activity of SltF. These mutant proteins are unable to support swimming, indicating that the physical interaction between SltF and FlgJ is central for flagellar formation. In a maximum likelihood tree of representative lytic transglycosylases, all of the flagellar SltF proteins cluster in subfamily 1F. From this analysis, it was also revealed that the lytic transglycosylases related to the type III secretion systems present in pathogens cluster with the closely related flagellar transglycosylases.IMPORTANCE Flagellar biogenesis is a highly orchestrated event where the flagellar structure spans the bacterial cell envelope. The rod diameter of approximately 4 nm is larger than the estimated pore size of the peptidoglycan layer; hence, its insertion requires the localized and controlled lysis of the cell wall. We found that a 47-residue domain of the C terminus of the lytic transglycosylase (LT) SltF of R. sphaeroides is involved in the recognition of the rod chaperone FlgJ. We also found that in many alphaproteobacteria, the flagellar cluster includes a homolog of SltF and FlgJ, indicating that association of an LT with the flagellar machinery is ancestral. A maximum likelihood tree shows that family 1 of LTs segregates into seven subfamilies.

Insight into adaptive remodeling of the rotor ring complex of the bacterial flagellar motor.

The bacterial flagellar motor rotates in both counterclockwise (CCW) and clockwise (CW) directions. FliG, FliM and FliN form the C ring on the cytoplasmic face of the MS ring made of a transmembrane protein, FliF. The C ring acts not only as a rotor but also as a switch of the direction of motor rotation. FliG consists of three domains: FliGN, FliGM and FliGC. FliGN directly binds to FliF. Intermolecular interactions between FliGM and FliGC drive FliG ring formation. FliGM is responsible for the interaction with FliM. FliGC is involved in the interaction with the stator protein MotA. Adaptive remodeling of the C ring occurs when the motor switches between the CCW and CW states. However, it remained unknown how. Here, we report the effects of a CW-locked deletion mutation (ΔPEV) in FliG of Thermotaoga maritia (Tm-FliG) on FliG-FliG and FliG-FliM interactions. The PEV deletion stabilized the intramolecular interaction between FliGM and FliGC, thereby suppressing the oligomerization of Tm-FliGMC in solution. This deletion also induced a conformational change of HelixMC connecting FliGM and FliGC to reduce the binding affinity of Tm-FliGMC for FliM. We will discuss adaptive remodeling of the C ring responsible for flagellar motor switching.

A triangular loop of domain D1 of FlgE is essential for hook assembly but not for the mechanical function.

The bacterial flagellar hook is a short, curved tubular structure made of FlgE. The hook connects the basal body as a rotary motor and the filament as a helical propeller and functions as a universal joint to smoothly transmit torque produced by the motor to the filament. Salmonella FlgE consists of D0, Dc, D1 and D2 domains. Axial interactions between a triangular loop of domain D1 (D1-loop) and domain D2 are postulated to be responsible for hook supercoiling. In contrast, Bacillus FlgE lacks the D1-loop and domain D2. Here, to clarify the roles of the D1-loop and domain D2 in the mechanical function, we carried out deletion analysis of Salmonella FlgE. A deletion of the D1-loop conferred a loss-of-function phenotype whereas that of domain D2 did not. The D1-loop deletion inhibited hook polymerization. Suppressor mutations of the D1-loop deletion was located within FlgD, which acts as the hook cap to promote hook assembly. This suggests a possible interaction between the D1-loop of FlgE and FlgD. Suppressor mutant cells produced straight hooks, but retained the ability to form a flagellar bundle behind a cell body, suggesting that the loop deletion does not affect the bending flexibility of the Salmonella hook.

Architecture of divergent flagellar promoters controlled by CtrA in Rhodobacter sphaeroides.

BACKGROUND: Rhodobacter sphaeroides has two sets of flagellar genes, fla1 and fla2, that are responsible for the synthesis of two different flagellar structures. The expression of the fla2 genes is under control of CtrA. In several α-proteobacteria CtrA is also required for the expression of the flagellar genes, but the architecture of CtrA-dependent promoters has only been studied in detail in Caulobacter crescentus. In many cases the expression of fla genes originates from divergent promoters located a few base pairs apart, suggesting a particular arrangement of the cis-acting sites. RESULTS: Here we characterized several control regions of the R. sphaeroides fla2 genes and analyzed in detail two regions containing the divergent promoters flgB2p-fliI2p, and fliL2p-fliF2p. Binding sites for CtrA of these promoters were identified in silico and tested by site directed mutagenesis. We conclude that each one of these promoter regions has a particular arrangement, either a single CtrA binding site for activation of fliL2p and fliF2p, or two independent sites for activation of flgB2p and fliI2p. ChIP experiments confirmed that CtrA binds to the control region containing the flgB2 and fliI2 promoters, supporting the notion that CtrA directly controls the expression of the fla2 genes. The flgB and fliI genes are syntenic and show a short intercistronic region in closely related bacterial species. We analyzed these regions and found that the arrangement of the CtrA binding sites varies considerably. CONCLUSIONS: The results in this work reveal the arrangement of the fla2 divergent promoters showing that CtrA promotes transcriptional activation using more than a single architecture.

An intrinsically disordered linker controlling the formation and the stability of the bacterial flagellar hook.

BACKGROUND: In a macro-molecular complex, any minor change may prove detrimental. For a supra-molecular nano-machine like the bacterial flagellum, which consists of several distinct parts with specific characteristics, stability is important. During the rotation of the bacterial flagellar motor, which is located in the membrane, the flagella rotate at speeds between 200 and 2000 rpm, depending on the bacterial species. The hook substructure of the bacterial flagellum acts as a universal joint connecting the motor to the flagellar filament. We investigated the formation of the bacterial flagellar hook and its overall stability between the FlgE subunits that make up the hook and attempted to understand how this stability differs between bacteria. RESULTS: An intrinsically disordered segment plays an important role for overall hook stability and for its structural cohesion during motor rotation. The length of this linker segment depends on the species of bacteria; for Salmonella enterica and Campylobacter jejuni it is approximately 37 and 54 residues, respectively. Few residues of the linker are conserved and mutating the conserved residues of the linker yields non-flagellated cells. In the case of Campylobacter, which rotates its flagella at a speed much higher than that of Salmonella, shortening the linker leads to a rupture of the hook at its base, decreasing cell motility. Our experiments show that this segment is required for polymerization and stability of the hook, demonstrating a surprising role for a disordered region in one of the most finely tuned and closely studied macromolecular machines. CONCLUSIONS: This study reveals a detailed functional characteristic of an intrinsically disordered segment in the hook protein. This segment evolved to fulfill a specific role in the formation of the hook, and it is at the core of the stability and flexibility of the hook. Its length is important in the case of bacteria with high-speed rotating flagella. Finding a way of disrupting this linker in Campylobacter might help in preventing infections.