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The hook length of the flagellum is controlled to about 55 nm in Salmonella. The flagellar type III protein export apparatus secretes FliK to determine hook length during hook assembly and changes its substrate specificity from the hook protein to the filament protein when the hook length has reached about 55 nm. Salmonella FliK consists of an N-terminal domain (FliKN, residues 1–207), a C-terminal domain (FliKC, residues 268–405) and a flexible linker (FliKL, residues 208–267) connecting these two domains. FliKN is a ruler to measure hook length. FliKC binds to a transmembrane export gate protein FlhB to undergo the export switching. FliKL not only acts as part of the ruler but also contributes to this switching event, but it remains unknown how. Here we report that FliKL is required for efficient interaction of FliKC with FlhB. Deletions in FliKL not only shortened hook length according to the size of deletions but also caused a loose length control. Deletion of residues 206–265 significantly reduced the binding affinity of FliKC for FlhB, thereby producing much longer hooks. We propose that an appropriate length of FliKL is required for efficient interaction of FliKC with FlhB.

The FliI ATPase of the flagellar type III protein export apparatus forms the FliH2FliI complex along with its regulator FliH. The FliH2FliI complex is postulated to bring export substrates from the cytoplasm to the docking platform made of FlhA and FlhB although not essential for flagellar protein export. Here, to clarify the role of the FliH2FliI complex in flagellar assembly, we analysed the effect of FliH and FliI deletion on flagellar protein export and assembly. The hook length was not controlled properly in the ∆fliH-fliI flhB(P28T) mutant compared to wild-type cells, whose hook length is controlled to about 55 nm within 10% error. The FlhA(F459A) mutation increased the export level of the hook protein FlgE and the ruler protein FliK by about 10-fold and 3-fold, respectively, and improved the hook length control in the absence of FliH and FliI. However, the ∆fliH-fliI flhB(P28T) flhA(F459A) mutant did not produce flagellar filaments efficiently, and a large amount of flagellin monomers were leaked out into the culture media. Neither the hook length control nor flagellin leakage was affected by the FlhB(P28T) and FlhA(F459A) mutations. We will discuss a hierarchical protein export mechanism of the bacterial flagellum.

The bacterial flagellar hook connects the helical flagellar filament to the rotary motor at its base. Bending flexibility of the hook allows the helical filaments to form a bundle behind the cell body to produce thrust for bacterial motility. The hook protein FlgE shows considerable sequence and structural similarities to the distal rod protein FlgG; however, the hook is supercoiled and flexible as a universal joint whereas the rod is straight and rigid as a drive shaft. A short FlgG specific sequence (GSS) has been postulated to confer the rigidity on the FlgG rod, and insertion of GSS at the position between Phe-42 and Ala-43 of FlgE actually made the hook straight. However, it remains unclear whether inserted GSS confers the rigidity as well. Here, we provide evidence that insertion of GSS makes the hook much more rigid. The GSS insertion inhibited flagellar bundle formation behind the cell body, thereby reducing motility. This indicates that the GSS insertion markedly reduced the bending flexibility of the hook. Therefore, we propose that the inserted GSS makes axial packing interactions of FlgE subunits much tighter in the hook to suppress axial compression and extension of the protofilaments required for bending flexibility.

The flagellar motor of Bacillus subtilis possesses two distinct H+-type MotAB and Na+-type MotPS stators. In contrast to the MotAB motor, the MotPS motor functions efficiently at elevated viscosity in the presence of 200 mM NaCl. Here, we analyzed the torque-speed relationship of the Bacillus MotAB and MotPS motors over a wide range of external loads. The stall torque of the MotAB and MotPS motors at high load was about 2,200 pN nm and 220 pN nm, respectively. The number of active stators in the MotAB and MotPS motors was estimated to be about ten and one, respectively. However, the number of functional stators in the MotPS motor was increased up to ten with an increase in the concentration of a polysaccharide, Ficoll 400, as well as in the load. The maximum speeds of the MotAB and MotPS motors at low load were about 200 Hz and 50 Hz, respectively, indicating that the rate of the torque-generation cycle of the MotPS motor is 4-fold slower than that of the MotAB motor. Domain exchange experiments showed that the C-terminal periplasmic domain of MotS directly controls the assembly and disassembly dynamics of the MotPS stator in a load- and polysaccharide-dependent manner.

Rotation of bacterial flagellar motor is driven by the interaction between the stator and rotor and the driving energy is supplied by ion influx through the stator channel. The stator is composed of the MotA and MotB proteins, which form a hetero-hexameric complex with a stoichiometry of four MotA and two MotB molecules. MotA and MotB are four- and single-transmembrane proteins, respectively. To generate torque, the MotA/MotB stator unit changes its conformation in response to the ion influx and interacts with the rotor protein FliG. Here, we overproduced and purified MotA of the hyperthermophilic bacterium Aquifex aeolicus. A chemical crosslinking experiment revealed that MotA formed a multimeric complex, most likely a tetramer. The three-dimensional structure of the purified MotA, reconstructed by electron microscopy single particle imaging, consisted of a slightly elongated globular domain and a pair of arch-like domains with spiky projections, likely to correspond to the transmembrane and cytoplasmic domains, respectively. We show that MotA molecules can form a stable tetrameric complex without MotB and for the first time, demonstrate the cytoplasmic structure of the stator.

A periplasmic flagellar chaperone protein, FlgA, is required for P-ring assembly in bacterial flagella of taxa such as Salmonella enterica or Escherichia coli. The mechanism of chaperone-mediated P-ring formation is poorly understood. Here we present the open and closed crystal structures of FlgA from Salmonella enterica serovar Typhimurium, grown under different crystallization conditions. An intramolecular disulfide cross-linked form of FlgA caused a dominant negative effect on motility of the wild-type strain. Pull-down experiments support a specific protein-protein interaction between FlgI, the P-ring component protein, and the C-terminal domain of FlgA. Surface plasmon resonance and limited-proteolysis indicate that flexibility of the domain is reduced in the covalently closed form. These results show that the structural flexibility of the C-terminal domain of FlgA, which is related to the structural difference between the two crystal forms, is intrinsically associated with its molecular chaperone function in P-ring assembly.

A novel bacterial type III secretion effector, VopV, from the enteric pathogen Vibrio parahaemolyticus has been identified as a key factor in pathogenicity due to its interaction with cytoskeletal actin. One of the repeat units in the long repetitive region of VopV, named VopVrep1, functions as an actin-binding module. Despite its importance in pathogenesis, the manner in which the effector binds to actin and the subsequent effects on actin dynamics remain unclear. Here, we report the molecular basis of the VopVrep1/actin interaction. VopVrep1 exists as an unstructured protein in solution but potently and specifically binds filamentous actin (F-actin) and not globular actin (G-actin). The F-actin/VopVrep1 complex was directly visualized at 9.6-Å resolution using electron cryomicroscopy (cryoEM) and helical image reconstitution. The density map revealed the binding site of VopVrep1 at the interface between two actin strands, which is close to the binding site of the bicyclic heptapeptide toxin phalloidin. Consistent with this observation, VopVrep1 alone prevented the depolymerization of F-actin. Overall, VopVrep1 demonstrated unique characteristics in comparison to known actin-binding proteins, but was relatively similar to phalloidin. The phalloidin-like behavior, targeting the interstrand region of actin filaments to stabilize the filament structure, likely contributes to the pathogenicity of V. parahaemolyticus.

For self-assembly of the bacterial flagellum, a specific protein export apparatus utilizes ATP and proton motive force (PMF) as the energy source to transport component proteins to the distal growing end. The export apparatus consists of a transmembrane PMF-driven export gate and a cytoplasmic ATPase complex composed of FliH, FliI and FliJ. The FliI(6)FliJ complex is structurally similar to the α(3)β(3)γ complex of F(O)F(1)-ATPase. FliJ allows the gate to efficiently utilize PMF to drive flagellar protein export but it remains unknown how. Here, we report the role of ATP hydrolysis by the FliI(6)FliJ complex. The export apparatus processively transported flagellar proteins to grow flagella even with extremely infrequent or no ATP hydrolysis by FliI mutation (E211D and E211Q, respectively). This indicates that the rate of ATP hydrolysis is not at all coupled with the export rate. Deletion of FliI residues 401 to 410 resulted in no flagellar formation although this FliI deletion mutant retained 40% of the ATPase activity, suggesting uncoupling between ATP hydrolysis and activation of the gate. We propose that infrequent ATP hydrolysis by the FliI6FliJ ring is sufficient for gate activation, allowing processive translocation of export substrates for efficient flagellar assembly.

Bacterial pathogens use an injectisome to deliver virulence proteins into eukaryotic host cells. The bacterial flagellum and injectisome export their component proteins for self-assembly. These two systems show high structural similarities and are classified as the type III secretion system, but it remains elusive how similar they are in situ because the structures of these complexes isolated from cells and visualized by electron cryomicroscopy have shown only the export channel and housing for the export apparatus. Here we report in situ structures of Salmonella injectisome and flagellum by electron cryotomography. The injectisome lacks the flagellar basal body C-ring, but a wing-like disc and a globular density corresponding to the export gate platform and ATPase hexamer ring, respectively, are stably attached through thin connectors, revealing yet unidentified common architectures of the two systems. The ATPase ring is far from the disc, suggesting that both apparatuses are observed in an export-off state.