species are Gram-negative rod-shaped bacteria that are ubiquitous and often highly motile in aqueous environments. swimming motility is driven by a polar flagellum covered with a membranous sheath, but this sheathed flagellum is not well understood at the molecular level because of limited structural information. Here, we use as a model system to study the sheathed flagellum in intact cells by combining cryoelectron tomography (cryo-ET) and subtomogram analysis with a genetic approach. We reveal striking differences between sheathed and unsheathed flagella in cells, including a novel ring-like structure at the bottom of the hook that is associated with major remodeling of the outer membrane and sheath formation. Using mutants defective in flagellar motor components, we defined aspecific feature (also known as the T ring) as a distinctive periplasmic structure with 13-fold symmetry. The unique architecture of the T ring provides a static platform to recruit the PomA/B complexes, which are required to generate higher torques for rotation of the sheathed flagellum and fast motility of cells. Furthermore, the flagellar motor exhibits an intrinsic length variation between the inner and the outer membrane bound complexes, suggesting the outer membrane bound complex can shift slightly along the axial rod during flagellar rotation. Together, our detailed analyses of the polar flagella in intact cells provide insights into unique aspects of the sheathed flagellum and the distinct motility of species.

The bacterial flagellar motor switches rotational directions between counterclockwise (CCW) and clockwise (CW) to direct the migration of the cell. The cytoplasmic ring (C-ring) of the motor, which is composed of FliG, FliM, and FliN, is known for controlling the rotational sense of the flagellum. However, the mechanism underlying rotational switching remains elusive. Here, we deployed cryo-electron tomography to visualize the C-ring in two rotational biased mutants in Vibrio alginolyticus. We determined the C-ring molecular architectures, providing novel insights into the mechanism of rotational switching. We report that the C-ring maintained 34-fold symmetry in both rotational senses and the protein composition remained constant. The two structures show FliG conformational changes elicit a large conformational rearrangement of the rotor complex that coincides with rotational switching of the flagellum. FliM and FliN form a stable spiral-shaped base of the C-ring, likely stabilizing the C-ring during the conformational remodeling.

FliG, which is composed of three distinctive domains, N-terminal (N), middle (M), and C-terminal (C), is an essential rotor component that generates torque and determines rotational direction. To determine the role of FliG in determining flagellar rotational direction, we prepared rotational biased mutants of fliG in Vibrio alginolyticus. The E144D mutant, whose residue is belonging to the EHPQR-motif in FliGM, exhibited an increased number of switching events. This phenotype generated a response similar to the phenol-repellent response in chemotaxis. To clarify the effect of E144D mutation on the rotational switching, we combined the mutation with other che mutations (G214S, G215A and A282T) in FliG. Two of the double mutants suppressed the rotational biased phenotype. To gain structural insight into the mutations, we performed molecular dynamic simulations of the FliGMC domain, based on the crystal structure of Thermotoga maritima FliG and nuclear magnetic resonance analysis. Furthermore, we examined the swimming behavior of the fliG mutants lacking CheY. The results suggested that the conformation of FliG in E144D mutant was similar to that in the wild type. However, that of G214S and G215A caused a steric hindrance in FliG. The conformational change in FliGM triggered by binding CheY may lead to a rapid change of direction and may occur in both directional states.

The bacterial flagellum has evolved as one of the most remarkable nanomachines in nature. It provides swimming and swarming motilities that are often essential for the bacterial life cycle and pathogenesis. Many bacteria such as and species use flagella as an external propeller to move to favorable environments, whereas spirochetes utilize internal periplasmic flagella to drive a serpentine movement of the cell bodies through tissues. Here, we use cryo-electron tomography to visualize the polar sheathed flagellum of with particular focus on a-specific feature, the H-ring. We characterized the H-ring by identifying its two components FlgT and FlgO. We found that the majority of flagella are located within the periplasmic space in the absence of the H-ring, which are different from those of external flagella in wild-type cells. Our results not only indicate the H-ring has a novel function in facilitating the penetration of the outer membrane and the assembly of the external sheathed flagella but also are consistent with the notion that the flagella have evolved to adapt highly diverse needs by receiving or removing accessary genes. Flagellum is the major organelle for motility in many bacterial species. While most bacteria possess external flagella, such as the multiple peritrichous flagella found in and or the single polar sheathed flagellum in spp., spirochetes uniquely assemble periplasmic flagella, which are embedded between their inner and outer membranes. Here, we show for the first time that the external flagella in can be changed as periplasmic flagella by deleting two flagellar genes. The discovery here may provide new insights into the molecular basis underlying assembly, diversity, and evolution of flagella.

The bacterial flagellum is a biological nanomachine that rotates to allow bacteria to swim. For flagellar rotation, torque is generated by interactions between a rotor and a stator. The stator, which is composed of MotA and MotB subunit proteins in the membrane, is thought to bind to the peptidoglycan (PG) layer, which anchors the stator around the rotor. Detailed information on the stator and its interactions with the rotor remains unclear. Here, we deployed cryo-electron tomography and genetic analysis to characterize in situ structure of the bacterial flagellar motor in Vibrio alginolyticus, which is best known for its polar sheathed flagellum and high-speed rotation. We determined in situ structure of the motor at unprecedented resolution and revealed the unique protein-protein interactions among Vibrio-specific features, namely the H ring and T ring. Specifically, the H ring is composed of 26 copies of FlgT and FlgO, and the T ring consists of 26 copies of a MotX-MotY heterodimer. We revealed for the first time a specific interaction between the T ring and the stator PomB subunit, providing direct evidence that the stator unit undergoes a large conformational change from a compact form to an extended form. The T ring facilitates the recruitment of the extended stator units for the high-speed motility in Vibrio species. IMPORTANCE The torque of flagellar rotation is generated by interactions between a rotor and a stator; however, detailed structural information is lacking. Here, we utilized cryo-electron tomography and advanced imaging analysis to obtain a high-resolution in situ flagellar basal body structure in Vibrio alginolyticus, which is a Gram-negative marine bacterium. Our high-resolution motor structure not only revealed detailed protein-protein interactions among unique Vibrio-specific features, the T ring and H ring, but also provided the first structural evidence that the T ring interacts directly with the periplasmic domain of the stator. Docking atomic structures of key components into the in situ motor map allowed us to visualize the pseudoatomic architecture of the polar sheathed flagellum in Vibrio spp. and provides novel insight into its assembly and function.