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 bacterial life cycle and for pathogenesis. Many bacteria such as Salmonella and Vibrio species use flagella as an external propeller to move to favorable environments, while 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 Vibrio alginolyticus with particular focus on a Vibrio 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 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.

The marine bacterium Vibrio alginolyticus possesses a polar flagellum driven by a sodium ion flow. The main components of the flagellar motor are the stator and rotor. The C-ring and MS-ring, which are composed of FliG and FliF, respectively, are parts of the rotor. Here, we purified an MS-ring composed of FliF–FliG fusion proteins and solved the near-atomic resolution structure of the S-ring—the upper part of the MS-ring—using cryo-electron microscopy. This is the first report of an S-ring structure from Vibrio , whereas, previously, only those from Salmonella have been reported. The Vibrio S-ring structure reveals novel features compared with that of Salmonella , such as tilt angle differences of the RBM3 domain and the β-collar region, which contribute to the vertical arrangement of the upper part of the β-collar region despite the diversity in the RBM3 domain angles. Additionally, there is a decrease of the inter-subunit interaction between RBM3 domains, which influences the efficiency of the MS-ring formation in different bacterial species. Furthermore, although the inner-surface electrostatic properties of Vibrio and Salmonella S-rings are altered, the residues potentially interacting with other flagellar components, such as FliE and FlgB, are well structurally conserved in the Vibrio S-ring. These comparisons clarified the conserved and non-conserved structural features of the MS-ring across different species. IMPORTANCE: Understanding the structure and function of the flagellar motor in bacterial species is essential for uncovering the mechanisms underlying bacterial motility and pathogenesis. Our study revealed the structure of the Vibrio S-ring, a part of its polar flagellar motor, and highlighted its unique features compared with the well-studied Salmonella S-ring. The observed differences in the inter-subunit interactions and in the tilt angles between the Vibrio and Salmonella S-rings highlighted the species-specific variations and the mechanism for the optimization of MS-ring formation in the flagellar assembly. By concentrating on the region where the S-ring and the rod proteins interact, we uncovered conserved residues essential for the interaction. Our research contributes to the advancement of bacterial flagellar biology.

The bacterial flagellar motor is an intricate nanomachine that 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 (CCW-biased fliG -G214S and CW-locked fliG -G215A) in Vibrio alginolyticus . Sub-tomogram averaging was utilized to resolve two distinct conformations of the C-ring. Comparison of the C-ring structures in two rotational senses provide direct evidence that the C-ring undergoes major structural remodeling during rotational switch. Specifically, FliG conformational changes elicit a large rearrangement of the C-ring that coincides with rotational switching, whereas FliM and FliN form a spiral-shaped base of the C-ring, likely stabilizing the C-ring during the conformational remodeling.

The bacterial flagellum, a complex nanomachine composed of numerous proteins, is utilized by bacteria for swimming in various environments and plays a crucial role in their survival and infection. The flagellar motor is composed of a rotor and stator complexes, with each stator unit functioning as an ion channel that converts flow from outside of cell membrane into rotational motion. Paenibacillus sp. TCA20 was discovered in a hot spring, and a structural analysis was conducted on the stator complex using cryo-electron microscopy to elucidate its function. Two of the three structures (Classes 1 and 3) were found to have structural properties typical for other stator complexes. In contrast, in Class 2 structures, the pentamer ring of the A subunits forms a C-shape, with lauryl maltose neopentyl glycol (LMNG) bound to the periplasmic side of the interface between the A and B subunits. This interface is conserved in all stator complexes, suggesting that hydrophobic ligands and lipids can bind to this interface, a feature that could potentially be utilized in the development of novel antibiotics aimed at regulating cell motility and infection.

The nuclear magnetic resonance (NMR) measurements under high pressure conditions are powerful technique for characterizing protein properties and its conformational changes. Bacterial flagellum is a molecular nano-machine and bacteria move toward favorable condition for survive and growth to rotate flagellar motor, although the molecular mechanism of the motor rotation is still unclear. In this article, we introduce our recently published results that clarified the determination mechanism of the rotational direction by analyzing of FliG, a flagellar rotor protein. Combining the structural analysis of solution NMR and the measurement of high-pressure condition, we revealed that the rearrangement of interaction network in FliG determines the rotational direction.