In Situ Structure of the Vibrio Polar Flagellum Reveals a Distinct Outer Membrane Complex and Its Specific Interaction with the Stator

Abstract

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.

The flagellar motor of Vibrio alginolyticus undergoes major structural remodeling during rotational switching

“…Different from E. coli and Salmonella , V. alginolyticus possesses several Vibrio-specific features: H-, T-, and Orings. The O-ring is located on the outside of the outer membrane ( Zhu et al, 2017 ), the H-ring facilitates the outer membrane penetration of the flagellum ( Terashima et al, 2010 ; Zhu et al, 2018 ), and the T-ring contacts the H-ring and stators, presumably acting as a scaffold to hold the stators ( Terashima et al, 2006 ; Zhu et al, 2019 ).…”

Phylogenetic Distribution, Ultrastructure, and Function of Bacterial Flagellar Sheaths

“…For some bacteria, including B. bacteriovorus, B. melitensis, H. pylori, and V. fischeri, a bulb-like structure is typically observed at the distal end of the flagellar sheath [7,[27][28][29]. The detailed electron micrographs from the early studies suggested the flagellar sheath was contiguous with the outer membrane, and recent cryo-electron tomography (cryo-ET) studies of sheathed flagella from various bacteria have yielded detailed images that confirm the structural continuity between the outer membrane and flagellar sheath [30][31][32][33][34]. Figure 1 shows tomograms of V. cholerae and H. pylori sheathed flagella (authors' data).…”

“…Biomolecules 2020, 10, 363 3 of 15 membrane and flagellar sheath [30][31][32][33][34]. Figure 1 shows tomograms of V. cholerae and H. pylori sheathed flagella (authors' data).…”

Living in a Foster Home: The Single Subpolar Flagellum Fla1 of Rhodobacter sphaeroides

“…Briefly, it is important to mention that recruitment of these complexes to the basal body has been related to the interaction of the cytoplasmic loop of MotA with FliG, which is part of the C-ring ( Figure 1) [22][23][24]. Besides, in Vibrio the proteins MotY and MotX form the periplasmic T-ring that interacts with PomB (equivalent to MotB in Vibrio) and stabilize the stator complexes [25,26]. Activation of the proton channel requires extensive remodeling of the periplasmic region of MotB [27][28][29][30][31][32], and it has been proposed that the flagellar protein FliL participates in this process [33][34][35][36][37].…”