Shiwei Zhu

Conformational change in the periplamic region of the flagellar stator coupled with the assembly around the rotor

Significance Stator is the energy-converting membrane protein complex in the flagellar motor. Its ion-conducting activity is only activated when incorporated into the motor, but the mechanism for assembly-coupled activation remains a mystery. In this study, we solved the structure of a C-terminal fragment of the sodium-driven stator protein PomB (PomBC), the region responsible for anchoring the stator unit, at 2.0-Å resolution. In vivo disulfide cross-linking studies of PomB double-Cys mutants and their motility assay suggested that the N-terminal region of PomBC changes its conformation, which is expected for MotB, the counterpart of PomB in the proton-driven Salmonella motor, in the final step of the stator assembly around the rotor. The torque of the bacterial flagellum is generated by the rotor–stator interaction coupled with the ion flow through the channel in the stator. Anchoring the stator unit to the peptidoglycan layer with proper orientation around the rotor is believed to be essential for smooth rotation of the flagellar motor. The stator unit of the sodium-driven flagellar motor of Vibrio is composed of PomA and PomB, and is thought to be fixed to the peptidoglycan layer and the T-ring by the C-terminal periplasmic region of PomB. Here, we report the crystal structure of a C-terminal fragment of PomB (PomBC) at 2.0-Å resolution, and the structure suggests a conformational change in the N-terminal region of PomBC for anchoring the stator. On the basis of the structure, we designed double-Cys replaced mutants of PomB for in vivo disulfide cross-linking experiments and examined their motility. The motility can be controlled reproducibly by reducing reagent. The results of these experiments suggest that the N-terminal disordered region (121–153) and following the N-terminal two-thirds of α1(154-164) in PomBC changes its conformation to form a functional stator around the rotor. The cross-linking did not affect the localization of the stator nor the ion conductivity, suggesting that the conformational change occurs in the final step of the stator assembly around the rotor.

Structure, gene regulation and environmental response of flagella in Vibrio

Vibrio species are Gram-negative, rod-shaped bacteria that live in aqueous environments. Several species, such as V. harveyi, V. alginotyticus, and V. splendidus, are associated with diseases in fish or shellfish. In addition, a few species, such as V. cholerae and V. parahaemolyticus, are risky for humans due to infections from eating raw shellfish infected with these bacteria or from exposure of wounds to the marine environment. Bacterial flagella are not essential to live in a culture medium. However, most Vibrio species are motile and have rotating flagella which allow them to move into favorable environments or to escape from unfavorable environments. This review summarizes recent studies about the flagellar structure, function, and regulation of Vibrio species, especially focused on the Na+-driven polar flagella that are principally responsible for motility and sensing the surrounding environment, and discusses the relationship between flagella and pathogenicity.

FliL associates with the stator to support torque generation of the sodium-driven polar flagellar motor of Vibrio

Flagellar motors generate torque to rotate flagellar filaments and drive bacterial cells. Each motor is composed of a rotor and many stators. The stator is a force‐generating complex that converts ion flux into torque. Previous reports have suggested that the membrane protein FliL is located near the stator and is involved in torque generation. We investigated the role of FliL in the sodium‐driven polar flagellar motor of Vibrio alginolyticus. Our results revealed that FliL is a cytoplasmic membrane protein and is located at the base of flagellum. The deletion of fliL did not affect the cell morphology or flagellation but resulted in a significant decrease of swimming speed, especially at a higher load thus suggesting that FliL is important for torque generation at high load conditions. Furthermore, the polar localization of the stator was decreased in a ΔfliL mutant, but the sodium‐dependent assembly of the stator complex was still retained. The polar localization of FliL was lost in the absence of the stator complex, indicating that FliL interacts directly or indirectly with the stator. Our results suggest that FliL is localized along with the stator in order to support the motor functioning for swimming at high load conditions by maintaining the stator assembly.

Imaging the motility and chemotaxis machineries in Helicobacter pylori by cryo-electron tomography

Helicobacter pylori is a bacterial pathogen that can cause many gastrointestinal diseases including ulcers and gastric cancer. A unique chemotaxis-mediated motility is critical for H. pylori to colonize in the human stomach and to establish chronic infection, but the underlying molecular mechanisms are not well understood. Here we employ cryo-electron tomography to reveal detailed structures of the H. pylori cell envelope including the sheathed flagella and chemotaxis arrays. Notably, H. pylori possesses a distinctive periplasmic cage-like structure with 18-fold symmetry. We propose that this structure forms a robust platform for recruiting 18 torque generators, which likely provide the higher torque needed for swimming in high-viscosity environments. We also reveal a series of key flagellar assembly intermediates, providing structural evidence that flagellar assembly is tightly coupled with biogenesis of the membrane sheath. Finally, we determine the structure of putative chemotaxis arrays at the flagellar pole, which have implications for how direction of flagellar rotation is regulated. Together, our pilot cryo-ET studies provide novel structural insights into the unipolar flagella of H. pylori and lay a foundation for a better understanding of the unique motility of this organism. IMPORTANCE Helicobacter pylori is a highly motile bacterial pathogen that colonizes approximately 50% of the worlds population. H. pylori can move readily within the viscous mucosal layer of the stomach. It has become increasingly clear that its unique flagella-driven motility is essential for successful gastric colonization and pathogenesis. Here we use advanced imaging techniques to visualize novel in situ structures with unprecedented detail in intact H. pylori cells. Remarkably, H. pylori possesses multiple unipolar flagella, which are driven by one of the largest flagellar motors found in bacteria. These large motors presumably provide higher torque needed by the bacterial pathogens to navigate in viscous environment of the human stomach.

Molecular architecture of the sheathed polar flagellum in Vibrio alginolyticus

Significance Many important bacterial pathogens such as Vibrio cholerae and Helicobacter pylori use a sheathed flagellum as the main organelle for motility. Although bacterial flagella have been extensively studied in model systems such as Escherichia coli and Salmonella, relatively little is known about sheathed flagella. In this study, we use high-throughput cryoelectron tomography to visualize thousands of polar flagella in Vibrio alginolyticus. We not only reveal unprecedented details of sheathed flagella in V. alginolyticus but also uncover key differences between sheathed flagella and unsheathed flagella in the same Vibrio species. Our studies provide insight into the unique aspects of sheathed flagella, which are effectively used by many bacterial pathogens to establish infection and disseminate in humans and other mammalian hosts. Vibrio species are Gram-negative rod-shaped bacteria that are ubiquitous and often highly motile in aqueous environments. Vibrio 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 Vibrio alginolyticus 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 V. alginolyticus 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 a Vibrio-specific 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 Vibrio cells. Furthermore, the Vibrio 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 Vibrio species.

In Situ Structural Analysis of the Spirochetal Flagellar Motor by Cryo-Electron Tomography

The bacterial flagellar motor is a large multi-component molecular machine. Structural determination of such a large complex is often challenging and requires extensive structural analysis in situ. Cryo-electron tomography (cryo-ET) has emerged as a powerful technique that enables us to visualize intact flagellar motors in cells with unprecedented details. Here, we detail the procedure beginning with sample preparation, followed by data acquisition, tomographic reconstruction, sub-tomogram analysis, and ultimately visualization of the intact spirochetal flagellar motor in Borrelia burgdorferi. The procedure is applicable to visualize other molecular machinery in bacteria or other organisms.

Serine suppresses the motor function of a periplasmic PomB mutation in the Vibrio flagella stator.

The flagellar motor of Vibrio alginolyticus is made of two parts: a stator consisting of proteins PomA and PomB, and a rotor whose main component is FliG. The interaction between FliG and PomA generates torque for flagellar rotation. Based on cross‐linking experiments of double‐Cys mutants of PomB, we previously proposed that a conformational change in the periplasmic region of PomB caused stator activation. Double‐Cys mutants lost their motility due to an intramolecular disulfide bridge. In this study, we found that the addition of serine, a chemotactic attractant, to a PomB(L160C/I186C) mutant restored motility without cleaving the disulfide bridge. We speculate that serine changed the rotor (FliG) conformation, affecting rotational direction. Combined with the counterclockwise (CCW)‐biased mutation FliG(G214S), motility of PomB(L160C/I186C) was restored without the addition of serine. Likewise, motility was restored without serine in Che− mutants, in either a CCW‐locked or clockwise (CW)‐locked strain. In contrast, in a ΔcheY (CCW‐locked) strain, Vibrio (L160C/I186C) required serine to be rescued. We speculate that CheY affects stator conformation and motility restoration by serine is independent on the chemotaxis signaling pathway.

Biochemical characterization of the flagellar stator-associated inner membrane protein FliL from Vibrio alginolyticus

The flagellar motor is embedded in the cell envelope and rotates upon interaction between the stator and the rotor. The rotation is powered by ion flow through the stator. A single transmembrane protein named FliL is associated with torque generation in the flagellar motor. We established an Escherichia coli over-expression system for FliL of Vibrio alginolyticus, a marine bacterium that has a sodium-driven polar flagellum. We successfully expressed, purified, and crystallized the ca. 17 kDa full-length FliL protein and generated a construct that expresses only the ca. 14 kDa periplasmic region of FliL (ΔTM FliL). Biochemical characterization and NMR analysis revealed that ΔTM FliL weakly interacted with itself to form an oligomer. We speculate that the observed dynamic interaction may be involved in the role of FliL in flagellar motor function.

FliL association with flagellar stator in the sodium-driven Vibrio motor characterized by the fluorescent microscopy

Bacterial flagellar motor (BFM) is a protein complex used for bacterial motility and chemotaxis that involves in energy transformation, torque generation and switching. FliL is a single-transmembrane protein associated with flagellar motor function. We performed biochemical and biophysical approaches to investigate the functional roles of FliL associated with stator-units. Firstly, we found the periplasmic region of FliL is crucial for its polar localization. Also, the plug mutation in stator-unit affected the polar localization of FliL implying the activation of stator-unit is important for FliL recruitment. Secondly, we applied single-molecule fluorescent microscopy to study the role of FliL in stator-unit assembly. Using molecular counting by photobleaching, we found the stoichiometry of stator-unit and FliL protein would be 1:1 in a functional motor. Moreover, the turnover time of stator-units are slightly increased in the absence of FliL. By further investigation of protein dynamics on membrane, we found the diffusions of stator-units and FliL are independent. Surprisingly, the FliL diffusion rate without stator-units is unexpectedly slow indicating a protein-complex forming event. Our results suggest that FliL plays a supporting role to the stator in the BFM.