Many bacteria use the flagellum for locomotion and chemotaxis. Its bi-directional rotation is driven by the membrane-embedded motor, which uses energy from the transmembrane ion gradient to generate torque at the interface between stator units and rotor. The structural organization of the stator unit (MotAB), its conformational changes upon ion transport and how these changes power rotation of the flagellum, remain unknown. Here we present ~3 Å-resolution cryo-electron microscopy reconstructions of the stator unit in different functional states. We show that the stator unit consists of a dimer of MotB surrounded by a pentamer of MotA. Combining structural data with mutagenesis and functional studies, we identify key residues involved in torque generation and present a mechanistic model for motor function and switching of rotational direction.

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The flagellar stator unit is an oligomeric complex of two membrane proteins (MotA 5 B 2 ) that powers bi-directional rotation of the bacterial flagellum. Harnessing the ion motive force across the cytoplasmic membrane, the stator unit operates as a miniature rotary motor itself to provide torque for rotation of the flagellum. Recent cryo-electron microscopic (cryo-EM) structures of the stator unit provided novel insights into its assembly, function, and subunit stoichiometry, revealing the ion flux pathway and the torque generation mechanism. Furthermore, in situ cryoelectron tomography (cryo-ET) studies revealed unprecedented details of the interactions between stator unit and rotor. In this review, we summarize recent advances in our understanding of the structure and function of the flagellar stator unit, torque generation, and directional switching of the motor. The bacterial flagellum and its rotary motorMany bacteria, including Escherichia coli, Salmonella, and Bacillus spp., use flagella (see Glossary) to move through liquid environments and across surfaces. The flagellum is a supramolecular nanomachine that protrudes from the cell envelope and measures~5-20 μm in length. It is able to rotate in both clockwise (CW) and counterclockwise (CCW) directions to propel the bacterial cell body in different living environments [1,2]. Rotational switching between these two modes is regulated by chemotactic signaling, which is a rapid process that responds to environmental stimuli and biases movement of the cell toward attractants and away from repellents. Flagella-mediated chemotaxis further enables pathogenic bacteria to move toward cells to establish in vivo niches. [3,4]. Thus, flagella have fundamental roles in bacterial locomotion and virulence [5].The flagellum comprises more than 25 kinds of building blocks, which assemble in a highly ordered manner. The flagellar structure can be divided into three morphologically distinguishable parts: a cell envelope-spanning motor (basal body), a universal joint (hook), and a long, thin helical filament [6,7] (Figure 1). Among them, the most intricate part is the basal body, containing the components responsible for assembly of the flagellum [the flagellar-specific type-III secretion system (T3SS) [8]], torque generation (the stator units [9]), and rotational switching (binding of the response regulator CheY-P to the cytoplasmic C-ring [10,11]). Cryo-ET studies of the motor from different bacterial species show the variation of its structure, while the core components are conserved [7,12,13]. For example, in the Gram-negative bacteria Salmonella and E. coli, the flagellar motor contains four ring-like structures based on their distributions relative to the cell surface layers [lipopolysaccharide (L-)ring, peptidoglycan (P-)ring, inner membrane/ supramembrane (MS-)ring, and cytoplasmic (C-)ring] surrounding a central rigid rod [14][15][16][17]. Additional ring-like structures, H-and T-rings, located in the periplasmic space, have also been observed in Vibrio spp [18]. It i...

The betaine/γ-aminobutyric acid (GABA) transporter 1 (BGT1) is one of the four GABA transporters (GATs) involved in the termination of GABAergic neurotransmission. Although suggested to be implicated in seizure management, the exact functional importance of BGT1 in the brain is still elusive. This is partly owing to the lack of potent and selective pharmacological tool compounds that can be used to probe its function. We previously reported the identification of 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid (ATPCA), a selective substrate for BGT1 over GAT1/GAT3, but also an agonist for GABA receptors. With the aim of providing new functional insight into BGT1, we here present the synthesis and pharmacological characterization of the tritiated analogue, [H]ATPCA. Using traditional uptake assays at recombinant transporters expressed in cell lines, [H]ATPCA displayed a striking selectivity for BGT1 among the four GATs ( K and V values of 21 μM and 3.6 nmol ATPCA/(min × mg protein), respectively), but was also found to be a substrate for the creatine transporter (CreaT). In experiments with mouse cortical cell cultures, we observed a Na-dependent [H]ATPCA uptake in neurons, but not in astrocytes. The neuronal uptake could be inhibited by GABA, ATPCA, and a noncompetitive BGT1-selective inhibitor, indicating functional BGT1 in neurons. In conclusion, we report [H]ATPCA as a novel radioactive substrate for both BGT1 and CreaT. The dual activity of the radioligand makes it most suitable for use in recombinant studies.

Many bacteria use the flagellum for locomotion and chemotaxis. Its bi-directional rotation is driven by the membrane-embedded motor, which uses energy from the transmembrane ion gradient to generate torque at the interface between stator units and rotor. The structural organization of the stator unit (MotAB), its conformational changes upon ion transport and how 15 these changes power rotation of the flagellum, remain unknown. Here we present ~3 Å-resolution cryo-electron microscopy reconstructions of the stator unit in different functional states. We show that the stator unit consists of a dimer of MotB surrounded by a pentamer of MotA. Combining structural data with mutagenesis and functional studies, we identify key residues involved in torque generation and present a mechanistic model for motor function and switching of rotational 20 direction.One Sentence Summary: Structural basis of torque generation in the bidirectional bacterial flagellar motor

Bacteria swim using a flagellar motor that is powered by stator units. These stator units are energized by an ionic gradient across the membrane, typically proton or sodium. The presumed monodirectional rotation of the stator units allows the bidirectional rotation of the flagellar motor. However, how ion selectivity is attained, how ion transport triggers the directional rotation of the stator unit, and how the stator unit is incorporated into the motor remain largely unclear. Here we have determined by cryo-electron microscopy the structure of the Na+-driven type stator unit PomAB from the gram-negative bacterium Vibrio alginolyticus in both lipidic and detergent environments, at a resolution up to 2.5 Angstrom. The structure is in a plugged, auto-inhibited state consisting of five PomA subunits surrounding two PomB subunits. The electrostatic potential map uncovers sodium ion binding sites within the transmembrane domain, which together with functional experiments and explicit solvent molecular dynamics simulations, suggest a mechanism for ion translocation and selectivity. Resolved conformational isomers of bulky hydrophobic residues from PomA, in the vicinity of key determinant residues for sodium ion coupling of PomB, prime PomA for clockwise rotation. The rotation is tightly blocked by the trans-mode organization of the PomB plug motifs. The structure also reveals a conformationally dynamic helical motif at the C-terminus of PomA, which we propose regulates the distance between PomA subunit cytoplasmic domains and is involved in stator unit-rotor interaction, concomitant stator unit activation, and torque transmission. Together, our studies provide mechanistic insight for understanding flagellar stator unit ion selectivity and incorporation of the stator units into the motor.

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Bacteria swim using a flagellar motor that is powered by stator units. Vibrio spp. are highly motile bacteria responsible for various human diseases, the polar flagella of which are exclusively driven by sodium-dependent stator units (PomAB). However, how ion selectivity is attained, how ion transport triggers the directional rotation of the stator unit, and how the stator unit is incorporated into the motor remain largely unclear. Here we have determined by cryo-electron microscopy the structure of Vibrio PomAB. The electrostatic potential map uncovers sodium binding sites, which together with functional experiments and molecular dynamics simulations, reveal a mechanism for ion translocation and selectivity. Bulky hydrophobic residues from PomA, prime PomA for clockwise rotation. We propose a dynamic helical motif in PomA regulates the distance between PomA subunit cytoplasmic domains, stator unit activation, and torque transmission. Together, our studies provide mechanistic insights for understanding stator unit ion selectivity and motor incorporation.