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1.A.30 The H+- or Na+-translocating Bacterial Flagellar Motor/ExbBD Outer Membrane Transport Energizer (Mot-Exb) Superfamily

The Mot-Exb Superfamily consists of six distant families, each probably with a distinct physiological function, although all may function as H+/Na+ channels, driving an energy-requiring process. For example, flagellar motors of marine bacteria Halomonas are driven by both protons and sodium ions (Kita-Tsukamoto et al. 2004). The MotAB family energizes bacterial flagellar rotation while the ExbBD family energizes accumulation of large molecules (i.e. iron-siderophores, vitamin B12, DNA from phage, and colicins) from the external medium across the outer Gram-negative bacterial membrane into the periplasm. The AglRS system powers gliding motility while the SilAB systems energize gian adhesin export.  The function of a 5th family (TC# 1.A.30.5) is not known, but the ZorAB systems have been reported to function as parts of antiphage defense systems. The pmf (or smf) is probably the driving force in all cases. MotAB and PomAB are homologous to ExbBD and TolQR. MotAB of E. coli, the stator, is known to form a proton channel. This stator is composed of MotA and MotB proteins, which form a hetero-hexameric complex with a stoichiometry of four MotA and two MotB molecules. MotA can form a tetramer in the absence of MotB (Takekawa et al. 2016). Ion binding residues for Na+ flow in the stator complex of the Vibrio flagellar motor have been identified (Onoue et al. 2019). The structure and dynamics of the bacterial flagellum have been reviewed (Nakamura and Minamino 2019). The flagellar motor, which structurally resembles an artificial motor, is embedded within the cell envelop and spins at several hundred revolutions per second (Morimoto and Minamino 2021).

About 10 stators (MotA/MotB complexes) are docked around a rotor, and the stator recruitment depends on the load, ion motive force, and coupling ion flux. The MotA(M206I) mutation slows motor rotation and decreases the number of docked stators in Salmonella.  Suzuki et al. 2019 showed that lowering the external pH improves the assembly of the mutant stators. Neither the collapse of the ion motive force nor a mutation mimicking the proton-binding state inhibited stator localization to the motor. Thus, MotA-Met206 is involved in torque generation and proton translocation, and stator assembly is stabilized by protonation of the stator. Ancestral sequence reconstructions of MotB require MotA and give rise to pmf-dependent motility (Islam et al. 2020).

Yonekura et al. (2011) presented the first three-dimensional structure of the PomAB torque-generating stator unit analyzed by electron microscopy. The structure of PomAB revealed two arm domains, which contain the PG-binding site, connected to a large base made of the transmembrane and cytoplasmic domains. The arms lean downward to the membrane surface, likely representing a 'plugged' conformation, which would prevent ions leaking through the channel. They propose a model for how PomAB units are placed around the flagellar basal body to function as torque generators. 

Leu46 of MotB acts as the gate for hydronium ion permeation, which induces the formation of a water wire that may mediate the proton transfer to Asp32 on MotB. The free energy barrier for H3O+ permeation is consistent with the proton transfer rate deduced from the flagellar rotational speed and the number of protons per rotation, suggesting that gating is the rate-limiting step (Nishihara and Kitao 2015). Structure and dynamics of MotA/B with nonprotonated and protonated Asp32 suggested size-dependent ion selectivity. In MotA/B with the nonprotonated Asp32, the A3 segment in MotA maintains a kink whereas protonation induces a straighter shape. Assuming that the cytoplasmic domain not included in the atomic model moves as a rigid body, the protonation/deprotonation of Asp32 is inferred to induce a ratchet motion of the cytoplasmic domain, correlated with the motion of the flagellar rotor (Nishihara and Kitao 2015). 

ExbBD forms both hexameric and pentameric complexes that coexist, with the proportion of the hexamers increasing with pH. Channel current measurements and 2D crystallography thus support the existence of and transition between the two oligomeric states in membranes. The hexameric complex has been reported to consist of six ExbB subunits and three ExbD transmembrane helices enclosed within the central channel (Maki-Yonekura et al. 2018). TonB physically interacts with the nutrient-loaded transporter to exert a force that opens an import pathway across the outer membrane. Another group showed that five copies of ExbB are arranged as a pentamer around two copies of ExbD in the complex. The revised stoichiometry has implications for motor function (Celia et al. 2019).

As noted above, each flagellum is a supramolecular motility machine consisting of a bi-directional rotary motor, a universal joint and a helical propeller. The signal transducers transmit environmental signals to the flagellar motor through the cytoplasmic chemotactic signaling pathway. The flagellar motor is composed of a rotor and multiple stator units, each of which acts as a transmembrane proton channel to conduct protons and exert force on the rotor (Minamino et al. 2019). FliG, FliM and FliN form the C ring on the cytoplasmic face of the basal body MS ring made of the transmembrane protein FliF and act as the rotor. The C ring also serves as a switching device that enables the motor to spin in both counterclockwise (CCW) and clockwise (CW) directions. The phosphorylated form of the chemotactic signaling protein CheY binds to FliM and FliN to induce conformational changes of the C ring responsible for switching the direction of flagellar motor rotation from CCW to CW (Minamino et al. 2019).

References associated with 1.A.30 family:

Bulathsinghala, C.M., B. Jana, K.R. Baker, and K. Postle. (2013). ExbB cytoplasmic loop deletions cause immediate, proton motive force-independent growth arrest. J. Bacteriol. 195: 4580-4591. 23913327
Castillo, D.J., S. Nakamura, Y.V. Morimoto, Y.S. Che, N. Kami-Ike, S. Kudo, T. Minamino, and K. Namba. (2013). The C-terminal periplasmic domain of MotB is responsible for load-dependent control of the number of stators of the bacterial flagellar motor. Biophysics (Nagoya-shi) 9: 173-181. 27493556
Celia, H., I. Botos, X. Ni, T. Fox, N. De Val, R. Lloubes, J. Jiang, and S.K. Buchanan. (2019). Cryo-EM structure of the bacterial Ton motor subcomplex ExbB-ExbD provides information on structure and stoichiometry. Commun Biol 2: 358. 31925206
Doron, S., S. Melamed, G. Ofir, A. Leavitt, A. Lopatina, M. Keren, G. Amitai, and R. Sorek. (2018). Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359:. 29371424
Hosking, E.R., C. Vogt, E.P. Bakker, and M.D. Manson. (2006). The Escherichia coli MotAB proton channel unplugged. J. Mol. Biol. 364: 921-937. 17052729
Ishida, T., R. Ito, J. Clark, N.J. Matzke, Y. Sowa, and M.A.B. Baker. (2019). Sodium-powered stators of the bacterial flagellar motor can generate torque in the presence of phenamil with mutations near the peptidoglycan-binding region. Mol. Microbiol. [Epub: Ahead of Print] 30927553
Islam, M.I., A. Lin, Y.W. Lai, N.J. Matzke, and M.A.B. Baker. (2020). Ancestral Sequence Reconstructions of MotB Are Proton-Motile and Require MotA for Motility. Front Microbiol 11: 625837. 33424826
Ito, M., D.B. Hicks, T.M. Henkin, A.A. Guffanti, B.D. Powers, L. Zvi, K. Uematsu, and T.A. Krulwich. (2004). MotPS is the stator-force generator for motility of alkaliphilic Bacillus, and its homologue is a second functional Mot in Bacillus subtilis. Mol. Microbiol. 53: 1035-1049. 15306009
Jakobczak, B., D. Keilberg, K. Wuichet, and L. Søgaard-Andersen. (2015). Contact- and Protein Transfer-Dependent Stimulation of Assembly of the Gliding Motility Machinery in Myxococcus xanthus. PLoS Genet 11: e1005341. 26132848
Kita-Tsukamoto, K., M. Wada, K. Yao, T. Nishino, and K. Kogure. (2004). Flagellar motors of marine bacteria Halomonas are driven by both protons and sodium ions. Can. J. Microbiol. 50: 369-374. 15213745
Kitao, A. and Y. Nishihara. (2017). Structure of the MotA/B Proton Channel. Methods Mol Biol 1593: 133-145. 28389950
Klebba, P.E. (2016). ROSET Model of TonB Action in Gram-Negative Bacterial Iron Acquisition. J. Bacteriol. 198: 1013-1021. 26787763
Koerdt, A., A. Paulick, M. Mock, K. Jost, and K.M. Thormann. (2009). MotX and MotY are required for flagellar rotation in Shewanella oneidensis MR-1. J. Bacteriol. 191: 5085-5093. 19502394
Kojima, S., K. Imada, M. Sakuma, Y. Sudo, C. Kojima, T. Minamino, M. Homma, and K. Namba. (2009). Stator assembly and activation mechanism of the flagellar motor by the periplasmic region of MotB. Mol. Microbiol. 73: 710-718. 19627504
Kopp, D.R. and K. Postle. (2020). The Intrinsically Disordered Region of ExbD is Required for Signal Transduction. J. Bacteriol. [Epub: Ahead of Print] 31932309
Liew, C.W., R.M. Hynson, L.A. Ganuelas, N. Shah-Mohammadi, A.P. Duff, S. Kojima, M. Homma, and L.K. Lee. (2017). Solution structure analysis of the periplasmic region of bacterial flagellar motor stators by small angle X-ray scattering. Biochem. Biophys. Res. Commun. [Epub: Ahead of Print] 29197577
Lin, T.S., S. Zhu, S. Kojima, M. Homma, and C.J. Lo. (2018). FliL association with flagellar stator in the sodium-driven Vibrio motor characterized by the fluorescent microscopy. Sci Rep 8: 11172. 30042401
Lo, C.J., Y. Sowa, T. Pilizota, and R.M. Berry. (2013). Mechanism and kinetics of a sodium-driven bacterial flagellar motor. Proc. Natl. Acad. Sci. USA 110: E2544-2551. 23788659
Maki-Yonekura, S., R. Matsuoka, Y. Yamashita, H. Shimizu, M. Tanaka, F. Iwabuki, and K. Yonekura. (2018). Hexameric and pentameric complexes of the ExbBD energizer in the Ton system. Elife 7:. 29661272
Mignot, T. and M. Nöllmann. (2017). New insights into the function of a versatile class of membrane molecular motors from studies of Myxococcus xanthus surface (gliding) motility. Microb Cell 4: 98-100. 28357395
Minamino, T., M. Kinoshita, and K. Namba. (2019). Directional Switching Mechanism of the Bacterial Flagellar Motor. Comput Struct Biotechnol J 17: 1075-1081. 31452860
Minamino, T., N. Terahara, S. Kojima, and K. Namba. (2018). Autonomous control mechanism of stator assembly in the bacterial flagellar motor in response to changes in the environment. Mol. Microbiol. [Epub: Ahead of Print] 30069936
Morimoto, Y.V. and T. Minamino. (2014). Structure and function of the bi-directional bacterial flagellar motor. Biomolecules 4: 217-234. 24970213
Morimoto, Y.V. and T. Minamino. (2021). Architecture and Assembly of the Bacterial Flagellar Motor Complex. Subcell Biochem 96: 297-321. 33252734
Nakamura, S. and T. Minamino. (2019). Flagella-Driven Motility of Bacteria. Biomolecules 9:. 31337100
Nan, B., J. Chen, J.C. Neu, R.M. Berry, G. Oster, and D.R. Zusman. (2011). Myxobacteria gliding motility requires cytoskeleton rotation powered by proton motive force. Proc. Natl. Acad. Sci. USA 108: 2498-2503. 21248229
Nishihara Y. and Kitao A. (2015). Gate-controlled proton diffusion and protonation-induced ratchet motion in the stator of the bacterial flagellar motor. Proc Natl Acad Sci U S A. 112(25):7737-42. 26056313
Nishikino, T., H. Iwatsuki, T. Mino, S. Kojima, and M. Homma. (2019). Characterization of PomA periplasmic loop and sodium ion entering in stator complex of sodium-driven flagellar motor. J Biochem. [Epub: Ahead of Print] 31738405
O'Neill, J., M. Xie, M. Hijnen, and A. Roujeinikova. (2011). Role of the MotB linker in the assembly and activation of the bacterial flagellar motor. Acta Crystallogr D Biol Crystallogr 67: 1009-1016. 22120737
O'Neill, J., M. Xie, M. Hijnen, and A. Roujeinikova. (2011). Role of the MotB linker in the assembly and activation of the bacterial flagellar motor. Acta Crystallogr D Biol Crystallogr 67: 1009-1016. 18540076
Okabe, M., T. Yakushi, and M. Homma. (2005). Interactions of MotX with MotY and with the PomA/PomB sodium ion channel complex of the Vibrio alginolyticus polar flagellum. J. Biol. Chem. 280: 25659-25664. 15866878
Onoue, Y., M. Iwaki, A. Shinobu, Y. Nishihara, H. Iwatsuki, H. Terashima, A. Kitao, H. Kandori, and M. Homma. (2019). Essential ion binding residues for Na flow in stator complex of the Vibrio flagellar motor. Sci Rep 9: 11216. 31375690
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Santiveri, M., A. Roa-Eguiara, C. Kühne, N. Wadhwa, H. Hu, H.C. Berg, M. Erhardt, and N.M.I. Taylor. (2020). Structure and Function of Stator Units of the Bacterial Flagellar Motor. Cell. [Epub: Ahead of Print] 32931735
Suzuki, Y., Y.V. Morimoto, K. Oono, F. Hayashi, K. Oosawa, S. Kudo, and S. Nakamura. (2019). Effect of the MotA(M206I) Mutation on Torque Generation and Stator Assembly in the H-Driven Flagellar Motor. J. Bacteriol. 201:. 30642987
Takekawa, N., N. Terahara, T. Kato, M. Gohara, K. Mayanagi, A. Hijikata, Y. Onoue, S. Kojima, T. Shirai, K. Namba, and M. Homma. (2016). The tetrameric MotA complex as the core of the flagellar motor stator from hyperthermophilic bacterium. Sci Rep 6: 31526. 27531865
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Yakushi, T., S. Maki, and M. Homma. (2004). Interaction of PomB with the third transmembrane segment of PomA in the Na+-driven polar flagellum of Vibrio alginolyticus. J. Bacteriol. 186: 5281-5291. 15292129
Yonekura, K., S. Maki-Yonekura, and M. Homma. (2011). Structure of the flagellar motor protein complex PomAB: implications for the torque-generating conformation. J. Bacteriol. 193: 3863-3870. 21642461
Zhu S., Homma M. and Kojima S. (2012). Intragenic suppressor of a plug deletion nonmotility mutation in PotB, a chimeric stator protein of sodium-driven flagella. J Bacteriol. 194(24):6728-35. 23024347