Directional Switching Mechanism of the Bacterial Flagellar Motor

Bacteria sense temporal changes in extracellular stimuli via sensory signal transducers and move by rotating flagella towards into a favorable environment for their survival. 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 a 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. 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. In this mini-review, we will describe current understanding of the switching mechanism of the bacterial flagellar motor.


Introduction
Many bacteria possess flagella to swim in liquid media and move on solid surfaces. Escherichia coli and Salmonella enterica serovar Typhimurium (hereafter referred to Salmonella) are model organisms that have provided deep insights into the structure and function of the bacterial flagellum. The flagellum is composed of basal body rings and an axial structure consisting of at least three parts: the rod as a drive shaft, the hook as a universal joint and the filament as a helical propeller (Fig. 1A). The flagellar motor of E. coli and Salmonella consists of a rotor and a dozen stator units and is powered by an electrochemical potential of protons across the cytoplasmic membrane, namely proton motive force. Marine Vibrio and extremely alkalophilic Bacillus utilize sodium motive force as the energy source to drive flagellar motor rotation. The rotor is composed of the MS ring made of the transmembrane protein FliF and the C ring consisting of three cytoplasmic proteins, FliG, FliM and FliN. Each stator unit is composed of two transmembrane proteins, MotA and MotB, and acts as a transmembrane proton channel to couple the proton flow through the channel with torque generation (Fig. 1B) [1][2][3][4][5].
The flagellar motor rotates in either counterclockwise (CCW; viewed from the flagellar filament to the motor) or clockwise (CW) direction in E. coli and Salmonella. When all the motors rotate in the CCW direction, flagellar filaments together form a bundle behind the cell body to push the cell forward. Brief CW rotation of one or more flagellar motors disrupts the flagellar bundle, allowing the cell to tumble, followed by a change in the swimming direction. Sensory signal transducers sense temporal changes in extracellular stimuli such as chemicals, temperature and pH and transmit such extracellular signals to the flagellar motor via the intracellular chemotactic signaling network. The phosphorylated form of CheY (CheY-P), which serves as a signaling molecule, binds to FliM and FliN in the C ring to switch the direction of flagellar motor rotation from CCW to CW. Thus, the C ring acts as a switching device to switch between the CCW and CW states of the motor [2,5].
The stator complex is composed of four copies of MotA and two copies of MotB. The MotA 4 /MotB 2 complex is anchored to the peptidoglycan (PG) layer through direct interactions of the C-terminal periplasmic domain of MotB with the PG layer to become an active stator unit around the rotor [4]. A highly conserved aspartate residue of MotB (Asp-32 in the E. coli protein and Asp-33 in the Salmonella protein) is located in the MotA 4 /MotB 2 proton channel and is involved in the energy coupling mechanism [6,7]. The cytoplasmic loop between transmembrane helices 2 and 3 of MotA (MotA C ) contains highly conserved Arg-90 and Glu-98 residues and are important not only for torque generation but also for stator assembly around the rotor [8][9][10].
FliG is directly involved in torque generation [8]. Highly conserved Arg-281 and Asp-289 residues are located on the torque helix of FliG (Helix Torque ) [11] and interact with Glu-98 and Arg-90 of MotA C , respectively [8,10]. Since the elementary process of torque generation caused by sequential stator-rotor interactions in the flagellar motor is symmetric in the CCW and CW rotation, Helix Torque is postulated to rotate 180°r elative to MotA C in a highly cooperative manner when the motor switches between the CCW and CW states of the C ring [12]. This mini-review article covers current understanding of how such a cooperative remodeling of the C ring structure occurs.

Structure of the C Ring
FliF assembles into the MS ring within the cytoplasmic membrane [13]. The C ring consisting of a cylindrical wall and inner lobes is formed by FliG, FliM and FliN on the cytoplasmic face of the MS ring with the inner lobes connected to the MS ring ( Fig. 1B) [14]. FliF requires FliG to facilitate MS ring formation in the cytoplasmic membrane [15]. FliG binds to FliF with a one-to-one stoichiometry [16]. FliM and FliN together form the FliM 1 /FliN 3 complex consisting of one copy of FliM and three copies of FliN [17], and the FliM 1 /FliN 3 complex binds to the FliG ring structure through a one-to-one interaction between FliG and FliM to form the continuous C ring wall [18][19][20]. Most of the domain structures of FliG, FliM and FliN have been solved at atomic resolution ( Fig. 2), and possible models of their organization in the C ring have been proposed (Fig. 1B) [21,22].

FliG
FliG consists of three domains: N-terminal (FliG N ), middle (FliG M ) and C-terminal (FliG C ) domains ( Fig. 2A) [23]. FliG C is divided into two subdomains: FliG CN and FliG CC . FliG N is involved in the interaction with the C-terminal cytoplasmic domain of FliF (FliF C ) (Fig. 2B) [24,25]. Inter-molecular interactions between FliG N and FliG N and between FliG M and FliG CN are responsible for the assembly of FliG into the ring structure on the cytoplasmic face of the MS ring [26][27][28][29]. FliG M provides binding sites for FliM (Fig. 2C) [18][19][20]. A highly conserved EHPQR motif of FliG M is involved in the interaction with FliM [18,30]. FliG CC contains Helix Torque , and highly conserved Arg-284 and Asp-292 residues of Aquifex aeolicus FliG, which corresponds to Arg-281 and Asp-289 of E. coli FliG involved in the interactions with conserved charged residues of MotA C [8,11], are exposed to solvent on the surface of Helix Torque [23].

FliM
FliM consists of three domains: N-terminal (FliM N ), middle (FliM M ) and C-terminal (FliM C ) domains [31,32]. FliM N contains a well conserved LSQXEIDALL sequence, which is responsible for the interaction with CheY-P [33]. FliM N is intrinsically disordered, and the binding of CheY-P to FliM N allows FliM N to become structured [32]. FliM M has a compactly folded conformation (Fig. 2C), and side-by-side associations between FliM M domains are responsible for the formation of the C ring wall [32]. The binding of CheY-P to FliM N affects inter-molecular FliM M -FliM M interactions, thereby inducing a conformational change in the C ring responsible for switching the direction of flagellar motor rotation [34]. A well conserved GGXG motif of FliM M is involved in the interaction with FliG M (Fig. 2C) [18,30]. FliM C shows significant sequence and structural similarities with FliN and is responsible for the interaction with FliN (Fig. 2D) [35].

FliN
FliN is composed of an intrinsically disordered N-terminal region (FliN N ) and a compactly folded domain (FliN C ), which structurally looks similar to FliM C [36]. FliN exists as a dimer of dimer in solution (Fig. 2E) [37] and forms the FliM 1 /FliN 3 complex along with FliM through an interaction between FliM C and FliN [17]. CheY-P binds to FliN C in a FliM-dependent manner [38]. Leu-68, Ala-93, Val-113 and Asp-116 of E. coli FliN are responsible for the interaction with CheY-P (Fig. 2D) [38,39]. The binding of CheY-P to FliN affects interactions between FliM C and FliN, inducing the conformational change of the C ring responsible for directional switching of flagellar motor rotation [38]. FliN N seems to control the binding affinity of FliN C for CheY-P [38] although it is dispensable for the function of FliN [40]. FliN also provides binding sites for FliH, a cytoplasmic component of the flagellar type III protein export apparatus for efficient flagellar protein export and assembly  [36,[39][40][41][42]. Val-111, Val-112 and Val-113 of FliN are responsible for the interaction with FliH (Fig. 2D) [39,41].

Subunit Organization in the C Ring Structure
Electron cryomicroscopy (cryoEM) image analysis has shown that the C ring structures of the purified CCW and CW motors have rotational symmetry varying from 32-fold to 35-fold, and the diameter varies accordingly [43,44]. The C ring diameters of the CCW and CW motors with C34 symmetry are 416 Å and 407 Å, respectively, and so the unit repeat distance along the circumference of the C ring is closer in the CW motor than in the CCW motor [45]. The C ring produced by a Salmonella fliF-fliG deletion fusion strain missing FliF C and FliG N lacks the inner lobe, suggesting that FliF C and FliG N together form the inner lobe (Fig. 1) [45,46]. In agreement with this, cryoEM images of the C ring containing the N-terminally green fluorescent protein (GFP) tagged FliG protein show an extra density corresponding to the GFP probe near the inner lobe [47]. The fliF-fliG deletion fusion results in unusual switching behavior of the flagellar motor, suggesting that the inner lobe is required for efficient and robust switching in the direction of flagellar motor rotation in response to changes in the environment [45]. The upper part of the C ring wall is formed by FliG M and FliG C . FliG M binds to FliG CN of its adjacent FliG subunit to produce a domain-swap polymer of FliG to form a ring in both CCW and CW motors [26,27,29]. Since Helix Torque of FliG CC interacts with MotA C [8,10], FliG CC is located at the top of the C ring wall (Fig. 1). Since FliM M directly binds to FliG M (Fig. 2C) [18][19][20], the continuous wall of the C ring with a thickness of 4.0 nm and a height of 6.0 nm is formed by side-by-side associations of the FliM M domains (Fig. 1) [32]. A continuous spiral density with a diameter of 7.0 nm along the circumference at the bottom edge of the C ring is made of FliM C and FliN (Fig. 1) [17,36].

Structural Basis for the Rotational Switching Mechanism
In E. coli and Salmonella, the flagellar motor is placed in a default CCW state [3,5]. Mutations located in and around Helix MC of FliG, which connects FliG M and FliG CN , cause unusual switching behavior of the flagellar motor [48], suggesting that helix MC is involved in switching the direction of flagellar motor rotation. Helix MC is located at the FliG M -FliM M interface and contributes to hydrophobic interactions between FliG M and FliM M (Fig. 3A) [18,19]. In-frame deletion of three residues, Pro-Ala-Ala at positions 169 to 171 of Salmonella FliG, which are located in Helix MC , locks the motor in the CW state even in the absence of CheY-P (CW-locked deletion) [49,50]. The crystal structure of the FliG M and FliG C domains derived from Thermotaoga maritima (Tm-FliG MC ) with this CW-locked deletion have shown that the conformation of Helix MC is distinct from that of the wild-type [19,50,51]. In the wild-type Tm-FliG MC /Tm-FliM M complex, Val-172 of Helix MC of Tm-FliG MC makes hydrophobic contact with Ile-130 and Met-131 of Tm-FliM M (Fig. 3A) [18,19]. In contrast, disulfate crosslinking experiments have shown that Helix MC is dissociated from Tm-FliG M in the presence of the CWlocked deletion (Fig. 3A) [28]. Consistently, the CW-locked deletion of Tm-FliG reduces the binding affinity of Tm-FliG MC for Tm-FliM M by about 400-fold [28]. Therefore, it seems likely that the binding of CheY-P to FliM and FliN induces conformational rearrangements of the FliG M -FliM M interface, thereby causing dissociation of Helix MC from the interface to facilitate the remodeling of the FliG ring structure responsible for directional switching of the flagellar motor. Helix MC interacts with Helix NM connecting FliG N and FliG M ( Fig. 2A) [23]. The E95D, D96V/Y, T103S, G106A/C and E108K substitutions in Helix NM of Salmonella FliG result in a strong CW switch bias [52]. A homology model of Salmonella FliG built based on the crystal structure of FliG derived from A. aeolicus (PDB code: 3HJL) has suggested that Thr-103 of Helix NM may make hydrophobic contacts with Pro-169 and Ala-173 of Helix MC [45]. These observations lead to a plausible hypothesis that a change in the Helix NM -Helix MC interaction mode may be required for conformational rearrangements of the C ring responsible for directional switching of the flagellar motor. A FliF-FliG full length fusion results in a strong CW switch bias of the E. coli flagellar motor [27]. Intragenic suppressor mutations, which improve the chemotactic behavior of the E. coli fliF-fliG full-length fusion strain, are located at the FliG N -FliG N interface [27], suggesting that a change in inter-molecular FliG N -FliG N interactions may be required for flagellar motor switching. Therefore, there is the possibility that conformational rearrangements of the FliG M -FliM M interface caused by the binding of CheY-P to the C ring influence the Helix NM -Helix MC interaction, thereby inducing conformational rearrangements of FliG N domains responsible for the switching in the direction of flagellar motor rotation.
The elementary process of torque generation by stator-rotor interactions is symmetric in CCW and CW rotation [12]. A hinge connecting FliG CN and FliG CC has a highly flexible nature at the conserved MFXF motif, allowing FliG CC to rotate 180°relative to FliG CN to reorient Arg-281 and Asp-289 residues in Helix Torque to achieve a symmetric elementary process of torque generation in both CCW and CW rotation (Fig. 3B) [53][54][55][56]. Structural comparisons between Tm-FliG MC of the wild-type and Tm-FliG MC with the CW-locked deletion have shown that the CWlocked deletion induces a 90°rotation of FliG CC relative to FliG CN through the MFXF motif (Fig. 3A) [50]. Consistently, the binding of CheY-P to the C ring induces a tilting movement of FliM M , resulting in the rotation of FliG CC relative to FliG CN [34]. Therefore, it is possible that such a tilting movement of FliM M may promote a detachment of Helix MC from the FliG M -FliM M interface, resulting in the 180°rotation of FliG CC relative to FliG CN .

Adaptive remodeling of the C ring
FliM and FliN alternate their forms between localized and freely diffusing ones (Fig. 4), and the copy number of FliM and FliN in the CCW motor has been found to be about 1.3 times larger than that in the CW motor [57][58][59][60]. Consistently, fluorescence anisotropy techniques have shown that the CCW motor accommodate more FliM 1 /FliN 3 complexes without changing the spacing between FliM subunits [61]. Such exchanges depend on the direction of flagellar rotation but not on the binding of CheY-P to the C ring per se [58]. The timescale of this adaptive switch remodeling of the C ring structure is much slower (~1 min) than that of the rotational switching between the CCW and CW states (less than millisecond). Such a structural remodeling of the C ring is important for fine-tuning the chemotactic response to temporal changes in the environments [62][63][64][65]. The CW-locked deletion of FliG considerably reduces the binding affinity of FliG M for FliM M presumably due to detachment of Helix MC from the FliG M -FliM M interface (Fig. 3A) [28]. Because FliM binds to Helix MC of FliG in the E. coli CCW motor [27], the dissociation of Helix MC from the FliG M -FliM M interface may promote the dissociation of several weakly bound FliM 1 /FliN 3 complexes from the FliG ring when CheY-P binds to the C ring to switch from its CCW to CW states (Fig. 4).

Summary and Perspectives
Switching between the CW and CCW states of the flagellar motor is highly cooperative [66]. The cooperative switching mechanism can be explained by a conformational spread model, in which a switching event is mediated by conformational changes in a ring of subunits that spread from subunit to subunit via their interactions along the ring [67][68][69]. The binding of CheY-P to FliM and FliN affects subunitsubunit interactions between FliM M domains and between FliM C and FliN in the C ring to induce a 180°rotation of FliG CC relative to MotA C , thereby allowing the motor to rotate in CW direction [34]. Helix MC of FliG located at an interface between FliG M and FliM M plays an important role in highly cooperative remodeling of the FliG ring structure [28]. However, it remains unknown how Helix MC coordinates cooperative rearrangements of FliG subunits with changes in the direction of flagellar motor rotation. The C ring of the CCW motor can accommodate more FliM/FliN 3 complexes without changing inter-subunit spacing, and directional switching of the motor induces several weakly bound FliM/ FliN 3 complexes from the C ring [57][58][59][60]. Consistently, the CW-locked deletion weakens an interaction between FliG M and FliM M [28]. Because there is no difference in the rotational symmetry of the C ring between the purified CCW and CW motors [45], it remains unclear how several FliM 1 /FliN 3 complexes weakly associate with the C ring when the motor spins in the CCW direction.
The elementary process of the torque-generation cycle is symmetrical in CCW and CW directions [12]. However, the output characteristics of the CW motor are distinct from those of the CCW motor. Torque produced by the CCW motor remains almost constant in a high-load, lowload regime of the torque-speed curve and decreases sharply to zero in a low-load, high-speed regime. In contrast, torque produced by the CW motor linearly decreases with increasing motor speed [70]. This suggests that directional switching of the flagellar motor may affect stator-rotor interactions in a load-dependent manner. However, Fig. 4. Adaptive remodeling of the FliG ring in the CCW and CW motors. Inter-molecular interactions of FliG CN with FliG M of its neighboring subunit produce the CCW ring structure. Upon binding of CheY-P to the C ring, conformational rearrangements of the FliG M -FliG C interface occur, resulting in detachment of Helix MC from the interface. As a result, several weakly bound FliM 1 /FliN 3 complexes dissociate from the FliG ring.
nothing is known about the molecular mechanism. Furthermore, the switching rate of the flagellar motor also depends on the motor speed [71,72]. A recent non-equilibrium model of the flagellar motor switching has predicted that the motor sensitivity to CheY-P increases with an increase in motor torque [73]. However, it remains unknown how stator-rotor interactions modulate the binding affinity for CheY-P. High-resolution structural analysis of the C rings in the CCW and CW states by cryoEM image analysis will be essential to advance our mechanistic understanding of the directional switching mechanism of the flagellar motor.

Declaration of Competing Interest
The authors declare that they have no competing interests.