Novel Insights into Conformational Rearrangements of the Bacterial Flagellar Switch Complex

Abstract

The flagellar motor can spin in both counterclockwise (CCW) and clockwise (CW) directions. The flagellar motor consists of a rotor and multiple stator units, which act as a proton channel. The rotor is composed of the transmembrane MS ring made of FliF and the cytoplasmic C ring consisting of FliG, FliM, and FliN. The C ring is directly involved in rotation and directional switching. The Salmonella FliF-FliG deletion fusion motor missing 56 residues from the C terminus of FliF and 94 residues from the N terminus of FliG keeps a domain responsible for the interaction with the stator intact, but its motor function is reduced significantly. Here, we report the structure and function of the FliF-FliG deletion fusion motor. The FliF-FliG deletion fusion not only resulted in a strong CW switch bias but also affected rotor-stator interactions coupled with proton translocation through the proton channel of the stator unit. The energy coupling efficiency of the deletion fusion motor was the same as that of the wild-type motor. Extragenic suppressor mutations in FliG, FliM, or FliN not only relieved the strong CW switch bias but also increased the motor speed at low load. The FliF-FliG deletion fusion made intersubunit interactions between C ring proteins tighter compared to the wild-type motor, whereas the suppressor mutations affect such tighter intersubunit interactions. We propose that a change of intersubunit interactions between the C ring proteins may be required for high-speed motor rotation as well as direction switching.IMPORTANCE The bacterial flagellar motor is a bidirectional rotary motor for motility and chemotaxis, which often plays an important role in infection. The motor is a large transmembrane protein complex composed of a rotor and multiple stator units, which also act as a proton channel. Motor torque is generated through their cyclic association and dissociation coupled with proton translocation through the proton channel. A large cytoplasmic ring of the motor, called C ring, is responsible for rotation and switching by interacting with the stator, but the mechanism remains unknown. By analyzing the structure and function of the wild-type motor and a mutant motor missing part of the C ring connecting itself with the transmembrane rotor ring while keeping a stator-interacting domain for bidirectional torque generation intact, we found interesting clues to the change in the C ring conformation for the switching and rotation involving loose and tight intersubunit interactions.

Keywords: chemotaxis; flagellar motility; flagellar structure; torque generation.

FIG 1

Rotor structure of the flagellar motor. (A) CryoEM image of Salmonella basal body (BB) and its schematic diagram. Purified BB consists of the C, MS, L, and P rings and the rod. The type III protein export apparatus, which is composed of a transmembrane export gate complex and a cytoplasmic ATPase complex, and a dozen stator units are associated with the BB. However, these structures are missing during BB purification. The C ring consists of FliG, FliM, and FliN. The N-terminal domain of FliG (FliG_N) forms the inner lobe structure along with the C-terminal cytoplasmic domain of FliF (FliF_C). The C-terminal domain of FliG (FliG_C) is located in the upper part of the C ring. The middle domain of FliM (FliM_M) is located between the middle domain of FliG (FliG_M) and FliN and forms a continuous wall of the C ring. A continuous spiral density at the bottom edge of the C ring is made of the C-terminal domain of FliM (FliM_C) and FliN. (B) Homology model of Salmonella FliG. A model was built based on the crystal structure of FliG derived from Aquifex aeolicus (PDB code 3HJL). The Cα backbone is color coded from blue to red, going through the rainbow colors from the N terminus to the C terminus. FliG consists of FliG_N, FliG_M, and FliG_C domains and two helix linkers named Helix_NM and Helix_MC. FliG_C is divided into FliG_CN and FliG_CC subdomains. Arg281 and Asp289 residues in FliG_CC are responsible for interactions with the cytoplasmic domain of the stator unit.

FIG 2

CW bias and switching frequency of the flagellar motor. (A to D) CW bias and switching frequency of wild-type (A), FliFG_d-f (B), FliFG_d-f FliG(D124Y) (C), and FliFG_d-f FliM(F188L) (D) motors were analyzed at pH 7.0 (left panels), at pH 6.5 in the presence of 10 mM potassium benzoate (middle panels), or at pH 6.0 in the presence of 10 mM potassium benzoate (right panels). Rotation measurements of tethered cells were performed at room temperature for 30 s. CW bias versus switching frequency plots were generated from individual motors analyzed under each condition. Histograms of CW bias and switching frequency were shown on the bottom and left, respectively. The number of tethered cells analyzed under each condition are as follows: wild-type cells at pH 7.0, 154 cells; wild-type cells at pH 6.5 with potassium benzoate, 220 cells; wild-type cells at pH 6.0 with potassium benzoate, 156 cells; FliFG_d-f at pH 7.0, 136 cells; FliFG_d-f at pH 6.5 with potassium benzoate, 127 cells; FliFG_d-f at pH 6.0 with potassium benzoate, 106 cells; FliFG_d-f FliG(D124Y) at pH 7.0, 116 cells; FliFG_d-f FliG(D124Y) at pH 6.5 with potassium benzoate, 121 cells; FliFG_d-f FliG(D124Y) at pH 6.0 with potassium benzoate, 67 cells; FliFG_d-f FliM(F188L) at pH 7.0, 116 cells; FliFG_d-f FliM(F188L) at pH 6.5 with potassium benzoate, 145 cells; FliFG_d-f FliG FliM(F188L) at pH 6.0 with potassium benzoate, 55 cells.

FIG 3

Rotation measurements of the FliFG_d-f and its suppressor mutant motors over a wide range of external load. (A) Torque-speed relationship of the wild-type (circles), FliFG_d-f (triangles), FliFG_d-f FliG(D124Y) (squares), and FliFG_d-f FliM(F188L) (diamonds) motors. Rotation measurements were carried out at room temperature by tracking the positions of 1.5-μm (orange), 1.0-μm (red), 0.8-μm (purple), 0.6-μm (cyan), and 0.5-μm (light green) beads attached to the partially sheared sticky filament. Each bead image was recorded for 7 s. (B) Resurrection trace of a single flagellar motor of MM046mAB (indicated as WT), MM3278-46mAB (indicated as FliFG_d-f), MM3278-8-46mAB [indicated as FliFG_d-f FliG(D124Y)], or MM3278-1-46mAB [indicated as FliFG_d-f FliM(F188L)] expressing the MotAB complex from an arabinose-inducible promoter on the pBAD24 vector. The cells were grown in L-broth with shaking until the cell density had reached an OD₆₀₀ of ca. 0.6. After incubation with 0.002% arabinose at room temperature for 30 min, rotation measurements were carried out by tracking the positions of 1.0-μm beads attached to the partially sheared sticky filament in a motility buffer containing 0.2% arabinose. The traces and speed histograms are shown on the left and right, respectively. Speed histograms were fitted by multiple Gaussian functions to estimate a unit increment.

FIG 4

Effect of the FliF-FliG deletion fusion on speed stability of flagellar motor rotation. (A) Typical examples of motor speed versus time plots of the wild-type, FliFG_d-f , FliFG_d-f FliG(D124Y), and FliFG_d-f FliM(F188L) motors. Rotation measurements of 1.0-μm beads attached to the wild-type, FliFG_d-f, FliFG_d-f FliG(D124Y), and FliFG_d-f FliM(F188L) motors were carried out at room temperature for 300 s. Speed histograms are shown on the left of the traces. (B) Speed fluctuation of the wild-type, FliFG_d-f, FliFG_d-f FliG(D124Y), and FliFG_d-f FliM(F188L) motors. The values of the average speeds (ω_av) and their standard deviations (σ_ω) were calculated. Black dots indicate individual motors.

FIG 5

Locations of suppressor mutations of a FliF-FliG deletion fusion. (A) Homology model of Salmonella FliG_M-FliM_M complex and location of the FliG(D124Y) suppressor mutation. A homology model was built based on the crystal structure of Thermotoga maritima FliG_M-FliM_M complex (PDB code 3SOH). Cα ribbon drawing of FliG_M (cyan) and FliM_M (green). (B) Model of FliM subunit arrangement in the C ring and locations of the FliM(V186A), FliM(F188L), and FliM(I217T) suppressor mutations. A homology model of Salmonella FliM_M was built based on the crystal structure of Thermotoga maritima FliM_M (PDB code 3SOH). (C) Crystal structure of Salmonella FliM_C-FliN_N fusion protein (PDB code 4YXB) and location of the FliN(E95G) suppressor mutation. FliM_C and FliN_N subunits are shown by green and cyan Cα ribbon models, respectively.

FIG 6

CryoEM image analysis of the C ring structure. (A) Projection images (top) and 3D volume maps (bottom) of the MS-C ring complex of the CCW, CW, and FliFG_d-f motors with C34, C34, and C31 symmetries, respectively. (B) Side views of representative 2D class averages of the MS-C ring complex of the FliFG_d-f FliG(D124Y) motor. A histogram of the C ring diameter is shown on the right.