A molecular conveyor belt-associated protein controls the rotational direction of the bacterial type 9 secretion system

Abstract

Many bacteria utilize the type 9 secretion system (T9SS) for gliding motility, surface colonization, and pathogenesis. This dual-function motor supports both gliding motility and protein secretion, where rotation of the T9SS plays a central role. Fueled by the energy of the stored proton motive force and transmitted through the torque of membrane-anchored stator units, the rotary T9SS propels an adhesin-coated conveyor belt along the bacterial outer membrane like a molecular snowmobile, thereby enabling gliding motion. However, the mechanisms controlling the rotational direction and gliding motility of T9SS remain elusive. Shedding light on this mechanism, we find that in the gliding bacterium Flavobacterium johnsoniae, deletion of the C-terminus of the conveyor belt-associated protein GldJ controls and, in fact, reverses the rotational direction of T9SS from counterclockwise (CCW) to clockwise (CW). This suggests that the interface between the conveyor belt-associated protein GldJ and the T9SS ring protein GldK plays an important role in controlling the directionality of T9SS, potentially by modulating its interaction with the stator complex GldLM, which drives motor rotation. Combined with MD simulation of the T9SS stator units GldLM, we suggest a "tri-component gearset" model where GldJ controls the rotational direction of its driver, the T9SS, thus providing adaptive sensory feedback to influence the motility of the gliding bacterium.

Importance: The type 9 secretion system (T9SS) is fundamental to bacterial gliding motility, pathogenesis, and surface colonization. Our findings reveal that the C-terminal region of the conveyor belt-associated protein GldJ functions as a molecular switch which is capable of reversing the rotational direction of T9SS. Through the coordinated actions of the T9SS stator units (akin to a driving motor), the GldK ring (the gear that converts rotational energy into linear movement), and GldJ, this machinery forms a smart conveyor belt system reminiscent of flexible or cognitive mechanical conveyors. Such advanced conveyors can alter their direction to adapt to shifting demands. Here, we show that the bacterial T9SS similarly adjusts its rotational bias based on feedback from the conveyor belt-associated protein GldJ. This dual-role feedback mechanism underscores an evolved, controllable biological snowmobile, offering new avenues for studying how bacteria fine-tune motility in dynamic environments.

Keywords: Flavobacterium; bacterial motility; bacterial protein secretion; chemotaxis; gliding motility; molecular motors; type 9 secretion system (T9SS).

Fig 1

The C-terminal region of a conveyor belt-associated protein, GldJ, controls the rotational direction of T9SS. (A) A cartoon showing the current rack and pinion model, where the GldK ring of rotary T9SS (pinion) drives the GldJ, part of conveyor belt (rack). (B) Frequency distribution of rotational speed shows that the outer portion of the T9SS in wild-type cells (n = 33) rotates primarily in the counterclockwise (CCW) direction with a mean speed of 1.3 Hz, whereas T9SS of cells lacking the C-terminal region of GldJ (GldJ ΔC8, n = 21) (CJ2443) rotates in the clockwise (CW) direction with a mean speed of 0.6 Hz. The red dotted line represents the mean rotational speed, and the + and − signs indicate CCW and CW rotational directions, respectively.

Fig 2

Experimental evolution restored gliding motility in cells that lack the C-terminal region of GldJ. (A) Cells lacking the C-terminal region of GldJ (GldJ ΔC8) do not swarm on agar. Experimental evolution of the GldJ ΔC8 strain (CJ2443) resulted in partial restoration of swarming. Spontaneous mutations that restored motility in the evolved strains were in the T9SS ring protein GldK. Scale bar = 6 mm. (B) Frequency distribution of gliding speed (μm/sec) for wild-type and mutant cells on a glass surface. The average gliding speeds for wild-type, GldJ ΔC8, GldK R73S GldJ ∆C8 (FJASU_21), M77L + GldJ ∆C8 (FJASU_20), and N307S + GldJ ∆C8 (FJASU_19) cells were 2 µm/s, 0.4 µm/s, 0.89 µm/s, 0.64 µm/s, and 0.88 µm/s, respectively. (C) Trajectories of wild-type and mutant cells gliding over a glass surface (n > 30), color-coded by time (seconds). The average farthest displacement for cells of wild-type, GldJ ∆C8, GldK R73S + GldJ ∆C8, M77L + GldJ ∆C8, and N307S + GldJ ∆C8 strains was 27 µm, 1.79 µm, 6.23 µm, 4.87 µm, and 8.14 µm, respectively.

Fig 3

Motion of the cell-surface adhesin SprB is partially restored in GldK N307S carrying GldJ ΔC-terminal cells. (A through C) A representative trajectory of fluorescently labeled SprB moving along the conveyor belt of wild-type and mutant cell (FJASU_19). (D) A box plot showing speeds of at least six SprB molecules for each strain. Statistical significance was analyzed via two-tailed Mann–Whitney test. P value: *P < 0.05, 0.001 < **P ≤ 0.01, ***P < 0.001. (E) Predicted structure of GldK showing the proximity of R73, M77, and N307 identified via the suppressor screen. In the surface representation, M77 is not visible, but the cartoon view (inset) displays all three residues. Docking of the conveyor belt-associated protein GldJ (cyan) with the T9SS rotor protein GldK (gray) shows that the GldJ C8 region fits into a GldK cleft region that contains R73 and N307 with M77 in the vicinity.

Fig 4

MD simulations suggest a preference for clockwise rotation of the driver gear, GldM, in the tri-component gearset. (A) Equilibrium MD simulations and nonbonded energy calculations show that the central arginine residue of GldM interacts more favorably with the glutamate residue of GldL in the CW direction. The kernel density estimation (KDE) method was used to estimate the probability density function of the nonbonded energy from the simulation trajectory. (B) Central: unaltered equilibrium configuration of the initial structure, excluding the membrane for the sake of clarity. Left: structure after a CCW GldM trans-membrane helix (TMH) rotation of −36°. Right: result after +36° CW rotation. The plot illustrates the nonequilibrium energetics from 10 consecutive SMD simulations (360° total in each direction), which reveal a lower work value for CW GldM TMH rotation. (C) Three-dimensional representation of the central structure shown in panel B. A top-down view highlights the plane used for the statistical analysis of post-MELD simulations. The spatial coordinates of the unbound arginine residue are mapped in a contour plot, with lighter shades indicating higher density, which suggests an unbiased preference for CW rotation. The arrows indicate the trajectory of the positional displacements of the unbound arginine. A green circle superimposed on the plot serves as the boundary where the probability of a datum falling within the enclosed region was computed.

Fig 5

A model for directional switching of T9SS. (A) A cartoon showing the tri-component gearset with GldM stator unit as the driver of the T9SS ring and conveyor belt. (B) An overlay of frames collected for the final 50 ns of the MD simulation for the wild-type GldJ (red) and GldK (blue) system with the GldJ C8 region highlighted in green. (C) Upon deletion of the C8 region of GldJ, an appreciable expansion of GldK is observed in contrast to the wild-type system shown in panel B. (D) An overlay of the GldK from the wild-type and GldJ∆C8 systems depicts conformational changes that are >1 nm. (E) A cross-sectional cartoon of the gliding machinery shows the proton motive force (pmf) powered driver gear, GldM, rotating unidirectionally clockwise (CW) and pushing the T9SS ring. This action results in the movement of the GldJ and SprB adhesin either towards the viewer or away from the current image plane. Images are not to scale. (F) A cartoon summarizing the cross-section of different conformational states (class 1, 2, and 3) of T9SS ring as observed by cryo-ET (25). (G) A top-down view of class 1 and class 3 T9SS motors as observed by cryo-ET (25) supports a physical model of T9SS directional switching. When the wild-type conveyor belt-associated protein GldJ interacts strongly with the GldK ring, T9SS maintains a conformation where GldM pushes the outer periphery of the GldK ring, causing T9SS to rotate CCW. Conversely, when the interaction energy between GldJ and GldK is reduced (GldJ∆C8), the GldK ring changes conformation. Consequently, GldM pushes near the inner periphery of the GldK ring, resulting in CW rotation of the T9SS.