Single pilus motor forces exceed 100 pN

Abstract

Force production by type IV pilus retraction is critical for infectivity of Neisseria gonorrhoeae and DNA transfer. We investigated the roles of pilus number and the retraction motor, PilT, in force generation in vivo at the single-molecule level and found that individual retraction events are generated by a single pilus fiber, and only one PilT complex powers retraction. Retraction velocity is constant at low forces but decreases at forces greater than 40 pN, giving a remarkably high average stall force of 110 +/- 30 pN. Further insights into the molecular mechanism of force generation are gained from the effect of ATP-depletion, which reduces the rate of retraction but not the stall force. Energetic considerations suggest that more than one ATP is involved in the removal of a single pilin subunit from a pilus. The results are most consistent with a model in which the ATPase PilT forms an oligomer that disassembles the pilus by a cooperative conformational change.

Fig 1.

(A) Sketch of the setup. For retraction experiments, 3-μm silica beads (Polysciences) were coated with poly(l-lysine) and adsorbed to glass cover slides by centrifugation. Subsequently, 1.5-μm silica beads or 2-μm carboxylated latex beads (Polysciences) were added without further treatment to a suspension of gonococci, mounted on a microscope slide, and sealed. (B) Histogram of stalling force for MS11. (C and E) Deflection of the bead as a function of time (orange line). The fit (•) is a polygon fit with an average over 30 ms, as described in Materials and Methods. (D and F) Velocity vs. force for the event shown in C and E, respectively. (C and D) MS11C9.10 at 0.1 mM IPTG. (E and F) MS11.

Fig 2.

Retraction kinetics of pilE mutant under load. Electron micrograph of WT MS11 (A) and pilE mutant MS11C9.10 (B) at 0.01 mM IPTG. (C) Frequency of retraction events and number of pili visible by electron microscopy. Only those cells were counted that retracted/showed pili. (D) Average stall force of WT compared with pilE mutant at varying IPTG concentrations. (E) Velocity as a function of force for varying expression level of pilin subunits. WT, black triangle; pilE, 10 mM IPTG, blue diamond; pilE, 0.1 mM IPTG, red square; pilE, 0.01 mM IPTG, yellow circle; averaged over K > 90 retraction events for each IPTG concentration. Note that many retraction events were terminated by breakage before the stalling force was reached.

Fig 3.

Retraction kinetics of pilT mutant. (A) Frequency of retraction events. (B) Average stall force of WT compared with pilT mutant at varying IPTG concentration. (C) Retraction rate for varying IPTG concentrations. WT, black triangle; pilE, 0.01 mM IPTG, yellow circle; pilE, 0.1 mM IPTG, blue square; averaged over K > 34 retraction events for each IPTG concentration.

Fig 4.

Depletion of ATP. (A) Retraction rate at varying concentrations of CCCP (yellow circle) and 1 mM DNP (blue diamond) at 30 pN. (B) Rate at varying concentrations of NaN₃ at 30 pN. (C) Comparison of the velocity vs. force curve at 0 mM (blue circle) and 5 mM NaN₃ (yellow square); averaged over K > 10 retraction events for each concentration. (D) Average stall force for 5 and 7 mM NaN₃.