A force-dependent switch reverses type IV pilus retraction

Abstract

Type IV pilus dynamics is important for virulence, motility, and DNA transfer in a wide variety of prokaryotes. The type IV pilus system constitutes a very robust and powerful molecular machine that transports pilus polymers as well as DNA through the bacterial cell envelope. In Neisseria gonorrhoeae, pilus retraction is a highly irreversible process that depends on PilT, an AAA ATPase family member. However, when levels of PilT are reduced, the application of high external forces (F = 110 +/- 10 pN) induces processive pilus elongation. At forces of >50 pN, single pili elongate at a rate of v = 350 +/- 50 nm/s. For forces of <50 pN, elongation velocity depends strongly on force and relaxation causes immediate retraction. Both pilus retraction and force-induced elongation can be modeled by chemical kinetics with same step length for the rate-limiting translocation step. The model implies that a force-dependent molecular switch can induce pilus elongation by reversing the retraction mechanism.

Fig. 1.

Type IV pili elongate at high external force. (a) Setup. N. gonorrhoeae was immobilized on a glass coverslide by attachment to a poly(l-lysine) coated bead. A 1.5- or 2-μm latex bead was approached to the bacterium by using an optical trap. When a pilus bound the bead and retracted, the deflection of the bead from the center of the laser trap was measured by using a quadrant photodiode. (b) Example for pilus dynamics in the derepressible pilT mutant (MS11-600) at 0.1 mM IPTG. Pilus retraction deflects the bead from the center of the optical trap. When the maximum force is reached, the bead is moved back into the center of the optical trap. (c) Detail of the time series shown in a. The black line is a fit to the raw data (orange) with an average time period of 33 ms. (d) Velocity of bead deflection derived from the fit shown in c. (e) At 11.5 s the optical trap was turned off, and the pilus retracted immediately until the bead stuck to the bacterium (n = 2).

Fig. 2.

Pilus elongation occurs only at reduced concentration of PilT. Typical pilus retraction events for derepressible pilT mutant at 0.1 mM IPTG (a), WT (b), and pilU mutant (c). (d) Distribution of pausing period before breaking event for WT and pilU mutant and distribution of pausing time before elongation event for derepressible pilT mutant. Gray bar, derepressible pilT at 0.1 mM IPTG (n = 43); red bar, WT (n = 22); blue bar, pilU (n = 24). (e) Frequency of retraction events. (f) Relative frequency of elongation events per total stalling events.

Fig. 3.

Force-dependent kinetics of pilus retraction. (a) Distribution of maximum forces of derepressible pilT (MS11-600) at 0.1 mM IPTG (n = 43; gray bar), WT (n = 22; red bar), and pilU (n = 24; blue bar). (b) Velocity-vs.-force relationship for pilus retraction for derepressible pilT at 0.1 mM IPTG (triangle), WT (square), and pilU (circle).

Fig. 4.

A force-dependent switch. (a) Averaged velocity-vs.-force relationship for pilus elongation (MS11-600). The fit according to Eq. 1 yielded a_e = 0.07 ± 0.02 nm and A_e = 0.9 ± 0.2 (χ² = 13, v = 13). (b) Averaged negative velocity-vs.-force relationship for pilus retraction in derepressible pilT mutant (MS11-600). To be able to fit the data, one data curve with a long stalling event at 50 pN was removed and data were averaged over a smaller force range (filled circles). The fit according to Eq. 1 yielded a_r = 0.10 ± 0.02 nm/s and A_r = 0.02 ± 0.01 (χ² = 5, v = 8). (c) Molecular model of pilus formation.