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. 2009 Jul;191(14):4633-8.
doi: 10.1128/JB.00396-09. Epub 2009 May 8.

High-force generation is a conserved property of type IV pilus systems

Martin Clausen  1 , Vladimir Jakovljevic, Lotte Søgaard-Andersen, Berenike Maier
Affiliations

High-force generation is a conserved property of type IV pilus systems

Martin Clausen et al. J Bacteriol. 2009 Jul.

Abstract

The type IV pilus (T4P) system of Neisseria gonorrhoeae is the strongest linear molecular motor reported to date, but it is unclear whether high-force generation is conserved between bacterial species. Using laser tweezers, we found that the average stalling force of single-pilus retraction in Myxococcus xanthus of 149 +/- 14 pN exceeds the force generated by N. gonorrhoeae. Retraction velocities including a bimodal distribution were similar between M. xanthus and N. gonorrhoeae, but force-dependent directional switching was not. Force generation by pilus retraction is energized by the ATPase PilT. Surprisingly, an M. xanthus mutant lacking PilT apparently still retracted T4P, although at a reduced frequency. The retraction velocity was comparable to the high-velocity mode in the wild type at low forces but decreased drastically when the force increased, with an average stalling force of 70 +/- 10 pN. Thus, M. xanthus harbors at least two different retraction motors. Our results demonstrate that the major physical properties are conserved between bacteria that are phylogenetically distant and pursue very different lifestyles.

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Figures

FIG. 1.
FIG. 1.
Experimental setup and force generation during T4P retraction. (a) Sketch of the experimental setup. A cell was immobilized on a polystyrene-coated cover slide. When a T4P bound to the bead in the laser trap and retracted, it displaced the bead by a distance d from the center of the trap. At a preset threshold, dset, the force feedback (Fig. 3a) could be triggered by moving the piezo table by a distance x to maintain d at a constant value. (b and c) Typical deflection, d, of the bead during pilus retraction as a function of time in the wt (DK1622). F, force; t, time.
FIG. 2.
FIG. 2.
Histogram of stalling forces of T4P in the wt (DK1622). Stalling events were classified into (a) 61 events containing no pause between onset of the retraction and the final plateau (Fig. 1b) and (b) 17 events containing a pause at an intermediate force level (Fig. 1c). Forces (F) exceeding 220 pN are systematically underestimated due to limited force generation of the laser tweezers. p, normalized number of stalling events.
FIG. 3.
FIG. 3.
Retraction velocities at constant force. (a) Typical length change x of a single T4P of the wt (DK1622) as a function of time (t) at a fixed force of 150 pN. When a T4P bound to the bead in the laser trap and retracted, it displaced the bead by a distance d from the center of the trap. At a predefined distance dset corresponding to a force F, the feedback was triggered, which moved the piezo table to keep d constant. (b) Histogram of velocities at constant force for the wt from 98 retraction curves. The average track length was about 1 second. (c) Retraction velocities obtained from panel b by fitting of a double Gaussian (15 and 30 pN) and a single Gaussian (60 pN) and by fitting of a line to all curves and averaging (120 and 150 pN). The error bars indicate standard deviations of the mean velocities.
FIG. 4.
FIG. 4.
T4P retraction velocity in the ΔpilT mutant. (a) Histogram of retraction velocities at constant force. DK1622 (wt), filled gray bars; DK10409 (ΔpilT), open bars. (b) Velocities obtained from panel a by fitting of a Gaussian (15 and 30 pN) and by fitting of a line to all curves and averaging (60 pN). DK10409, thick-bordered open bars; DK1622 (low-velocity peak), thin-bordered open bars; DK1622 (high-velocity peak), filled gray bars. The error bars indicate standard deviations. (c) Histogram of the stalling forces. DK1622, filled gray bars; DK10409, open bars.
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References

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