Kinetics of DNA uptake during transformation provide evidence for a translocation ratchet mechanism

Abstract

Horizontal gene transfer can speed up adaptive evolution and support chromosomal DNA repair. A particularly widespread mechanism of gene transfer is transformation. The initial step to transformation, namely the uptake of DNA from the environment, is supported by the type IV pilus system in most species. However, the molecular mechanism of DNA uptake remains elusive. Here, we used singlemolecule techniques for characterizing the force-dependent velocity of DNA uptake by Neisseria gonorrhoeae. We found that the DNA uptake velocity depends on the concentration of the periplasmic DNA-binding protein ComE, indicating that ComE is directly involved in the uptake process. The velocity-force relation of DNA uptake is in very good agreement with a translocation ratchet model where binding of chaperones in the periplasm biases DNA diffusion through a membrane pore in the direction of uptake. The model yields a speed of DNA uptake of 900 bp·s −1 and a reversal force of 17 pN. Moreover, by comparing the velocityforce relation of DNA uptake and type IV pilus retraction, we can exclude pilus retraction as a mechanism for DNA uptake. In conclusion, our data strongly support the model of a translocation ratchet with ComE acting as a ratcheting chaperone.

Bacterial Translocation Ratchets: Shared Physical Principles with Different Molecular Implementations

“…[33] Recently, we obtained direct evidence for the role of ComE as a DNA binding chaperone of a translocation ratchet from singlemolecule experiments measuring the kinetics of DNA import into the periplasm. [3] The concentration of ComE directly affects the velocity of DNA import, in agreement with theoretical predictions for a chaperone-assisted translocation ratchet. [34] Most importantly, Peskin et al predicted an analytical expression for the velocity versus force relation of DNA uptake [35] (Box 2).…”

“…Single molecule experiments have shown that fragments as long as 10 kbp or 3.4 mm can be imported without interruption. [3] Uptake of DNA from the environment (cis side) into the periplasm (trans side) occurs independent of transport through the inner membrane. However, no ATP is present within the periplasm and furthermore, no ion gradient is maintained over the outer membrane that may power molecular motors.…”

“…Recent biophysical experiments provide strong evidence that periplasmic chaperones bias Brownian motion in the direction of uptake. [3] DNA secretion by the T4SS requires secretion of DNA-binding proteins into the cytoplasm by the host cell. Biophysical experiments in conjunction with cell biological approaches suggest that chaperones are secreted early during the infection cycle, generating a chemical potential difference for biasing DNA diffusion into the host.…”

“…First, the speed and maximum force of T4P retraction exceed the speed and maximum force of DNA uptake by orders of magnitude in Neisseria gonorrhoeae. [3] Second, experiments with T4P mutants Figure 1. The translocation ratchet.…”

“…[3] Under these conditions, the uptake of a 10 kbp DNA fragment would take t % 6 h ( Figure 1D); a DNA uptake event would not be finished within one cell cycle.…”