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 single-molecule 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 velocity–force 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.