Bacterial gene transfer studied at the single molecule level

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Transport of macromolecules through nanometer-sized membrane pores is a ubiquitous theme in cell biology. Examples include the linear import of precursor proteins into mitochondria and DNA transport during bacterial gene transfer. During the previous project phase, we obtained strong evidence that the uptake of DNA during bacterial transformation is driven by a translocation ratchet mechanism; binding of ComE chaperones in the periplasm biases DNA diffusion through a membrane pore in the direction of DNA uptake. The translocation ratchet mechanism has been discussed in biophysical textbooks, but to our knowledge, there is little experimental data on force generation by this basic molecular motor. In the next project phase, we propose characterizing the biophysical properties of the translocation ratchet by combining single molecule tools with molecular biology. In particular, we will assess how variation of the chaperone dissociation constants affects velocity, force generation, and thermodynamic efficiency of the motor. We will further scrutinize the translocation ratchet model by generating bacterial strains with an artificial translocation ratchet by replacing the native chaperones ComE by other DNA-binding proteins. This approach will allow us to systematically tune the dissociation constants and the density of binding sites on the DNA. Finally, we will investigate the coupling of the translocation ratchet transporting DNA through the outer membrane to the motor that transports DNA into the cytoplasm.