Understanding the mechanism of genetic transformation

Natural transformation in bacteria is an important mode of horizontal gene transfer that contributes to bacterial evolution, including the spread of antibiotic resistance and virulence genes. This proposal will use Bacillus subtilis as a model to address the largely conserved mechanisms that enable the uptake of environmental DNA. It will address the traversal of transforming DNA through the cell wall, the translocation of DNA across the cell membrane, and steps during the recombination of this DNA with the recipient genome. Our studies will provide broad insights into molecular details of transformation and will bring us closer to the long-term goal of understanding the mechanism of DNA uptake. Using single molecule and bulk biochemical methods, it is proposed to investigate the ComFA ATPase that powers translocation to the cytoplasm, together with its binding partner ComFC. We will investigate CoiA, a conserved protein required for recombination of transforming DNA with the recipient chromosome that has been neglected, despite its major role in transformation. Finally, we will test and extend our hypothesis that ComEA is a force-generating protein that condenses incoming DNA, pulling it into the periplasm. These aims will be pursued using the tools of cell biology, genetics, biochemistry, single-molecule biophysics, and structural biology.