ABSTRACT Natural transformation in bacteria is an important mode of horizontal gene transfer. Generally, it contributes to bacterial evolution and specifically to the spread of antibiotic resistance and virulence genes. This proposal will use Bacillus subtilis as a model to address the fundamental, largely conserved mechanisms that enable the uptake of environmental DNA. It will address the binding of DNA to the surface of the cell, traversal through the cell wall and translocation of DNA across the cell membrane. This plan 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. It is proposed to investigate the energy sources for DNA uptake, the proteins involved, some of their structures and their interactions as components of a molecular machine. These aims will be pursued using the tools of cell biology, genetics, biochemistry and structural biology. A common feature of these original aims is a dependence on membrane protein purification. To aid us in the purification of the membrane proteins in preparation for cryoEM (or, in some cases, X-ray crystallography in lipidic cubic phase) we propose to purchase the Prometheus Panta instrument manufactured by NanoTemper Technologies.