Investigation of the Mechanism That Powers DNA Translocation During Bacterial Natural Transformation

Abstract

If deoxyribonucleic acids are the building blocks of life, perhaps the proteins that move, shape, and assemble nucleic acids should be called architects. These proteins come in every size and shape and have vastly different roles and mechanisms, but they share one thing in common: they help determine what life looks like by their interactions with DNA and RNA. Several of these protein architects play integral roles in the spread of genetic material among bacteria in natural transformation, the process by which bacteria take up DNA from the environment. Natural transformation has recently come into the limelight, as researchers have demonstrated its involvement in the spread of genes conferring antibiotic resistance. In spite of this, we have little understanding of the mechanism behind natural transformation. A multitude of proteins participate in natural transformation, but a few are considered to be at the heart of the process, since without them, transformation is abolished, or nearly so. ComEC and ComFA are two of these critical proteins. ComEC is the putative DNA uptake channel and is absolutely essential for transformation. The role of ComFA is less clear, but this helicase-like protein is thought to be involved in powering DNA uptake in Gram-positive bacteria. I used a genetic approach to explore the possible oligomerization of ComEC, finding that ComEC likely forms homo-oligomers. I also provided some of the first evidence that ComFA may indeed be involved in powering DNA uptake, since it has single-stranded DNA-stimulated ATPase activity, as well as 5’ to 3’ translocase activity. Lastly, I report a previously unexplored motif present in the C-terminal domain of ComFA that is important for protein function. Collectively, my work reveals new insights into interacting proteins and the use of energy in the competence mechanism.