| TraM is a plasmid-encoded protein that is essential for bacterial conjugation of the F episome. TraM mutants are able to form stable mating pairs, but do not initiate DNA transfer. I have explored the physiology of TraM protein to further elucidate how it may function in conjugation. Indirect immunofluorescence studies indicate that TraM is localized throughout the cytoplasm in donor cells. This localization is the same regardless of whether or not the donor cells are in contact with recipients, suggesting that there is no large redistribution of TraM protein during conjugation. Biochemical fractionation studies corroborate the cytoplasmic localization of TraM but also indicate that a small fraction of TraM associates with the membrane, consistent with previous observations that TraM can interact with inner membrane protein TraD in vitro. Despite the large cytoplasmic concentration of TraM transfer efficiency increases when TraM is overexpressed in donors, indicating that TraM is limiting for conjugation. To complement these studies, I have performed a thermodynamic analysis of TraM stability and oligomerization. The reversible unfolding transition of TraM is three-state. Analytical ultracentrifugation experiments indicate that the first phase of unfolding involves dissociation of the tetramer into folded monomers, which are subsequently unfolded to the denatured state. A C-terminal domain isolated by limited proteolysis is tetrameric in solution, like the full-length protein, and its loss of structure correlates with dissociation of the TraM tetramer. However, the domain tetramer is less stable than that of the full length protein. Furthermore, unfolding of individual domains indicate that the N- and C-terminal regions act cooperatively to stabilize the full-length protein. Together, these experiments suggest structural overlap of regions important for oligomerization and DNA binding. I propose a model in which modulating the oligomerization equilibrium of TraM in vivo regulates its essential activity in bacterial conjugation. In this scenario, mating pair stabilization increases the concentration of active TraM by stabilizing the tetrameric state. This allows TraM to simultaneously interact with the episome and Trap, thereby initiating DNA transfer. |
