Insights into the regulation of F and ColE1 plasmid transfer

Abstract

Multidrug resistant bacteria pose a threat to human health and economic burden for the healthcare system. Transmission of antibiotic resistance genes can occur by the conjugative transfer of the F plasmid between bacteria. The transferosome forms a channel linking the two cells together, while formation of the relaxosome at the oriT nicks and unwinds DNA for transfer. This thesis explores the interaction of the hexameric coupling protein and transferosome component, TraD, with TraM, the DNA binding protein of the relaxosome. Via mating assays, we determined that TraD-TraM interaction does not depend on specific sequences upstream of the terminal 8 residues within the TraD C-terminal domain (CTD). Rather, this region of the TraD CTD functions as a flexible unstructured tether linking the ATPase domain to the terminal 8 residues. These tethers must be more than 44 residues long in order to make multiple simultaneous contacts with TraM and form a stable TraD-TraM complex through avidity effects. Mobilizable plasmids, such as ColE1 encode the relaxase, but lack genes encoding coupling proteins and components for mating pair formation. For this reason, their transmission relies on the presence of conjugative plasmids to encode the necessary conjugation machinery. Expression constructs were made to further structurally and functionally characterize the ColE1-encoded relaxase, MbeA and accessory protein MbeC, which together with MbeB form the relaxosome. EMSA indicated that MbeC binds double-stranded oriT ColE1 DNA in a non-specific manner.