STRUCTURE AND FUNCTION STUDIES OF MICROBIAL CONJUGATIVE DNA TRANSFER & GI DRUG REACTIVATION PROCESSES

Antimicrobial resistance in Staphylococcus aureus presents an increasing threat to human health. This resistance is often encoded on mobile plasmids, such as pSK41; however, the transfer mechanism of these plasmids is not well understood. In this study, we examine key protein features of the relaxase enzyme, NES, which initiates and terminates the transfer of the multidrug resistance plasmid pSK41. This work establishes that both a novel C-terminal domain and two loops of the NES protein, hairpin loops 1 and 2, are essential for proper DNA cleavage and religation by the full 665-residue NES protein in vitro. Second, we show that NES is able to bind, cleave, and religate the oriT sequences of non-conjugative plasmids pSK156 and pCA347. These data indicate that the conjugative relaxase in trans mechanism recently described for the pWBG749 family of plasmids also applies to the pSK41 family of plasmids, further heightening the potential significance of this mechanism in the horizontal transfer of staphylococcal plasmids. Finally, we use the knowledge of important NES features to design polyamide inhibitors that disrupt key protein-DNA interactions and chelator fragments that target a required coordinated metal ion. The efficacy of these inhibitors suggests that disrupting NES function may be a viable option for disrupting conjugative plasmid transfer. β-glucuronidase (GUS) enzymes are responsible for the severe drug toxicity associated with the chemotherapy drug irinotecan. Previous characterization of E. coli GUS identified inhibitors whose efficacy is dependent on interaction with an active site loop termed Loop 1. Analysis of the Human Microbiome Project sequencing data establishes a catalog of GUS sequences present in the human gastrointestinal tract. Sequences in this catalog are classified according to the presence and size of an active site loop and we identify at least one sequence from each class for further characterization. Characterization confirms the β-glucuronidase activity of seven new enzymes and establishes the ability of six GUS enzymes to process SN-38G. We also confirm the importance of pH for optimal enzyme function. This data expands current understanding of the β-glucuronidase enzyme family and provides additional GUS enzymes against which to optimize inhibitors to prevent SN-38G toxicity.Doctor of Philosoph