Abstract 1782 Characterization of Conjugative Proteins from Salmonella Typhimurium pCU1

Increased antibiotic resistance in bacteria poses a significant public health concern. The spread of antibiotic resistant genes between bacteria occurs primarily through a horizontal gene transfer process called conjugative plasmid transfer, or CPT. During CPT a protein complex called the relaxosome processes the plasmid DNA for transfer. Studies into the structure and function of this key complex are currently lacking. pCU1 is a conjugative plasmid isolated from Salmonella Typhimurium that encodes a relaxosome composed of three proteins: an integrative host factor, IHF, a relaxase/helicase, TraI, and an accessory protein, TraK. Here, we present studies to characterize the pCU1 relaxosome. To examine the structure, function, and interactions of the pCU1 relaxosome, each protein was overexpressed in Escherichia coli BL21(DE3), extracted, and purified using various methods of chromatography. These methods yielded pure TraK, TraI, and IHF. Following purification, the components of the relaxosome were explored individually through methods such as crystallization and circular dichroism and in combination with each other through methods such as isothermal calorimetry and atomic force microscopy. Furthermore, interactions between components of the relaxosome and DNA were explored through DNA binding assays. We have successfully crystallized TraK and our assay results have begun to identify the interactions within the pCU1 relaxosome and, along with successful circular dichroism results, shed light on some of its structural features. Understanding the relaxosome during CPT, helps provide insight to the spread of antibiotic resistance in bacteria. We would like to thank the Research Corporation for Science Advancement for their support of this project.