Abstract 1447 Investigation of Conjugative Protein Orf90 from S. aureus pSK41

The spread of antibiotic resistance is a significant challenge for healthcare and public health systems. Antibiotic resistant genes are primarily spread through conjugative plasmid transfer (CPT). Conjugative plasmids harbor resistance genes as well as genes necessary for their maintenance in the cell. One such plasmid is pSK41, which is found in Staphylococcus aureus. Multidrug resistant S. aureus causes thousands of deaths each year, and so understanding conjugative plasmids like pSK41 could provide insight into the spread of resistance. In this study, we aimed to conduct a structural and functional characterization of pSK41 encoded protein Orf90. Sequence analysis showed that pSK41 Orf90 has no predicted conserved domains, however it may be involved in transcription regulation or recombination. We successfully expressed and purified recombinant Orf90 tagged with maltose-binding protein (His-MBP-Orf90), and screened for crystallization conditions using microbatch under oil, identifying three potential conditions. Circular dichroism was used to investigate secondary structure elements of untagged Orf90. Electrophoretic Mobility Shift Assays (EMSA) of Orf90 binding to a consensus sequence found on pSK41 support our hypothesis that Orf90 can bind DNA. Further structural characterization of Orf90 using experimental and computational techniques will provide new insights into the function of unstudied plasmid-encoded genes on pSK41, and support ongoing efforts to combat the spread of antibiotic resistance. This research is generously supported by the Research Corporation for Science Advancement.