Construction of Green Fluorescent Protein Transcriptional and Translational Sortase Fusions in Enterococcus faecalis

Alice Gu and Scott Hultgren

Department of Molecular Microbiology, Washington University School of Medicine

            Antibiotic resistance in Gram-positive bacteria is one of the major problems in today’s society.  The Gram-positive bacterium, Enterococcus faecalis, is commonly found to cause urinary tract infections, endocarditis, intraabdominal infections, and bacteremia. To devise more practical and cost-effective solutions to treat Enterococcal infections, a better understanding is needed of basic E. faecalis processes and functions on a molecular level.  E. faecalis uses an enzyme called sortase to perform important functions, such as attaching secreted proteins to cell wall precursors.  Sortase is thought to group together in a single area on the bacterial cell.  Learning how the site of sortase localization is formed and how it is coordinated are significant to aid in uncovering novel drug targets.  Therefore, the goal of my study was to confirm that sortase localizes to a single spot on whole cells using a methodology of green fluorescent protein (GFP) fusions.  To confirm the ability to express GFP in E. faecalis, since GFP is not widely used in gram-positive pathogens, I first created a transcriptional fusion between the sortase A promoter and GFP on a plasmid.  E. faecalis containing the transcriptional fusion fluoresced green. The transcriptional fusion will be useful for measuring sortase expression throughout the cell cycle. To study sortase localization in whole cells, I am creating translational fusions between the sortase enzyme and GFP.  I have fused sortase and GFP using PCR and have been working on inserting DNA fragment into a plasmid and subsequently transforming the plasmid into competent E. faecalis cells.  Ultimately, we expect to observe a green fluorescent glow from a single spot in E. faecalis cells, signifying that the sortase protein is expressed at a single site at some point during the cell cycle.