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MSc in Biochemistry Dissertation Project – 2nd Cycle Student´s Name: Student email address: No. Supervisor(s): Rita Sobral and Inês Grilo Supervisor(s) email address:[email protected] and [email protected] Lab/Institution: Lab of Molecular Microbiology of Bacterial Pathogens (333), UCIBIO, UNL TITLE:Presence of extracellular DNA and its regulation of the activity of the major autolysin of Staphylococcus aureus Atl BACKGROUND Staphylococcus aureus is a remarkably versatile microorganism that is usually a commensal of the human’s microbiota, but can also become invasive when the host’s defenses are breached, becoming a very important and successful pathogen, with high morbidity and mortality rates across the globe. In S. aureus, the major murein hydrolase is Atl, a bifunctional autolysin with amidase (AM) and glucosaminidase (GL) catalytic domains, primarily involved in cell separation, as well as in cell wall turnover and lysis. This protein has also been implicated in biofilm formation in S. aureus, not only due to its role in autolysis and subsequent genomic DNA release, but also because of its intrinsic adhesive properties. Both domains of the Atl protein are translated as a single pro-peptide that is exported to the extracellular milieu, where the proteolytic processing into the two mature AM and GL domains occurs, in a thus far uncharacterized manner. We have previously reported the DNA-binding capacity of the GL domain of Atl, and we have shown that Atl binds DNA at the cell surface, while we hypothesize that the presence of extracellular DNA (eDNA) could induce the differential proteolysis and/or activity of Atl. Furthermore, our results have shown that the GL and AM catalytic domains have distinct functions in biofilm formation and are likely involved in other cellular processes differently. OBJECTIVES We aim to characterize the expression of the atl gene in several conditions (temperature, pH, medium, presence of eDNA, etc) and to correlate it with the differential expression and activity of the AM and GL domains, in various strains of S. aureus. 1 MSc in Biochemistry Dissertation Project – 2nd Cycle PROJECT DESCRIPTION The transcriptional expression of the atl gene will be assayed using a promoter-GFP reporter system. Several strains with diverse genetic backgrounds will be used(Task 1), as well as different conditions, including the presence of eDNA, mimicking different environments(Task 2). The expression of the AM and GL catalytic domains of Atl will be assessed by Western blotting with specific antibodies(Task 3), while the activity of these proteins will be characterized using hydrolysis assays in microtiter plates and zymography with different cell wall substrates in various conditions(Task 4). The presence of extracellular DNA in particular will be tested to assess if the presence of this molecule affects the expression and activity of Atl(Task 5). TIMELINE(use fill tool for the cells) Month 1 Month 2 Month 3 Month 4 Month 5 Month 6 Month 7 Month 8 Month 9 Month 10 Task 1 Task 2 Task 3 Task 4 Task 5 Thesis 2