Researcher(s)
- Percival Tran, Biological Sciences, University of Delaware
Faculty Mentor(s)
- Mona Batish, Medical and Molecular Sciences, University of Delaware
Abstract
Bacteria adapt to environmental changes using two-component systems, which are composed of two parts, a sensor kinase and a response regulator. The sensor kinase senses external stimuli while the response regulator enacts a change such as altering gene expression to help the bacteria adapt and survive. In Staphylococcus aureus, the KdpD/KdpE (KdpDE) two-component system (TCS) senses potassium ion concentration and triggers KdpE phosphorylation and subsequent gene expression changes. The function of the USP domain in KdpD histidine kinase of this TCS is unknown. Our in-vitro data shows that the USP subunit binds directly to DNA. Cyclic di-AMP (CDA) is a secondary messenger that also binds USP and inhibits its binding to DNA in vitro. However, whether CDA affects the DNA binding by USP in vivo is a question that remains unanswered. To address this question, two mutants were created. 619 mutant has a deletion in GdpP which naturally degrades CDA. This deletion leads to elevated CDA levels in the cell. 814 mutant harbors a mutation in the DNA binding motif (K335A, R336A, R337A) in the USP subunit which would hinder DNA binding to USP. Wild-type KdpD was deleted in cells and replaced with GFP-tagged KdpD. Bacterial cells were grown in CS2 media overnight and were then immobilized on poly-l-lysine-coated cover glass. Colocalization of GFP signal (KdpD) and DNA (labeled with SiR-DNA dye) was analyzed and quantified in FIJI. The effects of these mutations on the oligomerization of KdpD as indicated by signal area and intensity was assessed using MATLAB. Interestingly, both mutants resulted in significantly higher KdpD oligomerization. Furthermore, consistent with previous reports, both higher levels of CDA as well as mutations in USP’s DNA binding motif significantly decreased colocalization of KdpD with DNA indicating lower nucleoid association. It would be interesting to see how these and other changes in KdpD might affect its nucleoid and membrane association.