Golemi-Kotra, Dasantila2015-08-282015-08-282014-01-172015-08-28http://hdl.handle.net/10315/29821Staphylococcus aureus is a major gram-positive pathogen, because of its remarkable capacity to develop resistance against different antimicrobial agents. It has been shown that in S. aureus two component systems (TCS) play an important role in responding to stress induced by antibiotics. Given the regulatory importance of TCS, herein we investigate in vitro the signal transduction mechanism of the WalKR, and the LytSR TCSs. These TCSs play a role in susceptibility towards cell wall active antibiotics and cationic antimicrobial agents, respectively. We found that both kinases, WalK and LytS, are active in vitro and capable of catalyzing ATP-dependent autophosphorylation and subsequent phosphoryl transfer reactions to their cognate response regulators WalR and LytR. In contrast to LytS, the autokinase activity of WalK is highly dependent on K+ and Ca2+ ions. We found that LytR undergoes phosphorylation by small molecule phosphate donors such as acetyl phosphate. Phosphorylation of LytR leads to dimerization of the N-terminal domain. Furthermore, we show that LytR has the ability to bind to the promoter region of lrgAB in its unphosphorylated state. Taken together, these data provide in vitro proof of phosphorylation mediated signal flow in the WalKR and LytSR TCSs.enAuthor owns copyright, except where explicitly noted. Please contact the author directly with licensing requests.BiochemistryCellular biologyChemistryElectronic Thesis or Dissertation2015-08-28Staphylococcus aureusTwo Component SystemsLytSRWalKRAutophosphorylationPhosphotransferCloningExpressionPurificationElectromobility Shift AssayPhos-tag gel