SUMMARY/ABSTRACTWe have identified a novel signaling mechanism in contemporary epidemic causing strains of Staphylococcusaureus that controls expression of blaZ and mecA, the genes that code for ?-lactamase and penicillin-bindingprotein 2a, respectively, important mediators of ?-lactam resistance in the bacteria. Our results suggest thatStk1 and Stp1, a serine/threonine kinase and phosphatase respectively, mediate this novel signaling mechanismin a yin-yang manner. While deletion of stk1 gene (?stk1) significantly attenuates bacterial survival upon ?-lactam treatment and is unable to induce blaZ and mecA expression, deletion of stp1 (?stp1) facilitates bacterialsurvival upon ?-lactam treatment compared to their isogenic wild type (Wt) strain. Complementation of ?stk1and ?stp1 strains with Wt stk1 and Wt stp1, respectively, restore Wt phenotypes. In addition, our results suggestthat Stk1 and Stp1 mediate another novel mode of ?-lactam resistance, also in a yin-yang manner, which isindependent of blaZ and mecA, though the mechanism is currently unknown. Generally, blaZ and mecA expression in S. aureus is known to be classically controlled by two separatepathways, BlaR1-BlaI and MecR1-MecI, respectively. Both pathways sense drugs through their cognate sensorinducers (BlaR1/MecR1) that leads to proteolytic degradation of their cognate repressors (BlaI/MecI) to de-repress blaZ and mecA expression. Contemporary epidemic strains of S. aureus however have a defectiveMecR1-MecI pathway. Our results suggest that the BlaR1-BlaI regulatory pathway in these strains also regulatesmecA expression. Our data further show that BlaR1-BlaI mediated expression of blaZ and mecA is dependenton the Stk1 and Stp1 signaling mechanism that we discovered in S. aureus. Stk1 and Stp1 mediated control ofBlaR1-BlaI pathway provides superior induction of blaZ and mecA expression, which likely provides better fitnessto contemporary epidemic strains of S. aureus. Besides the BlaR1-BlaI mediated pathway described above, blaZ expression is thought to be controlled byan enigmatic second regulator, BlaR2, initially recognized in the 1960s, whose identity and mode of action remainunknown. Several pieces of evidence from our study indicate that Stp1 could code for this enigmatic, long soughtBlaR2 in S. aureus. Three aims are proposed to decipher the mechanism through which Stk1 and Stp1 controls expression ofblaZ and mecA and to identify how they mediate the newly identified blaZ and mecA independent mode of ?-lactam resistance in S. aureus. Our study will help determine the mechanistic basis of how Stk1 and Stp1 corroborates ?-lactam resistanceand explain the evolutionary basis for the selection of the BlaR1-BlaI pathway over the MecR1-MecI pathway inmecA expression that could help identify alternate and better ways to treat S. aureus infections.