SUMMARY/ABSTRACT We have identified a novel signaling mechanism in contemporary epidemic causing strains of Staphylococcus aureus that controls expression of blaZ and mecA, the genes that code for β-lactamase and penicillin-binding protein 2a, respectively, important mediators of β-lactam resistance in the bacteria. Our results suggest that Stk1 and Stp1, a serine/threonine kinase and phosphatase respectively, mediate this novel signaling mechanism in a yin-yang manner. While deletion of stk1 gene (Δstk1) significantly attenuates bacterial survival upon β- lactam treatment and is unable to induce blaZ and mecA expression, deletion of stp1 (Δstp1) facilitates bacterial survival upon β-lactam treatment compared to their isogenic wild type (Wt) strain. Complementation of Δstk1 and Δstp1 strains with Wt stk1 and Wt stp1, respectively, restore Wt phenotypes. In addition, our results suggest that Stk1 and Stp1 mediate another novel mode of β-lactam resistance, also in a yin-yang manner, which is independent 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 separate pathways, BlaR1-BlaI and MecR1-MecI, respectively. Both pathways sense drugs through their cognate sensor inducers (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 defective MecR1-MecI pathway. Our results suggest that the BlaR1-BlaI regulatory pathway in these strains also regulates mecA expression. Our data further show that BlaR1-BlaI mediated expression of blaZ and mecA is dependent on the Stk1 and Stp1 signaling mechanism that we discovered in S. aureus. Stk1 and Stp1 mediated control of BlaR1-BlaI pathway provides superior induction of blaZ and mecA expression, which likely provides better fitness to contemporary epidemic strains of S. aureus. Besides the BlaR1-BlaI mediated pathway described above, blaZ expression is thought to be controlled by an enigmatic second regulator, BlaR2, initially recognized in the 1960s, whose identity and mode of action remain unknown. Several pieces of evidence from our study indicate that Stp1 could code for this enigmatic, long sought BlaR2 in S. aureus. Three aims are proposed to decipher the mechanism through which Stk1 and Stp1 controls expression of blaZ and mecA and to identify how they mediate the newly identified blaZ and mecA independent mode of β- lactam resistance in S. aureus. Our study will help determine the mechanistic basis of how Stk1 and Stp1 corroborates β-lactam resistance and explain the evolutionary basis for the selection of the BlaR1-BlaI pathway over the MecR1-MecI pathway in mecA expression that could help identify alternate and better ways to treat S. aureus infections.