Vancomycin is a potent antibiotic and used widely to treat methicillin resistant Staphylococcus aureus (MRSA) infections. Occasionally, vancomycin therapy fails, and patients are left with difficult-to-eradicate, life-threatening, persistent MRSA. We have studied such clinical MRSA isolates and observed that an amino acid substitution in the WalKR locus – the only essential two-component regulator (TCR) among the 16 TCRs in S. aureus - reduced susceptibility to vancomycin and increased intracellular persistence. To understand the basis for these phenotypes we created a powerful ensemble of bespoke, molecular and cell-biology tools for S. aureus. We show that like orthologous systems in Bacillus spp. and other Firmicutes, S. aureus WalKR controls autolysins involved in peptidoglycan biosynthesis, but it also regulates genes involved in other essential cell systems. We observed too, that mutations leading to WalKR upregulation increased MRSA susceptibility to vancomycin and resensitised the bacteria to beta-lactam antibiotics. Based on these observations, we undertook a 340,000-compound library screen to try and find small molecules that specifically activate WalKR and synergize with vancomycin. We have taken an obscure clinical observation to create (i) a deep understanding of a poorly understood central regulatory system in S. aureus and (ii) an opportunity to build new therapeutic options for otherwise untreatable infections.