has evolved a complex regulatory network to coordinate expression of virulence factors, including cytolytic toxins, with host environmental signals. Central to this network are two-component systems (TCSs), in which a histidine kinase senses an external signal and activates a response regulator via phosphorylation, leading to changes in gene expression. Using a comprehensive screen of transposon mutants in each of the non-essential histidine kinase and response regulator genes in , we demonstrate that 11 of these 16 systems regulate cytotoxicity. Further characterization of the phosphate-sensing PhoPR system revealed that PhoP affects cytotoxicity in a manner mediated through the Agr quorum-sensing system. Notably, we found that unphosphorylated PhoP is an activator of Agr activity, whilst phosphorylated PhoP also acts as a weak activator of Agr activity in high-phosphate environments but as a repressor in low-phosphate environments. Furthermore, overexpression of PhoP resulted in upregulation of -type phenol-soluble modulins, which may also contribute to the cytotoxicity phenotype observed in the mutant. Overall, we have demonstrated that phosphate sensing through PhoPR is a novel regulator of cytotoxicity in . Moreover, our study challenges the canonical model of TCSs as simple on/off systems and highlights the importance of unphosphorylated response regulators in gene regulation.