Detection of endogenous MazF enzymatic activity in Staphylococcus aureus

The mazEF_Sa toxin-antitoxin (TA) system is ubiquitous in clinical isolates of Staphylococcus aureus, yet its physiological role is unclear. MazF_Sa is a sequence-specific endoribonuclease that inhibits the growth of S. aureus and Escherichia coli on ectopic overexpression. MazF_Sa preferentially cleaves RNA at UACAU sites, which are overrepresented in genes encoding pathogenicity factors. The exploitation of the inherent toxicity of MazF_Sa by artificial toxin activation has been proposed as an antibacterial strategy; however, enzymatic activity of endogenous MazF_Sa has never been detected, and tools for such analyses are lacking. Here we detail methods for detection of the ribonuclease activity of MazF_Sa, including a continuous fluorometric assay and a gel-based cleavage assay. Importantly, these methods allowed for the first detection of endogenous MazF_Sa enzymatic activity in S. aureus lysate. These robust and sensitive assays provide a toolkit for the identification, analysis, and validation of stressors that induce MazF enzymatic activity and should assist in the discovery of artificial activators of the mazEF_Sa TA system.