title: Detection of endogenous MazF enzymatic activity in Staphylococcus aureus.
abstract: The mazEFSa toxin-antitoxin (TA) system is ubiquitous in clinical isolates of Staphylococcus aureus, yet its physiological role is unclear. MazFSa is a sequence-specific endoribonuclease that inhibits the growth of S. aureus and Escherichia coli on ectopic overexpression. MazFSa preferentially cleaves RNA at UACAU sites, which are overrepresented in genes encoding pathogenicity factors. The exploitation of the inherent toxicity of MazFSa by artificial toxin activation has been proposed as an antibacterial strategy; however, enzymatic activity of endogenous MazFSa has never been detected, and tools for such analyses are lacking. Here we detail methods for detection of the ribonuclease activity of MazFSa, including a continuous fluorometric assay and a gel-based cleavage assay. Importantly, these methods allowed for the first detection of endogenous MazFSa 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 mazEFSa TA system.
authors.affiliation: Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Julia J
authors.initials: JJ
authors.lastname: van Rensburg
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Hergenrother
mesh_terms: D000992:Antitoxins / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D057075:Enzyme Assays; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial; D010761:Phosphorus Radioisotopes; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D000992:Antitoxins; D001427:Bacterial Toxins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; C422955:MazF protein, E coli; D010761:Phosphorus Radioisotopes; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D004722:Endoribonucleases
keywords: Enzyme assay; MazF(Sa); Ribonuclease; Toxin–antitoxin