A continuous fluorometric assay for the assessment of MazF ribonuclease activity

Abstract

Plasmids maintain themselves in their bacterial host through several different mechanisms, one of which involves the synthesis of plasmid-encoded toxin and antitoxin proteins. When the plasmid is present the antitoxin binds to and neutralizes the toxin. If a plasmid-free daughter cell arises, however, the labile antitoxin is degraded (and not replenished) and the toxin kills the cell from within. These toxin-antitoxin (TA) systems thereby function as post-segregational killing systems, and the disruption of the TA interaction represents an intriguing antibacterial strategy. It was recently discovered that the genes for one particular TA system, MazEF, are ubiquitous on plasmids isolated from clinical vancomycin-resistant enterococci (VRE) strains. It thus appears that small molecule disruptors of the MazEF interaction have potential as antibacterial agents. The MazF toxin protein is known to be a ribonuclease. Unfortunately, traditional methods for the assessment of MazF activity rely on the use of radiolabeled substrates followed by analysis with polyacrylamide gel electrophoresis. Described herein is a simple and convenient continuous assay for the assessment of MazF activity. This assay utilizes an oligonucleotide with a fluorophore on the 5′-end and a quencher on the 3′-end, and processing of this substrate by MazF results in a large increase in the fluorescence signal. Through this assay we have for the first time determined a K M and V max for this enzyme, and have also found that MazF is not inhibited by standard ribonuclease inhibitors. This assay will be useful to those interested in the biochemistry of the MazF family of toxins and the disruption of MazE-MazF.

RETRACTED: ChpK and MazF of the toxin–antitoxin modules are involved in the virulence of Leptospira interrogans during infection

“…Considerable evidence has shown that TA modules in bacteria act as suicide systems in response to environmental stimuli such as nutritional deficiency, and protect bacteria from phage infection [39,51,56,57]. Interestingly, bacterial TA modules may be involved in the action against antibacterial agents in S. aureus and vancomycin-resistant enterococci [28,58]. However, several studies have revealed that the toxic proteins from prokaryotic TA modules cause the death of eukaryotic and mammalian host cells [38,59,60], suggesting the possibility that the TA modules in bacterial pathogens to play roles during host infection.…”

MazF-induced Growth Inhibition and Persister Generation in Escherichia coli

“…This was used as a substrate for WT and E24A MazF. RNase inhibitor was added to a final concentration of 1 unit/l to inactivate RNase A (32). MazF (0.01 or 0.05 g) was incubated with 6 g of RNA in PBS buffer, pH 7.4, at 37°C for 45 min.…”

Determination of ribonuclease sequence-specificity using Pentaprobes and mass spectrometry

“…Fluorogenic substrates have previously been used to analyze the reaction kinetics of ribonucleases (Park et al 2001;Wang and Hergenrother 2007) and generally consist of single RNA bases flanked by short DNA sequences. These mixed oligonucleotides are labeled with a fluorophore at one end and a quencher at the other.…”

“…Once the specificity of VapC was known, detailed kinetic analyses were carried out using a sensitive fluorometric substrate similar to that of the method described by Kelemen et al (1999) and Wang and Hergenrother (2007) for quantitation of VapC activity and kinetic analyses of these enzymes.…”