AbstractProkaryotes have extraordinary ways to deal with stress in general and antibiotics in particular. Such non-hereditary method is called persistence. Persistence is the survival of a certain percentage of bacteria in a stressful environment by entering a dormant state. Toxin-antitoxin modules are able to induce persistence via a regulation mechanism that involves the toxin’s activity. They have been found to be present on plasmids but also on the bacterial chromosomes. Most toxin-antitoxins operons consist of an antitoxin gene preceding the toxin gene but other organisations exist. The paaR2-paaA2-parE2 toxin-antitoxin module in E. coli O157 is an example of a three-component module in which three proteins are translated: a toxin (ParE2), an antitoxin (PaaA2) and a regulator (PaaR2). This regulator has been found to repress transcription of the toxin-antitoxin module.
During this master thesis, the expression and purification of the PaaR2 regulator have been optimized and its characteristics were examined with emphasis on the oligomeric state of the protein in solution and its DNA binding activity.
To simplify the purification, a His-tagged version of the PaaR2 protein with a cysteine-to-serine mutation, PaaR2HisC120S, was used. First, the expression, solubility and purification of this protein were optimized using small-scale expression tests. To obtain the highest amount of soluble protein, the cells were incubated for four hours at 37°C after induction with IPTG and lysed using a sonicator in a high salt lysis buffer. Purification using immobilized metal ion affinity chromatography and size exclusion chromatography resulted in a yield of 3-4 mg per liter LB medium. Next, it has been shown that the regulator forms a complex consisting of eight monomers and possesses little to no disorder. Finally, binding experiments have shown that the regulator PaaR2HisC120S binds the operator region and a secondary binding region. The binding site overlaps with predicted binding boxes and presumably one protein binds several boxes at once. It has been shown that the regulator binds the toxin-antitoxin complex and that this binding inhibits binding of the regulator to the DNA.
|Date of Award||29 Jun 2015|
|Supervisor||Remy Loris (Promotor) & Ariel Talavera Perez (Advisor)|
- bacterial stress response
- Toxin-antitoxin module
- Structural biology