Samenvatting
Toxin-antitoxin (TA) modules are ubiquitous in the genomes and on plasmids of free living bacteria. These operons generally have a characteristic genetic organisation: a gene coding for a small intracellular toxin is preceded by a gene encoding its antitoxin, a protein that can antagonise the toxic activity. The ccd operon on the F plasmid of Escherichia coli functions as a plasmid maintenance element, as plasmid-free progeny is killed by the toxin, CcdB, when the short-lived antitoxin, CcdA, is not replenished. This operon is negatively auto-regulated at the transcriptional level. The antitoxin can bind the operator DNA using its N-terminal domain and the toxin can either function as a co-repressor or a de-repressor for CcdA, depending on the toxin:antitoxin ratio. This mechanism, which is common to many TA modules, is called conditional cooperativity.During this master thesis, the auto-regulation of TA modules in general and the ccd operon in particular were examined quantitatively, focusing on the importance of the amount of binding sites for the antitoxin on the operator DNA and combining in silico, in vivo and in vitro methods.
First, the auto-regulation was translated into a general mathematical model. Stochastic simulations based upon this model showed that a higher amount of binding sites on the operator of the TA module allows the toxin level to be controlled more tightly in the presence of less antitoxin. Furthermore, ccd-lacZYA promoter/operator fusions and expression plasmids for CcdA and the non-toxic CcdBI101K were constructed. These constructs allow the transcription from and the repression of the ccd promoter/operator to be monitored in vivo during transcription assays. Finally, electrophoretic mobility shift assays indicated that the interaction of CcdB with CcdA's C-terminal domain had an allosteric effect on the DNA binding by CcdA's N-terminal domain. Moreover, they led to the proposal of a modified model for the auto-regulation of the ccd operon by conditional co-operativity, in which the complex with the highest affinity for the operator DNA, and hence the strongest repression activity, consists of CcdB and CcdA dimers in a 1:2 toxin:antitoxin ratio instead of in equimolar amounts.
| Datum prijs | 2012 |
|---|---|
| Originele taal | English |
| Begeleider | Remy Loris (Promotor), Jan Danckaert (Co-promotor), Henri De Greve (Co-promotor) & Daniel Charlier (Co-promotor) |