This PhD thesis consists of three research projects revolving around the common thread of investigation of the properties and biological functions of Toxin-Antitoxin loci. Toxin-Antitoxin (TA) loci are transcriptionally regulated via an auto-inhibition mechanism called conditional cooperativity, based on cooperative binding of toxin-antitoxin complexes to operator DNA that depends on the stoichiometric ratio between the toxin and the antitoxin. More specifically, toxin and antitoxin can form heteromers with different stoichiometric ratios, and the complex with the intermediate ratio works best as a transcription repressor. This allows transcription at low toxin level, strong repression at intermediate toxin level, and then again transcription at high toxin level
In the first project, we reveal the biological function of conditional cooperativity
by constructing a mathematical model of one particular E.coli TA system,
the relBE locus. We show that the model reproduces the experimentally observed response to nutritional stress. We further demonstrate that conditional cooperativity stabilizes the level of antitoxin in rapidly growing cells such that random induction of relBE is minimized. At the same time it enables quick removal of free toxin when the starvation is terminated.
In the second project, we explore the features and the potential biological role
of conditional cooperativity, in a more general perspective, that can be applied to the regulation of chromosome encoded TA loci in E.coli in general. In this context, we will neglect the cooperativity in the binding, and focus on the fact that the regulation depends on the ratio between the toxin and the antitoxin. For this reason, we talk about conditional regulation instead of conditional cooperativity. Such regulation has two interesting features: first, it provides a non-monotonous response to the concentration of one of the proteins, and second, it allows ultrasensitive response mediated by the sequestration of the active heteromers. We study conditional regulation in simple feedback motifs, and show that it can provide bistability for a wide range of parameters. We then demonstrate that the conditional cooperativity in toxin-antitoxin systems combined with the growthinhibition activity of free toxin can mediate bistability between a growing state and a dormant state.
The final project aims at unraveling the connection between stochasticity in
the expression of TA loci in E.coli and the phenomenon of bacterial persistence. Persistence is a form of antimicrobial tolerance that is not associated with DNA mutation that confers resistance, but rather with a spontaneous switch of a cell to a physiological state characterized by slow or non-growth that impairs the effectiveness of antibiotics. The action of TA loci has often been invoked in the attempt of explaining the mechanism of persisters formation. We suggest a stochastic description of the activity of chromosome-encoded TA loci, aiming at providing insights about the mechanisms that support the stochasticity in persister formation.