Athina Eleftheraki: A matter of life or death: Regulation of type I toxin-antitoxin systems in bacteria

Abstract

Bacteria live in constantly changing environments and face diverse stresses, including bacteriophage attacks. To survive, they must rapidly adjust their physiology through precise gene regulation. Post-transcriptional control is a central player to this adaptation, as it dictates the translation and degradation of transcripts. RNA structural elements, and small non-coding RNAs (sRNAs), often together with RNA-binding proteins, serve as key regulators of messenger RNAs (mRNAs). Among the most tightly controlled transcripts are those of type I toxin-antitoxin (T1TA) systems. Here, a self-acting toxin mRNA is produced in an inactive form, and its cognate sRNA antitoxin inhibits translation of the active transcript. Despite their importance, many regulatory and functional aspects of these systems remain unclear. This thesis aims to characterize the post-transcriptional regulation of the recently identified Salmonella T1TA system timP/ timR and to examine the role of chromosomally encoded T1TA systems in Escherichia coli during bacteriophage infection. 

We first show that the Salmonella timP mRNA, unlike many type I toxin transcripts, does not undergo enzymatic processing. Instead, it is regulated through a non-canonical mechanism of ‘structural processing’ The active mRNA adopts a pseudoknot structure essential for translation initiation, and the antitoxin sRNA TimR binds preferentially to this active conformation, destabilizing the pseudoknot and thereby inhibiting translation. We further demonstrate that the pseudoknot enables a long-range interaction that exposes the Shine-Dalgarno sequence. An alternative interaction prevents pseudoknot formation, maintains sequestration of the Shine-Dalgarno sequence in a stable stem-loop, and inhibits translation. Finally, we show that a T1TA system encoded within an Escherichia coli cryptic prophage promotes bacteriophage infection in cooperation with a major bacterial immune system, revealing an unexpected role for T1TA systems in host-phage interaction.

Overall, this thesis broadens our understanding of RNA-mediated regulation by uncovering a structural RNA switch that governs toxin expression and revealing an alternative mechanism of translation initiation in bacteria. Furthermore, it expands the functional repertoire of T1TA systems by demonstrating that they can influence host-bacteriophage interactions in previously unrecognized ways.