Minigene as a Novel Regulatory Element in Toxin-Antitoxin Systems

The axe-txe type II toxin-antitoxin (TA) system is characterized by a complex and multilayered mode of gene expression regulation. Precise and tight control of this process is crucial to keep the toxin in an appropriate balance with the cognate antitoxin until its activation is needed for the cell. In this report, we provide evidence that a minigene encoded within the axe-txe operon influences translation of the Txe toxin. This is the first example to date of such a regulatory mechanism identified in the TA modules. Here, in a series of genetic studies, we employed translational reporter gene fusions to establish the molecular basis of this phenomenon. Our results show that translation of the two-codon mini-ORF displays an in cis mode of action, and positively affects the expression of txe, possibly by increasing its mRNA stability through protection from an endonuclease attack. Moreover, we established that the reading frame in which the two cistrons are encoded, as well as the distance between them, are critical parameters that affect the level of such regulation. In addition, by searching for two-codon ORFs we found sequences of several potential minigenes in the leader sequences of several other toxins belonging to the type II TA family. These findings suggest that this type of gene regulation may not only apply for the axe-txe cassette, but could be more widespread among other TA systems.