Mutational Analysis of the Antitoxin in the Lactococcal Type III Toxin-Antitoxin System AbiQ
ABSTRACTThe lactococcal abortive phage infection mechanism AbiQ recently was classified as a type III toxin-antitoxin system in which the toxic protein (ABIQ) is regulated following cleavage of its repeated noncoding RNA antitoxin (antiQ). In this study, we investigated the role of the antitoxin in antiphage activity. The cleavage ofantiQby ABIQ was characterized using 5′ rapid amplification of cDNA ends PCR and was located in an adenine-rich region ofantiQ. We next generated a series of derivatives with point mutations withinantiQor with various numbers ofantiQrepetitions. These modifications were analyzed for their effect on the antiphage activity (efficiency of plaquing) and on the endoribonuclease activity (Northern hybridization). We observed that increasing or reducing the number ofantiQrepeats significantly decreased the antiphage activity of the system. Several point mutations had a similar effect on the antiphage activity and were associated with changes in the digestion profile ofantiQ. Interestingly, a point mutation in the putative pseudoknot structure ofantiQmutants led to an increased AbiQ antiphage activity, thereby offering a novel way to increase the activity of an abortive infection mechanism.