A phage-encoded RNA-binding protein inhibits the antiviral activity of a toxin-antitoxin system

Bacteria harbor diverse mechanisms to defend themselves against their viral predators, bacteriophages. In response, phages can evolve counter-defense systems, most of which remain poorly understood. In T4-like phages, the gene tifA prevents bacterial defense by the type III toxin-antitoxin (TA) system toxIN, but the mechanism by which TifA inhibits toxIN remains unclear. Here, we show that TifA directly binds both the endoribonuclease ToxN and RNA, leading to the formation of a high molecular weight ribonucleoprotein complex in which ToxN is inhibited. The RNA binding activity of TifA is necessary for its interaction with and inhibition of ToxN. Thus, we propose that TifA inhibits ToxN during phage infection by trapping ToxN on cellular RNA, particularly the abundant 16S rRNA, preventing cleavage of phage transcripts. Taken together, our results reveal a novel mechanism underlying inhibition of a phage-defensive RNase toxin by a small, phage-encoded protein. E. coli toxI and chitin binding domain-tagged ToxN were overexpressed with bacteriophage RB69 TifA (wild-type or the RNA-binding mutant TifA(K55A R56A R59A) in E. coli T7 express cells (NEB). Lysate was then purified through chitin resin to isolate RNAs and proteins associated with ToxN during the co-expression. RNAs from whole cell lysates and following chitin purification were then deep sequenced to identify the RNA species that co-purify with ToxN when it is co-expressed with TifA.