Physical and Functional Interplay between MazF₁^Bif and Its Noncognate Antitoxins from Bifidobacterium longum

Bifidobacterium longum strain JDM301, a widely used commercial strain in China, encodes at least two MazEF-like modules and one RelBE-like toxin-antitoxin (TA) system in its chromosome, designated MazE₁F₁^Bif, MazE₂F₂^Bif, and RelBE^Bif, respectively. Bacterial TA systems play an important role in several stress responses, but the relationship between these TA systems is largely unknown. In this study, the interactions between MazF₁^Bif and MazE₂^Bif or RelB^Bif were assessed in B. longum strain JDM301. MazF₁^Bif caused the degradation of tufA^Bif mRNA, and its toxicity was inhibited by forming a protein complex with its cognate antitoxin, MazE₁^Bif Notably, MazF₁^Bif toxicity was also partially neutralized when jointly expressed with noncognate antitoxin MazE₂^Bif or RelB^Bif Our results show that the two noncognate antitoxins also inhibited mRNA degradation caused by MazF₁^Bif toxin. Furthermore, the physical interplay between MazF₁^Bif and its noncognate antitoxins was confirmed by immunoprecipitation. These results suggest that MazF₁^Bif can arrest cell growth and that MazF₁^Bif toxicity can be neutralized by its cognate and noncognate antitoxins. These results imply that JDM301 uses a sophisticated toxin-antitoxin interaction network to alter its physiology when coping with environmental stress.IMPORTANCE Although toxin-antitoxin (TA) systems play an important role in several stress responses, the regulatory mechanisms of multiple TA system homologs in the bacterial genome remain largely unclear. In this study, the relationships between MazE₁F₁^Bif and the other two TA systems of Bifidobacterium longum strain JDM301 were explored, and the interactions between MazF₁^Bif and MazE₂^Bif or RelB^Bif were characterized. In addition, the mRNA degradation activity of MazF₁^Bif was demonstrated. In particular, the interaction of the toxin with noncognate antitoxins was shown, even between different TA families (MazF₁^Bif toxin and RelB^Bif antitoxin) in JDM301. This work provides insight into the regulatory mechanisms of TA systems implicated in the stress responses of bifidobacteria.