Characterization of DinJ-YafQ toxin-antitoxin module in Tetragenococcus halophilus: activity, interplay, and...

Tetragenococcus halophilus is a moderately halophilic lactic acid bacterium widely used in high-salt food fermentation because of its coping ability under various stress conditions. Bacterial toxin-antitoxin (TA) modules are widely distributed and play important roles in stress response, but those specific for genus Tetragenococcus have never been explored. Here, a bona fide TA module named DinJ₁-YafQ₁^tha was characterized in T. halophilus. The toxin protein YafQ₁^tha acts as a ribonuclease, and its overexpression severely inhibits Escherichia coli growth. These toxic effects can be eliminated by introducing DinJ₁^tha, indicating that YafQ₁^tha activity is blocked by the formed DinJ₁-YafQ₁^tha complex. In vivo and in vitro assays showed that DinJ₁^tha alone or DinJ₁-YafQ₁^tha complex can repress the transcription of dinJ₁-yafQ₁^tha operon by binding directly to the promoter sequence. In addition, dinJ₁-yafQ₁^tha is involved in plasmid maintenance and stress response, and its transcriptional level is regulated by various stresses. These findings reveal the possible roles of DinJ₁-YafQ₁^tha system in the stress adaptation processes of T. halophilus during fermentation. A single antitoxin DinJ₂^tha without a cognate toxin protein was also found. Its sequence shows low similarity to that of DinJ₁^tha, indicating that this antitoxin may have evolved from a different ancestor. Moreover, DinJ₂^tha can cross-interact with noncognate toxin YafQ₁^tha and cross-regulate with dinJ₁-yafQ₁^tha operon. In summary, DinJ-YafQ^tha characterization may be helpful in investigating the key roles of TA systems in T. halophilus and serves as a foundation for further research. KEY POINTS: • dinJ₁-yafQ₁^tha is the first functional TA module characterized in T. halophilus and upregulated significantly upon osmotic and acidic stress. • DinJ₂^tha can exhibit physical and transcriptional interplay with DinJ₁-YafQ₁^tha. • dinJ₂^tha may be acquired from bacteria in distant affiliation and inserted into the T. halophilus genome through horizontal gene transfer.