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

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 DinJ-YafQ was characterized in T. halophilus. The toxin protein YafQ acts as a ribonuclease, and its overexpression severely inhibits Escherichia coli growth. These toxic effects can be eliminated by introducing DinJ, indicating that YafQ activity is blocked by the formed DinJ-YafQ complex. In vivo and in vitro assays showed that DinJ alone or DinJ-YafQ complex can repress the transcription of dinJ-yafQ operon by binding directly to the promoter sequence. In addition, dinJ-yafQ is involved in plasmid maintenance and stress response, and its transcriptional level is regulated by various stresses. These findings reveal the possible roles of DinJ-YafQ system in the stress adaptation processes of T. halophilus during fermentation. A single antitoxin DinJ without a cognate toxin protein was also found. Its sequence shows low similarity to that of DinJ, indicating that this antitoxin may have evolved from a different ancestor. Moreover, DinJ can cross-interact with noncognate toxin YafQ and cross-regulate with dinJ-yafQ operon. In summary, DinJ-YafQ 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: • dinJ-yafQ is the first functional TA module characterized in T. halophilus and upregulated significantly upon osmotic and acidic stress. • DinJ can exhibit physical and transcriptional interplay with DinJ-YafQ. • dinJ may be acquired from bacteria in distant affiliation and inserted into the T. halophilus genome through horizontal gene transfer.