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 1 -YafQ 1 tha was characterized in T. halophilus . The toxin protein YafQ 1 tha acts as a ribonuclease, and its overexpression severely inhibits Escherichia coli growth. These toxic effects can be eliminated by introducing DinJ 1 tha , indicating that YafQ 1 tha activity is blocked by the formed DinJ 1 -YafQ 1 tha complex. In vivo and in vitro assays showed that DinJ 1 tha alone or DinJ 1 -YafQ 1 tha complex can repress the transcription of dinJ 1 -yafQ 1 tha operon by binding directly to the promoter sequence. In addition, dinJ 1 -yafQ 1 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 DinJ 1 -YafQ 1 tha system in the stress adaptation processes of T. halophilus during fermentation. A single antitoxin DinJ 2 tha without a cognate toxin protein was also found. Its sequence shows low similarity to that of DinJ 1 tha , indicating that this antitoxin may have evolved from a different ancestor. Moreover, DinJ 2 tha can cross-interact with noncognate toxin YafQ 1 tha and cross-regulate with dinJ 1 -yafQ 1 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 • dinJ 1 -yafQ 1 tha is the first functional TA module characterized in T. halophilus and upregulated significantly upon osmotic and acidic stress. • DinJ 2 tha can exhibit physical and transcriptional interplay with DinJ 1 -YafQ 1 tha . • dinJ 2 tha may be acquired from bacteria in distant affiliation and inserted into the T. halophilus genome through horizontal gene transfer.