Mechanistic insights into HipA‐mediated bacterial persistence: Role of initial cellular states and toxin expression kinetics

Abstract Persistent bacteria subpopulations showing multidrug tolerance pose significant challenges to antibiotic therapy. While the HipBA toxin–antitoxin (TA) system is known to play a role in persister formation, existing studies predominantly focus on its kinase activity and intracellular regulatory pathways, with limited attention to the environmental or conditional factors. In this study, we administered controlled induction of HipA across varied growth conditions in Escherichia coli , establishing that conditional constraints fundamentally modulate HipA's capacity to induce persistence. We demonstrated that cells in the lag phase, rather than in the exponential or stationary phase, were more likely to enter the persistent state upon HipA upregulation. Moreover, our results indicated that the accumulation rate of HipA, rather than the absolute HipA level, was a crucial determinant of persister frequency. Finally, we confirmed the essential role of RelA in HipA‐mediated formation of persistent bacteria. These findings highlight the importance of previously unexplored conditional factors and expression kinetics in HipA‐mediated persistence, providing mechanistic insights that link toxin kinetics, growth phase regulation, and stringent response signaling in bacterial antibiotic tolerance.