The bacterial antitoxin HipB establishes a ternary complex with operator DNA and phosphorylated toxin HipA to regulate bacterial persistence

Nearly all bacteria exhibit a type of phenotypic growth described as persistence that is thought to underlie antibiotic tolerance and recalcitrant chronic infections. The chromosomally encoded high-persistence (Hip) toxin-antitoxin proteins HipA_SO and HipB_SO from Shewanella oneidensis, a proteobacterium with unusual respiratory capacities, constitute a type II toxin-antitoxin protein module. Here we show that phosphorylated HipA_SO can engage in an unexpected ternary complex with HipB_SO and double-stranded operator DNA that is distinct from the prototypical counterpart complex from Escherichia coli. The structure of HipB_SO in complex with operator DNA reveals a flexible Cterminus that is sequestered by HipA_SO in the ternary complex, indicative of its role in binding HipA_SO to abolish its function in persistence. The structure of HipA_SO in complex with a non-hydrolyzable ATP analogue shows that HipA_SO autophosphorylation is coupled to an unusual conformational change of its phosphorylation loop. However, HipA_SO is unable to phosphorylate the translation factor Elongation factor Tu, contrary to previous reports, but in agreement with more recent findings. Our studies suggest that the phosphorylation state of HipA is an important factor in persistence and that the structural and mechanistic diversity of HipAB modules as regulatory factors in bacterial persistence is broader than previously thought.