Ectopic Overexpression of Wild-Type and Mutant <i>hipA</i> Genes in <i>Escherichia coli</i> : Effects on Macromolecular Synthesis and Persister Formation

<jats:title>ABSTRACT</jats:title> <jats:p> Persistence is an epigenetic trait that allows a small fraction of bacteria, approximately one in a million, to survive prolonged exposure to antibiotics. In <jats:italic>Escherichia coli</jats:italic> an increased frequency of persisters, called “high persistence,” is conferred by mutations in the <jats:italic>hipA</jats:italic> gene, which encodes the toxin entity of the toxin-antitoxin module <jats:italic>hipBA</jats:italic> . The high-persistence allele <jats:italic>hipA7</jats:italic> was originally identified because of its ability to confer high persistence, but little is known about the physiological role of the wild-type <jats:italic>hipA</jats:italic> gene. We report here that the expression of wild-type <jats:italic>hipA</jats:italic> in excess of <jats:italic>hipB</jats:italic> inhibits protein, RNA, and DNA synthesis in vivo. However, unlike the RelE and MazF toxins, HipA had no effect on protein synthesis in an in vitro translation system. Moreover, the expression of wild-type <jats:italic>hipA</jats:italic> conferred a transient dormant state (persistence) to a sizable fraction of cells, whereas the rest of the cells remained in a prolonged dormant state that, under appropriate conditions, could be fully reversed by expression of the cognate antitoxin gene <jats:italic>hipB</jats:italic> . In contrast, expression of the mutant <jats:italic>hipA7</jats:italic> gene in excess of <jats:italic>hipB</jats:italic> did not markedly inhibit protein synthesis as did wild-type <jats:italic>hipA</jats:italic> and yet still conferred persistence to ca. 10% of cells. We propose that wild-type HipA, upon release from HipB, is able to inhibit macromolecular synthesis and induces a bacteriostatic state that can be reversed by expression of the <jats:italic>hipB</jats:italic> gene. However, the ability of the wild-type <jats:italic>hipA</jats:italic> gene to generate a high frequency of persisters, equal to that conferred by the <jats:italic>hipA7</jats:italic> allele, may be distinct from the ability to block macromolecular synthesis. </jats:p>