Improved Growth of Escherichia coli in Aminoglycoside Antibiotics by the zor-orz Toxin-Antitoxin System

Type I toxin-antitoxin systems consist of a small protein (under 60 amino acids) whose overproduction can result in cell growth stasis or death, and a small RNA that represses translation of the toxin mRNA. Despite their potential toxicity, type I toxin proteins are increasingly linked to improved survival of bacteria in stressful environments and antibiotic persistence. While the interaction of toxin mRNAs with their cognate antitoxin sRNAs in some systems are well characterized, additional translational control of many toxins and their biological roles are not well understood. Using an ectopic overexpression system, we show that the efficient translation of a chromosomally encoded type I toxin, ZorO, requires mRNA processing of its long 5' untranslated region (UTR; A28 UTR). The severity of ZorO-induced toxicity on growth inhibition, membrane depolarization, and ATP depletion was significantly increased if expressed from the &28 UTR versus the full-length UTR. ZorO did not form large pores, as was evident via a liposomal leakage assay, in vivo morphological analyses, and measurement of ATP loss. Further, increasing the copy number of the entire zor-orz locus significantly improved the growth of bacterial cells in the presence of kanamycin and increased the minimum inhibitory concentration (MIC) against kanamycin and gentamicin; however, no such benefit was observed against other antibiotics. This supports a role for the zor-orz locus as a protective measure against specific stress agents and is likely not part of a general stress response mechanism. Combined, these data shed more insights into the possible native functions for type I toxin proteins. IMPORTANCE Bacterial species can harbor gene pairs known as type I toxin-antitoxin systems, in which one gene encodes a small protein that is toxic to the bacteria producing it and a second gene that encodes a small RNA antitoxin to prevent toxicity. While artificial overproduction of type I toxin proteins can lead to cell growth inhibition and cell lysis, the endogenous translation of type I toxins appears to be tightly regulated. Here, we show that translational regulation controls production of the ZorO type I toxin and prevents subsequent negative effects on the cell. Further, we demonstrate a role for zorO and its cognate antitoxin in the improved growth of Escherichia coli in the presence of aminoglycoside antibiotics.