Multilayered Regulation of the Type I Toxin ZorO in EHEC

A bacterial type I toxin-antitoxin system contains two genes: one encodes a small toxic protein and the second, a small regulatory RNA (sRNA) that inhibits toxin production. To date, very few type I loci have been described thoroughly in regards to the regulation of toxin and the function of the toxin at endogenous levels. In this study, I demonstrated that the zor-orz locus of Escherichia coli O157:H7 is composed of two highly homologous type I toxin-antitoxin systems: zorO-orzO and zorP-orzP. The zor genes encode the toxins and the orz genes encode the antitoxin sRNAs. Overexpression of zorO is toxic to E. coli and causes bacterial growth stasis or cell death; however, co-expression of orzO neutralizes this toxicity and restores normal bacterial growth. Rapid membrane depolarization was observed upon ZorO overproduction, suggesting that ZorO targets the membrane. Replacement of two charged amino acids in ZorO, glutamic acid at the 16^th position and arginine at the 23^rd position, can impair ZorO toxicity.

Given its inherent toxicity, production of ZorO is tightly controlled by both its 5’ untranslated region (UTR) and the antitoxin OrzO. The zorO 5’ UTR harbors two distinct regions that modulate zorO translation. One is a putative ribosome standby site that is exposed only upon processing of the 5’ UTR and likely promotes translation by facilitating ribosome preloading onto the mRNA. The other region spans from +35 to +50 of zorO and is required for optimal translation of zorO, although the underlying mechanism remains unclear. The OrzO sRNA inhibits ZorO production by reducing both the stability and the translation of the zorO mRNA. Specifically, OrzO base pairs to the putative standby site of zorO and impedes translation, potentially through competition with ribosome for this site. Successful base pairing of zorO by OrzO requires at least 15 nucleotides of perfect sequence complementarity. Once paired, the RNA duplex can be degraded by RNase III, rendering the zorO mRNA untranslatable. Collectively, this multilayered control of ZorO production limits its toxicity. Hence, my work provides new insights into regulation of type I toxins by their antitoxin sRNAs and beyond the sRNAs.