Regulation, Mechanism of Action, and Function of a Small Toxin Protein in EHEC

The zor-orz locus identified in the chromosome of Escherichia coli O157:H7 EDL933 is a type I toxin-antitoxin system that consists of two homologous gene pairs zorO-orzO and zorP-orzP. The zor genes encode for small toxin proteins and the orz genes encode for small RNAs. Previously it was shown that overproduction of ZorO results in cell growth stasis whereas co-expression of orzO rescues cellular growth. Within, I demonstrate that in addition to growth inhibition, ZorO overproduction results in membrane depolarization and ATP depletion but does not impact the gross morphology of E. coli. In vivo translation and subsequent impacts of ZorO is significantly increased if the 5’ untranslated region (UTR) of the zorO mRNA is processed.

When the zor-orz locus or the zorO-orzO gene pair was cloned on a medium copy plasmid and transformed into E. coli, it significantly improved bacterial growth in the presence of several aminoglycoside antibiotics. An increase in the minimum inhibitory concentration (MIC) for gentamycin and kanamycin was observed linking, for the first-time, a type I toxin-antitoxin system with an increase in MIC. The transcription profile of EDL933 cells harboring pBR-zor-orz after kanamycin treatment showed upregulation of nitrogen metabolism and anerobic respiration genes whereas flagella associated genes were downregulated.

Mutation of the charged residues within ZorO revealed that several charged residues are critical for ZorO induced toxicity. Using one of the non-toxic mutants of ZorO (arginine at the 23^rd position replaced by leucine), we tested its impact on cell growth in the presence of kanamycin. Unexpectedly, this non-toxic mutant was also able to improve cell growth, indicating that toxic effects observed during ZorO overproduction are unrelated to protection against kanamycin. Ongoing work in the lab challenges the hypothesis that zorO-orzOmediated improved cell growth is due to the ZorO protein. Additional work on this can change our perspective as to how type I toxin-antitoxin systems function natively and better appreciate their complex nature.