Small bacterial proteins under 50 amino acids were generally overlooked in the past but now have been shown to play important roles in numerous cellular pathways throughout a variety of species. However, their characterization and functional analyses remain challenging. We are investigating the function of a 29 amino acid protein (ZorO) encoded in the chromosome of enterohemorrhagic Escherichia coli (EHEC), a worldwide foodborne pathogen. We previously showed ZorO overproduction results in cell growth stasis but is repressed at the post-transcriptional level via complementary base pairing by a small RNA (OrzO). Thus, the zorO-orzO gene pair is an example of a type I toxin antitoxin system. Nonetheless, the mechanism of ZorO toxicity, its effects and function when expressed endogenously in EHEC is not well understood. Here, using an ectopic overproduction system, we show this protein localizes to the bacterial inner membrane, causes membrane depolarization and reduces cellular ATP levels without significantly affecting cellular morphology. We also observed that specific charged amino acid residues are critical for ZorO induced toxicity. Preliminary data supports a model for ZorO oligomerizing in the membrane leading to leakage of ions. We are also assessing the conditions under which ZorO is produced to elucidate when the protein naturally functions. To address not only its own expression but also that of its antitoxin OrzO, we designed a pFA6a plasmid vector expressing two different reporter proteins under the control of the zorO and orzO promoters. These studies demonstrated that zorO transcription is upregulated under nutrient limiting conditions, suggesting a role for ZorO when the cells encounter adverse growth conditions. Further studies are aimed to elucidate the contribution of the gene pair in bacterial persistence and pathogenesis.