| Summary: | Hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) is a common effector of defense mechanisms against pathogenic infections. However, bacterial factors involved in H<sub>2</sub>O<sub>2</sub> tolerance remain unclear. Here we used transposon-directed insertion-site sequencing (TraDIS), a technique allowing the screening of the whole genome, to identify genes implicated in H<sub>2</sub>O<sub>2</sub> tolerance in <i>Escherichia coli</i>. Our TraDIS analysis identified 10 mutants with fitness defect upon H<sub>2</sub>O<sub>2</sub> exposure, among which previously H<sub>2</sub>O<sub>2</sub>-associated genes (<i>oxyR</i>, <i>dps</i>, <i>dksA</i>, <i>rpoS</i>, <i>hfq</i> and <i>polA</i>) and other genes with no known association with H<sub>2</sub>O<sub>2</sub> tolerance in <i>E. coli</i> (<i>corA</i>, <i>rbsR</i>, <i>nhaA</i> and <i>gpmA</i>). This is the first description of the impact of <i>gpmA</i>, a gene involved in glycolysis, on the susceptibility of <i>E. coli</i> to H<sub>2</sub>O<sub>2</sub>. Indeed, confirmatory experiments showed that the deletion of <i>gpmA</i> led to a specific hypersensitivity to H<sub>2</sub>O<sub>2</sub> comparable to the deletion of the major H<sub>2</sub>O<sub>2</sub> scavenger gene <i>katG</i>. This hypersensitivity was not due to an alteration of catalase function and was independent of the carbon source or the presence of oxygen. Transcription of <i>gpmA</i> was upregulated under H<sub>2</sub>O<sub>2</sub> exposure, highlighting its role under oxidative stress. In summary, our TraDIS approach identified <i>gpmA</i> as a member of the oxidative stress defense mechanism in <i>E. coli</i>.
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