Significance and Impact of the Study: Bicarbonate (NaHCO 3 ) can enhance the activity of various antibiotics. This work investigated its action mechanism. We carried out a transcriptional analysis in Escherichia coli with the aim of defining initial bacterial changes potentially linked to the enhancing activity of NaHCO 3 . Our approach differed from the longer term exposure to NaHCO 3 recently used by other researchers, who noticed changes in the bacterial proton motive force. Based on our analysis, we propose two routes possibly linked to the effect of NaHCO 3 . Conceivably, those routes are potential targets that could be manipulated by alternative means to augment the effect of antibiotics. AbstractWe have reported that bicarbonate (NaHCO 3 ) potentiates the activity of aminoglycosides in Escherichia coli, but the action mechanism was not identified. To eventually understand how NaHCO 3 can potentiate antibiotics, we thought that a rational first step was to examine the effect of NaHCO 3 separately and to inspect initial gene expression changes triggered by it. In this work, we started by confirming that NaHCO 3 can reduce the number of viable E. coli bacteria. We then investigated, via RNAseq, gene expression changes induced by NaHCO 3 . There were upregulated and downregulated genes, among the top upregulated genes c. 10-fold increase in expression) was tnaA, the gene encoding tryptophanase, the enzyme that degrades tryptophan to indole. Considering that higher expression of tnaA likely led to increases in indole, we tested the effect of indole and found both growth inhibition and synergy with NaHCO 3 . We suggest that indole may participate in growth inhibition of E. coli. The RNAseq analysis also revealed upregulation (≥4-fold) of genes encoding proteins for the acquisition of iron and downregulation (≥16-fold) of genes encoding iron-sulphur-holding proteins; hence NaHCO 3 apparently triggered also an iron-deficit response. We suggest that iron deficiency may also be involved in growth inhibition by NaHCO 3 .