GSE110493
The antibiotics potentiator bicarbonate causes upregulation of tryptophanase and iron acquisition proteins in Escherichia coli
We have reported that bicarbonate (NaHCO3) potentiates the activity of aminoglycosides in Escherichia coli, but the action mechanism was not identified. To eventually understand how NaHCO3 can potentiate antibiotics, we thought that a rational first step was to examine the effect of NaHCO3 separately and to inspect initial gene expression changes triggered by it. In this work we started by confirming that NaHCO3 can reduce the number of viable E. coli bacteria. We then investigated, via RNAseq, gene expression changes induced by NaHCO3. There were upregulated and downregulated genes, among the top upregulated genes (~10-fold increase in expression) was tnaA, the gene encoding tryptophanase (TnaA), 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 NaHCO3. 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-sulfur-holding proteins, hence NaHCO3 apparently triggered also an iron deficit response. We suggest that iron deficiency may also be involved in growth inhibition by NaHCO3. Overall design: Liquid LB cultures of E. coli were followed throughout the growth curve by periodic optical density readings, usually every 20 min. When cultures were in the logarithmic phase of growth, they were exposed to 40 mM NaHCO3 and the incubation was continued for 40 minutes. The control cultures were treated exactly as the experimental cultures but they were exposed to 40 mM NaCl in lieu of NaHCO3. The treatment with NaCl was intended to standardize the ion molarity in the control cultures and in the NaHCO3-treated cultures. It should be noted however, that the LB medium already contained 1% NaCl, or a 170 mM concentration of NaCl, thus after addition of 40 mM NaCl to the control cultures the extra NaCl contributed approximately 20% to the final molarity. The extra NaCl had no apparent effect on E. coli responses, including sensitivity to antibiotics (unpublished data). At the end of the incubation period, cultures were transferred to ice and bacterial cells were collected at 4°C by centrifugation at 10,000 x g for a 10 min period. The collected cells were then subjected to RNA extraction. Sequencing was performed by the specialist company Otogenetics (Norcross, GA). According to the contracted services, the RNA-seq protocol included initial sample quality control (QC), rRNA depletion, random primed cDNA synthesis/QC; Illumina sequencing library preparation/QC; PE100 and HiSeq2000/2500 sequencing, designated 10 million reads; paired-end 2 x 100nt.