A critical role for iron and zinc homeostatic systems in the evolutionary adaptation of E. coli to metal restriction

Host nutritional immunity utilises metal deprivation to help prevent microbial infection. To investigate bacterial adaptation to such restrictive conditions, we conducted experimental evolution with two metal sequestering agents. EDTA and DTPMP were selected as ligands because they differentially affect cellular levels of iron, manganese and zinc in E. coli. Mutants were isolated after cultivation at sub-MIC chelant levels and genetic changes potentially responsible for tolerance were characterised. In EDTA-selected strains, mutations in the promoter region of yeiR resulted in elevated gene expression. The yeiR product, a zinc-specific metallochaperone, was confirmed to be primarily responsible for EDTA resistance. In DTPMP-selected strains, increased expression of fepA or entD, encoding components of the ferric-enterobactin uptake pathway, improved E. coli growth in the presence of this chelant. Additional mutations in the cadC gene product, an acid-response regulator, preserve the neutrality of the growth medium by constitutively activating expression of the cadAB regulon. This study uncovers specific resistance mechanisms for zinc and iron starvation that could emerge by selection against host nutritional immunity or competition with heterologous metallophores. It also provides insight into the specific metals affected by these two widely used chelators that are critical for their antibacterial mode of action.