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

Host nutritional immunity utilizes metal deprivation to help prevent microbial infection. To investigate bacterial adaptation to such restrictive conditions, we conducted experimental evolution with two metal sequestering agents. Ethylenediaminetetraacetic acid (EDTA) and diethylenetriamine pentamethylene phosphonic acid (DTPMP) were selected as ligands because they differentially affect cellular levels of iron, manganese and zinc in . Mutants of strain BW25113 were isolated after cultivation at sub-minimum inhibitory concentration (MIC) chelant levels and genetic changes potentially responsible for tolerance were identified by whole-genome sequencing. In EDTA-selected strains, mutations in the promoter region of resulted in elevated gene expression. The product, a zinc-specific metallochaperone, was confirmed to be primarily responsible for EDTA resistance. Similarly, in two of the DTPMP-selected strains, a promoter mutation increased expression of the operon, which encodes components of the ferric-enterobactin uptake pathway. However, in this case improved DTPMP tolerance was only detectable following overexpression of FepA or EntD . Additional mutations in the gene product, an acid-response regulator, preserved the neutrality of the growth medium by constitutively activating expression of the 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 critical for their antibacterial mode of action.