A co-conserved gene pair supports Caulobacter iron homeostasis during chelation stress

Synthetic metal chelators are widely used in industrial, clinical, and agricultural settings, leading to their accumulation in the environment. Although the effects of chelators on metal solubility and bioavailability are well documented, their impact on microbial physiology is not well defined. We measured the growth of Caulobacter crescentus , a common soil and aquatic bacterium, in the presence of the ubiquitous chelator ethylenediaminetetraacetic acid (EDTA) and found that it restricts growth by reducing intracellular iron levels. Using barcoded transposon sequencing, we identified an operonic gene pair, cciT–cciO , that is required to maintain iron homeostasis during EDTA challenge. cciT encodes one of four TonB-dependent transporters that are regulated by the ferric uptake repressor (Fur) and stands out among this group of genes in its ability to support Caulobacter growth across diverse media conditions. The function of CciT strictly requires cciO , which encodes a cytoplasmic Fe _II dioxygenase-family protein. Our results thus define a functional partnership between an outer membrane iron receptor and a cytoplasmic dioxygenase that are broadly co-conserved in Proteobacteria. We expanded our analysis of the cciT–cciO system to natural environments by measuring the growth of mutant strains in freshwater from two ecologically distinct lakes, which have significantly different nutritional and geochemical profiles compared to standard laboratory media. cciT and cciO were not required for growth in lake water, regardless of EDTA presence, highlighting the iron acquisition versatility of Caulobacter in bona fide environments and underscoring the conditional toxicity of EDTA. This study defines a conserved iron acquisition system and bridges laboratory-based physiology studies to real-world environments.