A co-conserved gene pair supports Caulobacter iron homeostasis and resistance to chelation stress
Bacteria assimilate a diverse set of metals that enable metabolic functions essential for growth and survival, but metal homeostasis can be disrupted by natural and synthetic chelators in the environment. We have identified genes that influence fitness of the common soil and aquatic bacterium, Caulobacter crescentus, in the presence of a growth-limiting concentration of the synthetic chelator ethylenediaminetetraacetic acid (EDTA). Among the identified genes important in EDTAacclimationthere are several controlled by the metal-responsive regulators Fur, Zur, and UzcRS. However, a subset of genes in the iron-responsive Fur regulon emerged as the primary fitness determinants during EDTA challenge. EDTA treatment reduced intracellular iron levels and a syntenic gene pair, cciT and cciO, that supports Caulobacter iron homeostasis is necessary for growth in EDTA. cciT encodes one of four Fur-regulated TonB-dependent transporters (TBDTs) and – of these regulators – uniquely supports wild-type levels of growth on defined and complex lab media. The iron acquisition and EDTA resistance functions of the CciT transporter specifically require the presence of cciO, which encodes a 2-oxoglutarate-Fe(II) dioxygenase. Thus, there is a critical functional partnership between an outer membrane transporter and a cytoplasmic dioxygenase that are broadly co-conserved in the phylum Proteobacteria. cciT and cciO mutants cultivated in natural freshwater were not impacted by an equivalent EDTA treatment, demonstrating robustness of the highly redundant set of Caulobacter iron acquisition systems in an ecosystem context. This study advances our understanding of genes involved in metal acquisition and chelation stress survival, and illuminates the complex interactions between the chemical environment and the processes that regulate intracellular metal homeostasis. Overall design: To investigate the relationship between the genes cciO (CCNA_00027), and cciT (CCINA_00028) to iron homeostasis, we grew wildtype CB15 and mutants lacking cciO or cciT in M2X broth with 10 µM FeCl3 for 2.5 hours. RNA was extracted using TRIzol (Invitrogen) ___________. RNA-sequencing libraries were prepared utilizing the Illumina TruSeq Stranded RNA Kit and sequenced on the Illumina NextSeq 500 platform at SeqCoast Genomics (Portsmouth, NH).