The AadR-FixK hierarchy coordinates iron-responsive metabolism via Fur-family regulators in Rhodopseudomonas palustris TIE-1

Rhodopseudomonas palustris TIE-1 (TIE-1) is a metabolically versatile environmental bacterium that flourishes across gradients of iron, oxygen, and light. This versatility necessitates extensive regulatory control, exemplified by the aerobic-anaerobic metabolic shift controlled by the hierarchy of CRP/FNR-family regulators AadR and FixK. Many anaerobic metabolic pathways demand expression of iron cofactor-intensive proteins, and TIE-1 in particular can generate energy through phototrophic iron oxidation via the PioABC system. However, TIE-1 lacks canonical iron-sensing regulators: IscR, ancestral Fe(II)-sensing Fur, and Fe(II)-sensing RirA of Rhizobiaceae, leaving it unclear how TIE-1 coordinates expression of these iron-requiring metabolisms with bioavailable iron levels. Here, we demonstrate that the AadR-FixK hierarchy plays a previously underappreciated role in iron regulation in TIE-1 by comparing growth and transcription in wild-type and regulatory mutants across wetland-inspired naturomimetic conditions. {Delta}aadR and {Delta}fixK showed defects in iron-dependent growth and Fe(II) oxidation, and the {Delta}aadR{Delta}fixK double mutant was synthetically lethal under anaerobiosis. The regulatory hierarchy of FixK and AadR influences expression of Fur-family regulators: the two irr paralogs were oppositely regulated in the presence of AadR, and absence of AadR perturbed iron-responsive expression of mur. Furthermore, the AadR regulon was significantly enriched for iron-related and iron-containing proteins. Despite initial predictions that AadR directly regulates pioABC, we found no conclusive evidence for direct AadR activity at the pioABC promoter, refining the search for pio regulators. Together, these findings establish AadR as a central integrator of oxygen and iron signals to coordinate iron-requiring anaerobic metabolism in TIE-1.