Ferrosome Organelles Spatially Insulate a Redox-Active Ferrous Phosphate Biomineral from Cytosolic ROS Chemistry

Ferrosomes are recently discovered lipid-bound bacterial organelles that store iron as iron-phosphate biominerals, yet the chemical nature and physiological consequences of ferrosome-stored iron remain poorly understood. Here, we combined X-ray absorption spectroscopy (XAS), electron microscopy, inductively coupled plasma mass spectrometry (ICP-MS), and physiological analyses to characterize ferrosome iron in Clostridioides difficile. XAS analysis of isolated ferrosomes revealed an amorphous iron-phosphate biomineral containing mixed Fe(II)/Fe(III), consistent with partial oxidation during aerobic isolation. In contrast, whole-cell XAS of intact anaerobically maintained cells demonstrated that ferrosomes predominantly contain a structurally disordered ferrous phosphate biomineral with local Fe-O-P coordination features similar to those of vivianite. Upon air exposure, this ferrous biomineral rapidly oxidized to a ferric phosphate-like state, revealing a highly oxygen-sensitive iron-storage phase. Despite containing abundant redox-active Fe(II), ferrosome-stored iron contributed minimally to the cytosolic labile iron pool. Consistent with this observation, isolated ferrosomes exhibited little ROS-generating activity, and ferrosome-overproducing cells displayed no substantial increase in sensitivity to oxygen, peroxide, or paraquat stress relative to ferrosome-deficient controls. Together, these results establish ferrosomes as iron-storage organelles that sequester redox-active Fe(II) in a mineralized ferrous phosphate phase, limiting its participation in cytosolic ROS chemistry and providing a mechanism for the safe storage of reactive iron. Significance StatementIron is essential for life but can also damage cells because ferrous iron drives oxidative stress. How cells store large amounts of ferrous iron while limiting toxicity therefore remains a fundamental biological question. Ferrosomes are recently discovered bacterial organelles that store iron as iron-phosphate biominerals, but the chemical nature and physiological consequences of ferrosome-associated iron remained unknown. Using Fe K-edge X-ray absorption spectroscopy, we show that ferrosomes in Clostridioides difficile contain a redox-sensitive ferrous phosphate biomineral. Physiological analyses demonstrate that this iron is largely inaccessible to cytosolic reactive oxygen species (ROS) chemistry. These findings reveal that bacteria can combine biomineralization and subcellular compartmentalization to maintain large intracellular iron reservoirs while limiting iron-dependent oxidative damage.