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ABSTRACT Iron (Fe) is a trace nutrient required by nearly all organisms. As a result of the demand for Fe and the toxicity of non-chelated cytosolic ionic Fe, regulatory systems have evolved to tightly balance Fe acquisition and usage while limiting overload. In most bacteria, including the mammalian pathogenStaphylococcus aureus, the ferric uptake regulator (Fur) is the primary transcriptional regulator controlling the transcription of genes that code for Fe uptake and utilization proteins. Fpa (formerly YlaN) was demonstrated to be essential inBacillus subtilisunless excess Fe is added to the growth medium, suggesting a role in Fe homeostasis. Here, we demonstrate that Fpa is essential inS. aureusupon Fe deprivation. Nullfuralleles bypassed the essentiality of Fpa. The absence of Fpa abolished the derepression of Fur-regulated genes during Fe limitation. Bioinformatic analyses suggest thatfpawas recruited to Gram-positive bacteria and, once acquired, was maintained in the genome as it co-evolved with Fur. Consistent with a role for Fpa in alleviating Fur-dependent repression, Fpa and Fur interactedin vivo, and Fpa decreased the DNA-binding ability of Furin vitro. Fpa bound Fe(II)in vitrousing oxygen or nitrogen ligands with an association constant that is consistent with a physiological role in Fe homeostasis. These findings have led to a model wherein Fpa is an Fe(II) binding protein that influences Fur-dependent regulation through direct interaction.IMPORTANCEIron (Fe) is an essential nutrient for nearly all organisms. If Fe homeostasis is not maintained, Fe may accumulate in the cytosol, which can be toxic. Questions remain about how cells efficiently balance Fe uptake and usage to prevent overload. Iron uptake and proper metalation of proteins are essential processes in the mammalian bacterial pathogenStaphylococcus aureus. Understanding the gene products involved in the genetic regulation of Fe uptake and usage and the physiological adaptations thatS. aureususes to survive in Fe-depleted conditions provides insight into pathogenesis. Herein, we demonstrate that the DNA-binding activity of the ferric uptake regulator transcriptional repressor is alleviated under Fe limitation, but uniquely, inS. aureus, alleviation requires the presence of Fpa. 

To gain a better insight of how Copper (Cu) ions toxify cells, metabolomic analyses were performed inS.aureusstrains that lacks the described Cu ion detoxification systems (ΔcopBLΔcopAZ;cop^-). Exposure of thecop^-strain to Cu(II) resulted in an increase in the concentrations of metabolites utilized to synthesize phosphoribosyl diphosphate (PRPP). PRPP is created using the enzyme phosphoribosylpyrophosphate synthetase (Prs) which catalyzes the interconversion of ATP and ribose 5-phosphate to PRPP and AMP. Supplementing growth medium with metabolites requiring PRPP for synthesis improved growth in the presence of Cu(II). A suppressor screen revealed that a strain with a lesion in the gene coding adenine phosphoribosyltransferase (apt) was more resistant to Cu. Apt catalyzes the conversion of adenine with PRPP to AMP. Theaptmutant had an increased pool of adenine suggesting that the PRPP pool was being redirected. Over-production ofapt, or alternate enzymes that utilize PRPP, increased sensitivity to Cu(II). Increasing or decreasing expression ofprsresulted in decreased and increased sensitivity to growth in the presence of Cu(II), respectively. We demonstrate that Prs is inhibited by Cu ionsin vivoandin vitroand that treatment of cells with Cu(II) results in decreased PRPP levels. Lastly, we establish thatS.aureusthat lacks the ability to remove Cu ions from the cytosol is defective in colonizing the airway in a murine model of acute pneumonia, as well as the skin. The data presented are consistent with a model wherein Cu ions inhibits pentose phosphate pathway function and are used by the immune system to preventS.aureusinfections.