SummaryThe small RNA RyhB has recently been shown to negatively regulate a number of mRNAs encoding dispensable iron-using proteins in Escherichia coli. The resulting decrease in the synthesis of iron-using proteins is thought to spare iron in order to ensure its availability for iron-requiring proteins that are indispensable. Indeed, the expression of RyhB from a heterologous promoter activates the iron-sensing repressor Fur, which suggests an increase in the pool of free intracellular iron (iron-sparing). In accordance with these observations, we report here that RyhB expression increases the concentration of free intracellular iron, as shown by direct measurements of the metal in whole cells by electron paramagnetic resonance spectroscopy. Our data also suggest that ironsparing originates from rapid uptake of extracellular iron and not from already internalized metal. Furthermore, RyhB is shown to be essential for normal bacterial growth and survival during iron starvation, which is consistent with previous data describing the function of the small RNA. Overall, our data demonstrate that, by regulating synthesis of nonessential iron-using proteins, the small RNA RyhB ensures that the iron is directed towards the iron-requiring enzymes that are indispensable.