The czcD (NiCo) Riboswitch Responds to Iron(II)

Iron is essential for nearly every organism, and mismanagement of its intracellular concentrations (either deficiency or excess) contributes to diminished virulence in human pathogens, necessitating intricate metalloregulatory mechanisms. To date, although several metal-responsive riboswitches have been identified in bacteria, none has been shown to respond to Fe^II. The czcD riboswitch, present in numerous human gut microbiota and pathogens, was recently shown to respond to Ni^II and Co^II but thought not to respond to Fe^II, on the basis of aerobic, in vitro assays; its function in vivo is not well understood. We constructed a fluorescent sensor using this riboswitch fused to the RNA aptamer, Spinach2. When assayed anaerobically, the resulting sensor responds in vitro to Fe^II, as well as to Mn^II, Co^II, Ni^II, and Zn^II, but only in the cases of Fe^II and Mn^II do the apparent K_d values (0.4 and 11 μM, respectively) fall within the range of labile metal concentrations maintained by known metalloregulators. We also show that the sensor-which is, to the best of our knowledge, the first reversible genetically encoded fluorescent sensor for Fe^II-responds to iron in Escherichia coli cells. Finally, we demonstrate that the putative metal exporters directly downstream of two czcD riboswitches efficiently rescue iron toxicity in a heterologous expression system. Together, our results indicate that iron merits consideration as a plausible physiological ligand for czcD riboswitches, although a response to general metal stress cannot be ruled out at present.