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. The czcD riboswitch, present in numerous human gut microbiota and pathogens, was recently shown to respond to Ni(II) and Co(II) but claimed 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). However, of all of these metals, only in the case of Fe(II) does the apparent Kd (0.4 µM) fall within the range of labile metal concentrations maintained by known metalloregulators. These results are supported by biophysical characterization of metal binding to the riboswitch itself. 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 E. 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 strongly suggest that iron (and not nickel or cobalt) is the most likely physiological ligand for czcD riboswitches under most cellular conditions, constituting a new mechanism of bacterial iron regulation.

Reclassification of the NiCo riboswitch as an iron-responsive riboswitch: Discovery of a new mechanism of bacterial iron regulation

ACS Fall 2021

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