Reconsidering the czcD (NiCo) riboswitch as iron (II) riboswitch
Restricted (Penn State Only)
- Author:
- Xu, Jiansong
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 09, 2022
- Committee Members:
- Xin Zhang, Major Field Member
Joseph Cotruvo, Chair & Dissertation Advisor
Philip Bevilacqua, Major Field Member
Philip Bevilacqua, Program Head/Chair
Emily Weinert, Outside Unit & Field Member - Keywords:
- Irving-William series
metalloregulation
RNA
iron
metal selectivity
riboswitch
metal sensor
czcD riboswitch
NiCo riboswitch
metal overload - Abstract:
- 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 FeII. The czcD (or NiCo) 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 demonstrate that Spinach-based RNA sensors generated from both the E. bacterium (Eba) and Listeria monocytogenes (Lmo) czcD riboswitches respond to the biologically relevant divalent first-row transition metals according to the Irving-Williams series. By studying the native riboswitches from three organisms (Eba, Lmo, and Clostridium cellulolyticum) using isothermal titration calorimetry (ITC), we observed similar affinities for Fe(II), Co(II) and Ni(II), weaker affinity to Zn(II), and no response for Mn(II). By comparing these affinities to the physiological labile concentrations of these metals, only the Fe(II) affinity falls within the physiological concentration range. When expressed in E. coli, the Eba sensor responds reversibly to Fe(II) in a dose-dependent fashion; furthermore, it exhibits a basal response, suggesting that the riboswitch is tuned to cellular labile FeII concentrations. Finally, Eba3544 and Lmo3448, the metal transporters putatively controlled by the Eba and Lmo czcD riboswitches, were shown to effectively rescue iron toxicity in a heterologous system, but not toxicity of cobalt and nickel. We conclude that Fe(II) is the most plausible physiological ligand for the czcD class of riboswitch and the associated downstream metal transporters.