Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States.
Biochemistry. 2020 Apr 21;59(15):1508-1516. doi: 10.1021/acs.biochem.0c00074. Epub 2020 Apr 13.
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. The riboswitch, present in numerous human gut microbiota and pathogens, was recently shown to respond to Ni and Co but thought not to respond to Fe, 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, as well as to Mn, Co, Ni, and Zn, but only in the cases of Fe and Mn do the apparent 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-responds to iron in cells. Finally, we demonstrate that the putative metal exporters directly downstream of two riboswitches efficiently rescue iron toxicity in a heterologous expression system. Together, our results indicate that iron merits consideration as a plausible physiological ligand for riboswitches, although a response to general metal stress cannot be ruled out at present.
铁对于几乎所有生物体都是必不可少的，其细胞内浓度（无论是缺乏还是过量）的管理不当都会导致人类病原体的毒力减弱，这就需要复杂的金属调控机制。迄今为止，尽管已经在细菌中鉴定出了几种金属响应型核糖开关，但没有一种被证明可以响应 Fe。最近，该核糖开关（存在于许多人类肠道微生物群和病原体中）被证明可以响应 Ni 和 Co，但基于有氧体外测定，据认为它不会响应 Fe；其在体内的功能尚未得到很好的理解。我们使用该核糖开关融合到 RNA 适体 Spinach2 构建了一个荧光传感器。在进行厌氧测定时，所得传感器在体外对 Fe 以及 Mn、Co、Ni 和 Zn 作出响应，但只有在 Fe 和 Mn 的情况下，表观值（分别为 0.4 和 11 μM）才在已知金属调控器维持的可利用金属浓度范围内。我们还表明，该传感器（据我们所知，是第一个用于 Fe 的可逆转遗传编码荧光传感器）在细胞中对铁有响应。最后，我们证明了两个 核糖开关下游的假定金属外排泵在异源表达系统中有效地挽救了铁毒性。总之，我们的结果表明，尽管目前不能排除对一般金属应激的反应，但铁值得考虑作为 核糖开关的一种合理的生理配体。
