The trace metal ion manganese (Mn) in excess is toxic. Therefore, a small subset of factors tightly maintains its cellular level, among which an efflux protein MntP is the champion. Multiple transcriptional regulators and a manganese-dependent translational riboswitch regulate the MntP expression in Escherichia coli. As riboswitches are untranslated RNAs, they are often associated with the Rho-dependent transcription termination in bacteria. Here, performing in vitro transcription and in vivo reporter assays, we demonstrate that Rho efficiently terminates transcription at the mntP riboswitch region. Excess manganese activates the riboswitch, restoring the coupling between transcription and translation to evade Rho-dependent transcription termination partially. RT-PCR and western blot experiments revealed that the deletion of the riboswitch abolishes Rho-dependent termination and thereby overexpresses MntP. Interestingly, deletion of the riboswitch also renders bacteria sensitive to manganese. This manganese sensitivity is linked with the overexpression of MntP. Further spot assays, confocal microscopy, and flow cytometry experiments revealed that the high level of MntP expression was responsible for slow growth, cell filamentation, and reactive oxygen species (ROS) production. We posit that manganese-dependent transcriptional activation of mntP in the absence of Rho-dependent termination leads to excessive MntP expression, a membrane protein, causing membrane protein toxicity. Thus, we show a regulatory role of Rho-dependent termination, which prevents membrane protein toxicity by limiting MntP expression.

中文翻译：

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mntP 基因表达的 Rho 和核糖开关依赖性调节逃避了锰和膜毒性。


过量的痕量金属离子锰 （Mn） 有毒。因此，一小部分因子紧密维持其细胞水平，其中外排蛋白 MntP 是冠军。多个转录调节因子和一个锰依赖性翻译核糖开关调节大肠杆菌中 MntP 的表达。由于核糖开关是非翻译 RNA，因此它们通常与细菌中的 Rho 依赖性转录终止有关。在这里，进行体外转录和体内报告基因测定，我们证明 Rho 有效地终止了 mntP 核糖开关区域的转录。过量的锰激活核糖开关，恢复转录和翻译之间的耦合，以部分逃避 Rho 依赖性转录终止。RT-PCR 和蛋白质印迹实验表明，核糖开关的缺失消除了 Rho 依赖性终止，从而过表达了 MntP。有趣的是，核糖开关的缺失也使细菌对锰敏感。这种锰敏感性与 MntP 的过表达有关。进一步的斑点测定、共聚焦显微镜和流式细胞术实验表明，高水平的 MntP 表达是导致生长缓慢、细胞细丝化和活性氧 （ROS） 产生的原因。我们假设在没有 Rho 依赖性终止的情况下 mntP 的锰依赖性转录激活导致 MntP 过度表达，一种膜蛋白，导致膜蛋白毒性。因此，我们显示了 Rho 依赖性终止的调节作用，它通过限制 MntP 表达来防止膜蛋白毒性。