Rho and riboswitch-dependent regulations of mntP gene expression evade manganese and membrane toxicities

Abstract

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.

Keywords: Escherichia coli (E. coli); MntP; ROS; Rho; gene regulation; manganese; membrane protein; riboswitch; transcription termination; translation.