A riboswitch-controlled manganese exporter (Alx) tunes intracellular Mn2+concentration inE. coliat alkaline pH

Abstract

AbstractCells use transition metal ions as structural components of biomolecules and cofactors in enzymatic reactions, making transition metals vital cellular components. The buildup of a particular metal ion in certain stress conditions becomes harmful to the organism due to the misincorporation of the excess ion into biomolecules, resulting in perturbed enzymatic activity or metal-catalyzed formation of reactive oxygen species. Organisms optimize metal concentration by regulating the expression of proteins that import and export that metal, often in a metal concentration-dependent manner. One such regulation mechanism is via riboswitches, which are 5’-untranslated regions (UTR) of an mRNA that undergo conformational changes to promote or inhibit the expression of the downstream gene, commonly in response to a ligand. TheyybP-ykoYfamily of bacterial riboswitches shares a conserved aptamer domain that binds manganese (Mn2+). InE. coli, theyybP-ykoYriboswitch precedes and regulates the expression of two genes:mntP, which based on extensive genetic evidence encodes an Mn2+exporter, andalx, which encodes a putative metal ion transporter whose cognate ligand is currently in question. Expression ofalxis upregulated by both elevated intracellular concentrations of Mn2+and alkaline pH. With metal ion measurements and gene expression studies, we demonstrate that the alkalinization of media increases cytoplasmic Mn2+content, which in turn enhancesalxexpression. Alx then exports excess Mn2+to prevent toxic buildup of the metal inside the cell, with the export activity maximal at alkaline pH. Using mutational and complementation experiments, we pinpoint a set of acidic residues in the predicted transmembrane segments of Alx that play a crucial role in its Mn2+export. We propose that Alx-mediated Mn2+export provides a primary protective layer that fine-tunes the cytoplasmic Mn2+levels, especially during alkaline stress.