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

Abstract Cells 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. The yybP-ykoY family of bacterial riboswitches shares a conserved aptamer domain that binds manganese (Mn 2+ ). In E. coli , the yybP-ykoY riboswitch precedes and regulates the expression of two genes: mntP , which based on extensive genetic evidence encodes an Mn 2+ exporter, and alx , which encodes a putative metal ion transporter whose cognate ligand is currently in question. Expression of alx is upregulated by both elevated intracellular concentrations of Mn 2+ and alkaline pH. With metal ion measurements and gene expression studies, we demonstrate that the alkalinization of media increases cytoplasmic Mn 2+ content, which in turn enhances alx expression. Alx then exports excess Mn 2+ 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 Mn 2+ export. We propose that Alx-mediated Mn 2+ export provides a primary protective layer that fine-tunes the cytoplasmic Mn 2+ levels, especially during alkaline stress.