A riboswitch-controlled TerC family transporter Alx tunes intracellular manganese concentration in at alkaline pH.

Cells use transition metal ions as structural components of biomolecules and cofactors in enzymatic reactions, making transition metal ions integral cellular components. Organisms optimize metal ion concentration to meet cellular needs by regulating the expression of proteins that import and export that metal ion, often in a metal ion concentration-dependent manner. One such regulation mechanism is via riboswitches, which are 5'-untranslated regions of an mRNA that undergo conformational changes to promote or inhibit the expression of the downstream gene, commonly in response to a ligand. The family of bacterial riboswitches shares a conserved aptamer domain that binds manganese ions (Mn). In , the riboswitch precedes and regulates the expression of two different genes: , which based on genetic evidence encodes an Mn exporter, and , which encodes a putative metal ion transporter whose cognate ligand is currently in question. The expression of is upregulated by both elevated concentrations of Mn and alkaline pH. With metal ion measurements and gene expression studies, we demonstrate that the alkalinization of media increases the cytoplasmic manganese pool, which, in turn, enhances expression. The Alx-mediated Mn export prevents the toxic buildup of the cellular manganese, with the export activity maximal at alkaline pH. We pinpoint a set of acidic residues in the predicted transmembrane segments of Alx that play a critical role in Mn export. We propose that Alx-mediated Mn export serves as a primary protective mechanism that fine tunes the cytoplasmic manganese content, especially during alkaline stress.IMPORTANCEBacteria use clever ways to tune gene expression upon encountering certain environmental stresses, such as alkaline pH in parts of the human gut and high concentration of a transition metal ion manganese. One way by which bacteria regulate the expression of their genes is through the 5'-untranslated regions of messenger RNA called riboswitches that bind ligands to turn expression of genes on/off. In this work, we have investigated the roles and regulation of  and , the two genes in  regulated by the   riboswitches, in alkaline pH and high concentration of Mn. This work highlights the intricate ways through which bacteria adapt to their surroundings, utilizing riboregulatory mechanisms to maintain Mn levels amidst varying environmental factors.