A reversible YbbR-domain hinge switch senses extracellular pH to control bacterial cyclic-di-AMP signaling

Acidification is a principal antimicrobial host defence: during infection, bacteria have to survive the stomach, the vaginal tract or the phagolysosome of immune cells, where the pH falls below 5. To persist, bacteria must sense and adapt to extracellular pH. In Gram-positive bacteria, the membrane protein CdaR, a regulator of the c-di-AMP-synthesizing enzyme CdaA, has been proposed as a pH sensor, but the molecular basis of this sensing remains unknown. Here we show, using the model organism Bacillus subtilis, that CdaR is essential for growth below pH 5. SEC-MALS, mass photometry and circular dichroism demonstrate that its extracellular YbbR domains neither oligomerize nor unfold at low pH. Instead, tryptophan and ANS fluorescence reveal a reversible conformational change that requires at least two tandem YbbR domains and occurs between pH 4.5 and 5.5. Crystallography, molecular dynamics and solution NMR localize this transition to a conserved hinge between YbbR domains 1 and 2, where a network of charged residues rearranges to rigidify it and restrict inter-domain motion. Mutagenesis identifies a highly conserved aspartate whose substitution abolishes the conformational switch in vitro and accelerates adaptation to low pH in vivo. These findings define a structural mechanism by which CdaR senses extracellular acidification through its YbbR domains and couples this signal to intracellular c-di-AMP signaling. As the hinge and its aspartate are the most conserved features of the YbbR family, found in pathogens including Staphylococcus aureus and Listeria monocytogenes, we propose this as a general mechanism of acid adaptation in Gram-positive bacteria.