A Cytoplasmic pH Sensor Redefines Gating in Bacterial Urea Channels and Acid Resistance

Bacterial survival in acidic environments depends on urea channels like UreI, which neutralize protons after importing urea and hydrolyzing it to ammonia and carbon dioxide. While pH-dependent gating was attributed to periplasmic histidines, the role of cytoplasmic residues remained unexplored. Using yeast complementation assays, in vitro permeability measurements, systematic sequence analysis, and molecular dynamics simulations, we identify a conserved cytoplasmic glutamic acid residue (E136 in Streptococcus salivarius, E135 in Helicobacter hepaticus, E159 in Helicobacter pylori) as a pH sensor in UreI channels. Protonation of this residue disrupts its interaction with a conserved partner (R20 in S. salivarius and H. hepaticus, T170 in H. pylori), increasing cytoplasmic-loop flexibility and enhancing conformational sampling of a conserved aromatic triad in the selectivity filter, thereby facilitating urea permeation. Mutagenesis across S. salivarius, H. pylori, and H. hepaticus confirms the residue´s indispensable and conserved role, revealing a Gram-type-specific gating mechanism: Gram-positive bacteria rely solely on this glutamic acid sensor, while Gram-negative pathogens employ a dual-sensor model integrating both cytoplasmic glutamic acid and periplasmic histidines. By overturning the paradigm of exclusive periplasmic pH sensing, this work reveals a conserved yet adaptable mechanism for bacterial acid resistance, offering a new molecular target for precision antimicrobial development.