Abstract
Microorganisms face environmental threats from fluoride anions (present at 100μM in soil) that inhibit enolase and pyrophosphatase (Ki~100μM), and it was believed that fluoride toxicity could be more troublesome under acidic pH level, because membrane permeant HF (pKa ~3.4) from the acidic extracellular fluid diffuses into the cytoplasm where it dissociates at the neutral pH, and F- becomes trapped and accumulates. This pH-driven anion accumulation effect is thought to be governed by the relationship [F-]in/[F-]out=[H+]out/[H+]in. We sought to directly test the validity of this thermodynamic principle in E. coli and measure whether this F- ion trapping mechanism governs the F- load that bacteria endure under acid stress. Using the F--selective LaF3/EuF3 electrode, we continuously monitored external F- concentrations of cultures of E. coli with its native F- channel knocked out (ΔFluc) during external pH changes and found that under weakly acidic environment, fluoride anions accumulate in E. coli lacking F- efflux pathways, and the actual cytoplasmic F- concentrations are in accord with predictions from the weak acid accumulation hypothesis [F-]in=[F-]out×10^(pHin-pHout). This F- accumulation reaches equilibrium in ~30 min, has bacteriostatic effects on growth, and prolongs the lag-time before bacterial growth recovers after removal of acidic stress. The same F- dependent delay on growth-recovery were observed for ΔFluc cells after 2 hours of extreme acid stress (pH 2.5), suggesting that fluoride anion accumulation in bacteria also holds under extremely acidic environments. \r We found representatives from the two families of F− membrane transport proteins—Fluoride anion channel (Fluc) and CLCF-type F−/H+ antiporter (Pst from Piruella staleyi)–were both effective at preventing cytoplasmic F- accumulation. Furthermore, amino group crosslinking by glutaraldehyde treatment of S214K mutant of Pst homologue revealed the homodimeric structure of the CLCF subclade, in harmony with the dimeric quaternary structure of canonical CLC transporters.