Osmolytes Contribute to pH Homeostasis of Escherichia coli

Background: Cytoplasmic pH homeostasis in Escherichia coli includes numerous mechanisms involving pH-dependent catabolism and ion fluxes. An important contributor is transmembrane K(+) flux, but the actual basis of K(+) compensation for pH stress remains unclear. Osmoprotection could mediate the pH protection afforded by K(+) and other osmolytes. Methods and Principal Findings: The cytoplasmic pH of E. coli K-12 strains was measured by GFPmut3 fluorimetry. The wild-type strain Frag1 was exposed to rapid external acidification by HCl addition. Recovery of cytoplasmic pH was enhanced equally by supplementation with NaCl, KCl, proline, or sucrose. A triple mutant strain TK2420 defective for the Kdp, Trk and Kup K(+) uptake systems requires exogenous K(+) for steady-state pH homeostasis and for recovery from sudden acid shift. The K(+) requirement however was partly compensated by supplementation with NaCl, choline chloride, proline, or sucrose. Thus, the K(+) requirement was mediated in part by osmolarity, possibly by relieving osmotic stress which interacts with pH stress. The rapid addition of KCl to strain TK2420 suspended at external pH 5.6 caused a transient decrease in cytoplasmic pH, followed by slow recovery to an elevated steady-state pH. In the presence of 150 mM KCl, however, rapid addition of another 150 mM KCl caused a transient increase in cytoplasmic pH. These transient effects may arise from secondary K(+) fluxes occurring through other transport processes in the TK2420 strain. Conclusions: Diverse osmolytes including NaCl, KCl, proline, or sucrose contribute to cytoplasmic pH homeostasis in E. coli, and increase the recovery from rapid acid shift. Osmolytes other than K(+) restore partial pH homeostasis in a strain deleted for K(+) transport.