MH1, a second-site revertant of an Escherichia coli mutant lacking Na+/H+ antiporters (ΔnhaAΔnhaB), regains Na+ resistance and a capacity to excrete Na+ in a Δμ̃H+-independent fashion

Abstract

The Escherichia coli mutant ΔnhaAΔnhaB (EP432), which lacks the two specific Na+/H+ antiporter genes, is incapable of efficiently excreting Na+. Accordingly at low K+ (6 mM) medium, its intracellular Na+ concentration is only slightly lower (1.5-2x) than the extracellular concentration (50 mM), explaining the high sensitivity to Na+ (≥30 mM) of the mutant. This Na+ sensitivity is shown to be a powerful selection for spontaneous second-site suppressor mutations that allow growth on high Na+ (≤0.6 M) with a rate similar to that of the wild type. One such mutation, MH1, maps at 25.7 min on the E. coli chromosome. It confers Na+ but not Li+ resistance upon ΔnhaAΔnhaB cells and exposes a Na+-excreting capacity, maintaining a Na+ gradient of about 8-10 (at 50 mM extracellular Na+), which is similar to that of the wild type. Although lower, Na+ excretion capacity is also observed in the ΔnhaAΔnhaB mutant when grown in medium containing higher K+ (70 mM). This capacity is accompanied with a shift in the sensitivity of the mutant to higher Na+ concentrations (≥300 mM). Whereas Na+ excretion by a wild type carrying Δunc is uncoupler sensitive, that of MH1Δunc is dependent on respiration in an uncoupler-insensitive fashion. It is concluded that under some conditions (high K+ in the medium or in MH1-like mutants), a primary pump driven by respiration is responsible for Na+ extrusion when the Na+/H+ antiporters are not active.