MH1, A 2ND-SITE REVERTANT OF ALL ESCHERICHIA-COLI MUTANT LACKING NA+/H+ ANTIPORTERS (DELTA-NHAA-DELTA-NHAB), REGAINS NA+ RESISTANCE AND A CAPACITY TO EXCRETE NA+ IN A DELTA-MU(H)+-INDEPENDENT FASHION

The Escherichia coli mutant Delta nhaA Delta nhaB (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+ (less than or equal to 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+ (less than or equal to 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 Delta nhaA Delta nhaB cells and exposes a Nac-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 Delta nhaA Delta nhaB 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 (greater than or equal to 300 mar). Whereas Na+ excretion by a wild type carrying Delta unc is uncoupler sensitive, that of MH1 Delta unc is dependent on respiration in an uncoupler-insensitive fashion. It is concluded that under some conditions (high HC 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.