A POINT MUTATION (G338S) AND ITS SUPPRESSOR MUTATIONS AFFECT BOTH THEPH RESPONSE OF THE NHAA-NA+ H+ ANTIPORTER AS WELL AS THE GROWTH PHENOTYPE OF ESCHERICHIA-COLI/

pH controls the activity of the NhaA Na+/H+ antiporter of Escherichia coli. In the present work we show that replacement of glycine 338 of N haA with serine (G338S) alleviates the pH control of the antiporter. M onitoring Na+-dependent collapse of Delta pH, to assess antiporter act ivity in isolated membrane vesicles, shows that the mutant protein is practically independent of pH, between pH 7 and 9, and even at pH 6 is 70% active. Similarly the purified reconstituted mutant protein catal yzes pH-independent passive efflux of Na-22 from proteoliposomes as we ll as Delta pH-driven influx. Whereas the native NhaA in isolated memb rane vesicles is exposed to digestion by trypsin only above pH 7, the mutated protein is degraded already at pH 6.5. Delta nhaA Delta nhaB c ells transformed with a plasmid encoding the pH-independent antiporter are sensitive to Na+ but not to K+ at alkaline pH, while growing in t he presence of both ions at neutral pH. Several possibilities that cou ld explain the Na+ sensitivity of the mutant at alkaline pH were exclu ded; Western analysis and measurement of Na+/H+ antiporter activity in membrane vesicles, isolated from cells shifted to the non-permissive growth conditions, showed neither reduced expression of G338S-NhaA nor defective activity. The finding that the mutated protein is electroge nic led to the retraction of the idea that the protein is active in vi tro but not in vivo at alkaline pH, when only Delta psi exists in the cells. The Na+ concentration needed for half-maximal activity of G338S in isolated everted membrane vesicles is similar to that of the wild type. Therefore an increase in intracellular Na+ due to a reduced anti porter affinity could not explain the results. It is suggested that th e loss of growth. at alkaline pH in the presence of Na+ is due to the loss of the pH control of the mutated NhaA, Indeed, in the four mutati ons suppressing G338S phenotype, growth at alkaline pH was restored to gether with the pH regulation of NhaA, Three of the four suppressor mu tations cluster in helix IV,whereas the original mutation is in helix XI, suggesting that the two helixes interact.