FUNCTIONAL EXPRESSION OF THE ENTEROCOCCUS-HIRAE NAH-ANTIPORTER IN ESCHERICHIA-COLI

We recently described the cloning of napA, the putative structural gen e for the NaH-antiporter of Enterococcus hirae (Waser, M., Bienz-Hess, D., Davies, K., and Solioz, M. (1992) J. Biol. Chem 267, 5396-5400). To analyze the gene product of napA, we expressed it in Escherichia co li. When placed under the control of a T7 promotor, napA could be tran scribed and labeled specifically with [S-35]methionine. The resultant gene product exhibited an apparent M(r) of 3,4 x 10(4) when subjected to sodium dodecyl sulfate-gel electrophoresis. The function of NapA wa s tested by expressing it from its own promotor in the E. coli mutant EP432. This mutant lacks both of the endemic NaH-antiporters, NhaA and NhaB; its growth is thus very sensitive to Na+ and Li+ and membranes derived from this strain do not exhibit NaH-antiport activity. When co mplemented with napA, EP432 gained tolerance to Na+ or Li+. Membranes prepared from the complemented mutant exhibited NaH-antiport activity. The properties of this activity were determined by acridine fluoresce nce measurements on vesicles energized with lactate. The NaH-antiporte r expressed by napA exhibited a K(m) of 1 mM for Na+ and 0.1 mM for Li + at pH 7.5. At pH 8.5, the relative rate of NaH-antiport activity was 50%, with little change in the K(m), and approached zero at pH 9. The se results demonstrate that napA is the structural gene for the NaH-an tiporter of E. hirae. NapA exhibits properties different from those of the two E. coli NaH-antiporters encoded by nhaA and nhaB, yet functio nally complements a defect in these genes.