Functional expression of the Enterococcus hirae NaH-antiporter in Escherichia coli.
We recently described the cloning of napA, the putative structural gene 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 coli. When placed under the control of a T7 promoter, napA could be transcribed and labeled specifically with [35S]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 was tested by expressing it from its own promoter 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 complemented 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 fluorescence measurements on vesicles energized with lactate. The NaH-antiporter expressed by napA exhibited a Km 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 Km, and approached zero at pH 9. These results demonstrate that napA is the structural gene for the NaH-antiporter of E. hirae. NapA exhibits properties different from those of the two E. coli NaH-antiporters encoded by nhaA and nhaB, yet functionally complements a defect in these genes.
Strausak
D
D
Department of Clinical Pharmacology, University of Berne, Switzerland.
Waser
M
M
Solioz
M
M
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D016983:Enterococcus / Q000254:growth & development / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics*
D017923:Sodium-Hydrogen Exchangers