Cloning and disruption of a putative NaH-antiporter gene of Enterococcus hirae.
When growing in a sodium-rich environment, wild-type Enterococcus hirae extrudes sodium by two mechanisms, ATP-driven sodium extrusion, and NaH-antiport. Mutant 7683 is unable to grow on sodium-rich media. This is due to two mutations, one inactivating ATP-driven sodium transport and a second rendering NaH-antiport inoperative. 7683 was transformed by electroporation with a gene bank, derived from E. hirae, in an Escherichia coli-E. hirae shuttle vector. Transformants which had regained the ability to grow on sodium-rich media were selected for and the transforming plasmids analyzed. A gene able to restore NaH-antiport activity in 7683 was identified. This gene was named napA. It codes for an extremely hydrophobic protein of 383 amino acids. Hydropathy analysis of this protein indicates that it probably forms 12 transmembraneous helices. In a mutant, possessing only the NaH-antiporter, the napA gene was disrupted by homologous recombination. The resultant strain failed to grow in sodium-rich media, and vesicles isolated from these cells exhibited a defect in sodium proton antiport activity. We conclude that the napA gene codes for a NaH-antiporter. The NapA protein does not exhibit significant homology to any protein in the EMBL genetic data bank.
Waser
M
M
Department of Clinical Pharmacology, University of Berne, Switzerland.
Hess-Bienz
D
D
Davies
K
K
Solioz
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D013293:Enterococcus faecalis / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; D012964:Sodium