Cloning and Sequencing of the Genes Encoding Vacuolar-type Na^+-ATPase in Enterococcus hirae

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1. A 1000-bp fragment of Enterococcus hirae genomic DNA was amplified by polymerase chain reaction, using the oligonucleotide primers designed from amino acid sequences of both amino-terminal and a tryptic fragment of the Na^+-ATPase A subunit in this organism. DNA sequencing of this product revealed that the amino acid sequence of this subunit is highly homologous to the corresponding sequences of large (a) subunits of vacuolar(archaebacterial) type H^+-ATPases.2. The eubacterium Enterococcus hirae ATCC 9790 possesses a H^+-translocating ATPase, and the deduced amino acid sequences of the genes coding for this enzyme have indicated that it is a typical F_0F_1-type ATPase. We cloned the ntpA and ntpB genes coding for the A and B subunits, respectively, of Na^+-ATPase from the same bacterium, and the full amino acid sequences of the two subunits were deduced from the nucleotide sequence. The A (593 amino acid residues) and B (458 amino acid residues) subunits were highly homologous (48-60 % identical) to the A (large or a) and the B (small or b) subunits, respectively, of various vacuolar-type H^+-ATPases which have been found in eukaryotic endomembrane systems and archaebacterial cell membranes. The A and B subunits of Na^+-ATPase showed about 23-28 % identities with the b and a subunits of E. hirae F_1-ATPase and of Escherichia coli F_1-ATPase, respectively. These results indicate that E. hirae Na^+-ATPase belongs to the vacuolar-type ATPase. This is the first demonstration that both genes for V- and F-type ATPases are functionally expressed in one bacterial cell.