Complementation of an Enterococcus hirae (Streptococcus faecalis) mutant in the alpha subunit of the H(+)-ATPase by cloned genes from the same and different species.
We isolated an Enterococcus hirae (formerly Streptococcus faecalis) mutant, designated MS117, in which 'G' at position 301 of the alpha-subunit gene of the F1F0 type of H(+)-ATPase was deleted. MS117 had low H(+)-ATPase activity, was deficient in the regulatory system of cytoplasmic pH, and was unable to grow at pH 6.0. When the alpha-subunit gene of E. hirae H(+)-ATPase was ligated with the shuttle vector pHY300PLK at the downstream region of the tet gene of the vector, it was expressed without its own promoter in MS117, and the mutation of MS117 was complemented; the mutant harbouring the plasmid had the ability to maintain a neutral cytoplasm and grew at pH 6.0. We next transformed MS117 with pHY300PLK containing the alpha-subunit gene of Bacillus megaterium F1F0-ATPase constructed in the same way. The transformant grew at pH 6.0, and the ATP hydrolysis activity was recovered. These results suggested that an active hybrid H(+)-ATPase containing the B. megaterium alpha subunit was produced, and that the hybrid enzyme regulated the enterococcal cytoplasmic pH, although the function of the B. megaterium enzyme did not include pH regulation. Thus, our present results support the previous proposal that the enterococcal cytoplasmic pH is regulated by the F1F0 type of H(+)-ATPase.
Suzuki
T
T
Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Shibata
C
C
Yamaguchi
A
A
Igarashi
K
K
Kobayashi
H
H
D001410:Bacillus megaterium / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D016983:Enterococcus / Q000235:genetics*; D013293:Enterococcus faecalis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009154:Mutation; D006180:Proton-Translocating ATPases / Q000235:genetics*; D013045:Species Specificity
D001426:Bacterial Proteins; D006180:Proton-Translocating ATPases