Characterization of semi-uncoupled hybrid Escherichia coli-Bacillus megaterium F1F0 proton-translocating ATPases.
Cloned atp genes for the proton-translocating ATPase of the obligate aerobe Bacillus megaterium have been demonstrated to be capable of complementing Escherichia coli ATPase (unc) mutants (Hawthorne, C. A., and Brusilow, W. S. A. (1986) J. Biol. Chem. 261, 5245-5248). To determine the minimum subunit requirements for cross-species complementation, we constructed all combinations of B. megaterium atpA, G, D, and C genes (coding for the alpha, gamma, beta, and epsilon subunits, respectively) and tested their abilities to complement two uncA (alpha subunit) and two uncD (beta subunit) mutants of E. coli. The results indicated that complementation of either uncD mutant required atpD (beta) only. Complementation of one of the uncA (alpha) mutants required atpA, G, and D (alpha, gamma, and beta) and possibly atpE (epsilon) as well. The other uncA mutant was not complemented by any combination of B. megaterium ATPase genes. Complementation of a beta mutant by atpD (beta) or atpD and C (beta epsilon) produced cells which could grow aerobically on a nonfermentable carbon source (succinate) but not anaerobically on rich medium containing glucose. These E. coli therefore had become obligate aerobes. The ability to grow anaerobically could be restored to the mutant complemented by atpD alone by growth at pH 7.5 or pH 8 in the presence of 0.1 M potassium.
Scarpetta
M A
MA
Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201.
Hawthorne
C A
CA
Brusilow
W S
WS
D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005816:Genetic Complementation Test; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D006180:Proton-Translocating ATPases