Ion mixture (1 ml), 5 ml of 200 mM ATP was added. The fluorescence intensity from ACMA was measured with excitation and emission wavelengths of 410 nm and 490 nm, respectively.Results Enzyme Activities in the F1Fo-ATPase MutantsOxidative phosphorylation is mediated by the respiratory 1379592 chain and the F1Fo-ATPase in E. coli [33]. E. coli F1Fo-ATPase Ebselen site consists of two parts, F1 and Fo, which contain five subunits (a, b, c, d, and e) and three subunits (a, b, and c), respectively [34,35]. We used mutants deficient in atpD (SE020) and atpE (SE023) in this study. atpD and atpE encode the b and c subunits, respectively [36]. We also used DK8 [16] in which all genes for the F1Fo-ATPase were deleted. Since the optimum pH of the ATP hydrolysis activity was 9.0 [24], the ATP hydrolysis activity was measured at pH 9.0. The activity was 0.5260.17 mmol Pi/min/mg protein in the wild type, while the activity was not detected (less than 0.01 mmol Pi/min/ mg protein) in any of the F1Fo-ATPase mutants at pH 9.0. The proton pumping activity was impaired in these mutants (Fig. 1). The wild type strain grew in the E medium containing lactic acid instead of glucose at pH 5.5, but none of the F1Fo-ATPase mutants grew under this condition, indicating that the oxidative phosphorylation was still active at pH 5.5 in the wild type strain but not in the F1Fo-ATPase mutants. These results suggested that the F1Fo-ATPase activity was negligible in these mutants.Measurement of the Membrane Permeability to ProtonsThe membrane permeability to protons was measured as described previously [2,28] with the following modifications. The E. coli cells cultured overnight in LBG medium were diluted 1000fold with EG medium of pH 7.5 and then were grown at 37uC until OD600 reached 0.3,0.4. The cells were resuspended in the same volume of EG medium at pH 5.5, and cultured for 4 h at 37uC for the acidic adaptation. The adapted cells were collected by centrifugation at 10,0006g for 5 min at room temperature, washed with H2O and resuspended in 3 ml H2O containing 20 mM MgCl2 at 56109 cells per ml. The measurement was carried out at 25uC with stirring. After 20 ml of 0.2 M HCl had been added, the change of pH was measured with a pH meter connected to a computer. The membrane permeability to protons was represented as one pH unit change per min per mg protein [25]. The measurement was repeated three times using separate culture, and the mean value and the standard deviation were calculated.The AR of Mutants Deficient in a Gene for the F1FoATPase or Heme SynthesisThe stationary-phase cells may be resistant to various stresses [4]. To minimize the responses to stresses other than acidic stress, cells growing logarithmically were used in the present study. 15826876 F1FoATPase mutants SE020, SE023 and DK8 CAL-120 showed AR of E. coli decreased about 20-fold compared with that of the wild type W3110 after the cells were challenged at pH 2.5 for 1 h (Fig. 2), and none of these mutants survived after 2 h challenge at pH 2.5 (data not shown). The mutant deficient in hemA encoding glutamyl tRNA reductase (SE022) had a low ability to survive at pH 2.5. Glutamyl tRNA reductase is the enzyme that synthesizes glutamate 1-semialdehyde in E. coli [37,38]. Delta-aminolevulinic acid (ALA) is a precursor of heme biosynthesis and is synthesized from glutamate 1-semialdehyde. Therefore, ALA was added to produce heme in the hemA mutant as indicated. When glutamate or arginine was added, the survival of the ATPase mutants was inc.Ion mixture (1 ml), 5 ml of 200 mM ATP was added. The fluorescence intensity from ACMA was measured with excitation and emission wavelengths of 410 nm and 490 nm, respectively.Results Enzyme Activities in the F1Fo-ATPase MutantsOxidative phosphorylation is mediated by the respiratory 1379592 chain and the F1Fo-ATPase in E. coli [33]. E. coli F1Fo-ATPase consists of two parts, F1 and Fo, which contain five subunits (a, b, c, d, and e) and three subunits (a, b, and c), respectively [34,35]. We used mutants deficient in atpD (SE020) and atpE (SE023) in this study. atpD and atpE encode the b and c subunits, respectively [36]. We also used DK8 [16] in which all genes for the F1Fo-ATPase were deleted. Since the optimum pH of the ATP hydrolysis activity was 9.0 [24], the ATP hydrolysis activity was measured at pH 9.0. The activity was 0.5260.17 mmol Pi/min/mg protein in the wild type, while the activity was not detected (less than 0.01 mmol Pi/min/ mg protein) in any of the F1Fo-ATPase mutants at pH 9.0. The proton pumping activity was impaired in these mutants (Fig. 1). The wild type strain grew in the E medium containing lactic acid instead of glucose at pH 5.5, but none of the F1Fo-ATPase mutants grew under this condition, indicating that the oxidative phosphorylation was still active at pH 5.5 in the wild type strain but not in the F1Fo-ATPase mutants. These results suggested that the F1Fo-ATPase activity was negligible in these mutants.Measurement of the Membrane Permeability to ProtonsThe membrane permeability to protons was measured as described previously [2,28] with the following modifications. The E. coli cells cultured overnight in LBG medium were diluted 1000fold with EG medium of pH 7.5 and then were grown at 37uC until OD600 reached 0.3,0.4. The cells were resuspended in the same volume of EG medium at pH 5.5, and cultured for 4 h at 37uC for the acidic adaptation. The adapted cells were collected by centrifugation at 10,0006g for 5 min at room temperature, washed with H2O and resuspended in 3 ml H2O containing 20 mM MgCl2 at 56109 cells per ml. The measurement was carried out at 25uC with stirring. After 20 ml of 0.2 M HCl had been added, the change of pH was measured with a pH meter connected to a computer. The membrane permeability to protons was represented as one pH unit change per min per mg protein [25]. The measurement was repeated three times using separate culture, and the mean value and the standard deviation were calculated.The AR of Mutants Deficient in a Gene for the F1FoATPase or Heme SynthesisThe stationary-phase cells may be resistant to various stresses [4]. To minimize the responses to stresses other than acidic stress, cells growing logarithmically were used in the present study. 15826876 F1FoATPase mutants SE020, SE023 and DK8 showed AR of E. coli decreased about 20-fold compared with that of the wild type W3110 after the cells were challenged at pH 2.5 for 1 h (Fig. 2), and none of these mutants survived after 2 h challenge at pH 2.5 (data not shown). The mutant deficient in hemA encoding glutamyl tRNA reductase (SE022) had a low ability to survive at pH 2.5. Glutamyl tRNA reductase is the enzyme that synthesizes glutamate 1-semialdehyde in E. coli [37,38]. Delta-aminolevulinic acid (ALA) is a precursor of heme biosynthesis and is synthesized from glutamate 1-semialdehyde. Therefore, ALA was added to produce heme in the hemA mutant as indicated. When glutamate or arginine was added, the survival of the ATPase mutants was inc.
