Reased, but the survival rate was still lower than that of the wild type strain (Fig. 2). The addition of glutamate increased the survival of the hemA mutant, but surprisingly the addition of arginine decreased survival in the hemA mutant (Fig. 2). The reason for this decrease is still 1379592 unknown. We next constructed a double mutant deficient in both atpD and hemA. After the double mutant had been cultured overnight in LBG with 100 mg/ml ALA, the cells were transferred to the EG medium at pH 7.5 and then to pH 5.5 medium without the addition of ALA. Although the double mutant could grow in the medium at both 7.5 and 5.5 at a slower rate than that of the single mutant, the double mutant could not survive after 1 h challenge at pH 2.5. Even if glutamate or arginine was added, the survival of the double mutant was very low (less than 0.0001 , Fig. 2). These results suggest that either respiration, or the F1Fo-ATPase, is essential for survival at pH 2.5 in E. coli since both could not be eliminated simultaneously.Western Blot Analysis of ATPase Subunits in the MembranesWestern blot analysis of the membrane fraction was carried out as described previously [29,30] using rabbit antiserum against F1 part of E. coli F1Fo-ATPase which was donated by M. Futai (buy GDC-0068 School of Pharmacy, Iwate medical University, Iwate, Japan). The protein content in the membrane fraction was quantified as described below. Two mg of membrane proteins were mixed with Ganetespib web 46SDS-PAGE sample buffer (125 mM Tris?HCl, pH 6.8, 20 glycerol, 4 SDS, 10 b-mercaptoethanol, and 0.05 bromophenol blue), boiled for 90 seconds, and then applied to a 10 polyacrylamide gel containing 0.1 SDS. Proteins separated by the gel electrophoresis were transferred to a PVDF membrane at 50 volt/cm for 60?0 min. After the PVDF membrane had been incubated with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.4 mM KH2PO4, pH 7.4) containing 3 BSA for blocking, the membrane was overlaid with 1 ml of antibody diluent solution (3 mM Tris-HCl buffer containing 45 mM NaCl, 3 BSA, and 10 FBS, pH 7.6) containing 1 ml of antiserum against F1 part of E. coli F1Fo-ATPase. The membrane was washed 2 times with TBS-Tween (10 mM Tris-HCl buffer containing 150 mM NaCl and 0.1 Tween 20, pH 7.6) and overlaid with 1 ml of antibody diluent containing 5 ml of anti-rabbit antibodies conjugated with alkaline phosphatase (Biosource, USA). After the membrane was washed 2 times by TBS-Tween, staining was carried out as described previously [29.30].Respiration and F1Fo-ATPase Enhance AR in E. coliFigure 1. Proton pumping activity of the mutants and the wild type strain. W3110 (wild type, parent strain of SE mutants), DK8, SE023 (atpE), and SE020 (atpD) were grown, and proton pumping activity was measured as described in Materials and Methods. ATP (1 mM) was added at zero time. doi:10.1371/journal.pone.0052577.gATP Content of the Mutants Deficient in the F1Fo-ATPase and Heme ProteinIn order to examine whether the ATPase mutants and the respiratory chain mutant affect the ATP content, we investigated the ATP content in the mutants. The ATP content was decreased at pH 7.5 in the F1Fo-ATPase mutants, but not at pH 5.5 (Fig. 3). In contrast, the ATP content of the hemA mutant was lower than that of its parent strain at pH 5.5 (Fig. 3). These data indicated that the ATP synthetic activity of glycolysis is enough to compensate the ATP consumption at pH 5.5 but the activity of oxidative phosphorylation is not. The ATP content of th.Reased, but the survival rate was still lower than that of the wild type strain (Fig. 2). The addition of glutamate increased the survival of the hemA mutant, but surprisingly the addition of arginine decreased survival in the hemA mutant (Fig. 2). The reason for this decrease is still 1379592 unknown. We next constructed a double mutant deficient in both atpD and hemA. After the double mutant had been cultured overnight in LBG with 100 mg/ml ALA, the cells were transferred to the EG medium at pH 7.5 and then to pH 5.5 medium without the addition of ALA. Although the double mutant could grow in the medium at both 7.5 and 5.5 at a slower rate than that of the single mutant, the double mutant could not survive after 1 h challenge at pH 2.5. Even if glutamate or arginine was added, the survival of the double mutant was very low (less than 0.0001 , Fig. 2). These results suggest that either respiration, or the F1Fo-ATPase, is essential for survival at pH 2.5 in E. coli since both could not be eliminated simultaneously.Western Blot Analysis of ATPase Subunits in the MembranesWestern blot analysis of the membrane fraction was carried out as described previously [29,30] using rabbit antiserum against F1 part of E. coli F1Fo-ATPase which was donated by M. Futai (School of Pharmacy, Iwate medical University, Iwate, Japan). The protein content in the membrane fraction was quantified as described below. Two mg of membrane proteins were mixed with 46SDS-PAGE sample buffer (125 mM Tris?HCl, pH 6.8, 20 glycerol, 4 SDS, 10 b-mercaptoethanol, and 0.05 bromophenol blue), boiled for 90 seconds, and then applied to a 10 polyacrylamide gel containing 0.1 SDS. Proteins separated by the gel electrophoresis were transferred to a PVDF membrane at 50 volt/cm for 60?0 min. After the PVDF membrane had been incubated with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.4 mM KH2PO4, pH 7.4) containing 3 BSA for blocking, the membrane was overlaid with 1 ml of antibody diluent solution (3 mM Tris-HCl buffer containing 45 mM NaCl, 3 BSA, and 10 FBS, pH 7.6) containing 1 ml of antiserum against F1 part of E. coli F1Fo-ATPase. The membrane was washed 2 times with TBS-Tween (10 mM Tris-HCl buffer containing 150 mM NaCl and 0.1 Tween 20, pH 7.6) and overlaid with 1 ml of antibody diluent containing 5 ml of anti-rabbit antibodies conjugated with alkaline phosphatase (Biosource, USA). After the membrane was washed 2 times by TBS-Tween, staining was carried out as described previously [29.30].Respiration and F1Fo-ATPase Enhance AR in E. coliFigure 1. Proton pumping activity of the mutants and the wild type strain. W3110 (wild type, parent strain of SE mutants), DK8, SE023 (atpE), and SE020 (atpD) were grown, and proton pumping activity was measured as described in Materials and Methods. ATP (1 mM) was added at zero time. doi:10.1371/journal.pone.0052577.gATP Content of the Mutants Deficient in the F1Fo-ATPase and Heme ProteinIn order to examine whether the ATPase mutants and the respiratory chain mutant affect the ATP content, we investigated the ATP content in the mutants. The ATP content was decreased at pH 7.5 in the F1Fo-ATPase mutants, but not at pH 5.5 (Fig. 3). In contrast, the ATP content of the hemA mutant was lower than that of its parent strain at pH 5.5 (Fig. 3). These data indicated that the ATP synthetic activity of glycolysis is enough to compensate the ATP consumption at pH 5.5 but the activity of oxidative phosphorylation is not. The ATP content of th.
