FIG. 3.

GadC is the antiporter for glutamate-dependent acid resistance. (A) Western blot assay. Cells were grown to stationary phase in minimal EG medium, and extracts were prepared with a French press. Crude extracts were separated into soluble and insoluble membrane pellets by ultracentrifugation. Samples of each fraction were electrophoresed through a 10% polyacrylamide gel electrophoresis gel and probed with anti-GadC antibody. (B) GadC is required for intact cells to convert glutamic acid to GABA. Cells were grown in LBG to stationary phase, washed in minimal EG pH 7.0 medium, and resuspended in EG at different pH values. Radiolabeled glutamic acid was added to suspensions of intact cells placed at pH 2.5 and to suspensions of cells permeabilized with 0.1% Triton X at pH 4.4, the reported internal pH of acid-challenged cells. Substrate (striped bars) and product (solid bars) present in filtered supernatants were separated by paper chromatography and identified by staining with 0.3% ninhydrin. WT, EK227; gadC, EF962; gadAB, EF522.