FIG. 4.

The gyrA751 mutant is unable to transport succinate. Succinate uptake was measured with radiolabeled [2,3-¹⁴C]succinate. Cultures were grown in minimal no-carbon E medium supplemented with 1 mM MgSO₄, 24 mM glycerol, and 16 mM fumarate. Cells were harvested in mid-exponential phase and washed twice in 3 ml no-carbon E buffer. Succinate transport assays were performed at 37°C and contained the following: 100 mM potassium phosphate (KP) buffer (pH 7.4), 100 mM MgSO₄, 0.2 mM succinate (2.4 nCi/nmol), and approximately 7 × 10⁷ CFU in a total volume of 0.520 ml. Aliquots of 0.1 ml were taken every 15 seconds after the addition of succinate, applied to a 25-mm, 0.4-μm-pore-size nitrocellulose membrane, and washed twice in rapid succession with 3 ml KP · MgSO₄ buffer over a vacuum manifold. Filters were dried, and radioactivity retained on filters was measured by standard methods. Representative transport levels for DM8306 [gyrA751 ompC396::Tn10d(Tc)] (▵), DM8307 [gyrA⁺ ompC396::Tn10d(Tc)] (♦), and FM2 (dct-71) (16) (▪) in a representative experiment are shown.