The Role of Energy Coupling in the Transport of β-Galactosides by Escherichia coli

Abstract Lactose and o-nitrophenylgalactoside (NPG) were transported against considerable concentration gradients by ML 308-225, a mutant of Escherichia coli which lacks β-galactosidase but possesses a constitutive transport system for β-galactosides. The kinetics of influx of NPG during this accumulation were found to be the same as those found for this galactoside moving down a concentration gradient into ML 308, i.e. transport into the parent cell containing β-galactosidase. When cells of ML 308-225 were exposed to azide, azide plus iodoacetate, or dinitrophenol transport of these galactosides against a concentration gradient was completely abolished. However, the presence of functional membrane carriers in such inhibited cells was indicated by (a) very rapid equilibration of external and internal concentrations of galactoside, (b) inhibition of the rate of this equilibration by chemical analogues, and (c) transientaccumulation of substrate by washed cells preloaded with galactoside. All the evidence was consistent with the hypothesis that the same membrane carriers were involved in active transport by control cells and facilitated diffusion by poisoned cells. The most striking finding was that the addition of metabolic inhibitors reduced the Kt of exit about two orders of magnitude, whereas the Kt of entrance remained constant. It was inferred from these studies that energy coupling reduced the affinity of the carrier for its substrate on the inner surface of the plasma membrane. The possible physiological control of energy coupling of transport was discussed in the light of the present observations.