Evidence for the Involvement of Proton Motive Force in the Transport of Glucose by a Mutant of Streptococcus mutans Strain DR0001 Defective in Glucose-Phosphoenolpyruvate Phosphotransferase Activity

Abstract

Streptococcus mutans DR0001 and a glucose-phosphotransferase (PTS)-defective mutant, DR0001/6, were grown anaerobically in a chemostat with a glucose limitation at dilution rates ( D ) of 0.04 to 0.6 h −1 (mean generation time, 17 to 1.2 h). The mutant possessed only 15% of glucose-PTS activity of the wild type and gave cell yields (19%) less than those of the wild type. Glucose-PTS activity in strains DR0001 was maximum at D = 0.1 h −1 and was adequate to account for transport in the chemostat at all dilution rates except D = 0.6 h −1 , at which it was 80% of the actual glucose uptake activity. The mutant DR0001/6, on the other hand, possessed only sufficient glucose-PTS activity to sustain growth at below D = 0.1 h −1 , indicating the presence of an alternate transport activity. This was confirmed in glycolytic rate experiments with washed cells, which demonstrated that the mutant showed rates 11- to 27-fold higher than that accountable via glucose-PTS activity alone. The wild-type organism contained both a high ( K s , 6.7 to 8.0 μM)- and a low ( K s , 57 to 125 μM)-affinity transport system, whereas the glucose-PTS-defective mutant contained only the low-affinity system ( K s , 62 to 133 μM). The glucose-PTS was shown to be the high-affinity system. Glucose uptake by the mutant was unaffected by 8 mM sodium arsenate, 10 mM azide, and 10 mM dinitrophenol but was completely inhibited by 0.05 mM sodium iodoacetate. Glycolysis in the organism was almost completely inhibited by 0.25 mM N ′, N ′ -dicyclohexylcarbodiimide (DCCD), indicating the involvement of an ATPase in glucose uptake. The ionophores carbonylcyanide- m -chlorophenylhydrazone and tetrachlorosali-cylanilide were inhibitory at concentrations of 10 μM, suggesting that a proton gradient was important in the transport process. Higher levels of DCCD and the ionophores were required to inhibit the wild-type organism to the same degree. A mechanism is proposed for the alternative transport system whereby proton motive force is created by the extrusion of protons by the DCCD-sensitive ATPase and glucose is transported down a proton gradient in a symport with protons.