Examination of glucose transport in Clostridium beijerinckii NCIMB 8052 and the solvent hyper-producing mutant BA101

Glucose uptake and accumulation by Clostridium beijerinckii BA101, a butanol hyper-producing mutant was examined during various stages of growth. Glucose uptake in C. beijerinckii BA101 was repressed 20% by 2-deoxyglucose (2-DG) and 25% by mannose, while glucose uptake in C. beijerinckii 8052 was repressed 52% and 28% by these sugars, respectively. We confirmed the presence of a phosphoenolpyruvate (PEP) dependent phosphotransferase system (PTS) associated with cell-free extracts of C. beijerinckii BA101 by glucose phosphorylation by PEP. The PTS activity associated with C. beijerinckii BA101 was 50% of that observed for C. beijerinckii 8052. C. beijerinckii BA101 also demonstrated lower PTS activity for fructose and glucitol. Glucose phosphorylation by cell-free extracts derived from both C. beijerinckii BA101 and 8052 was also dependent on the presence of ATP, consistent with the presence of glucokinase activity in C. beijerinckii extracts. ATP-dependent glucose phosphorylation was predominant during the solventogenic stage when PEP-dependent glucose phosphorylation was dramatically repressed. A nearly two-fold greater ATP-dependent phosphorylation rate was observed for solventogenic stage C. beijerinckii BA101 than for solventogenic stage C. beijerinckii 8052. These results suggest that C. beijerinckii BA101 is defective in PTS activity and that C. beijerinckii BA101 compensates for this defect with enhanced glucokinase activity resulting in an ability to transport and utilize glucose during the solventogenic stage. Glucose uptake by both acidogenic and solventogenic stage C. beijerinckii BA 101 was inhibited by proton conductors, ATPase inhibitors and non-PTS substrates in contrast with what was observed for acidogenic stage C. beijerinckii 8052. However, it was observed that these compounds were also potent inhibitors of glucose uptake by solventogenic C. beijerinckii 8052. ATP-dependent glucose phosphorylation was dramatically induced during solventogenic stage when PEP-dependent glucose phosphorylation was decreased. Approximately a two-fold greater ATP-dependent phosphorylation rate was observed during all growth stages for C. beijerinckii BA101 versus C. beijerinckii 8052. These results suggest that an alternative glucose transport mechanism is present in C. beijerinckii other than PTS, which is predominant during the solventogenic stage. The alternative transport mechanism together with enhanced glucokinase activity may allow C. beijerinckii BA101 to more completely utilize glucose in spite of a defective PTS being associated with this strain.