Transport of sugars via two anomer‐specific sites on mannose–phosphotransferase system in Lactococcus cremoris: In vivo study of mechanism, kinetics, and adaptation

Abstract

Glucose uptake in Lactococcus lactis subsp. cremoris FD1 occurs via the mannose phosphotransferase system (Man–PTS), which is quite unspecific and allows transport of many different sugars and sugar analogues. It was previously shown (Benthin, S., Nielsen, J., Villadsen, J. Biotechnol. Bioeng. 40:137–146, 1992) that the kinetics of in vivo glucose uptake in a glucose-limited chemostat culture is best described by assuming that the glucose transport system has two anomer-specific sites with a relative uptake rate of 36% through the α-site. In the present study, the existence of anomer-specific sites on Man–PTS is shown by experiments where α-glucose, β-glucose, mannose, and 2-deoxyglucose are added to glucose-limited chemostat cultures. A quantitative description of the competitive uptake of the involved sugars at the two sites is given. In a mannose-limited chemostat culture, the relative glucose flux via the α-site is 50%, corresponding to a change toward the equilibrium composition of mannose (68%). Furthermore, when the feed to a mannose-limited chemostat culture is changed to glucose, the rate of change of relative glucose flux through the α-site corresponds to constitutive synthesis of Man–PTS with 36% α-site stoichiometry in new cells. When N-acetylglucosamine (73% α-anomer at equilibrium) is the limiting substrate, the relative glucose flux through the α-site is also 48% to 50%. With a feed of α-glucose generated enzymatically from nonmetabolizable sucrose the relative glucose flux through the α-site can be as high as 78%. Finally, growth in the presence of nonmetabolizable α-methylglucoside leads to formation of cells with a relative glucose flux through the α-site of 29% to 30%. The adaptation of the flux distribution between the α- and β-site is tentatively explained by the hypothesis that two integral membrane proteins of Man–PTS are involved in this process. © 1993 John Wiley & Sons, Inc.