TRANSPORT OF SUGARS VIA 2 ANOMER-SPECIFIC SITES ON MANNOSE PHOSPHOTRANSFERASE SYSTEM IN LACTOCOCCUS-CREMORIS - IN-VIVO STUDY OF MECHANISM, KINETICS, AND ADAPTATION

Glucose uptake in Lactococcus lactis subsp. cremoris FD1 occurs via th e mannose phosphotransferase system (Man-PTS), which is quite unspecif ic and allows transport of many different sugars and sugar analogues. It was previously shown (Benthin, S., Nielsen, J., Villadsen, J. Biote chnol. Bioeng. 40:137-146, 1992) that the kinetics of in vivo glucose uptake in a glucose-limited chemostat culture is best described by ass uming that the glucose transport system has two anomer-specific sites with a relative uptake rate of 36% through the alpha-site. In the pres ent study, the existence of anomer-specific sites on Man-PTS is shown by experiments where alpha-glucose, beta-glucose, mannose, and 2-deoxy glucose are added to glucose-limited chemostat cultures. A quantitativ e description of the competitive uptake of the involved sugars at the two sites is given. In a mannose-limited chemostat culture, the relati ve glucose flux via the alpha-site is 50%, corresponding to a change t oward 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 alpha-site co rresponds to constitutive synthesis of Man-PTS with 36% alpha-site sto ichiometry in new cells. When N-acetylglucosamine (73% alpha-anomer at equilibrium) is the limiting substrate, the relative glucose flux thr ough the alpha-site is also 48% to 50%. With a feed of alpha-glucose g enerated enzymatically from nonmetabolizable sucrose the relative gluc ose flux through the alpha-site can be as high as 78%. Finally, growth in the presence of nonmetabolizable alpha-methylglucoside leads to fo rmation of cells with a relative glucose flux through the alpha-site o f 29% to 30%. The adaptation of the flux distribution between the alph a- and beta-site is tentatively explained by the hypothesis that two i ntegral membrane proteins of Man-PTS are involved in this process. (C) 1993 John Wiley & Sons, Inc.