AGROBACTERIUM-RADIOBACTER AND RELATED ORGANISMS TAKE UP FRUCTOSE VIA A BINDING-PROTEIN-DEPENDENT ACTIVE-TRANSPORT SYSTEM

Washed cells of Agrobacterium radiobacter prepared from a fructose-lim ited continuous culture (D 0.045 h(-1)) transported D-[U-C-14]fructose in a linear manner for up to 4 min at a rate several-fold higher than the rate of fructose utilization by the growing culture. D-[U-C-14]Fr uctose transport exhibited a high affinity for fructose (K-T < 1 mu M) and was inhibited to varying extents by osmotic shock, by the uncoupl ing agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and by u nlabelled sugars (D-fructose/D-mannose > D-ribose > D-sorbose > D-gluc ose/D-galactose/D-xylose; no inhibition by D-arabinose). Prolonged gro wth of A. radiobacter in fructose-limited continuous culture led to th e selection of a novel strain (AR100) which overproduced a fructose-bi nding protein (FBP) and showed an increased rate of fructose transport . FBP was purified from osmotic-shock fluid using anion-exchange fast protein liquid chromatography (FPLC). the monomeric protein (M(r) 34 2 00 by SDS-PACE and 37 700 by gel-filtration FPLC) bound D-[U-C-14]fruc tose stoichiometrically (1.17 nmol nmol FBP-1) and with high affinity (K-D 0.49 mu M) as shown by equilibrium dialysis. Binding of D-[U-C-14 ]fructose by FBP was variably inhibited by unlabelled sugars (D-fructo se/D-mannose > D-ribose > D-sorbose; no inhibition by D-glucose, D-gal actose or D-arabinose). The N-terminal amino acid sequence of FBP (ADT SVCLI-) was similar to that of several sugar-binding proteins from oth er species of bacteria. Fructose transport and FBP were variably induc ed in batch cultures of A. radiobacter by growth on different carbon s ources (D-fructose > D-ribose/D-mannose > D-glucose; no induction by s uccinate). An immunologically similar protein to FBP was produced by A grobacterium tumefaciens and various species of Rhizobium following gr owth on fructose. It is concluded that fructose is transported into A. radiobacter and related organisms via a periplasmic fructose/mannose- binding-protein-dependent active-transport system, in contrast to the phosphotransferase system used by many other species of bacteria.