REGULATION OF BACTERIAL SUGAR H-DEPENDENT ENZYME-I HPR-MEDIATED PHOSPHORYLATION( SYMPORT BY PHOSPHOENOLPYRUVATE)

The lactose-H+ symport protein (LacS) of Streptococcus thermophilus ha s a C-terminal hydrophilic domain that is homologous to IIA protein(s) domains of the phosphoenolpyruvate:sugar phosphotransferase system (P TS). C-terminal truncation mutants were constructed and expressed in E scherichia coli and their properties were analyzed. Remarkably, the en tire IIA domain (160 amino acids) could be deleted without significant effect on lactose-H+ symport and galactoside equilibrium exchange. An alysis of the LacS mutants in S. thermophilus cells suggested that tra nsport is affected by PTS-mediated phosphorylation of the IIA domain. For further studies, membrane vesicles of S. thermophilus were fused w ith cytochrome c oxidase-containing liposomes, and, when appropriate, phosphoenolpyruvate (PEP) plus purified enzyme I and heat-stable prote in HPr were incorporated into the hybrid membranes. Generation of a pr otonmotive force (Delta p) in the hybrid membranes resulted in accumul ation of lactose, whereas uptake of the PTS sugar sucrose was not obse rved. With PEP and the energy-coupling proteins enzyme I and HPr of th e PTS on the inside, high rates of sucrose uptake were observed, where as Delta-driven lactose uptake by wild-type LacS was inhibited. This i nhibition was not observed with LacS(Delta 160) and LacS(H552R), indic ating that PEP-dependent enzyme I/HPr-mediated phosphorylation of the IIA domain (possibly the conserved His-552 residue) modulates lactose- H+ symport activity.