Sugar transport by the bacterial phosphotransferase system. Preparation and characterization of membrane vesicles from mutant and wild type Salmonella typhimurium.

Modifications of published procedures (reviewed by Kaback, H. R. (1974) Science (Wash. D. C.) 186, 882-892) were developed for preparing membrane vesicles from Salmonella typhimurium. The preparations consisted largely of closed, unilamellar structures and contained inner membrane with little to no contamination by outer membrane or cell wall. A variety of cytoplasmic proteins was assayed in the membrane preparations, and they were found to be present at low to trace levels, whereas other proteins known to be associated with membranes were found at high levels (with respect to specific activities) in the vesicle preparations. At least 90% of the vesicles appeared to be oriented right-side-out; we do not know whether the remaining 10% represents closed vesicles oriented inside-out or "leaky" right-side-out vesicles. The vesicle preparations were impermeable to both low and high molecular weight solutes, for example, to both intra- and extravesicular sucrose. In double label experiments, the vesicle volumes were found to be about 6 microliters/mg of protein for preparations isolated from the wild type strain, and about 4.5 microliters/mg of protein for vesicles isolated from a mutant, SB2950, deleted in ptsH, ptsI, and crr genes (proteins HPr, Enzyme I, and IIIGlc, respectively). One advantage of S. typhimurium over Escherichia coli for these studies is that the former can be induced to take up phosphoenolpyruvate. This may be the reason that S. typhimurium vesicles transported methyl alpha-glucoside at 4- to 100-fold the rates reported for vesicles from E. coli, while uptake rates of proline were comparable in the two types of preparations. Vesicles from strain SB2950 were unable to take up methyl alpha-glucoside, but the transport (and phosphorylating) system was reconstituted in the vesicles by trapping the soluble purified proteins inside the vesicles during preparation of the latter. All three proteins were required for reconstruction. Studies with intra- and extravesicular soluble proteins of the phosphoenolpyruvate:glucose phosphotransferase system showed that the IIMan complex, which phosphorylates glucose, 2-deoxyglucose, and other sugars, is symmetrically oriented in the membranes. That is, this complex could phosphorylate 2-deoxyglucose when supplemented with Enzyme I and HPr either inside or outside of the membranes, and the sugar phosphate was found on the same side of the membranes as the soluble phosphotransferase system proteins. The integral membrane protein, II-BGlc, which phosphorylates glucose and methyl alpha-glucoside, showed contrasting behavior. Methyl alpha-glucoside phosphate was formed (intravesicularly) only when the soluble proteins (Enzyme I, HPr, and IIIGlc) were located inside the vesicles. Thus, II-BGlc appears to be asymmetrically oriented in the membranes.