Restoration of phosphate transport by the phosphate-binding protein in spheroplasts of Escherichia coli

Abstract

Reconstitution of phosphate transport in Escherichia coli was demonstrated. Conversion ofE. coli K10 cells to spheroplasts decreased phosphate transport to about 2%. Addition of purified phosphate-binding protein at physiological levels to these spheroplasts caused a mean 14-fold increase in phosphate transport rate. Crude shock fluid fractions were also stimulatory but not if the shock fluid was obtained from mutants lacking phosphate-binding protein. The effect of the binding protein was abolished by its specific antibody. The phosphate was shown to have entered the cell, where it became esterified. Reconstitution was not possible with cold-shocked or osmotically shocked cells. The role of osmotically shockable binding proteins in metabolite transport of Escherichia coli, and of other species as well, has often been questioned (6). Direct proof of the involvement of binding proteins in transport requires a demonstration of restoration, by the protein, of a specific transport function in cells that have lost it. Although several binding-protein preparations have been reported to stimulate specific transport systems (1, 2, 4, 13, 21), the evidence so far obtained seems to be unconvincing in some instances (6) and, in the case of previous work from this laboratory (13), cannot be repro

Co-Regulation in Escherichia coli of a Novel Transport System for sn -Glycerol-3-Phosphate and Outer Membrane Protein Ic (e, E) with Alkaline Phosphatase and Phosphate-Binding Protein

“…A mutation in phoRIc distinct from phoB turns off all of the P1 to P4 proteins, except part of the P4 band later named P4a, the phosphate-binding protein (42). Even though the phoS gene product, the phosphate-binding protein, is part of the PSTphosphate transport system (16,41), the absence of this protein in phoS mutants apparently only alters but does not abolish phosphate transport (43). In addition,phoT mutants that turn off the PST-phosphate transport system still synthesize the phosphate-binding protein (protein P4a) in large amounts (42,44).…”

Phosphate-specific transport system of Escherichia coli: nucleotide sequence and gene-polypeptide relationships

“…Garen and Otsuji (9) isolated the product of one of these genes (R2a), a protein of molecular weight about 40,000. This was eventually identified (10,21) as the periplasmic phosphate-binding protein of the Pst system and was shown to reconstitute phosphate transport in spheroplasts (11).…”

Reconstitution of binding protein dependent ribose transport in spheroplasts derived from a binding protein negative escherichia coli K12 mutant and from salmonella typhimurium

“…Lysozyme was added to maintain a concentration of 100 pg/ml throughout the spheroplasting steps. Complete (> 99%) spheroplasting was observed within 10 minutes as judged by phase contrast microscopy, and was confirmed by a tenfold dilution and plating on nutrient agar [4] . The spheroplast solution was supplemented with MgS04 to a fina!…”

“…At present, although the binding proteins are well characterized and additional components have been identified in the cytoplasmic membrane [ 8 , 9 ] , the nature of the protein interactions and ligand transfer steps remains obscure. Reconstitution of binding-protein-dependent phosphate transport in E coli spheroplasts, reported by Gerdes et a1 [4], providedthe first reliable biochemical assay for the interaction of the binding protein with its membrane receptor(s). Earlier reports of reconstitution in cells depleted of binding protein by osmotic shock proved intractable during repeated studies in other laboratories [lo, 111 .…”

“…Earlier reports of reconstitution in cells depleted of binding protein by osmotic shock proved intractable during repeated studies in other laboratories [lo, 111 . The procedure of Gerdes et a1 [4] has proven to be reproducible in the case of the reconstitution of ribose uptake in spheroplasts derived from E coli K12 [ I ] . This system is a favorable general model for binding-protein-dependent systems, since ribose-binding protein-deficient strains are devoid of other systems for ribose uptake.…”