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

Robb, Frank T.; Furlong, Clement E.

doi:10.1002/jss.400130206

Cited by 9 publications

(2 citation statements)

References 14 publications

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“…Under these conditions, even proteins can be imported into the periplasm, as demonstrated by the reconstitution of maltose transport in the nonpolar deletion strain HS3018 (AmalE maW) by the addi-tion of maltose-binding protein (MBP). Previously, the reconstitution of binding protein-dependent transport had only been achieved by the addition of binding protein to spheroplasts or isolated membrane vesicles of mutant strains that were free of this protein (4,15,16,20,28). Reconstitution experiments with whole cells, which are much easier and highly reproducible, reflect a more natural situation.…”

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confidence: 99%

Reconstitution of maltose transport in Escherichia coli: conditions affecting import of maltose-binding protein into the periplasm of calcium-treated cells

Brass¹,

Ehmann²,

Bukau³

1983

J Bacteriol

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The reconstitution of active transport by the Ca2+-induced import of exogenous binding protein was studied in detail in whole cells of a malE deletion mutant lacking the periplasmic maltose-binding protein. A linear increase in reconstitution efficiency was observed by increasing the Ca2+concentration in the reconstitution mixture up to 400 mM. A sharp pH optimum around pH 7.5 was measured for reconstitution. Reconstitution efficiency was highest at 0°C and decreased sharply with increasing temperature. The time necessary for optimal reconstitution at 0°C and 250 mM Ca was about 1 min. The competence for reconstitution was highest in exponentially growing cultures with cell densities up to 1 x 109/ml and declined when the cells entered the stationary-growth phase. The apparent Km for maltose uptake was the same as that of wild-type cells (1 to 2 puM). Vmax at saturating maltose-binding protein concentration was 125 pmol per min per 7.5 x 107 cells (30% of the wild-type activity). The concentration of maltose-binding protein required for half-maximal reconstitution was about 1 mM. The reconstitution procedure appears to be generally applicable. Thus, galactose transport in Escherichia coli could also be reconstituted by its respective binding protein. Maltose transport in E. coli was restored by maltose-binding protein isolated from Salmonella typhimurium. Finally, in S. typhimurium, histidine transport was reconstituted by the addition of shock fluid containing histidinebinding protein to a hisJ deletion mutant lacking histidine-binding protein. The method is fast and general enough to be used as a screening procedure to distinguish between transport mutants in which only the binding protein is affected and those in which additional transport components are affected.

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confidence: 99%

Reconstitution of maltose transport in Escherichia coli: conditions affecting import of maltose-binding protein into the periplasm of calcium-treated cells

Brass¹,

Ehmann²,

Bukau³

1983

J Bacteriol

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“…Restoration of transport by binding protein was dependent upon the induction of the ribose transport system, but it did not require the metabolizing of ribose since reconstitution could be achieved with a strain that was constitutive for the transport system but lacked ribokinase. In recent work with this system, it was shown that binding protein dependent transport could be restored in spheroplasts from a mutant strain which is defective in the ribose binding protein but normal in the other component(s) of the ribose transport system (Robb et al, 1980).…”

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confidence: 99%

Reconstitution of binding protein dependent active transport of glutamine in spheroplasts of Escherichia coli

Masters¹,

Hong²

1981

Biochemistry

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In order to directly prove that the periplasmic glutamine binding protein is an essential component of the osmotic shock sensitive active transport system for glutamine in Escherichia coli, we demonstrated the reconstitution of binding protein dependent glutamine transport in spheroplasts of that organism. It was shown by arsenate inhibition that the reconstituted transport system was energy dependent, and the use of azaserine, an inhibitor of glutamine-utilizing enzymes, indicated that the restoration of transport by binding protein did not require the metabolizing of the transport substrate. Furthermore, the binding protein dependent transport of glutamine was shown to require at least one other macromolecular component, presumably membrane bound, which was absent in a strain containing a deletion of the genes coding for the glutamine transport system but was present in a strain carrying a mutation only in the structural gene for the glutamine binding protein.

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Bacterial Amino Acid Transport Systems

Landick

Oxender

Ames

1985

The Enzymes of Biological Membranes

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

Cited by 9 publications

References 14 publications

Reconstitution of maltose transport in Escherichia coli: conditions affecting import of maltose-binding protein into the periplasm of calcium-treated cells

Reconstitution of maltose transport in Escherichia coli: conditions affecting import of maltose-binding protein into the periplasm of calcium-treated cells

Reconstitution of binding protein dependent active transport of glutamine in spheroplasts of Escherichia coli

Bacterial Amino Acid Transport Systems

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