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

Abstract

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.

Interspecific reconstitution of maltose transport and chemotaxis in Escherichia coli with maltose-binding protein from various enteric bacteria

“…The present experiments extend the application of the reconstitution procedure with calcium-permeabilized cells (5,6) in one significant way. Until now, proteins introduced into the periplasm by this technique have always been rather acidic, and hence negatively charged at the pH (7.0) used for reconstitution (6). MBP from E. aerogenes and S. marcescens, however, is basic ( Fig.…”

Characterization of the Salmonella typhimurium mgl operon and its gene products

“…2). Strains LA5440 (mglBSSO) and LA6221 (mglBSOI), which carry missense mutations in mglB (7,8), were complemented to mgl+ by plasmid pHG15 by using the criterion of restoration of wild-type transport activity (data not shown). This result indicated that GBP from S. typhimurium could replace E. coli GBP and functionally interacts with membrane components of the E. coli mgl transport system.…”

Reconstitution of maltose chemotaxis in Escherichia coli by addition of maltose-binding protein to calcium-treated cells of maltose regulon mutants

“…30 mM). This concentration of MBP gives good transport rates when maltose transport is reconstituted (11). The suspension was shaken at 0°C for 30 min, and the cells were then gently washed with 1 ml of 0.9% NaCl at room temperature.…”

“…We have recently developed a technique whereby soluble substrate-binding proteins can be introduced into the periplasmic space after Ca2+induced permeabilization of the outer membrane of gramnegative bacteria. Import and function of these proteins can be demonstrated by the reconstitution of transport in mutants lacking binding protein (10,11).…”