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首页> 外文期刊>Journal of bacteriology >Reconstitution of maltose transport in Escherichia coli: conditions affecting import of maltose-binding protein into the periplasm of calcium-treated cells.
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Reconstitution of maltose transport in Escherichia coli: conditions affecting import of maltose-binding protein into the periplasm of calcium-treated cells.

机译：大肠杆菌中麦芽糖运输的重构：影响麦芽糖结合蛋白导入钙处理细胞周质的条件。

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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 degree C and decreased sharply with increasing temperature. The time necessary for optimal reconstitution at 0 degree C and 250 mM Ca2+ was about 1 min. The competence for reconstitution was highest in exponentially growing cultures with cell densities up to 1 X 10(9)/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 microM). Vmax at saturating maltose-binding protein concentration was 125 pmol per min per 7.5 X 10(7) 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 histidine-binding 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.
机译：在缺少周质麦芽糖结合蛋白的malE缺失突变体的全细胞中，详细研究了Ca2 +诱导的外源结合蛋白的导入对主动转运的重构。通过将重构混合物中的Ca2 +-浓度增加至400 mM，可以观察到重构效率的线性增加。测量到在pH 7.5附近的最适pH值以进行重构。重构效率在0摄氏度时最高，并且随着温度的升高而急剧下降。在0摄氏度和250 mM Ca2 +下最佳重构所需的时间约为1分钟。在细胞密度高达1 X 10（9）/ ml的指数增长培养物中，重构的能力最高，而当细胞进入静止生长阶段时，重构能力则下降。麦芽糖摄取的表观Km与野生型细胞的表观Km（1至2 microM）相同。饱和麦芽糖结合蛋白浓度下的Vmax为每7.5 X 10（7）细胞每分钟125 pmol（野生型活性的30％）。半最大重构所需的麦芽糖结合蛋白的浓度为约1mM。重建程序似乎普遍适用。因此，在大肠杆菌中的半乳糖运输也可以通过其各自的结合蛋白来重构。通过从鼠伤寒沙门氏菌中分离的麦芽糖结合蛋白恢复了大肠杆菌中的麦芽糖运输。最后，在鼠伤寒沙门氏菌中，通过向缺乏组氨酸结合蛋白的hisJ缺失突变体中添加含有组氨酸结合蛋白的休克液来重建组氨酸转运。该方法快速且通用，足以用作筛选程序，以区分仅影响结合蛋白的转运突变体和影响其他转运组分的转运突变体。

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