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首页> 外文期刊>Journal of bacteriology >Ca2+-induced permeabilization of the Escherichia coli outer membrane: comparison of transformation and reconstitution of binding-protein-dependent transport.
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Ca2+-induced permeabilization of the Escherichia coli outer membrane: comparison of transformation and reconstitution of binding-protein-dependent transport.

机译：Ca2 +诱导大肠杆菌外膜的渗透性：转化和结合蛋白依赖性转化的比较。

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Ca2+ treatment renders the outer membrane of Escherichia coli reversibly permeable for macromolecules. We investigated whether Ca2+-induced uptake of exogenous protein into the periplasm occurs by mechanisms similar to Ca2+-induced uptake of DNA into the cytoplasm during transformation. Protein import through the outer membrane was monitored by measuring reconstitution of maltose transport after the addition of shock fluid containing maltose-binding protein. DNA import through the outer and inner membrane was measured by determining the efficiency of transformation with plasmid DNA. Both processes were stimulated by increasing Ca2+ concentrations up to 400 mM. Plasmolysis was essential for a high efficiency; reconstitution and transformation could be stimulated 5- and 40-fold, respectively, by a high concentration of sucrose (400 mM) in cells incubated with a suboptimal Ca2+ concentration (50 mM). The same divalent cations that promote import of DNA (Ca2+, Ba2+, Sr2+, Mg2+, and Ni2+) also induced import of protein. Ca2+ alone was found to be inefficient in promoting reconstitution; successive treatment with phosphate and Ca2+ ions was essential. Transformation also was observed in the absence of phosphate, but could be stimulated by pretreatment with phosphate. The optimal phosphate concentrations were 100 mM and 1 to 10 mM for reconstitution and transformation, respectively. Heat shock, in which the cells are rapidly transferred from 0 to 42 degrees C, affected the two processes differently. Incubation of cells at 0 degrees C in Ca2+ alone allows rapid entry of protein, but not of DNA. Transformation was observed only when exogenous DNA was still present during the heat shock. Shock fluid containing maltose-binding protein inhibited transformation (with 6 microgram of DNA per ml, half-maximal inhibition occurred at around 300 microgram of shock fluid per ml). DNA inhibited reconstitution (with 5 microgram of shock fluid per ml, half-maximal inhibition occurred at around 3 mg of DNA per ml).
机译：Ca2 +治疗使大肠杆菌的外膜可逆地渗透到大分子。我们研究了Ca2 +诱导外源蛋白的摄取到周质中是否通过类似于Ca2 +诱导的DNA摄取到转化期间的细胞质中的机制发生。通过在加入含有麦芽糖结合蛋白的休克流体后测量麦芽糖传输的重构来监测通过外膜的蛋白质进口蛋白质。通过用质粒DNA的转化效率测量通过外膜和内膜的DNA进口。通过增加高达400mm的Ca 2 +浓度来刺激两种方法。质子溶解对于高效率至关重要;通过与次优Ca2 +浓度（50mM）温育的细胞中，可以分别通过高浓度的蔗糖（400mm）来刺激5-和40倍的重构和转化。促进DNA进口（CA2 +，BA2 +，SR2 +，MG2 +和Ni2 +）的相同二价阳离子也诱导了蛋白质的进口。仅发现CA2 +在促进重建方面是效率低下;磷酸盐和Ca2 +离子的连续处理是必需的。在没有磷酸盐的情况下也观察到转化，但可以通过用磷酸盐进行预处理来刺激。重构和转化分别最佳磷酸盐浓度为100mm，1至10mm。热冲击，其中细胞从0到42℃迅速转移，不同地影响了两种过程。在Ca2 +中在0℃下孵育细胞允许快速进入蛋白质，但不是DNA。仅当在热休克期间仍存在外源DNA时，才观察到转化。含有麦芽糖结合蛋白的休克液抑制转化（每mL 6微克DNA，每mL的约300微克休克液发生半最大抑制）。 DNA抑制重构（每mL的5微克休克液，半最大抑制在每mL的约3mg DNA）上发生。

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