Escherichia coli membranes during electrotransformation: an electron microscopy study.

Structural changes undergone by Escherichia coli cell envelope membranes under the conditions of electrically induced gene (DNA) transfer (exponential pulse of about 13 kV/cm, tau = 5 ms) were studied by freeze-fracture electron microscopy. Special device similar to that of Stenger and Hui [1986) J. Membr. Biol. 93, 43-53), that allowed cryofixation of samples almost simultaneously with application of electric pulse, was employed to examine the cells within a short time (less than or equal to 1 s) after the pulse. Extensive blebbing of cells was observed immediately after the pulse. At later times (30-40 s after the pulse) blebbing was not detected, instead infrequent cellular membrane fusion and formation of large membrane 'opening' or pores were observed. An attempt to relate the observed membrane changes with cellular viability and permeability to exogenous DNA failed. Challenge of cells with a plasmid DNA 10 s after the pulse application resulted in a dramatic loss (at least four orders of magnitude) of the number of transformants compared to cells pulsed in the presence of DNA. On the other hand the results on additional pulsing of cell prior to the main electrotransformation procedure suggested that the life-time of membrane defects is at least no less than 2 min. Possible ways to reconcile the results are suggested.

Electro-stimulated transformation of E. coli cells pretreated by EDTA solution.

The phenomenon of transformation of E. coli cells under electric treatment has been studied. The cells of strains MH 1, HB 101 and DH 1 after EDTA treatment in an isotonic medium were transformed with DNA pBR322 by applying a single exponential pulse (E = 10 kV/cm, T = 1.5 ms) to the suspension. The maximum transformation efficiency obtained was 4 X 10(6) colonies/micrograms DNA. The maximum transformation frequency was 0.4% at a DNA concentration of 15 micrograms/ml.