Introduction of an active enzyme into permeable cells of Escherichia coli: acquisition of ultraviolet light resistance by uvr mutants on introduction of T4 endonuclease V.
Plasmolysed cells of Escherichia coli N212 (uvr A recA) acquired ultraviolet resistance when the cells were exposed to high concentrations of T4 endonuclease V. With increasing concentrations of T4 enzyme, survivals of plasmolysed cells after ultraviolet irradiation increased while colony-forming ability of unirradiated plasmolysed cells was not significantly affected by the enzyme treatment. Under appropriate conditions more than 200 fold increase in survivals was observed. When plasmolysed cells were treated with a pre-heated enzyme preparation or enzyme fractions derived from T4v1 (endonuclease V-deficient mutant)-infected cells, only little or no reactivation took place. Permeabilization of cells prior to the enzyme treatment was essential for the effective reactivation. Treatment of intact cells with the T4 enzyme did not cause any reactivation. Cells treated with 20 mM EGTA or 50 mM CaCl2 in cold were reactivated to certain extents by the enzyme, but the extents of the reactivation were far less compared to those of plasmolysed cells. Plasmolysed cells of strains carrying a mutation in one of uvrA, uvrB and uvrC genes were reactivated by introduction of T4 endonuclease V, as was the uvrA recA double mutant. UvrD mutants were also reactivated, but rather slightly. However, wild type strain as well as strains having a mutation in recA or polA gene were not reactivated. From these results it was suggested that T4 endonuclease V, taken up into permeable cells, can function in vivo to replace defective functions, which are controlled by the uvr genes. The conditions established in the present study may be used for introduction of other proteins into viable bacterial cells.
D002463:Cell Membrane Permeability; D003090:Coliphages / Q000201:enzymology*; D004307:Dose-Response Relationship, Radiation; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases / Q000494:pharmacology*; D004926:Escherichia coli / Q000502:physiology / Q000528:radiation effects*; D008722:Methods; D011836:Radiation Tolerance; D014466:Ultraviolet Rays
D004720:Endonucleases