Role of membrane potential on artificial transformation of E. coli with plasmid DNA.
The standard method of transformation of Escherichea coli with plasmid DNA involves two important steps: cells are first suspended in 100mM CaCl(2) at 0 degrees C (in which DNA is added), followed by the administration of a heat-pulse from 0 to 42 degrees C for 90s [Cohen, S., Chang, A., Hsu, L., 1972. Nonchromosomal antibiotic resistance in bacteria. Proc. Natl. Acad. Sci. U.S.A., 69, 2110-2114]. The first step makes the cells competent for uptake of DNA and the second step is believed to facilitate the DNA entry into the cells by an unknown mechanism. In this study, the measure of membrane potential of the intact competent cells, at different steps of transformation process, either by the method of spectrofluorimetry or that of flow cytometry, indicates that the heat-pulse step (0-->42 degrees C) heavily decreases the membrane potential. A subsequent cold shock (42-->0 degrees C) raises the potential further to its original value. Moreover, the efficiency of transformation of E. coli XL1 Blue cells with plasmid pUC19 DNA remains unaltered when the heat-pulse step is replaced by the incubation of the DNA-adsorbed competent cells with 10 microM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for 90s at 0 degrees C. Since the CCCP, a well-known protonophore, reduces membrane potential by dissipating the proton-motive-force (PMF) across E. coli plasma membrane, our experimental results suggest that the heat-pulse step of the standard transformation procedure facilitates DNA entry into the cells by lowering the membrane potential.
Panja
Subrata
S
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741 235, West Bengal, India.
Saha
Swati
S
Jana
Bimal
B
Basu
Tarakdas
T
D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology*; D004247:DNA / Q000493:pharmacokinetics*; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology; D005434:Flow Cytometry; D006361:Heating; D008564:Membrane Potentials / Q000502:physiology*; D010957:Plasmids / Q000235:genetics* / Q000502:physiology; D013050:Spectrometry, Fluorescence; D014169:Transformation, Bacterial / Q000502:physiology*
D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D004247:DNA