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Escherichia coli membranes during electrotransformation: an electron microscopy study
A G Sabelnikov1, E S Cymbalyuk, G Gongadze
1All Union Research Institute of Protein Biosynthesis, Moscow, U.S.S.R.
Biochimica Et Biophysica Acta
|July 1, 1991
Summary
Electrically induced gene transfer in Escherichia coli causes immediate cell membrane blebbing and later pore formation. However, introducing DNA after pulsing significantly reduces transformation efficiency, suggesting transient membrane defects.
Area of Science:
- Microbiology
- Cell Biology
- Biophysics
Background:
- Electroporation is a key method for gene transfer in bacteria.
- Understanding the dynamic structural changes in cell membranes during electroporation is crucial for optimizing transformation protocols.
Purpose of the Study:
- To investigate the structural alterations of Escherichia coli cell envelope membranes following electric pulse application.
- To correlate observed membrane changes with cellular viability and DNA permeability.
Main Methods:
- Utilized freeze-fracture electron microscopy to visualize membrane structures.
- Employed a specialized cryofixation device for near-simultaneous sample fixation after electric pulsing.
- Assessed bacterial transformation efficiency with plasmid DNA at various time points post-pulsing.
Main Results:
- Immediate electric pulsing induced extensive cell membrane blebbing.
- Later observations (30-40s post-pulse) revealed membrane fusion and pore formation.
- DNA introduction 10s after pulsing drastically reduced transformant numbers (by >10^4-fold).
- Additional pulsing suggested membrane defect longevity of at least 2 minutes.
Conclusions:
- Electric pulses induce transient, dynamic structural changes in the E. coli cell envelope.
- The timing of DNA introduction relative to the electric pulse is critical for successful electrotransformation.
- Observed membrane alterations do not directly explain the significant reduction in transformation efficiency when DNA is added post-pulse.