improved protocol for electroporation of Oenococcus oeni ATCC BAA-1163 using ethanol as immediate membrane fluidizing agent

• Published in: Letters in applied microbiology Vol. 47; no. 4; pp. 333 - 338
• Main Authors: Assad-García, J.S, Bonnin-Jusserand, M, Garmyn, D, Guzzo, J, Alexandre, H, Grandvalet, C
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01.10.2008; Blackwell Publishing Ltd; Blackwell Science; Oxford University Press
• Subjects: Bacterial Proteins - genetics; Biological and medical sciences; Cell Membrane - drug effects; Cell Membrane Permeability - drug effects; DNA, Bacterial - analysis; DNA, Bacterial - genetics; electroporation; Electroporation - methods; Ethanol - pharmacology; Fundamental and applied biological sciences. Psychology; Gram-Positive Asporogenous Rods - drug effects; Gram-Positive Asporogenous Rods - genetics; Life Sciences; membrane fluidizing agent; Microbiology; Microbiology and Parasitology; Oenococcus oeni; Plasmids - genetics; Lactic acid bacteria; Ethanol; Applied microbiology; Bacteria; Electroporation; Membrane; membrane fluidizing agent; Oenococcus oeni; ELECTROPORATION; MEMBRANE FLUIDIZING AGENT; OENOCOCCUS OENI
• ISSN: 0266-8254; 1472-765X
• DOI: 10.1111/j.1472-765X.2008.02435.x

To finalize an effective and reproducible electroporation procedure to transform Oenococcus oeni ATCC BAA-1163 strain. The vector pGID052 was selected to optimize the electroporation procedure. Transformation efficiency was 5·8 x 10³ per μg of DNA. Transformation was improved when competent cells were prepared with exponential phase cultures; optimum electroporation parameters were an electric pulse of 12·5 kV cm⁻¹, under a resistance of 200 Ω and the presence of 10% (v/v) ethanol in the electroporation buffer (EPB). An effective protocol to transform O. oeni ATCC BAA-1163 strain by electroporation has been obtained by addition of ethanol to the EPB. A heterologous expression was obtained in O. oeni ATCC BAA-1163 by introducing a recombinant vector encoding a truncated form of ClpL2 protein. This is the first report of a successful electroporation of O. oeni ATCC BAA-1163. The major improvement was the addition of ethanol to the EPB, which has never been reported before as means of enhancing the incorporation of foreign DNA molecules into prokaryote cells by electroporation. This method constitutes a useful tool for the genetic study of this lactic bacterium.