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Previously reported techniques for the electrotransfer of foreign DNA into pediococci yield only a small number of transformants/mug DNA, especially when using undomesticated strains. This study reports an improved protocol for the electrotransformation of pediococci, based on trials using Pediococcus acidilactici P60 and the plasmid pRS4C1. The improved protocol yields from 2 to 3 log units more transformants than the previously reported methods, with up to (9.1+/-1.3)x10(4) transformants/mug of foreign DNA under the best conditions identified. The most important modifications proposed are an increase in electric field strength during electroporation (from 12.5 to 20kV/cm) and a reduction in lysozyme concentration during the preparation of electrocompetent cells (from 4000 to 2000U/ml): together, these two modifications greatly improve transformant yield. In addition, increasing cell culture time (from OD(600nm)=0.6 to OD(600nm)=1.0-1.2) and increasing dl-threonine concentration in the growth medium (from 20 to 40mM) also contribute to improved electrotransformation efficiency.

An improved method for the electrotransformation of Lactobacillus plantarum CECT 220 (ATCC 8014) with plasmid DNA isolated from Escherichia coli is described. The two main modifications with respect to existing methods are: (i) isolation of plasmid DNA from E. coli JM110 grown in minimal medium and (ii) in vitro modification of the DNA by cell-free extracts of the host L. plantarum. Optimal electrotransformation was obtained with exponentially growing cells of L. plantarum concentrated to 6x10(9) cfu ml-1, with electric pulses of 13 kV cm-1 in cuvettes with 1 mm inter-electrode distance. We consider that this method constitutes a useful tool for routine manipulation of L. plantarum, and can probably be extended to other lactic acid bacteria.

Abstract: An improved method for the electrotransformation of Lactobacillus plantarum CECT 220 (ATCC 8014) with plasmid DNA isolated from Escherichia coli is described. The two main modifications with respect to existing methods are: (i) isolation of plasmid DNA from E. coli JM110 grown in minimal medium and (ii) in vitro modification of the DNA by cell-free extracts of the host L. plantarum. Optimal electrotransformation was obtained with exponentially growing cells of L. plantarum concentrated to 6×109 cfuml−1, with electric pulses of 13 kVcm−1 in cuvettes with 1 mm inter-electrode distance. We consider that this method constitutes a useful tool for routine manipulation of L. plantarum, and can probably be extended to other lactic acid bacteria. [Copyright &y& Elsevier]

Two strains of Oenococcus oeni, RS1 (which carries the plasmid pRS1) and RS2 (which carries the plasmids pRS2 and pRS3), were grown in the presence of different curing agents and at different temperatures. Sublethal temperature together with acriflavine generated all possible types of cured strains, i.e., lacking pRS1 (from strain RS1), and lacking pRS2, pRS3, or both (from strain RS2). Sublethal temperature together with acridine orange only generated cured strains lacking pRS3. These results suggest that acriflavine is a better curing agent than acridine orange for O. oeni, and that pRS3 is the most sensitive to these curing agents. We also observed spontaneous loss of pRS2 or both pRS2 and pRS3 by electroporation. The ability to cure O. oeni strains of plasmids provides a critical new tool for the genetic analysis and engineering of this commercially important bacterium. [Copyright &y& Elsevier]