Electrotransformation of Streptococcus pneumoniae: evidence for restriction of DNA on entry

Laboratoire de Microbiologie et Génétique Moléculaire du CNRS and Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex, France 2 Author for correspondence: J. Lefrançois. Tel: +33 61 33 59 71. Fax: +33 61 33 58 86. e-mail: Jacques@IBCG.biotoul.fr ABSTRACT Electrotransformation is a method generally used in biotechnology to introduce recombinant DNA into a wide range of bacteria. However, the mechanism of DNA entry is poorly understood. We report that in Streptococcus pneumoniae, a naturally transformable species, electrotransformation efficiently introduces a plasmid replicon. DNA is strongly restricted by the restriction-modification systems Dpnl and Dpnll which degrade methylated and non-methylated DNA, respectively, at GATC sequences. This suggests that in electrotransformation double-stranded DNA penetrates into these bacteria without a single-stranded DNA step in contrast to natural transformation. Single-stranded DNA by Itself is able to electrotransform very weakly and linearized double-stranded plasmid DNA yields barely detectable levels of transformants. Keywords: Streptococcus pneumoniae , electrotransformation, restriction, DNA uptake