Transformation of Acinetobacter calcoaceticus RAG-1 by electroporation

A method is described for transforming Acinetobacter calcoaceticus RAG-1 with a transposon-delivery plasmid, pLOFPttKm, and the broad-host-range cloning vector pKT230. Kanamycin-resistant transformants isolated following electroporation with pLOFPttKm were shown to have lost the plasmid, and contained the transposon mini-Tn10PttKm in at least five unique, single sites. Transformation with pKT230 was confirmed by plasmid screening and acquisition of the kanamycin-resistant, streptomycin-resistant phenotype. By optimizing conditions of electric field strength and pulse length for electroporation, we obtained a maximum efficiency of 4.36 × 102/(μg DNA∙109 cells) for insertion of the mini-Tn10PttKm transposon, and 9.88 × 104/(μg DNA∙109 cells) for transformation with plasmid pKT230. Our results indicate that electroporation is a suitable method for introducing transposon and cloning vectors into A. calcoaceticus RAG-1, and possibly other Acinetobacter spp. The availability of a transformation system should enhance the molecular genetic and physiological analysis of these bacteria, particularly the vast majority of strains that are not competent for transformation with DNA.