Letters in Applied Microbiology 1997, 24, 33–36 An efficient transformation system for Bifidobacterium spp. M. Rossi, P. Brigidi and D. Matteuzzi Department of Pharmaceutical Sciences, Interdepartmental Center for Biotechnology, University of Bologna, Bologna, Italy 1161/96: received 15 May 1996 and accepted 6 June 1996 M. ROSSI, P. BRIGIDI AND D. MATTEUZZI. 1997. This study describes a broad host transformation protocol that enables the uptake of plasmid DNA into 10 different species of Bifidobacterium, some of which have never been transformed before. The vector pNC7 (4·9 kb) was used to optimize the electroporation protocol. Transformation efficiencies ranged from 3·6×10 1 to 1·2×10 5 transformations per mg DNA. The impact of growth medium composition and electric field strength on transformation efficiency were independently optimized. Electrocompetent cells were grown in Iwata medium broth enriched with Actilight ® P 16%, harvested during the early exponential growth phase, and pulsed at 12·5 kV cm -1 , 100 V and 25 mF. INTRODUCTION to develop an efficient electroporation protocol for several Bifidobacterium species. Bifidobacteria constitute an important part of the normal intestinal microflora in humans and other animals. They play a health-promoting role by maintaining a correct balance between intestinal bacteria (Ibrahim and Bezkorovainy 1993), MATERIALS AND METHODS stimulation of the immune response (Lee et al. 1993), anti- carcinogenic activity (Reddy and Rivenson 1993) and pro- Bacterial strains and cultural conditions tection against virus infections (Saavedra et al. 1994). Due to All the Bifidobacterium strains used in this study (Table 1) this wide probiotic activity, bacteria of the genus Bifido- came from the collection of the Institute of Agricultural bacterium have large industrial and medical importance. The Microbiology, University of Bologna, Italy. Bifidobacteria possibility of introducing and expressing genes encoding were grown anaerobically at 37°C in Iwata medium (IM) activities of technological importance is fundamental to (Iwata and Morishita 1989). Chloramphenicol was added to improve the characteristics of some strains. One reason for the appropriate medium, when required, at a final con- difficulties encountered in developing a transformation sys- centration of 5 mg ml -1 . tem for Bifidobacterium spp. has been a shortage of genetic information available for this genus. Progress towards this goal has been hampered by the lack of the basic tools for genetic manipulation, such as stable vectors and efficient Plasmids DNA delivery systems. The B. longum plasmid pMB1 was the only one completely sequenced and information from its The transformation experiments were performed with the sequence has enabled the characterization of this replicon and plasmid pNC7 (Rossi et al. 1996), extracted from B. animalis the construction of a family of Bifidobacterium cloning vectors MB209 according to Le Blanc and Lee (1979), and purified (Rossi et al. 1996). Up to now, no conjugation or transfection by caesium chloride–ethidium bromide density gradient cen- systems have been available for this genus and only recently trifugation. pNC7 (4·9 kb), based on the replicon pMB1, was have there been two reports of successful transformation by able to replicate only in Bifidobacterium. The presence of electroporation of Bifidobacterium strains (Missich et al. 1994; pNC7 in the transformants was confirmed by electrophoresis Argnani et al. 1996). Therefore, this study was prompted in 0·9% agarose gels of isolated plasmid DNA and by Sou- thern blotting and hybridization with a digoxigenin-labelled Correspondence to: Dr M. Rossi, Department of Pharmaceutical Sciences, Inter- pNC7 probe, according to the supplier (Boehringer departmental Center for Biotechnology, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy (e-mail: E056MR01@AREA.CNR.BA.IT). Mannheim Biochimica). © 1997 The Society for Applied Bacteriology