AN IMPROVED METHOD FOR THE TRANSFORMATION OF LACTOBACILLUS STRAINS USING ELECTROPORATION

Because of their widespread industrial and medical importance, there i s considerable interest in the manipulation and improvement of Lactoba cillus strains using modern genetic engineering techniques. However, m ost reports have focused on industrial strains and often have resulted in non-reproducible transformation efficiencies. We have developed an optimised protocol for electroporating foreign plasmid DNA into clini cal strains of lactobacilli. Treatment of the recipient lactobacilli w ith either lysozyme, glycine or penicillin improved electrotransformat ion efficiencies up to 480-fold. A critical step in achieving efficien t and reproducible electrotransformation of clinical lactobacilli with the plasmid pSA3 was the requirement for a post-pulse recovery time o f 2-3 h, combined with the use of sub-inhibitory concentrations of ant ibiotics in the selective plates. While pNZ17 transformants also benef ited from a post-pulse recovery period, good transformation efficienci es could be achieved when plated directly onto selective concentration s of chloramphenicol. We also observed significant differences in elec trotransformation efficiencies between our guinea pig vaginal Lactobac illus isolates (maximum of 4.8 x 10(4) transformants/mu g pNZ17 DNA) a nd the human L. casei strain ATCC 393 (3.7 x 10(6) transformants/mu g pNZ17 DNA). An optimised procedure for the electroporation of plasmid DNA into lactobacilli is described.