Plasmids with Homology to pFX3 and pCI3340 in Starter Lactococci and Lactobacilli and Electroporation of Selected Strains with these Vectors

Abstract

An important consideration in the genetic manipulation of lactic acid bacteria is whether or not strains retain their normal industrial properties after transformation with plasmid DNA. A survey of plasmid types in various lactic acid bacteria showed that pE194-type rolling circle (RCR) plasmids were present in 24% of 70 commercial Lactococcus lactis cheese starters but not in any of 38 strains representing nine species of Lactobacillus. By contrast, theta plasmids showing homology to the pCI305 rep gene were present in most (though not all) lactococcal strains and not in any of the lactobacilli. Plasmids not belonging to either group were also present in some Lc. lactis and Lactobacillus strains. Six selected Lc. lactis subsp. cremoris strains were transformable with pFX3 (a 4.5 kb pE194-type rolling circle vector), yet four of these strains had significantly lower or zero transformation frequencies with pCI3340 (a 5.7 kb pCI305-based theta vector), possibly due to plasmid incompatibility. However, all transformants maintained full milkcoagulating activity. In addition to Lc. lactis, strains of Lc. garviae, Enterococcus faecalis and Lactobacillus reuteri were able to be transformed with pCI3340, making this a potentially useful vector for these species.