A method for genetic transformation of nonprotoplasted Streptococcus lactis

Abstract

Plasmid transformation of whole cells of Streptococcus lactis LM0230 was demonstrated. The procedure required polyethylene glycol and incubation in hypertonic media, but did not require enzymatic cell wall digestion. Conditions were optimized, yielding 5 x 105 transformants per ,ug of pSA3 DNA. Variables tested for effect on transformation efficiency included molecular weight, concentration, and pH of polyethylene glycol; cell density; plating media; DNA concentration; heat shock; and incubation of cells in hypertonic buffer. DNAs transformed included pSA3, pVA856, pTV1, and c2(. Transformation from DNA-DNA ligation mixes, with DNA not purified through density gradients, and with previously frozen cells was also achieved. The method described here for transformation of nonprotoplasted cells of LM0230 is unique, and to date has not been applied successfully to other lactic acid bacteria. * Corresponding author. were obtained for intact, cesium chloride gradient-purified pSA3 plasmid DNA into S. lactis LM0230. This paper describes the first report of a whole-cell plasmid transformation system for lactic acid bacteria.

High-Frequency Transformation, by Electroporation, of Lactococcus lactis subsp. cremoris Grown with Glycine in Osmotically Stabilized Media

“…It is likely that the present method to obtain competent cells for electrotransformation is applicable to other grampositive organisms. Incubation in sucrose itself is known to cause a weakening of the cell wall in several bacteria (11,14), and this may explain the finding that in some strains, glycine tolerance was not enhanced by the presence of sucrose during growth. In most strains, however, this effect of sucrose did not appear to significantly reduce the recovery rate of transformants.…”

In vivo genetic systems in lactic acid bacteria

“…It has been developed into a sophisticated genetic tool for use in B. subtilis where Tn917 based transposition can be used not only to mutate genes but also to generate transcriptional fusions to lacZ or cat86 reporter genes thereby facilitating the study of gene expression [86][87][88][89]. The considerable potential of this transposon for use in lactic acid bacteria has yet to be realized although the integration of Tn917 into different sites within the chromosomes of L. lactis [90] and Lb. plantarum [91] was achieved by direct transformation with vector pTV1.…”

Evidence for the conjugal transfer of the broad host range plasmid pIP501 into strains of Lactobacillus helveticus

“…In this respect there has been much recent. progress towards the development of a transformation system and vectors for lactic streptococci (Simon et al 1986;Sanders & Nicholson 1987;Woskow & Kondo 1987;de Vos 1987). In contrast, the bacteria of the genus Lactobacillus appear less amenable to genetic manipulation.…”