Application of conjugation to produce genetically modified lactobacilli

Abstract

Lactic acid bacteria play a crucial role in the production of dairy food products, as well as in human and animal health as part of the microbiota. In particular, lactobacilli strains are well-known for their properties as probiotics and hence, they constitute an ideal candidate for therapeutic molecules delivery in humans. Despite their potential in the industry and biomedical applications, the genetic modification of this group is challenging due to their low transformation efficiency and complex diversity, especially in wild type strains. Recently, bacterial conjugation from Escherichia coli to lactobacilli strains has been successfully performed, opening a wide range of new possibilities; however, conjugation frequencies obtained were low. In this work we have tried to optimize this conjugation protocol to lactobacilli strains. First, several parameters of the protocol were modified such as the growth phases and the ratios between donors and recipients. During these experiments we have not seen an increase of the conjugation frequency; in fact, a decrease was observed under some conditions. We have also studied the effect of DNA methylation in bacterial conjugation frequencies using as donor an E. coli strain with a mutation in the DNA-cytosine methyltransferase system, in order to avoid restriction systems in the recipient. No increase in the transfer frequency of unmethylated DNA was observed. Finally, we have designed two different plasmid systems in order to combine bacterial conjugation with homologous recombination techniques to perform specific gene modifications in the recipient cell.