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Lactic acid bacteria (LAB) belonging to the genus classically known as  Lactobacillus , recently split into 25 different genera, include many relevant species for the food industry. The well-known properties of lactobacilli as probiotics make them an attractive model also for vaccines and therapeutic proteins delivery in humans. However, scarce tools are available to accomplish genetic modification of these organisms, and most are only suitable for laboratory strains. Here, we test bacterial conjugation as a new tool to introduce genetic modifications into many biotechnologically relevant laboratory and wild type lactobacilli. Using mobilizable shuttle plasmids from a donor  Escherichia coli  carrying either RP4 or R388 conjugative systems, we were able to get transconjugants to all tested  Lactocaseibacillus casei  strains, including many natural isolates, and to several other genera, including  Lentilactobacillus parabuchneri , for which no transformation protocol has been reported. Transconjugants were confirmed by the presence of the  oriT  and 16S rRNA gene sequencing. Serendipitously, we also found transconjugants into researcher-contaminant  Staphylococcus epidermidis . Conjugative DNA transfer from  E. coli  to  S. aureus  was previously described, but at very low frequencies. We have purified this recipient strain and used it in standard conjugation assays, confirming that both R388 and RP4 conjugative systems mediate mobilization of plasmids into  S. epidermidis . This protocol could be assayed to introduce DNA into other Gram-positive microorganisms which are resistant to transformation.

Conjugative DNA Transfer From E. coli to Transformation-Resistant Lactobacilli