Development of Conjugal Systems for Gene Transfer and Allelic Exchange in

Oenococcus oeni (formerly Leuconostoc oenos) is a common starter culture used to achieve the malolactic fermentation (MLF) in commercial wine production. To date, basic research on O. oeni, especially molecular analysis of genes involved in growth on grape juice, has been hampered by the lack of efficient gene transfer and allelic exchange systems. One potential mechanism for genetic manipulation of starter cultures is the use of conjugal systems indigenous to lactic acid bacteria (LAB). Previous work on the conjugal element pRSOl from the dairy starter Lactococcus lactis identified several regions involved in conjugative transfer and localized the conjugative origin of transfer (oriT). In an effort to develop a generally applicable system for gene transfer, we examined the host range of pRSOl, and several oriT-containing plasmid derivatives, with a range of LAB recipients. Conjugal transfer was demonstrated into Leuconostoc mesenteriodes subsp. cremoris, Enterococcus faecalis, Streptococcus thermophilus, Pediococcus cerevisiae and 0. oeni. Broad host range transfer of pRSOl and mobilization of oriT-containing plasmid derivatives provides a novel means for molecular analysis of a variety of LAB, such as O. oeni, that are resistant to current methods of genetic manipulation. Current work is focused on the development of the appropriate mobilization and allelic exchange vectors for genetic analysis of O. oeni.