Identification of endogenous genetic elements in Oenococcus oeni for the development of novel molecular tools

Oenococcus oeni is a lactic acid bacterium associated to the wine-microbiota. It is considered the main actor of the malolactic fermentation in wine, contributing to the complexity of the final product. Besides its contribution to the winemaking process, this species is able to produce interesting aromas and functional compounds (i.e., melatonin). Due to the lack of efficient tools for its genetic manipulation, the molecular mechanisms underlying its capability to survive in the oenological environment and to develop compounds of interest are still poorly investigated. Several studies suggest that O. oeni genetic variability is associated to genetic elements for horizontal transfer, both natural plasmids and oenophages, which are known to contain adaptive advantage traits. For this reason, the aim of this work is the identification of genetic elements for the construction of new molecular tools exploitable in genetic functional studies and potential future biotechnological applications. Seventy-one O. oeni strains (isolated from Valtellina, Aosta Valley and Valpolicella wines or wineries) were investigated for the presence of these genetic elements. Three natural plasmids (pOeA, pOeB and pOeC) showing a high percentage of similarity with the well-known O. oeni pOENI-1 and pOENI-1v2 plasmids were isolated and sequenced. By combining the amplification of five target genes (tauE, oye, oye_parB, repAlys), nine strains were found to putatively contain phages. In particular, the genetic analysis of the four groups of integrases (A, B, C or D) showed that four strains could belong to the group A, one to the group C, while one responded positively both to group A and C. Finally, phylogenetic analyses using the lys gene sequences were performed to evaluate the evolutionary relationship of the putative phages. In conclusion, the selected strains and identified genetic elements represent a valuable resource to shed light in the O. oeni genetics and metabolism.