A Plasmid-Encoded Surface Polysaccharide Partly Blocks Ceduovirus Infection in Lactococci

Abstract

Bacteriophages (or phages) remain the leading cause of failure in dairy fermentations. Thereby, phage-resistant Lactococcus lactis and Lactococcus cremoris dairy starters arein continuous demand. In this work, our goal was to identify phage defense mechanismsagainst ceduoviruses encoded by two wild isolates of dairy origin named L. lactis IPLA517and IPLA1064. These strains were previously subjected to experimental evolution to selectderivatives that are resistant to the bacteriocin Lcn972. It was observed that the Lcn972Rderivatives became sensitive to phage infection; however, the underlying mechanism wasnot defned. The long-read sequencing technologies applied in this work reveal that all ofthe Lcn972R derivatives shared the loss of a 41 kb endogenous plasmid (p41) that harbors aputative exopolysaccharide (EPS) gene cluster with signifcant homology to one describedin Lactococcus garvieae. Using a CRISPR-Cas9-based approach, p41 was selectively curedfrom L. lactis IPLA1064. Phage infection assays with three ceduoviruses demonstrated thatcuring p41 restored phage sensitivity at levels comparable to the Lcn972R-IPLA1064 derivatives. Phage adsorption to ∆p41 cells was also increased, consistent with the hypothesis ofEPS production hindering access to the phage receptor protein Pip. Our results reinforcethe role of EPSs in protecting Lactococcus against phage infection, a phenomenon that israrely reported for ceduoviruses. Moreover, the results also exemplify the likely horizontalgene transfer that can occur between L. lactis and L. garvieae in a dairy environment.