Production and immunity of the two-component lantibiotic lacticin 3147 is encoded by the 60-2 kb lactococcal plasmid pMRC01. A 12.6 kb region of this plasmid, containing ten genes in two divergently arranged gene clusters, has been subcloned in Lactococcus lactis subsp. cremoris MG1363 and has been shown to confer both lacticin 3147 production and immunity. Further subcloning revealed that the smaller of the two clusters (ItnRIFE) confers immunity. Although the ItnF and E genes are homologous to ABC transporters which confer immunity to other lantibiotics, deletion analysis indicates that they do not play a role in the immunity exhibited by this subclone in L. lactis subsp. cremoris MG1363. Also, a deletion in ItnR (which resembles a family of transcriptional repressors) had no effect on immunity. The remaining gene, Itnl, encodes a 116 amino acid protein with a predicted membrane location which bears no homology to other bacteriocin immunity proteins. Confirmation of its role in immunity was obtained when it was observed that disruption of Itnl resulted in a complete loss of immunity. When Itnl was cloned into the expression vector pMG36e, the resulting construct conferred levels of immunity comparable to pMRC01. This confirmed that under the control of a strong promoter, the Itnl gene product alone is sufficient to confer lacticin immunity. In addition, heterologous expression of Itnl was observed in Enterococcus faecalis OG1X. On cloning Itnl behind a nisin-inducible promoter, it was observed that the level of immunity was dependent on nisin concentration. Using this construct, the authors have demonstrated a potential role for Itnl as food-grade selectable marker. Thus, Ltnl appears to represent a new class of lantibiotic immunity proteins.