Identification and overexpression of ltnl, a novel gene which confers immunity to the two-component lantibiotic lacticin 3147

Production and immunity of the two-component lantibiotic lacticin 3147 is e ncoded 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, ha s been subcloned in Lactococcus lactis subsp. cremoris MG1363 and has been shown to confer both lacticin 3147 production and immunity, Further subclon ing revealed that the smaller of the two clusters (ItnRIFE) confers immunit y, Although the ItnF and E genes are homologous to ABC transporters which c onfer 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 ge ne, Itnl, encodes a 116 amino acid protein with a predicted membrane locati on which bears no homology to other bacteriocin immunity proteins. Confirma tion of its role in immunity was obtained when it was observed that disrupt ion of Itnl resulted in a complete loss of immunity, When Itnl was cloned i nto the expression vector pMG36e, the resulting construct conferred levels of immunity comparable to pMRCO1, This confirmed that under the control of a strong promoter, the Itnl gene product alone is sufficient to confer lact icin immunity. In addition, heterologous expression of Itnl was observed in Enterococcus faecalis OG1X, On cloning Itnl behind a nisin-inducible promo ter, it was observed that the level of immunity was dependent on nisin conc entration, Using this construct, the authors have demonstrated a potential role for Itnl as food-grade selectable marker, Thus, Ltnl appears to repres ent a new class of lantibiotic immunity proteins.