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This report describes the first functional analysis of a bacteriocin immunity gene from Propionibacterium freudenreichii and its use as a selection marker for food-grade cloning. Cloning of the pcfI gene (previously orf5 [located as part of the pcfABC propionicin F operon]) rendered the sensitive host 1,000-fold more tolerant to the propionicin F bacteriocin. The physiochemical properties of the 127-residue large PcfI protein resemble those of membrane-bound immunity proteins from bacteriocin systems found in lactic acid bacteria. The high level of immunity conferred by pcJl allowed its use as a selection marker for plasmid transformation in P. freudenreichii. Electroporation of P. freudenreichii 1F012426 by use of the pcft expression plasmid pSL1O2 and propionicin F selection (200 bacteriocin units/ml) yielded 107 transformants/μg DNA. The 2.7-kb P. freudenreichii food-grade cloning vector pSL104 consists of the pLME1O8 replicon, a multiple cloning site, and pcJl expressed from the constitutive PpampS promoter for selection. The pSL1O4 vector efficiently facilitated cloning of the propionicin T1 bacteriocin in P. freudenreichii. High-level propionicin T1 production (640 BU/mI) was obtained with the IFO12426 strain, and the food-grade propionicin T1 expression plasmid pSL1O6 was maintained by ~91% of the cells over 25 generations in the absence of selection. To the best of our knowledge this is the first report of an efficient cloning system that facilitates the generation of food-grade recombinant P. freudenreichii strains. [ABSTRACT FROM AUTHOR]