Pep5 is a cationic pore-forming lantibiotic produced by Staphylococcus epidermidis strain 5. The producer strain protects itself from the lethal action of its own bacteriocin through the 69-amino-acid immunity peptide PepI. The N-terminal segment of PepI contains a 20-amino-acid stretch of apolar residues, whereas the C terminus is very hydrophilic, with a net positive charge. We used green fluorescent protein (GFP)-PepI fusions to obtain information on its localization in vivo. PepI was found to occur outside the cytoplasm and to accumulate at the membrane-cell wall interface. The extracellular localization appeared essential for conferring immunity. We analyzed the functional role of the specific segments by constructing various mutant peptides, which were also fused to GFP. When the hydrophobic N-terminal segment of PepI was disrupted by introducing charged amino acids, the export of PepI was blocked and clones expressing such mutant peptides were Pep5 sensitive. When PepI was successively shortened at the C terminus, in contrast, its export properties remained unchanged whereas its ability to confer immunity was gradually reduced. The results show that the N-terminal part is required for the transport of PepI and that the C-terminal part is important for conferring the immunity phenotype. A concept based on target shielding is proposed for the PepI immunity mechanism.Lantibiotics, a subgroup of bacteriocins from gram-positive bacteria, are polycyclic peptides containing modified amino acids: in particular, the thioether amino acids lanthionine and 3-methyllanthionine and the dehydroamino acids 2,3-didehydroalanine and 2,3-didehydrobutyrine. Lantibiotics are ribosomally synthesized as precursor peptides, consisting of a leader sequence and a propeptide part (31). The precursor peptides are converted into the mature peptide by posttranslational modification followed by processing and export from the producing cell (for a review, see reference 27). The cationic lantibiotic Pep5 is produced by Staphylococcus epidermidis strain 5 (28) and is classified along with nisin, subtilin, and epidermin as a pore-forming type-A lantibiotic (27). The biosynthetic gene cluster of Pep5 contains the structural gene pepA as well as the genes for posttranslational modification (pepB and pepC), processing (pepP), transport (pepT), and immunity (pepI) (18).Bacteriocin production generally requires a self-protection mechanism for the producer strain (1, 29). Various immunity concepts have been elaborated by the different groups of bacteria; e.g., the immunity proteins of the channel-forming colicins (e.g., colicin A, E1, and B) reside in the cytoplasmic membrane and protect the producer by forming a stoichiometric complex with the respective colicin (10, 32, 36). For the lantibiotic bacteriocins of gram-positive bacteria generally two different mechanisms, which in some cases may even complement each other, are found: the small immunity peptides collectively defined as LanI and the ABC (ATP-binding cassette) transporters LanFEG. Th...