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The lantibiotic Pep5 is produced byStaphylococcus epidermidis 5. Within its biosynthetic gene cluster, the immunity genepepI , providing producer self-protection, is localized upstream of the structural genepepA . Pep5 production and the immunity phenotype have been found to be tightly coupled (M. Reis, M. Eschbach-Bludau, M. I. Iglesias-Wind, T. Kupke, and H.-G. Sahl, Appl. Environ. Microbiol. 60:2876–2883, 1994). To study this phenomenon, we analyzedpepA andpepI transcription and translation and constructed a number of strains containing various fragments of the gene cluster and expressing different levels of immunity. Complementation of apepA -expressing strain withpepI intrans did not result in phenotypic immunity or production of PepI. On the other hand, neitherpepA nor its product was found to be involved in immunity, since suppression of the translation of thepepA mRNA by mutation of the ATG start codon did not reduce the level of immunity. Moreover, homologous and heterologous expression ofpepI from a xylose-inducible promoter resulted in significant Pep5 insensitivity. Most important for expression of the immunity phenotype was the stability ofpepI transcripts, which in the wild-type strain, is achieved by an inverted repeat with a free energy of −56.9 kJ/mol, localized downstream ofpepA . We performed site-directed mutagenesis to study the functional role of PepI and constructed F13D PepI, I17R PepI, and PepI 1-65; all mutants showed reduced levels of immunity. Western blot analysis indicated that F13D PepI and PepI 1-65 were not produced correctly or were partially degraded, while I17R PepI apparently was less efficient in providing self-protection than the wild-type PepI.

Molecular Analysis of Expression of the Lantibiotic Pep5 Immunity Phenotype