Molecular analysis of expression of the lantibiotic Pep5 immunity phenotype

The lantibiotic Pep5 is produced by Staphylococcus epidermidis 5. Within it s biosynthetic gene cluster, the immunity gene pepI, providing producer sel f-protection, is localized upstream of the structural gene pepA, Pep5 produ ction 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, Ap pl. Environ. Microbiol. 60:2876-2883, 1994). To study this phenomenon, we a nalyzed pepA and pepI transcription and translation and constructed a numbe r of strains containing various fragments of the gene cluster and expressin g different levels of immunity. Complementation of a pepA-expressing strain with PepI in trans did not result in phenotypic immunity or production of PepI. On the other hand, neither pepA nor its product was found to be invol ved in immunity, since suppression of the translation of the pepA mRNA by m utation of the ATG start codon did not reduce the level of immunity. Moreov er, homologous and heterologous expression of pepI from a xylose-inducible promoter resulted in significant Pep5 insensitivity. Most important for exp ression of the immunity phenotype was the stability of pepI transcripts, wh ich in the wild-type strain, is achieved by an inverted repeat with a free energy of -56.9 kJ/mol, localized downstream of pepA. We performed site-dir ected mutagenesis to study the functional role of PepI and constructed F13D PepI, I17R PepI, and PepI 1-65; all mutants showed reduced levels of immun ity. Western blot analysis indicated that F13D PepI and PepI 1-65 were not produced correctly or were partially degraded, while I17R PepI apparently w as less efficient in providing self-protection than the wild-type PepI.