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Applied and Environmental Microbiology, February 1999, p. 591-598, Vol. 65, No. 2
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Molecular Analysis of Expression of the Lantibiotic Pep5 Immunity Phenotype

Ulrike Pag, Christoph Heidrich,dagger Gabriele Bierbaum, and Hans-Georg Sahl*

Institut für Medizinische Mikrobiologie und Immunologie der Universität Bonn, Bonn, Germany

Received 12 August 1998/Accepted 23 November 1998

The lantibiotic Pep5 is produced by Staphylococcus epidermidis 5. Within its biosynthetic gene cluster, the immunity gene pepI, providing producer self-protection, is localized upstream of the structural gene pepA. 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 analyzed pepA and pepI transcription and translation and constructed a number of strains containing various fragments of the gene cluster and expressing 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 involved in immunity, since suppression of the translation of the pepA mRNA by mutation of the ATG start codon did not reduce the level of immunity. Moreover, homologous and heterologous expression of pepI from a xylose-inducible promoter resulted in significant Pep5 insensitivity. Most important for expression of the immunity phenotype was the stability of pepI transcripts, which 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-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.

* Corresponding author. Mailing address: Institut für Medizinische Mikrobiologie und Immunologie der Universität Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany. Phone: 49 228 287 5704. Fax: 49 228 287 4808. E-mail: sahl{at}mibi03.meb.uni-bonn.de.

dagger Present address: Max-Planck-Institut für Entwicklungsbiologie, Abteilung Biochemie, Tübingen, Germany.

Applied and Environmental Microbiology, February 1999, p. 591-598, Vol. 65, No. 2
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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