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Applied and Environmental Microbiology, July 2003, p. 3777-3783, Vol. 69, No. 7
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.7.3777-3783.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Construction of an Expression System for Site-Directed Mutagenesis of the Lantibiotic Mersacidin

Christiane Szekat,1 Ralph W. Jack,2,{dagger} Dirk Skutlarek,3 Harald Färber,3 and Gabriele Bierbaum1*

Institut für Medizinische Mikrobiologie und Immunologie der Universität Bonn,1 Hygiene-Institut der Universität Bonn, D-53105 Bonn,3 Institut für Organische Chemie der Universität Tübingen, D-72076 Tübingen, Germany2

Received 26 December 2002/ Accepted 31 March 2003

The lantibiotic (i.e., lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide of 20 amino acids which is produced by Bacillus sp. strain HIL Y-85,54728. Mersacidin inhibits bacterial cell wall biosynthesis by binding to the precursor molecule lipid II. The structural gene of mersacidin (mrsA) and the genes for the enzymes of the biosynthesis pathway, dedicated transporters, producer self-protection proteins, and regulatory factors are organized in a biosynthetic gene cluster. For site-directed mutagenesis of lantibiotics, the engineered genes must be expressed in an expression system that contains all of the factors necessary for biosynthesis, export, and producer self-protection. In order to express engineered mersacidin peptides, a system in which the engineered gene replaces the wild-type gene on the chromosome was constructed. To test the expression system, three mutants were constructed. In S16I mersacidin, the didehydroalanine residue (Dha) at position 16 was replaced with the Ile residue found in the closely related lantibiotic actagardine. S16I mersacidin was produced only in small amounts. The purified peptide had markedly reduced antimicrobial activity, indicating an essential role for Dha16 in biosynthesis and biological activity of mersacidin. Similarly, Glu17, which is thought to be an essential structure in mersacidin, was exchanged for alanine. E17A mersacidin was obtained in good yields but also showed markedly reduced activity, thus confirming the importance of the carboxylic acid function at position 17 in the biological activity of mersacidin. Finally, the exchange of an aromatic for an aliphatic hydrophobic residue at position 3 resulted in the mutant peptide F3L mersacidin; this peptide showed only moderately reduced activity.

* Corresponding author. Mailing address: Institut für Medizinische Mikrobiologie und Immunologie, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany. Phone: 49-228-2879103. Fax: 49-228-2874808. E-mail: bierbaum{at}mibi03.meb.uni-bonn.de.

{dagger} Present address: Department of Microbiology, University of Otago, Dunedin, New Zealand.

Applied and Environmental Microbiology, July 2003, p. 3777-3783, Vol. 69, No. 7
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.7.3777-3783.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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