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Appl. Environ. Microbiol. doi:10.1128/AEM.00912-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Biosynthesis of Staphylococcus aureus autoinducing peptides using the Synechocystis DnaB mini-intein

Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242

^* To whom correspondence should be addressed. Email: alex-horswill{at}uiowa.edu.

	Abstract

The Agr quorum-sensing system of Staphylococcus aureus modulates the expression of virulence factors in response to autoinducing peptides (AIPs). The peptides are seven to nine residues in length and have the C-terminal five residues constrained in an unusual thiolactone ring. We have developed a new method to generate AIP structures using an engineered DnaB mini-intein from Synechocystis spp. PCC6803. In the method, an oligonucleotide encoding the AIP is ligated to the intein, and the fusion protein is expressed and purified by affinity chromatography. To produce the correct AIP structure, intein splicing is interrupted, allowing the cysteine side-chain to catalyze thiolactone ring formation and release AIP from the resin. The technique is simple and robust, and we have successfully produced the three main classes of AIPs using the intein system. The intein-generated AIPs possessed the correct thiolactone ring modification based on biochemical analysis, and importantly, all the samples were bioactive against S. aureus. The AIP activity was confirmed through Agr interference and activation profiling with developed S. aureus reporter strains. The simplicity of the method, benefits of intein DNA-encoding, and scaleable nature enables the production of S. aureus AIPs for many biological applications.