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The tyrosine recombinase MrpA and its target sequence: a mutational analysis of the recombination site mrpS resulting in a new left element/right element (LE/RE) deletion system

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Abstract

MrpA is the multimer resolution protein of the Streptomyces coelicolor A3(2) plasmid SCP2*. Previously, MrpA was found to be a site-specific tyrosine recombinase that acts with the 36-bp recombination site mrpS. The present report gives a comprehensive characterization of the composition as well as the position of the spacer and MrpA binding sites within mrpS. Experiments revealed a spacer consisting of 6 remarkably variable nucleotides in the middle of the mrpS-site. A reduction in the spacer to 5 nucleotides abolished recombination. Investigation of the MrpA binding sites showed the importance of its 15 nucleotides on an effective recombination. Among almost randomly exchangeable nucleotides, two nucleotides were identified as essential for MrpA binding. Alteration of either of these nucleotides led to a reduction in MrpA binding down to 2 % or even to no binding. Based on these results, a new left element/right element (LE/RE) deletion system was developed. The constructed heteromeric mrpS-sites are efficiently resolved by MrpA. The resulting double mutated (LE/RE) site can no longer be used as a recombination site by MrpA. The system has been successfully applied for the generation of multiple-targeted deletions in the genome of E. coli.

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Acknowledgments

The present work has been completed as part of a PhD thesis at Stuttgart University, Institute of Industrial Genetics (IIG). We would like to thank Prof. Dr. Ralf Mattes for his great and generous support during the past years of research.

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  1. Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, 70569, Stuttgart, Germany

    Lydia Warth & Josef Altenbuchner

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  1. Lydia Warth
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Correspondence to Josef Altenbuchner.

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Communicated by Jean-Luc Pernodet.

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Warth, L., Altenbuchner, J. The tyrosine recombinase MrpA and its target sequence: a mutational analysis of the recombination site mrpS resulting in a new left element/right element (LE/RE) deletion system. Arch Microbiol 195, 617–636 (2013). https://doi.org/10.1007/s00203-013-0910-x

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  • DOI: https://doi.org/10.1007/s00203-013-0910-x

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