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Applied and Environmental Microbiology, July 2007, p. 4234-4242, Vol. 73, No. 13
0099-2240/07/$08.00+0     doi:10.1128/AEM.00509-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Rapid Engineering of Bacterial Reporter Gene Fusions by Using Red Recombination{triangledown} ,{dagger}

Roman G. Gerlach, Stefanie U. Hölzer, Daniela Jäckel, and Michael Hensel*

Mikrobiologisches Institut, Universitätsklinikum Erlangen, Erlangen, Germany

Received 6 March 2007/ Accepted 7 May 2007

Reporter gene fusions are essential tools for the investigation of gene regulation. Such fusions are traditionally generated by transposon mutagenesis and identified by a suitable selection procedure. Alternatively, specific reporter fusions can be generated by cloning of DNA fragments containing promoters or other regulatory elements in reporter plasmids. Here, we describe a novel approach for the rapid generation of reporter gene fusions in single copies at defined positions in bacterial genomes. This technique utilizes the Red recombinase for the homologous recombination of PCR-generated cassettes containing various currently used reporter genes, such as those for ß-galactosidase, luciferase, and green fluorescent protein. The approach allows the generation of transcriptional or translational reporter fusions in a single step without the requirement for recombinant DNA constructs and is applicable to various enterobacterial species. Generation of reporter fusions by Red recombination is rapid, overcomes the current limitations of transposon mutagenesis or reporter plasmids, and offers new options for the study of bacterial gene regulation.

* Corresponding author. Mailing address: Mikrobiologisches Institut, Universitätsklinikum Erlangen, Wasserturmstr. 3-5, D-91054 Erlangen, Germany. Phone: 49 (0)9131 8523640. Fax: 49 (0)9131 8522531. E-mail: hensel{at}mikrobio.med.uni-erlangen.de

{triangledown} Published ahead of print on 18 May 2007.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, July 2007, p. 4234-4242, Vol. 73, No. 13
0099-2240/07/$08.00+0     doi:10.1128/AEM.00509-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Gerlach, R. G., Jackel, D., Holzer, S. U., Hensel, M. (2009). Rapid Oligonucleotide-Based Recombineering of the Chromosome of Salmonella enterica. Appl. Environ. Microbiol. 75: 1575-1580 [Abstract] [Full Text]  
  • Gerlach, R. G., Jackel, D., Geymeier, N., Hensel, M. (2007). Salmonella Pathogenicity Island 4-Mediated Adhesion Is Coregulated with Invasion Genes in Salmonella enterica. Infect. Immun. 75: 4697-4709 [Abstract] [Full Text]  


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