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Applied and Environmental Microbiology, June 2006, p. 4329-4337, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.03072-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Construction of a Large Signature-Tagged Mini-Tn5 Transposon Library and Its Application to Mutagenesis of Sinorhizobium meliloti{dagger}

Nataliya Pobigaylo,1,2 Danijel Wetter,1,2 Silke Szymczak,3 Ulf Schiller ,4,{ddagger} Stefan Kurtz,4,§ Folker Meyer,5 Tim W. Nattkemper,3 and Anke Becker1*

Lehrstuhl für Genetik, Universität Bielefeld, 33501 Bielefeld, Germany,1 International Graduate School in Bioinformatics and Genome Research, Universität Bielefeld, 33501 Bielefeld, Germany,2 Angewandte Neuroinformatik, Technische Fakultät, Universität Bielefeld, 33501 Bielefeld, Germany,3 Praktische Informatik, Technische Fakultät, Universität Bielefeld, 33501 Bielefeld, Germany,4 Bioinformatics Resource Facility, Centrum für Biotechnologie, Universität Bielefeld, 33501 Bielefeld, Germany5

Received 30 December 2005/ Accepted 23 March 2006

Sinorhizobium meliloti genome sequence determination has provided the basis for different approaches of functional genomics for this symbiotic nitrogen-fixing alpha-proteobacterium. One of these approaches is gene disruption with subsequent analysis of mutant phenotypes. This method is efficient for single genes; however, it is laborious and time-consuming if it is used on a large scale. Here, we used a signature-tagged transposon mutagenesis method that allowed analysis of the survival and competitiveness of many mutants in a single experiment. A novel set of signature tags characterized by similar melting temperatures and G+C contents of the tag sequences was developed. The efficiencies of amplification of all tags were expected to be similar. Thus, no preselection of the tags was necessary to create a library of 412 signature-tagged transposons. To achieve high specificity of tag detection, each transposon was bar coded by two signature tags. In order to generate defined, nonredundant sets of signature-tagged S. meliloti mutants for subsequent experiments, 12,000 mutants were constructed, and insertion sites for more than 5,000 mutants were determined. One set consisting of 378 mutants was used in a validation experiment to identify mutants showing altered growth patterns.

* Corresponding author. Mailing address: Lehrstuhl für Genetik, Universität Bielefeld, Postfach 100131, 33501 Bielefeld, Germany. Phone: 49 521-106-4824. Fax: 49 521-106-5626. E-mail: Anke.Becker{at}genetik.uni-bielefeld.de.

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

{ddagger} Present address: MPI für Polymerforschung, 55128 Mainz, Germany.

§ Present address: Zentrum für Bioinformatik, Universität Hamburg, 20146 Hamburg, Germany.

Applied and Environmental Microbiology, June 2006, p. 4329-4337, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.03072-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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