This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pobigaylo, N.
Right arrow Articles by Becker, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pobigaylo, N.
Right arrow Articles by Becker, A.
Agricola
Right arrow Articles by Pobigaylo, N.
Right arrow Articles by Becker, A.

 Previous Article  |  Next Article 

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.



This article has been cited by other articles:

  • Patankar, A. V., Gonzalez, J. E. (2009). An Orphan LuxR Homolog of Sinorhizobium meliloti Affects Stress Adaptation and Competition for Nodulation. Appl. Environ. Microbiol. 75: 946-955 [Abstract] [Full Text]  
  • Shimoda, Y., Mitsui, H., Kamimatsuse, H., Minamisawa, K., Nishiyama, E., Ohtsubo, Y., Nagata, Y., Tsuda, M., Shinpo, S., Watanabe, A., Kohara, M., Yamada, M., Nakamura, Y., Tabata, S., Sato, S. (2008). Construction of Signature-tagged Mutant Library in Mesorhizobium loti as a Powerful Tool for Functional Genomics. DNA Res 15: 297-308 [Abstract] [Full Text]  
  • McIntosh, M., Krol, E., Becker, A. (2008). Competitive and Cooperative Effects in Quorum-Sensing-Regulated Galactoglucan Biosynthesis in Sinorhizobium meliloti. J. Bacteriol. 190: 5308-5317 [Abstract] [Full Text]  
  • Rossbach, S., Mai, D. J., Carter, E. L., Sauviac, L., Capela, D., Bruand, C., de Bruijn, F. J. (2008). Response of Sinorhizobium meliloti to Elevated Concentrations of Cadmium and Zinc. Appl. Environ. Microbiol. 74: 4218-4221 [Abstract] [Full Text]  
  • Sauviac, L., Philippe, H., Phok, K., Bruand, C. (2007). An Extracytoplasmic Function Sigma Factor Acts as a General Stress Response Regulator in Sinorhizobium meliloti. J. Bacteriol. 189: 4204-4216 [Abstract] [Full Text]  
  • MacLean, A. M., Finan, T. M., Sadowsky, M. J. (2007). Genomes of the Symbiotic Nitrogen-Fixing Bacteria of Legumes. Plant Physiol. 144: 615-622 [Full Text]  
  • Sharypova, L.A., Chataigne, G., Fraysse, N., Becker, A., Poinsot, V. (2006). Overproduction and increased molecular weight account for the symbiotic activity of the rkpZ-modified K polysaccharide from Sinorhizobium meliloti Rm1021. Glycobiology 16: 1181-1193 [Abstract] [Full Text]  
  • Cowie, A., Cheng, J., Sibley, C. D., Fong, Y., Zaheer, R., Patten, C. L., Morton, R. M., Golding, G. B., Finan, T. M. (2006). An Integrated Approach to Functional Genomics: Construction of a Novel Reporter Gene Fusion Library for Sinorhizobium meliloti. Appl. Environ. Microbiol. 72: 7156-7167 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»