S.meliloti mutant libraries

 

Library information

Functional genomics studies of S. meliloti are based on investigation of particular genes and their functions. Gene disruption with subsequent analysis of mutant phenotypes is a usual approach for such type of studies. However, most of these methods are laborious and time-consuming. Genome-wide mutagenesis projects are undertaken to generate mutant collections for screening approaches and a quick access to specific mutants.

 

Signature-tagged transposon mutants

Signature-tagged mutagenesis (STM) can be applied to analyze competitiveness and the symbiotic ability of many mutants in one experiment. A set of 412 miniTn5 transposons each carrying two individual sequence tags was generated. These tags are short DNA segments that contain a 24-bp variable central region flanked by invariant "arms" that facilitate the amplification and labeling of the central portions by PCR. When the tagged transposons are used for mutagenesis, each individual mutant of a set can be distinguished from every other mutant based on the different tags carried by the transposon in its genome. 30 mutant sets were generated in S. meliloti 2011 and about 5000 insertion sites were sequenced. Identification and quantification of the mutants can be performed by amplification and labeling of the tag sequences of mutants from a set and comparative hybridization of the mTn5-STM-1 microarray carrying the variable tag sequences.
Mutants can be browsed using the public S. meliloti GenDB genome browser.

A list of available mTn5-STM S. meliloti 2011 mutants can be downloaded here.

 

References

  • Pobigaylo N, Wetter D, Szymczak S, Schiller U, Kurtz S, Meyer F, Nattkemper TW, Becker A (2006) Construction of a large sequence signature-tagged miniTn5 transposon library and its application to mutagenesis of Sinorhizobium meliloti. Appl Environ Microbiol 72:4329-4337.
  • Pobigaylo N, Szymczak S, Nattkemper TW, Becker A (2008) Identification of genes relevant to symbiosis and competitiveness in Sinorhizobium meliloti using signature-tagged mutants. Mol Plant-Microbe Interact 21: 219-231.
     

Plasmid integration mutants

A second mutagenesis approach focuses on the directed mutagenesis of S. meliloti genes by plasmid integration. Internal fragments of the coding regions of protein-coding genes were cloned into a mobilizable suicide vector. The resulting plasmids were tranferred to S. meliloti 1021 by E. coli S17-1 mediated conjugation. Integration of the plasmids into the S. meliloti genome results in disruption of the targeted genes.
Mutants can be browsed using the public S. meliloti GenDB genome browser.
A list of available S. meliloti 1021 mutants can be downloaded here.
A list of available E. coli S17-1 carrying plasmids for integration mutagenesis can be downloaded here.

References

  • Becker A, Barnett MJ, Capela D, Dondrup M, Kamp P-B, Krol E, Linke B, Rüberg S, Runte K, Schroeder BK, Weidner S, Yurgel S, Batut J, Long SR, Pühler A, Finan TM, Goesmann A (2009) A portal for rhizobial genomes: RhizoGATE integrates a S. meliloti genome annotation update with postgenome data. J Biotechnol 140: 45-50.

 

Procedure for mutant requests

Write to Bettina Happel to request mTn5-STM S.meliloti 2011 or S.meliloti 1021 plasmid integration mutants. Use the subject "sm mutant request" and please provide your FedEx customer No. for payment of shipping by the recipient.

 

 

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