Construction of a large signature-tagged mini-Tn5 transposon library and its application to mutagenesis of Sinorhizobium meliloti.
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.
Pobigaylo
Nataliya
N
Lehrstuhl für Genetik, Universität Bielefeld, Postfach 100131, 33501 Bielefeld, Germany.
Wetter
Danijel
D
Szymczak
Silke
S
Schiller
Ulf
U
Kurtz
Stefan
S
Meyer
Folker
F
Nattkemper
Tim W
TW
Becker
Anke
A
D001483:Base Sequence; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D016680:Genome, Bacterial; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids; D015183:Restriction Mapping; D016962:Sinorhizobium meliloti / Q000235:genetics*
D004251:DNA Transposable Elements