TY - JOUR
T1 - Construction of a large signature-tagged mini-Tn5 transposon library and its application to mutagenesis of Sinorhizobium meliloti
AU - Pobigaylo, Nataliya
AU - Wetter, Danijel
AU - Szymczak, Silke
AU - Schiller, Ulf
AU - Kurtz, Stefan
AU - Meyer, Folker
AU - Nattkemper, Tim W.
AU - Becker, Anke
PY - 2006/6
Y1 - 2006/6
N2 - 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 metagenesis 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.
AB - 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 metagenesis 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.
UR - http://www.scopus.com/inward/record.url?scp=33745122953&partnerID=8YFLogxK
U2 - 10.1128/AEM.03072-05
DO - 10.1128/AEM.03072-05
M3 - Journal articles
C2 - 16751548
AN - SCOPUS:33745122953
SN - 0099-2240
VL - 72
SP - 4329
EP - 4337
JO - Applied and Environmental Microbiology
JF - Applied and Environmental Microbiology
IS - 6
ER -