PUB - Publikationen an der Universität Bielefeld

Sinorhizobium meliloti is a model organism for studies of plant-microbe interactions. This Gram-negative soil bacterium can enter an endosymbiosis with alfalfa plants through the formation of nitrogen-fixing nodules. S. meliloti genome sequence determination has provided a basis for different approaches of functional genomics to this bacterium. One of these approaches is gene disruption with subsequent analysis of mutant phenotypes. Usually, the selection of mutants that can survive under a certain condition is simple and efficient, and can be performed using a mixture of different mutants. However, the selection of mutants that have an attenuated phenotype in a tested condition is problematic, because all mutants have to be checked separately one by one. A microarray-based signature-tagged mutagenesis (STM) strategy can overcome this problem. Signature-tagged mutagenesis is based on a collection of mutants split into sets, where each mutant is modified by one or more different signature tags. The tags are short DNA segments that are unique for each mutant in a set and can be amplified using invariant or specific priming sites. Tagged mutants from the same set are pooled prior to an experiment, and each mutant in the mixture can be identified based on the unique tag in its genome. The presence of a particular tag in the mixture can be detected through a hybridization of amplified products to a microarray containing tag-specific probes. In order to integrate the signature tags into the genome, a strategy based on libraries of tag-carrying transposons can be used. In the transposon-based STM, the number of mutants that can be pooled together in one experiment depends on the quantity of transposons containing different tags. In this study, the construction of a novel set of 412 mTn5-based signature-tagged transposons was performed, which is the largest tagged transposon set reported so far, and this set was applied to the mutagenesis of S. meliloti. To achieve a high specificity of tag detection, each transposon was bar coded by two signature tags. In order to generate defined, non-redundant sets of signature-tagged S. meliloti mutants for subsequent experiments, 12000 mutants were constructed and insertion sites for more than 5000 mutants were determined. One set consisting of 378 mutants was used in a validation experiment to identify mutants showing altered growth patterns. This and one additional mutant set, which also contained 378 mutants, were used in the experiment for identification of genes in S. meliloti relevant to symbiosis and competitiveness. From all the tested mutants, 69 were found to be attenuated. For 25 mutants, attenuated phenotypes were checked by a different method. As a result, the attenuated competitiveness phenotype could be confirmed for 22 from 25 tested mutants. Four mutants were found to have additional symbiotic defects: Fix- phenotype (ccmC mutant), low efficiency of nodulation (asnO mutant) and delayed nodulation (cysG and metA mutants).