Building a Genetic Platform for S. meliloti, a Nitrogen-Fixing Symbiont of Alfalfa

Washington State University, Pullman WA

Investigators

Abstract

Nitrogen-fixing symbioses are ecologically and agronomically important and provide the major nitrogen input for many systems of sustainable agriculture. For scientific and historical reasons, the interaction between Sinorhizobium meliloti and a diploid alfalfa, Medicago truncatula, has become a model for this kind of nitrogen-fixing symbiosis. The DNA sequence of the S. meliloti genome has been determined and was predicted to encode about 6200 proteins. To investigate the genome further, an integrated genetic strategy is being developed that will provide a platform for many different types of genetic, biochemical, physiological and taxonomic analysis. The strategy uses high-throughput, genomic scale PCR and integrase technology to clone sequences that encode every predicted gene in the S. meliloti genome. These clones can be used immediately in creating DNA arrays for expression analysis and cross species comparisons and, using the clones, it should be possible to move smoothly to the construction of S. meliloti strains with reporter genes fused to each predicted promoter, to the isolation of mutant strains lacking precisely defined regions of the S. meliloti genome, and to the production of proteins for biochemical characterization. The strategy also provides built-in flexibility for incorporating technical advances in the future. Characterization of gene expression and symbiotic phenotypes of the resulting bacterial strains will allow investigators to investigate various bacterial traits important to a productive symbiosis, such as resistance to acid soils and heavy metals, cold tolerance, desiccation tolerance, competitiveness for nodulation and efficiency of nitrogen fixation. The information obtained by studying S. meliloti and its symbiosis with alfalfa will be relevant to understanding other symbioses and plant-microbe interactions in general. Resources will be developed for sharing this genetic material and information. Through both their immediate utility and their features designed to accommodate changes necessitated by future research needs, these constructs should provide a basis for many years of fruitful work. The strategy should be applicable to many other organisms important to society in areas ranging from agriculture and the environment to bioengineering.