Biology of the N2-fixing soil bacterium, Sinorhizobium meliloti

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The bacterium, Sinorhizobium meliloti, forms N2-fixing nodules on the roots of alfalfa. This symbiotic association represents the culmination of a series of complex interactions between plant and bacteria. Symbiotic N2-fixation (SNF) is of huge significance to agriculture as it by-passes the economic and environmental costs associated with nitrogen fertilizer. My NSERC program is directed towards understanding the S. meliloti-alfalfa symbiosis, particularly events occurring within bacteria in nodules and in free-living cells in the soil and root environment. Sinorhizobium meliloti is a model organism for studying symbiotic N2-fixation (SNF), soil-microbe biology and multipartite genome biology. The S. meliloti genome consists of a 3.5 Mb chromosome and two large replicons referred to as pSymA (1.34 Mb) and pSymB (1.67 Mb). During the past funding period, we manipulated the S. meliloti genome and generated sequential deletions that collectively removed the ~ 3000 genes present on the pSymA and pSymB replicons. We identified active toxin/antitoxin systems, a new symbiotic locus on pSymB and two genes (ArgtRNACCG and engA) that are essential for cell viability. These two essential genes are part of a 110 kb region (designated ETR) on the pSymB that translocated from the chromosome to pSymB in an ancestral strain. We have generated S. meliloti strains that lack the pSymA and pSymB replicons, and in which a complete ETR region is integrated into the chromosome. We now propose using this strain as the entry point or a basic genetic chassis onto which we wish to identify the minimal symbiotic genome that is, the minimal complement of genes that are necessary and sufficient for the formation of N2-fixing nodules on alfalfa. Work from many labs over the last 40 years identified many of the nodulation (nod), N2-fixation (nif) and symbiotic genes (fix) genes involved in SNF. Thus far, we have identified pSymA and pSymB regions that are required for SNF and in our future work, we propose to combine these regions to generate a minimal symbiotic genome and further reduce that gene complement through targeted removal of individual genes. This will serve as a platform for synthetic biology approaches to manipulating SNF and also for identification of other genes that are important to the specific SNF plant symbiosis and specific bacterial hosts. In related projects we propose to complete the analysis of the smb20752 locus that we recently identified. Our goal to determine the molecular bases for the defective SNF phenotype. In addition, in a project more related to the ecology of the bacteria in soil, we propose to analyze a locus on the pSymA replicon that is involved in restricting the growth of viruses that infect these bacteria. This research program addresses fundamental questions of broad interest and it has and will continue to generate high quality training for undergraduate, graduate and post-doctoral personnel.