Defining a minimal symbiotic genome of the legume symbiont Sinorhizobium meliloti

Abstract

Identifying and understanding bacterial genes involved in the formation of N2-fixing root nodules is of agricultural and environmental interest. Sinorhizobium meliloti is a model bacterium for studying the rhizobia-legume symbiosis. Most genes with direct functions in symbiotic nitrogen fixation (SNF) are harboured on two megaplasmids: pSymA (1354 kb) and pSymB (1683 kb). This thesis describes work on minimizing the pSymB replicon to establish a gene complement sufficient for a robust SNF phenotype. It also outlines my contribution towards establishing that 58 genes (63 kb) from pSymA are sufficient for a robust SNF phenotype. Megaplasmid pSymB is evolutionarily older and more chromosomal-like (i.e. a chromid) than the more recently acquired pSymA replicon. Both large-scale deletion analyses (top-down) and assembly-based methods (bottom-up) to minimize pSymB were conducted. These analyses revealed that minimizing pSymB results in a large symbiotic penalty. An initial minimization of pSymB to 261 kb (15%) resulted in SNF with a large degree of plant genotype-dependent variation. This served as a platform to demonstrate that additional regions housing undiscovered auxiliary genes are necessary for the efficient SNF. Accordingly, a minimized 673 kb replicating pSymB that facilitated consistent SNF was isolated. A cumulative deletion strategy refined this set to 276 kb (16% of pSymB) without further SNF impairment. In addition to the deletion approach, we developed a methodology that targeted the assembly of pSymB loci into discrete clusters followed by their iterative integration into a S. meliloti strain lacking pSymB. A set of 101 genes (114 kb) from pSymB proved capable of routinely forming nodules with SNF at 25% wild-type levels. By combining the minimized pSymA and iv pSymB sets, the smallest genome capable of forming root-nodule symbioses was established. This should serve as a powerful chassis for gain-of-function approaches to studying SNF.