Towards Synthesis of Minimal Plasmids for Symbiotic Nitrogen Fixation

When nitrogen is limited, many legume plants form beneficial associations with nitrogen-fixing rhizobia. Efficient reduction of atmospheric nitrogen by rhizobial nitrogenase is restricted to plant nodules, inside which rhizobia establish persistent colonies of intracellular bacteroids. Of all known nodulating rhizobia, Sinorhizobium fredii strain NGR234 fixes nitrogen with >130 legumes. Genetic basis for such symbiotic promiscuity was linked to a 536 kb plasmid called pNGR234a. In this study, we aimed at downsizing pNGR234a to a core of essential loci sufficient to sustain proficient interactions with legumes. Hence, synthetic plasmids carrying loci needed for the flavonoid-dependent synthesis of basic nodulation factor structures (pMiniSym2) and functions required for nitrogen fixation (pMiniSym4) were constructed. When mobilized into a derivative of NGR234 cured of pNGR234a or into non-symbiotic soil bacteria, pMiniSym2 conferred nodulation and infection abilities to recipient strains. However, pMiniSym4 failed to confer a robust SNF, suggesting additional work is needed to secure SNF.