Effect of Nir genes on nitrogen fixation by Rhizobium ‘hedysari’, symbiont of Hedysarum coronarium roots

Hedysrum coronarium is a forage legume present in all the mediterranean basin known and studied for its drought resistance, alkaline soil tolerance and the ability to give good agronomical performances in calcareous and semiarid soil. The root symbiont Rhizobium ‘hedysari’ HCNT1 has been recently studied for its ability to produce nodules on the roots of sulla in marginal soils, permitting H. coronarium to respond well as a pioneer plant. The specificity of this bacterium has been partially proved and a taxonomic characterization is under study. R. ‘hedysari’ HCNT1 was initially isolated because of its ability to reduce nitrite to nitrous oxide, suggesting this bacteria can grow anaerobically with nitrogen oxides as terminal electron acceptor. Further work showed that HCNT1 could not couple the reduction of nitrite to growth, unlike true denitrifiers. It has been suggested that the nitrite reduction is a detoxification mechanism used by the cell to prevent nitrite-dependent inhibition of nitrogenase or perhaps components of the electron transport chain. Recent work has demonstrated that it is nitric oxide, not nitrite, that is the direct inhibitor of the electron transport chain. Under specific low-oxygen growth conditions some strains of B. japonicum appear to have a nitrite reductase activity that is different than that found under anaerobic conditions. This low oxygen nitrite reductase activity has similarities to the activity of nitrite reductase in HCNT1.This suggests that nitrite reductase in rhizobia may have different physiological roles under different conditions. In this work the relationship of nitrite reductase activity toward nitrogenase has been investigated. In particular, the ability of nitrite reductase-deficient strains to nodulate Hedysarum coronarium roots, fix nitrogen and support plant development have been evaluated. The regulation and expression of genes encoding nitrite reductase in some R.'hedysari' strains which are naturally unable to reduce nitrogen oxides has been also investigated.