K512R mutation in the capsule biosynthesis regulator RkpI of the commercial nitrogen-fixing alfalfa inoculant Sinorhizobium meliloti B399 improves soil nitrogen content

Abstract

Alfalfa, known as the queen of forages, is a sustainable crop that reduces reliance on synthetic nitrogen fertilizers through symbiosis with nitrogen-fixing rhizobia. This biological fixation supports its high protein content and enriches soil nitrogen, benefiting crop rotations (e.g., alfalfa–wheat) and forage mixtures (e.g., alfalfa–fescue). However, both natural rhizobia and commercial inoculants typically possess active denitrification pathways, which can reduce net nitrogen inputand release nitrous oxide. In this context, we explored a novel genetic strategy to modulate denitrification in the commercial alfalfa inoculant B399. Specifically, we introduced targeted mutations in a conserved lysine residue (K512) within the C-terminal hypervariable domain of RkpI, a key regulator of capsule biosynthesis. All mutants displayed impaired denitrification activity, while most retained the ability to produce capsule to varying degrees. A positive association wasobserved between capsule synthesis and plant growth promotion, suggesting that capsule production is an important trait contributing to the symbiotic effectiveness of inoculants. Remarkably, the K512R variant enhanced soil nitrogen without compromising forage productivity. These results suggest that precise modulation of capsule production represents a promising strategy to enhance the agronomic and environmental performance of inoculants.