Bacillus populations restore amino acid metabolism in Mesorhizobium under saline–alkali stress to enhance nitrogen fixation efficiency

The root nodules formed by rhizobia and leguminous plants are specialized structures for nitrogen fixation. However, a large number of non-rhizobial endophytes also coexist within the nodules, and their contribution to nitrogen fixation under abiotic stress conditions remains unclear. Here, using the wild leguminous shrub Sophora davidii as model system, we identified an important NRE (Bacillus siamensis BT-9-1) by analyzing keystone taxa within the bacterial cooccurrence network of root nodules. This strain could improve the survival of Mesorhizobium metallidurans YC-39 under saline-alkali stress. A mechanistic investigation revealed that the expression of ilvA, ilvH, and ilvD was downregulated, and the contents of (2S)-isopropylmalate and succinic acid decreased in M. metallidurans YC-39 under saline-alkali conditions, whereas B. siamensis BT-9-1 presented increased accumulation of these metabolites. These findings indicate that B. siamensis BT-9-1 cross-feeds M. metallidurans YC-39 with these metabolites, rescuing the compromised branched-chain amino acid synthesis pathway and the tricarboxylic acid cycle in saline-alkali environments. Eventually, coinoculation with B. siamensis BT-9-1 and M. metallidurans YC-39, along with (2S)-isopropylmalate and succinic acid supplementation, increased nitrogenase activity of the symbionts. Our study reveals a novel mechanism by which non-rhizobial endophyte Bacillus species enhances the growth and nitrogen fixation efficiency of M. metallidurans under saline-alkali stress through the delivery of key metabolites.