Genome-wide association analysis and metabolic profiling reveal the high complexity of salt tolerance polymorphism in the nitrogen-fixing Sinorhizobium meliloti

Salinity is nowadays one of the largest constraints in agriculture impairing the legume-rhizobia symbiosis and in turn biological nitrogen fixation. Selecting NaCl-tolerant strains and improving native strains fitness under salt stress are key points to ameliorate rhizobium-legume symbiosis effectiveness under salt stress. Understanding the genetic basis of this complex phenotype offers promising targets for the selection of symbiotic systems adapted for saline soils. To identify genes that might be responsible for salt resistance in S. meliloti strains, we performed a k-mer-based GWA analysis on a pool of de novo-sequenced and well-characterized strains. In particular, the genome sequences of strains and the phenotypic matrix, gained with a thorough in vitro screening at progressively higher NaCl concentrations, were used to pinpoint specific k-mers significantly associated with salt-tolerance phenotype. The retrieved k-mers allowed us to identify several loci associated with the variability in salt tolerance and involved in various processes, including cell wall organization (peptidoglycan transport and degradation), LPS biosynthesis, quorum sensing, and carbohydrate transport and metabolism (as galactose metabolism, sorbitol biosynthesis, trehalose metabolic process, and cyclic-glucan synthesis). To confirm the involvement of carbohydrates, metabolic profiling of salt-tolerant and salt-sensitive strains were recorded on different carbon sources in presence and absence of salt. Salt-tolerant strains showed broader abilities in carbon sources exploitation under salt stress, showing comparable kinetics in both conditions on L-arabinose, D-arabinose, D-galactose and N-acetyl-D-Glucosamine sources, and confirming some key genetic determinants previously detected. Our study's findings provided new insights into the knowledge of the mechanisms of salt tolerance in S. meliloti, underscoring the multifaceted nature of salt tolerance in S. meliloti.