Metabolic and proteomic profiling during stringent response in Bradyrhizobium diazoefficiens

ITURRALDE, ESTEBAN TOMÁS; LAGARES, ANTONIO JR.; CASTELLANI, LUCAS GABRIEL; TORRES TEJERIZO, GONZALO; COLLA, DELFINA; LUCHETTI, ABRIL; PÉREZ-GIMÉNEZ, JULIETA

Online

Congreso; Reunión conjunta SAIB SAMIGE 2020; 2020

SAIB SAMIGE

When soybean symbiont Bradyrhizobium diazoefficiens is cultivated with low N, the production rate and the number of nodules in the roots are stimulated. Bacteria cultivated with low N are also more competitive to nodulate in comparison to those cultivated in N-sufficiency. In this condition of N-deficiency, the stringent response (SR) is triggered. SR is modulated by the amounts of the second messenger (p)ppGpp. This would suggest that the improvements of the symbiotic parameters are due to the expression of different genes regulated by the amount of (p)ppGpp. This hypothesis is supported by the fact that B. diazoefficiens LP5065, a mutant in rsh gene whose product regulates the synthesis of (p)ppGpp, is able to nodulate but fixes less N2. Consequently, we studied SR in B. diazoefficiens in order to correlate the symbiotic phenotype of these rhizobia with the differential expression of proteins and metabolites against the presence/absence of (p)ppGpp using different omics approaches.The proteins were analyzed by nanoHPLC coupled to mass spectrometry (MS) with Orbitrap technology. The resulting spectra were analyzed with the Proteome Discoverer software and the data obtained was processed with Perseus sotfware, applying a statistical analysis of Student´s t-test with a p-value ≤ 0.05. UniProt and eggNOG were used to analyze the results. The metabolites were ionized, separated, and identified by gas chromatography coupled to MS. The qualitative and quantitative analysis of the chromatograms and mass spectra were carried out with Xcalibur program. Finally, Metaboanalyst was used for the analysis and interpretation of metabolic data. The whole data was integrated with KEGG.Proteins and metabolites were obtained from B. diazoefficiens USDA 110, wild type, and LP5065 in two conditions: Treated (T) with mineral solution C and N free (in which SR would be triggered in the wild type), and Untreated (U). We identified an average of 2500 proteins and 44 metabolites per sample. The effects of (p)ppGpp were observed when we compared WT T vs WT U samples in both experiments. (p)ppGpp would be increased in WT T due to the nutritional stress imposed. In this comparison, a decreased in amino acid biosynthesis, pentose phosphate pathway, pantothenate and Coenzyme-A biosynthesis, Krebs cycle and β-oxidation was found. On the contrary, biosynthesis of fatty acids (FA) was increased. In order to study the effect of the mutation, we proceeded to compare WT U vs MUT U. In this analysis, the carbon flux towards pentose phosphate pathway was increased in WT U, thus also increasing the metabolism of purines. Further, fatty acid metabolism was affected.Overall, the SR response triggers several mechanisms at proteomic and metabolic levels. The development of a model of SR in B. diazoefficiens, and its relationship with soybean symbiosis will be a valuable tool for the improvement of the quality of the inoculants in order to get better agronomical yields.