RpoN (or σ) is the key sigma factor for the regulation of transcription of nitrogen fixation genes in diazotrophic bacteria, which include α- and β-rhizobia. Our previous studies showed that an mutant of the β-rhizobial strain STM815 formed root nodules on cv. Negro jamapa, which were unable to reduce atmospheric nitrogen into ammonia. In an effort to further characterize the RpoN regulon of , transcriptomics was combined with a powerful metabolomics approach. The metabolome of root nodules infected by a Fix mutant revealed statistically significant metabolic changes compared to wild-type Fix⁺ nodules, including reduced amounts of chorismate and elevated levels of flavonoids. A transcriptome analysis on Fix and Fix⁺ nodules-combined with a search for RpoN binding sequences in promoter regions of regulated genes-confirmed the expected control of σ on nitrogen fixation genes in nodules. The transcriptomic data also allowed us to identify additional target genes, whose differential expression was able to explain the observed metabolite changes in numerous cases. Moreover, the genes encoding the two-component regulatory system NtrBC were downregulated in root nodules induced by the mutant, and contained a putative RpoN binding motif in their promoter region, suggesting direct regulation. The construction and characterization of an mutant strain revealed impaired nitrogen assimilation in free-living conditions, as well as a noticeable symbiotic phenotype, as fewer but heavier nodules were formed on roots.