SRA

RpoN (s54) is the key sigma factor for the regulation of transcription of nitrogen fixation genes in diazotrophic bacteria, which include alpha- and beta-rhizobia. Our previous studies showed that a rpoN mutant of the beta-rhizobial strain Paraburkholderia phymatum formed root nodules on Phaseolus vulgaris that were unable to reduce atmospheric nitrogen into ammonia. In an effort to further characterize the RpoN regulon of P. phymatum, transcriptomics was combined with a powerful metabolomics approach. The metabolome of P. vulgaris root nodules infected by the P. phymatum rpoN 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 s54 on nitrogen fixation genes in nodules. The transcriptomic data also identified additional target genes, whose differential expression was able to explain the observed metabolite changes in a numerous cases. Moreover, the genes encoding the two-component regulatory system NtrBC were downregulated in root nodules induced by the rpoN mutant and contained a putative RpoN binding motif in their promoter region, suggesting direct regulation. The construction and characterization of an ntrB mutant strain revealed impaired nitrogen assimilation in free-living conditions, as well as a noticeable symbiotic phenotype by forming less but heavier nodules on P. vulgaris roots. Overall design: Unraveling the molecular basis of the nitrogen-fixing symbiosis between P. vulgaris and P. phymatum.