Metabolomics and Transcriptomics Identify Multiple Downstream Targets of Paraburkholderia phymatum σ54 During Symbiosis with Phaseolus vulgaris

Abstract

RpoN (or σ⁵⁴) 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 rpoN mutant of the β-rhizobial strain Paraburkholderia phymatum STM815^T formed root nodules on Phaseolus vulgaris cv. Negro jamapa, which 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 a P. phymatumrpoN 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 σ⁵⁴ 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 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, as fewer but heavier nodules were formed on P. vulgaris roots.

Keywords: RNA-sequencing; legumes; metabolome; nitrogen fixation; papilionoid; rhizobia; rpoN; sigma factor.

Figure 1

Principal component analysis (PCA) of metabolome datasets from P. vulgaris root nodules induced by wild-type P. phymatum (wt, blue) and rpoN mutant (rpoN mt, red) strains, or from uninfected P. vulgaris roots (yellow). Three biological replicates were analyzed, each injected twice by non-targeted metabolomics; #: number.

Figure 2

Functional categories of the top 500 differentially expressed genes in P. phymatum nodules infected by the wild type versus an rpoN mutant during symbiosis, with P. vulgaris (genes up-regulated in the rpoN mt are in black, those that were down-regulated are in grey) according to classification by eggNOG [40]. Percentages were calculated by dividing the number of significantly up-regulated (322) or down-regulated (178) genes in each category by the total number of retained genes in the same category. The asterisks (*) indicate statistical significance (Fischer test, p-value < 0.01).

Figure 3

Selected P. phymatum gene clusters harboring a putative but high-scoring RpoN binding box in their promoter region. The operon containing the ammonium transporter gene amtB (A), the operon containing the 2CRS NtrBC (B), the genes coding for a hydantoinase (C), and the cluster for urea transport (D) are shown. Gene names are indicated in italics, while the genes containing an RpoN binding box upstream are shown in bold. Genes present among the top 500 regulated genes (Table S3) are colored in grey, and their log₂ fold expression changes are shown below. Black arrows indicate the position of the RpoN box; the distance (in nucleotides) from the middle of the box to the translation start site is indicated below the arrow.

Figure 4

Comparison of the symbiotic properties of P. vulgaris plants inoculated with a ntrB deletion mutant (ΔntrB) with those of the P. phymatum nalidixic acid-resistant wild-type (wt nal^R) strain. Number of nodules per plant (A), dry weight per nodule (B), and relative nitrogenase activity (C) were determined at 21 days post-infection (dpi). Here, the combined results of two independent experiments are shown. Error bars indicate the standard error of the mean (SEM). The two columns were analyzed by an unpaired student t-test (p-values are indicated above the SEM, * indicates p-value < 0.05).

Figure 5

Utilization of selected nitrogen sources by P. phymatum wild-type (wt), nalidixic acid-resistant wild type (wt nal^R), and by a ntrB deletion mutant strain (ΔntrB). Growth was assessed with at least two independent replicates. Error bars indicate standard deviation (SD). For each group of columns, values with the same letter are not statistically different, while those with different letters are (ANOVA, Tukey’s test, p < 0.001).

Figure 6

Scheme of the main changes in metabolites and transcripts profile in P. vulgaris root nodules infected by a rpoN mutant, compared to wild-type nodules. Metabolites and reactions down- and up-regulated in nodules induced by the rpoN mutant are indicated in blue and green, respectively. Glu: glutamate; Gln: glutamine; OAA: oxaloacetate; Trp: tryptophan; Ala: alanine; Pyr: pyruvate; Arg: arginine; RND: resistance-nodulation-division; PG: peptidoglycan.