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

Martina Lardi,Gabriella Pessi,Gaetano Giudice,Yilei Liu,Nicola Zamboni,Christian Ahrens

doi:10.3390/ijms19041049

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 STM815T 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. 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 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.

Highlights

Paraburkholderia phymatum STM815T is a nitrogen-fixing soil bacterium that belongs to the β-proteobacteria (β-rhizobia); it is able to induce nodules on the roots of Mimosoid and Papilionoid legumes [1]
We have previously shown that the alternative sigma factor RpoN (σ54) of P. phymatum STM815T is required for the development of an effective symbiotic interaction between this β-rhizobial strain and the legume P. vulgaris [23]
To gain a better understanding of the molecules and metabolic pathways relevant during the P. vulgaris–P. phymatum symbiosis, we carried out two experiments: we first compared the metabolite levels measured in P. vulgaris nodules infected with

Summary

Introduction

Paraburkholderia phymatum STM815T is a nitrogen-fixing soil bacterium that belongs to the β-proteobacteria (β-rhizobia); it is able to induce nodules on the roots of Mimosoid and Papilionoid legumes [1]. Able to respond to specific flavonoid compounds secreted by the roots of compatible legume plants. They colonize the plants’ roots, which leads to the formation of specialized root organs called nodules. Several studies have relied on functional genomics technologies like transcriptomics, proteomics and metabolomics, and comparative genomics [11,12,13,14,15,16,17,18,19,20]

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