Project description:Paraburkholderia phymatum belongs to the β-subclass of proteobacteria. It has recently been shown to be able to nodulate and fix nitrogen in symbiosis with several mimosoid and papillionoid legumes. In contrast to symbiosis of legumes with α-proteobacteria, very little is known about the molecular determinants underlying the successful establishment of this mutualistic relationship with β-proteobacteria. In this study, we analyzed RNA-seq data of free-living P. phymatum growing under nitrogen replete and limited conditions, the latter partially mimicking the situation in nitrogen deprived soils. Among the genes up-regulated under nitrogen limitation, we found genes involved in exopolysaccharide production and motility, two traits relevant for plant root infection. Next, RNA-seq data of P. phymatum grown under free-living conditions and from symbiotic root nodules of Phaseolus vulgaris (common bean) were generated and compared. Among the genes highly up-regulated during symbiosis, we identified an operon encoding a potential cytochrome o ubiquinol oxidase (Bphy_3646-49). Bean root nodules induced by a cyoB mutant strain showed reduced nitrogenase and nitrogen fixation abilities suggesting an important role of the cytochrome for respiration inside the nodule. Analysis of mutant strains for RNA polymerase transcription factor rpoN (σ54) and its activator NifA indicated that – similar to the situation in α-rhizobia – P. phymatum RpoN and NifA are key regulators during symbiosis with P. vulgaris.
Project description:Paraburkholderia phymatum is a beta-proteobacterium, which lives in the soil and is able to enter nitrogen-fixing symbiosis with different legumes. The biological nitrogen fixation (BNF) process is of great ecological and agronomic importance. We previously showed that the expression of the key P. phymatum BNF enzyme – the nitrogenase –is regulated by the sigma factor σ54 (or RpoN) inside root nodules. This study focused on identifying the σ54 regulon of P. phymatum grown in nitrogen limited conditions using RNA-Sequencing. Among the genes significantly down-regulated in absence of σ54 we found those coding for a C4-dicarboxylate transport system (Bphy_0225-27), a flagellar biosynthesis cluster (Bphy_2926-64) and one of the two type 6 secretion system (T6SS-b) present in P. phymatum genome (Bphy_5978-97). Indeed, the σ54 mutant was unable to grow on C4 dicarboxylates (fumarate, malate and succinate) as the sole carbon source and was less motile compared to the wild-type strain. Both defects were complemented by adding rpoN in trans. Additionally, using reporter fusions we confirmed that T6SS-b expression is regulated by σ54. Finally, a σ54 mutant was less competitive than its parental strain against P. diazotrophica, suggesting a role of σ54 in controlling interbacterial competition.
Project description:RpoN (σ54) 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 σ54 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.
Project description:time-course salt stress experiment of model legume Medicago truncatula roots using Affymetrix Medicago Array, aimed to dig some useful gene for improve salt resistance for legumes and other crops
