Members of the diazeniumdiolate class of natural compounds show potential for drug development because of their antifungal, antibacterial, antiviral, and antitumor activities. Yet, their biosynthesis has remained elusive to date. Here, we identify a gene cluster directing the biosynthesis of the diazeniumdiolate compound fragin in Burkholderia cenocepacia H111. We provide evidence that fragin is a metallophore and that metal chelation is the molecular basis of its antifungal activity. A subset of the fragin biosynthetic genes is involved in the synthesis of a previously undescribed cell-to-cell signal molecule, valdiazen. RNA-Seq analyses reveal that valdiazen controls fragin biosynthesis and affects the expression of more than 100 genes. Homologs of the valdiazen biosynthesis genes are found in various bacteria, suggesting that valdiazen-like compounds may constitute a new class of signal molecules. We use structural information, in silico prediction of enzymatic functions and biochemical data to propose a biosynthesis route for fragin and valdiazen.

Members of the genus Burkholderia are versatile bacteria capable of colonizing highly diverse environmental niches. In this study, we investigated the global response of the opportunistic pathogen Burkholderia cenocepacia H111 to nitrogen limitation at the transcript and protein expression levels. In addition to a classical response to nitrogen starvation, including the activation of glutamine synthetase, PII proteins, and the two-component regulatory system NtrBC, B. cenocepacia H111 also upregulated polyhydroxybutyrate (PHB) accumulation and exopolysaccharide (EPS) production in response to nitrogen shortage. A search for consensus sequences in promoter regions of nitrogen-responsive genes identified a 54 consensus sequence. The mapping of the 54 regulon as well as the characterization of a 54 mutant suggests an important role of 54 not only in control of nitrogen metabolism but also in the virulence of this organism.

Members of the genus Burkholderia (β-proteobacteria) have only recently been shown to be able to establish a nitrogen-fixing symbiosis with several legumes, which is why they are also referred to as β-rhizobia. Therefore, very little is known about the competitiveness of these species to nodulate different legume host plants. In this study, we tested the competitiveness of several Burkholderia type strains (B. diazotrophica, B. mimosarum, B. phymatum, B. sabiae, B. symbiotica and B. tuberum) to nodulate four legumes (Phaseolus vulgaris, Macroptilium atropurpureum, Vigna unguiculata and Mimosa pudica) under our closely defined growth conditions. The assessment of nodule occupancy of these species on different legume host plants revealed that B. phymatum was the most competitive strain in the three papilionoid legumes (bean, cowpea and siratro), while B. mimosarum outcompeted the other strains in mimosa. The analysis of phenotypes known to play a role in nodulation competitiveness (motility, exopolysaccharide production) and additional in vitro competition assays among β-rhizobial strains suggested that B. phymatum has the potential to be a very competitive legume symbiont.

Bradyrhizobium diazoefficiens is a nitrogen-fixing endosymbiont, which can grow inside root-nodule cells of the agriculturally important soybean and other host plants. Our previous studies described B. diazoefficiens host-specific global expression changes occurring during legume infection at the transcript and protein level. In order to further characterize nodule metabolism, we here determine by flow injection–time-of-flight mass spectrometry analysis the metabolome of (i) nodules and roots from four different B. diazoefficiens host plants; (ii) soybean nodules harvested at different time points during nodule development; and (iii) soybean nodules infected by two strains mutated in key genes for nitrogen fixation, respectively. Ribose (soybean), tartaric acid (mungbean), hydroxybutanoyloxybutanoate (siratro) and catechol (cowpea) were among the metabolites found to be specifically elevated in one of the respective host plants. While the level of C4-dicarboxylic acids decreased during soybean nodule development, we observed an accumulation of trehalose-phosphate at 21 days post infection (dpi). Moreover, nodules from non-nitrogen-fixing bacteroids (nifA and nifH mutants) showed specific metabolic alterations; these were also supported by independent transcriptomics data. The alterations included signs of nitrogen limitation in both mutants, and an increased level of a phytoalexin in nodules induced by the nifA mutant, suggesting that the tissue of these nodules exhibits defense and stress reactions.

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 papilionoid legumes. In contrast to the 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 performed an RNA-sequencing (RNA-seq) analysis 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 upregulated under nitrogen limitation, we found genes involved in exopolysaccharides production and in 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 upregulated during symbiosis, we identified—besides the nif gene cluster—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. The analysis of mutant strains for the 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.