The induction mechanism of the flavonoid-responsive regulator FrrA.

Bradyrhizobium diazoefficiens, a bacterial symbiont of soybean and other leguminous plants, enters a nodulation-promoting genetic programme in the presence of host-produced flavonoids and related signalling compounds. Here, we describe the crystal structure of an isoflavonoid-responsive regulator (FrrA) from Bradyrhizobium, as well as cocrystal structures with inducing and noninducing ligands (genistein and naringenin, respectively). The structures reveal a TetR-like fold whose DNA-binding domain is capable of adopting a range of orientations. A single molecule of either genistein or naringenin is asymmetrically bound in a central cavity of the FrrA homodimer, mainly via C-H contacts to the π-system of the ligands. Strikingly, however, the interaction does not provoke any conformational changes in the repressor. Both the flexible positioning of the DNA-binding domain and the absence of structural change upon ligand binding are corroborated by small-angle X-ray scattering (SAXS) experiments in solution. Together with a model of the promoter-bound state of FrrA our results suggest that inducers act as a wedge, preventing the DNA-binding domains from moving close enough together to interact with successive positions of the major groove of the palindromic operator.

Nitrogen Assimilation and Transport by Ex Planta Nitrogen-Fixing Bradyrhizobium diazoefficiens Bacteroids Is Modulated by Oxygen, Bacteroid Density and l-Malate.

Symbiotic nitrogen fixation requires the transfer of fixed organic nitrogen compounds from the symbiotic bacteria to a host plant, yet the chemical nature of the compounds is in question. Bradyrhizobium diazoefficiens bacteroids were isolated anaerobically from soybean nodules and assayed at varying densities, varying partial pressures of oxygen, and varying levels of l-malate. Ammonium was released at low bacteroid densities and high partial pressures of oxygen, but was apparently taken up at high bacteroid densities and low partial pressures of oxygen in the presence of l-malate; these later conditions were optimal for amino acid excretion. The ratio of partial pressure of oxygen/bacteroid density of apparent ammonium uptake and of alanine excretion displayed an inverse relationship. Ammonium uptake, alanine and branch chain amino acid release were all dependent on the concentration of l-malate displaying similar K0.5 values of 0.5 mM demonstrating concerted regulation. The hyperbolic kinetics of ammonium uptake and amino acid excretion suggests transport via a membrane carrier and also suggested that transport was rate limiting. Glutamate uptake displayed exponential kinetics implying transport via a channel. The chemical nature of the compounds released were dependent upon bacteroid density, partial pressure of oxygen and concentration of l-malate demonstrating an integrated metabolism.

Microbiome signatures in neonatal central line associated bloodstream infections.

Neonates are at high risk for central line associated bloodstream infections (CLABSI). Biofilm formation is universal on indwelling catheters but why some biofilms seed the bloodstream to cause CLABSI is not clearly understood. With the objective to test the hypothesis that catheter biofilm microbiome in neonates with CLABSI differs than those without infection, we prospectively enrolled neonates (n = 30) with infected and uninfected indwelling central catheters. Catheters were collected at the time of removal, along with blood samples and skin swabs at the catheter insertion sites. Microbiomes of catheter biofilms, skin swabs and blood were evaluated by profiling the V4 region of the bacterial 16S rRNA gene using Illumina MiSeq sequencing platform. The microbial DNA load was higher from catheter biofilms of CLABSI patients without differences in alpha diversity when compared to that of the non-CLABSI neonates. Proteus and unclassified Staphylococcaceae were more abundant in infected catheter biofilms while Bradyrhizobium, Cloacibacterium, and Sphingomonas were more abundant in the uninfected catheters. A blood microbiome was detected in uninfected samples. The blood microbiome in CLABSI neonates clustered separately from the uninfected blood samples in beta diversity plots. We found that the microbiome signature in catheter biofilm and blood of neonates with CLABSI is different than the microbiomes of non-CLABSI neonates.

Classical Soybean (Glycine max (L.) Merr) Symbionts, Sinorhizobium fredii USDA191 and Bradyrhizobium diazoefficiens USDA110, Reveal Contrasting Symbiotic Phenotype on Pigeon Pea (Cajanus cajan (L.) Millsp).

Pigeon pea (Cajanus cajan (L.) Millspaugh) is cultivated widely in semiarid agricultural regions in over 90 countries around the world. This important legume can enter into symbiotic associations with a wide range of rhizobia including Bradyrhizobium and fast-growing rhizobia. In comparison with other major legumes such as soybean and common bean, only limited information is available on the symbiotic interaction of pigeon pea with rhizobia. In this study, we investigated the ability of two classical soybean symbionts-S. fredii USDA191 and B. diazoefficiens USDA110-and their type 3 secretion system (T3SS) mutants, to nodulate pigeon pea. Both S. fredii USDA191 and a T3SS mutant S. fredii RCB26 formed nitrogen-fixing nodules on pigeon pea. Inoculation of pigeon pea roots with B. diazoefficiens USDA110 and B. diazoefficiens Δ136 (a T3SS mutant) resulted in the formation of Fix- and Fix+ nodules, respectively. Light and transmission electron microscopy of Fix- nodules initiated by B. diazoefficiens USDA110 revealed the complete absence of rhizobia within these nodules. In contrast, Fix+ nodules formed by B. diazoefficiens Δ136 revealed a central region that was completely filled with rhizobia. Ultrastructural investigation revealed the presence of numerous bacteroids surrounded by peribacteroid membranes in the infected cells. Analysis of nodule proteins by one- and two-dimensional gel electrophoresis revealed that leghemoglobin was absent in B. diazoefficiens USDA110 nodules, while it was abundantly present in B. diazoefficiens Δ136 nodules. Results of competitive nodulation assays indicated that B. diazoefficiens Δ136 had greater competitiveness for nodulation on pigeon pea than did the wild type strain. Our results suggest that this T3SS mutant of B. diazoefficiens, due to its greater competitiveness and ability to form Fix+ nodules, could be exploited as a potential inoculant to boost pigeon pea productivity.

Transcriptional analysis of genes involved in competitive nodulation in Bradyrhizobium diazoefficiens at the presence of soybean root exudates.

Nodulation competition is a key factor that limits symbiotic nitrogen fixation between rhizobia and their host legumes. Soybean root exudates (SREs) are thought to act as signals that influence Bradyrhizobium ability to colonize roots and to survive in the rhizosphere, and thus they act as a key determinant of nodulation competitiveness. In order to find the competitiveness-related genes in B. diazoefficiens, the transcriptome of two SREs treated B. diazoefficiens with completely different nodulation abilities (B. diazoefficiens 4534 and B. diazoefficiens 4222) were sequenced and compared. In SREs treated strain 4534 (SREs-4534), 253 unigenes were up-regulated and 204 unigenes were down-regulated. In SREs treated strain 4534 (SREs-4222), the numbers of up- and down-regulated unigenes were 108 and 185, respectively. There were considerable differences between the SREs-4534 and SREs-4222 gene expression profiles. Some differentially expressed genes are associated with a two-component system (i.g., nodW, phyR-σEcfG), bacterial chemotaxis (i.g., cheA, unigene04832), ABC transport proteins (i.g., unigene02212), IAA (indole-3-acetic acid) metabolism (i.g., nthA, nthB), and metabolic fitness (i.g., put.), which may explain the higher nodulation competitiveness of B. diazoefficiens in the rhizosphere. Our results provide a comprehensive transcriptomic resource for SREs treated B. diazoefficiens and will facilitate further studies on competitiveness-related genes in B. diazoefficiens.

Metabolomic Profiling of Bradyrhizobium diazoefficiens-Induced Root Nodules Reveals Both Host Plant-Specific and Developmental Signatures.

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.

Mutualistic co-evolution of type III effector genes in Sinorhizobium fredii and Bradyrhizobium japonicum.

Two diametric paradigms have been proposed to model the molecular co-evolution of microbial mutualists and their eukaryotic hosts. In one, mutualist and host exhibit an antagonistic arms race and each partner evolves rapidly to maximize their own fitness from the interaction at potential expense of the other. In the opposing model, conflicts between mutualist and host are largely resolved and the interaction is characterized by evolutionary stasis. We tested these opposing frameworks in two lineages of mutualistic rhizobia, Sinorhizobium fredii and Bradyrhizobium japonicum. To examine genes demonstrably important for host-interactions we coupled the mining of genome sequences to a comprehensive functional screen for type III effector genes, which are necessary for many Gram-negative pathogens to infect their hosts. We demonstrate that the rhizobial type III effector genes exhibit a surprisingly high degree of conservation in content and sequence that is in contrast to those of a well characterized plant pathogenic species. This type III effector gene conservation is particularly striking in the context of the relatively high genome-wide diversity of rhizobia. The evolution of rhizobial type III effectors is inconsistent with the molecular arms race paradigm. Instead, our results reveal that these loci are relatively static in rhizobial lineages and suggest that fitness conflicts between rhizobia mutualists and their host plants have been largely resolved.

Low endotoxic activity of lipopolysaccharides isolated from Bradyrhizobium, Mesorhizobium, and Azospirillum strains.

The endotoxic activities of lipopolysaccharides (LPS) isolated from different strains of rhizobia and rhizobacteria (Bradyrhizobium, Mesorhizobium, and Azospirillum) were compared to those of Salmonella enterica sv. Typhimurium LPS. The biological activity of all the examined preparations, measured as Limulus lysate gelation, production of tumor necrosis factor (TNF), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6), and nitrogen oxide (NO) induction in human myelomonocytic cells (line THP-1), was considerably lower than that of the reference enterobacterial endotoxin. Among the rhizobial lipopolysaccharides, the activities of Mesorhizobium huakuii and Azospirillum lipoferum LPSs were higher than those of the LPS preparations from five strains of Bradyrhizobium. The weak endotoxic activity of the examined preparations was correlated with differences in lipid A structure compared to Salmonella.

Stimulation of adhesiveness, infectivity, and competitiveness for nodulation of Bradyrhizobium japonicum by its pretreatment with soybean seed lectin.

Soybean seed lectin stimulates adsorption of Bradyrhizobium japonicum to its host roots. Pretreatment of the rhizobia with soybean seed lectin for at least 6-12 h previous to their interaction with the plants was required to detect the stimulatory effect. This activity could be observed with as few as 1000 soybean seed lectin molecules per bacterium, and required specific carbohydrate binding. Infectivity and competitiveness for nodulation were also stimulated by preincubation of the rhizobia either with soybean seed meal extract or soybean seed lectin, the extract being more effective in enhancing competitiveness.