Project description:Sinorhizobium fredii HH103 is a fast-growing rhizobial strain that is able to nodulate legumes that develop determinate nodules, e.g., soybean, and legumes that form nodules of the indeterminate type. Here we present the genome of HH103, which consists of one chromosome and five plasmids with a total size of 7.22 Mb.
Project description:Here we report that the structure of the Sinorhizobium fredii HH103 exopolysaccharide (EPS) is composed of glucose, galactose, glucuronic acid, pyruvic acid, in the ratios 5∶2∶2∶1 and is partially acetylated. A S. fredii HH103 exoA mutant (SVQ530), unable to produce EPS, not only forms nitrogen fixing nodules with soybean but also shows increased competitive capacity for nodule occupancy. Mutant SVQ530 is, however, less competitive to nodulate Vigna unguiculata. Biofilm formation was reduced in mutant SVQ530 but increased in an EPS overproducing mutant. Mutant SVQ530 was impaired in surface motility and showed higher osmosensitivity compared to its wild type strain in media containing 50 mM NaCl or 5% (w/v) sucrose. Neither S. fredii HH103 nor 41 other S. fredii strains were recognized by soybean lectin (SBL). S. fredii HH103 mutants affected in exopolysaccharides (EPS), lipopolysaccharides (LPS), cyclic glucans (CG) or capsular polysaccharides (KPS) were not significantly impaired in their soybean-root attachment capacity, suggesting that these surface polysaccharides might not be relevant in early attachment to soybean roots. These results also indicate that the molecular mechanisms involved in S. fredii attachment to soybean roots might be different to those operating in Bradyrhizobium japonicum.
Project description:Sinorhizobium fredii USDA257 and S. fredii USDA191 are fast-growing rhizobia that form nitrogen-fixing nodules on soybean roots. In contrast to USDA191, USDA257 exhibits cultivar specificity and can form nodules only on primitive soybean cultivars. In response to flavonoids released from soybean roots, these two rhizobia secrete nodulation outer proteins (Nop) to the extracellular milieu through a type III secretion system. In spite of the fact that Nops are known to regulate legume nodulation in a host-specific manner, very little is known about the differences in the compositions of Nops and surface appendages elaborated by USDA191 and USDA257. In this study we compared the Nop profiles of USDA191 and USDA257 by one-dimensional (1D) and 2D gel electrophoresis and identified several of these proteins by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and liquid chromatography-tandem MS (LC-MS/MS). Examination of the surface appendages elaborated by these two strains of soybean symbionts by transmission electron microscopy revealed distinct differences in their morphologies. Even though the flagella produced by USDA191 and USDA257 were similar in their morphologies, they differed in their flagellin composition. USDA257 pili resembled long thin filaments, while USDA191 pili were short, rod shaped, and much thinner than the flagella. 2D gel electrophoresis of pilus-like appendages of USDA191 and USDA257 followed by mass spectrometry resulted in the identification of several of the Nops along with some proteins previously undetected in these strains. Some of the newly identified proteins show homology to putative zinc protease and a LabA-like protein from Bradyrhizobium sp. ORS278, fimbrial type 4 assembly proteins from Ralstonia solanacearum, and the type III effector Hrp-dependent protein from Rhizobium leguminosarum bv. trifolii.
Project description:Here we announce the complete genome sequence of the symbiotic and nitrogen-fixing bacterium Sinorhizobium fredii USDA257. The genome shares a high degree of sequence similarity with the closely related broad-host-range strains S. fredii NGR234 and HH103. Most strikingly, the USDA257 genome encodes a wealth of secretory systems.
Project description:Quorum sensing (QS) is a bacterial cell-to-cell signaling mechanism that collectively regulates and synchronizes behaviors by means of small diffusible chemical molecules. In rhizobia, QS systems usually relies on the synthesis and detection of N-acyl-homoserine lactones (AHLs). In the model bacterium Sinorhizobium meliloti functions regulated by the QS systems TraI-TraR and SinI-SinR(-ExpR) include plasmid transfer, production of surface polysaccharides, motility, growth rate and nodulation. These systems are also present in other bacteria of the Sinorhizobium genus, with variations at the species and strain level. In Sinorhizobium fredii NGR234 phenotypes regulated by QS are plasmid transfer, growth rate, sedimentation, motility, biofilm formation, EPS production and copy number of the symbiotic plasmid (pSym). The analysis of the S. fredii HH103 genomes reveal also the presence of both QS systems. In this manuscript we characterized the QS systems of S. fredii HH103, determining that both TraI and SinI AHL-synthases proteins are responsible of the production of short- and long-chain AHLs, respectively, at very low and not physiological concentrations. Interestingly, the main HH103 luxR-type genes, expR and traR, are split into two ORFs, suggesting that in S. fredii HH103 the corresponding carboxy-terminal proteins, which contain the DNA-binding motives, may control target genes in an AHL-independent manner. The presence of a split traR gene is common in other S. fredii strains.
Project description:The tricarboxylic acid (TCA) cycle plays an important role in generating the energy required by bacteroids to fix atmospheric nitrogen. Citrate synthase is the first enzyme that controls the entry of carbon into the TCA cycle. We cloned and determined the nucleotide sequence of the gltA gene that encodes citrate synthase in Sinorhizobium fredii USDA257, a symbiont of soybeans (Glycine max [L.] Merr.) and several other legumes. The deduced citrate synthase protein has a molecular weight of 48,198 and exhibits sequence similarity to citrate synthases from several bacterial species, including Sinorhizobium meliloti and Rhizobium tropici. Southern blot analysis revealed that the fast-growing S. fredii strains and Rhizobium sp. strain NGR234 contained a single copy of the gene located in the bacterial chromosome. S. fredii USDA257 gltA mutant HBK-CS1, which had no detectable citrate synthase activity, had diminished nodulation capacity and produced ineffective nodules on soybean. Light and electron microscopy observations revealed that the nodules initiated by HBK-CS1 contained very few bacteroids. The infected cells contained large vacuoles and prominent starch grains. Within the vacuoles, membrane structures that appeared to be reminiscent of disintegrating bacteroids were detected. The citrate synthase mutant had altered cell surface characteristics and produced three times more exopolysaccarides than the wild type produced. A plasmid carrying the USDA257 gltA gene, when introduced into HBK-CS1, was able to restore all of the defects mentioned above. Our results demonstrate that a functional citrate synthase gene of S. fredii USDA257 is essential for efficient soybean nodulation and nitrogen fixation.
Project description:Populations of genetically identical Sinorhizobium fredii NGR234 cells differ significantly in their expression profiles of autoinducer (AI)-dependent and AI-independent genes. Promoter fusions of the NGR234 AI synthase genes traI and ngrI showed high levels of phenotypic heterogeneity during growth in TY medium on a single-cell level. However, adding very high concentrations of N-(3-oxooctanoyl-)-l-homoserine lactone resulted in a more homogeneous expression profile. Similarly, the lack of internally synthesized AIs in the background of the NGR234-ΔtraI or the NGR234-ΔngrI mutant resulted in a highly homogenous expression of the corresponding promoter fusions in the population. Expression studies with reporter fusions of the promoter regions of the quorum-quenching genes dlhR and qsdR1 and the type IV pilus gene cluster located on pNGR234b suggested that factors other than AI molecules affect NGR234 phenotypic heterogeneity. Further studies with root exudates and developing Arabidopsis thaliana seedlings provide the first evidence that plant root exudates have strong effects on the heterogeneity of AI synthase and quorum-quenching genes in NGR234. Therefore, plant-released octopine appears to play a key role in modulation of heterogeneous gene expression.