The acidic exopolysaccharide is required for the establishment of symbiosis between the nitrogen-fixing bacterium Rhizobium leguminosarum bv. trifolii and clover. Here, we describe RosR protein from R. leguminosarum bv. trifolii 24.2, a homolog of transcriptional regulators belonging to the family of Ros/MucR proteins. R. leguminosarum bv. trifolii RosR possesses a characteristic Cys2His2 type zinc-finger motif in its C-terminal domain. Recombinant (His)6RosR binds to an RosR-box sequence located up-stream of rosR. Deletion analysis of the rosR upstream region resulted in identification of two -35 to -10 promoter sequences, two conserved inverted palindromic pentamers that resemble the cAMP-CRP binding site of Escherichia coli, inverted repeats identified as a RosR binding site, and other regulatory sequence motifs. When assayed in E. coli, a transcriptional fusion of the cAMP-CRP binding site containing the rosR upstream region and lacZ gene was moderately responsive to glucose. The sensitivity of the rosR promoter to glucose was not observed in E. coli deltacyaA. A rosR frame-shift mutant of R. leguminosarum bv. trifolii formed dry, wrinkled colonies and induced nodules on clover, but did not fix nitrogen. In the rosR mutant, transcription of pssA-lacZ fusion was decreased, indicating positive regulation of the pssA gene by RosR. Multiple copies of rosR in R. leguminosarum bv. trifolii 24.2 increased exopolysaccharide production.
Specific complex interactions between soil bacteria belonging to Rhizobium, Sinorhizobium, Mesorhizobium, Phylorhizobium, Bradyrhizobium and Azorhizobium commonly known as rhizobia, and their host leguminous plants result in development of root nodules. Nodules are new organs that consist mainly of plant cells infected with bacteroids that provide the host plant with fixed nitrogen. Proper nodule development requires the synthesis and perception of signal molecules such as lipochitooligosaccharides, called Nod factors that are important for induction of nodule development. Bacterial surface polysaccharides are also crucial for establishment of successful symbiosis with legumes. Sugar polymers of rhizobia are composed of a number of different polysaccharides, such as lipopolysaccharides (LPS), capsular polysaccharides (CPS or K-antigens), neutral β-1, 2-glucans and acidic extracellular polysaccharides (EPS). Despite extensive research, the molecular function of the surface polysaccharides in symbiosis remains unclear.This review focuses on exopolysaccharides that are especially important for the invasion that leads to formation of indetermined (with persistent meristem) type of nodules on legumes such as clover, vetch, peas or alfalfa. The significance of EPS synthesis in symbiotic interactions of Rhizobium leguminosarum with clover is especially noticed. Accumulating data suggest that exopolysaccharides may be involved in invasion and nodule development, bacterial release from infection threads, bacteroid development, suppression of plant defense response and protection against plant antimicrobial compounds. Rhizobial exopolysaccharides are species-specific heteropolysaccharide polymers composed of common sugars that are substituted with non-carbohydrate residues. Synthesis of repeating units of exopolysaccharide, their modification, polymerization and export to the cell surface is controlled by clusters of genes, named exo/ exs, exp or pss that are localized on rhizobial megaplasmids or chromosome. The function of these genes was identified by isolation and characterization of several mutants disabled in exopolysaccharide synthesis. The effect of exopolysaccharide deficiency on nodule development has been extensively studied. Production of exopolysaccharides is influenced by a complex network of environmental factors such as phosphate, nitrogen or sulphur. There is a strong suggestion that production of a variety of symbiotically active polysaccharides may allow rhizobial strains to adapt to changing environmental conditions and interact efficiently with legumes.
Rhizobium leguminosarum bv. trifolii exopolysaccharide (EPS) plays an important role in determining symbiotic competence. The pssA gene encoding the first glucosyl-IP-transferase and rosR encoding a positive transcriptional regulator are key genes involved in the biosynthesis and regulation of EPS production. Mutation in pssA resulted in deficiency in EPS production and rosR mutation substantially decreased the amount of EPS. Both mutants induced nodules but the bacteria were unable to fix nitrogen. Defective functions of pssA and rosR mutants were fully restored by wild type copies of the respective genes. Introduction of multiple rosR and pssA gene copies on the plasmid vector pBBR1MCS-2 into five R. leguminosarum bv. trifolii nodule isolates resulted in significantly increased growth rates, EPS production and the number of nodules on clover roots. Increase in fresh and dry shoot mass of clovers and nodule occupation was also statistically significant. Interestingly, additional copies of pssA but particularly rosR gene, increased strains' competitiveness in relation to the wild type parental strains nearly twofold. Overall, experimental evidence is provided that increased amount of EPS beneficially affects R. leguminosarum bv. trifolii competitiveness and symbiosis with clover.
Of 105 rhizobial isolates obtained from nodules of commonly cultivated legumes, we selected 19 strains on the basis of a high rate of symbiotic plant growth promotion. Individual strains within the species Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viciae, and Rhizobium etli displayed variation not only in plasmid sizes and numbers but also in the chromosomal 16S-23S internal transcribed spacer. The strains were tagged with gusA gene and their competitiveness was examined in relation to an indigenous population of rhizobia under greenhouse conditions. A group of 9 strains was thus isolated that were competitive in relation to native rhizobia in pot experiments. Nineteen selected competitive and uncompetitive strains were examined with respect to their ability to utilize various carbon and energy sources by means of commercial Biolog GN2 microplate test. The ability of the selected strains to metabolize a wide range of nutrients differed markedly and the competitive strains were able to utilize more carbon and energy sources than uncompetitive ones. A major difference concerned the utilization of amino and organic acids, which were metabolized by most of the competitive and only a few uncompetitive strains, whereas sugars and their derivatives were commonly utilized by both groups of strains. A statistically significant correlation between the ability to metabolize a broad range of substrates and nodulation competitiveness was found, indicating that metabolic properties may be an essential trait in determining the competitiveness of rhizobia.
Flavonoids play a crucial role as signal molecules in promoting the formation of nodules by symbiotic bacteria commonly known as rhizobia. The early interaction between flavonoids and NodD regulatory protein activates nod gene transcription and the synthesis of Nod factor that initiates nodule primordium. In this study, we assessed response to flavonoids as factors influencing competitiveness of rhizobia and their symbiotic activity. Rhizobium leguminosarum nodule isolates belonging to three biovars, trifolii, viciae and phaseoli characterized earlier as competitive or uncompetitive relative to native rhizobia, were used. Investigating nodA promoter induction using plasmid lacZ fusion, we found that competitive strains more readily responded to a wide range of synthetic flavonoids and seed exudates in comparison to uncompetitive strains, albeit some exceptions were noticed. Of all the synthetic flavonoids and seed exudates studied, naringenin, hespertin and clover and vetch exudates were the most effective inducers of nodA promoter in competitive strains. Only one of the nine examined uncompetitive strains was highly induced by clover seed exudate. Subsequently, the effect of preinduction of R. leguminosarum bv. trifolii with clover exudate was assessed. Out of 18 pre-activated strains, nine strains (including competitive ones) increased clover wet mass of shoots and nodule number when used as inoculants. Our results demonstrate a plausible approach of isolating and characterizing flavonoid-responsive field isolates that could be further developed into relevant legume inoculants.
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