Exopolysaccharide Characterization of Rhizobium favelukesii LPU83 and Its Role in the Symbiosis With Alfalfa

Abstract

One of the greatest inputs of available nitrogen into the biosphere occurs through the biological N₂-fixation to ammonium as result of the symbiosis between rhizobia and leguminous plants. These interactions allow increased crop yields on nitrogen-poor soils. Exopolysaccharides (EPS) are key components for the establishment of an effective symbiosis between alfalfa and Ensifer meliloti, as bacteria that lack EPS are unable to infect the host plants. Rhizobium favelukesii LPU83 is an acid-tolerant rhizobia strain capable of nodulating alfalfa but inefficient to fix nitrogen. Aiming to identify the molecular determinants that allow R. favelukesii to infect plants, we studied its EPS biosynthesis. LPU83 produces an EPS I identical to the one present in E. meliloti, but the organization of the genes involved in its synthesis is different. The main gene cluster needed for the synthesis of EPS I in E. meliloti, is split into three different sections in R. favelukesii, which probably arose by a recent event of horizontal gene transfer. A R. favelukesii strain devoided of all the genes needed for the synthesis of EPS I is still able to infect and nodulate alfalfa, suggesting that attention should be directed to other molecules involved in the development of the symbiosis.

Keywords: alfalfa; exopolysaccharide; nitrogen fixation; rhizobia; symbiosis.

FIGURE 1

Genetic organization of the EPS I and APS genes. The E. meliloti pSymB figure (middle) shows the organization of the cluster of genes involved in APS (left) and EPS I (right) synthesis. The upper box shows the distribution of orthologs to EPS I production genes, distributed among R. favelukesii LPU83 chromosome. The bottom box shows the distribution of orthologs to APS (left) and EPS I (right) production genes, distributed among plasmids pLPU83d and pLPU83a, respectively. Numbers at the beginning and at the end of each cluster indicate the position of each gene. Orthologs are marked and connected with similar colors. Percentages in bold stand for amino acidic identity of the proteins. Percentages below bold-numbers stand for query coverage. All the comparisons were made using E. meliloti 2011 exo genes as query. The striped lines within gray boxes indicate the deletions made in the exo cluster of the chromosome and the plasmid.

FIGURE 2

Phylogenetic analyses of EPS I genes of LPU83. Phylogenetic tree based on (A) ExoB (only in the chromosome of LPU83), (B) ExoH (in the chromosome of LPU83 and pLPU83a), (C) ExoY (only in the chromosome of LPU83), and (D) ExoV (only in the plasmid pLPU83a). Analyses were conducted by means of the Maximum Likelihood method. Support values (SH like × 100) greater than 50 are indicated at the nodes. Bars indicate substitution/site.

FIGURE 3

Composition and structural analyses of EPS. (A) Separation of monosaccharides obtained by acid hydrolysis of cetylpyridinium chloride (CPC) and ethanol precipitated culture supernatant of E. meliloti 2011 and R. favelukesii LPU83. The monosaccharides were separated by high-performance anion-exchange chromatography with pulsed amperometric detection on a Carbo Pac PA1 column. Only the relevant parts of the chromatograms are shown. (B) ¹H-NMR spectra of isolated acidic exopolysaccharides (EPS) from R. favelukesii LPU83 and E. meliloti 2011. The singlets at 2.1 and 2.7 ppm represent the methyl protons from pyruvate and acetyl groups, respectively. The triplets at 3.1 and 3.25 ppm arise from the methylene protons of the succinyl group, while the complex region between 3.9 and 5.5 ppm represents signals from the ring protons of the carbohydrate constituents. (C) Structure of E. meliloti EPS I (succinoglycan). Diagram showing the octasaccharide repeating unit of succinoglycan (OAc, acyl group; Pyr, pyruvyl group; OSuc, succinyl group).

FIGURE 4

Characterization of EPS production of LPU83 mutants. (A) EPS quantification by the anthrone method. The EPS amount normalized per mg of total proteins is shown for each evaluated strain of E. meliloti and R. favelukesii. (B) Phenotypic analyses of the LPU83 mutants. EPS production was evaluated in YEM with Congo Red (first line) and in YEM with calcofluor under visible light and under UV light (lines below). The presence of EPS I is evidenced in the last condition due to its fluorescence. The statistical analysis was done by a t-test using three independent biological replicates. Representative pictures of at least three different experiments are shown.

FIGURE 5

Analysis of the nodulation of LPU83 EPS I mutant strains. M. sativa plants infected by the indicated strains were harvested at 4 weeks post-inoculation. In the top panel, a representative plant was photographed. In second panel, zoom on the nodules of each plant was made. The number of nodules per plant were counted and weighted. Nodules were surfaced-sterilized, then crushed in sterile isotonic solution followed by plating on TY with the corresponding antibiotics and after 2 days CFU were counted. The shoot dry weight per plant was measured. Non-inoculated roots did not show nodules and the dry weight per plant was 3.98 ± 0.78^b. Results were statistically analyzed by the ANOVA and least significant difference tests (Box et al., 1978). Values followed by different letters differed significantly with p < 0.05.

FIGURE 6

Morphology and occupancy of M. sativa nodules generated by EPS-I mutants of LPU83. Plants infected by E. meliloti 2011 and the indicated EPS I mutant strains of R. favelukesii LPU83 were harvested at 4 weeks post-inoculation. Nodule sections of 60 μm were obtained by means of a vibratome, and then stained with Syto 9 (green fluorescence that indicates living cells) and Propidium Iodide (red fluorescence that indicates cells with a damage in the membrane, i.e., dead). Light (A–F) and fluorescence (G–L) micrographs of full nodule slices were acquired with a confocal microscope. The photographs are representative of nodules of different plants experiments.