Exo-oligosaccharides of Rhizobium sp. strain NGR234 are required for symbiosis with various legumes

Abstract

Rhizobia are nitrogen-fixing bacteria that establish endosymbiotic associations with legumes. Nodule formation depends on various bacterial carbohydrates, including lipopolysaccharides, K-antigens, and exopolysaccharides (EPS). An acidic EPS from Rhizobium sp. strain NGR234 consists of glucosyl (Glc), galactosyl (Gal), glucuronosyl (GlcA), and 4,6-pyruvylated galactosyl (PvGal) residues with beta-1,3, beta-1,4, beta-1,6, alpha-1,3, and alpha-1,4 glycoside linkages. Here we examined the role of NGR234 genes in the synthesis of EPS. Deletions within the exoF, exoL, exoP, exoQ, and exoY genes suppressed accumulation of EPS in bacterial supernatants, a finding that was confirmed by chemical analyses. The data suggest that the repeating subunits of EPS are assembled by an ExoQ/ExoP/ExoF-dependent mechanism, which is related to the Wzy polymerization system of group 1 capsular polysaccharides in Escherichia coli. Mutation of exoK (NGROmegaexoK), which encodes a putative glycanase, resulted in the absence of low-molecular-weight forms of EPS. Analysis of the extracellular carbohydrates revealed that NGROmegaexoK is unable to accumulate exo-oligosaccharides (EOSs), which are O-acetylated nonasaccharide subunits of EPS having the formula Gal(Glc)5(GlcA)2PvGal. When used as inoculants, both the exo-deficient mutants and NGROmegaexoK were unable to form nitrogen-fixing nodules on some hosts (e.g., Albizia lebbeck and Leucaena leucocephala), but they were able to form nitrogen-fixing nodules on other hosts (e.g., Vigna unguiculata). EOSs of the parent strain were biologically active at very low levels (yield in culture supernatants, approximately 50 microg per liter). Thus, NGR234 produces symbiotically active EOSs by enzymatic degradation of EPS, using the extracellular endo-beta-1,4-glycanase encoded by exoK (glycoside hydrolase family 16). We propose that the derived EOSs (and not EPS) are bacterial components that play a crucial role in nodule formation in various legumes.

FIG. 1.

(A) Repeating subunit of the acidic EPS from NGR234 (9). Additional acetyl groups are present at unidentified positions. (B) Genetic map of the exo cluster of pNGR234b (38). The exs genes and neighboring exoB gene are not shown. BamHI fragments were cloned into pBluescript II KS(+), yielding pExo8867, pExo9987, and pExo1888. Seven exo genes were mutated by insertion of Ω interposons at the restriction sites indicated. The pexoK plasmid is a pLAFR6 derivative carrying the 2.9-kb BamHI-ApaI fragment indicated. Computer analyses predicted that exoLAMON and exoFQZ form operons. Biochemical characteristics and symbiotic phenotypes of NGRΩexoK, NGRΩexoP, and NGRΩexoZ can be attributed to site-specific mutations.

FIG. 2.

Phenotypes of Exo⁻ mutants. (A) Parent strain NGR234 and Exo⁻ mutants were grown on GMS agar plates containing 5% (wt/vol) sucrose and Congo red. NGRΩexoF (ΩF), NGRΩexoL (ΩL), NGRΩexoP (ΩP), NGRΩexoQ (ΩQ), and NGRΩexoY (ΩY) formed nonmucoid (“dry”) streaks and absorbed Congo red. NGR234 (234), NGRΩexoK (ΩK), and NGRΩexoZ (ΩZ) formed mucoid colonies. (B) Typical nitrogen-fixing nodule of L. leucocephala cv. Peru induced by NGR234 and harvested 92 days postinoculation. Nodules of L. leucocephala are often branched at this stage. (C) L. leucocephala cultivated in Magenta jars and inoculated with NGR234, as well as NGRΩexoF, NGRΩexoK, NGRΩexoL, NGRΩexoP, NGRΩexoQ, NGRΩexoY, and NGRΩexoZ. Plants were photographed 61 days postinoculation. Only nodules containing NGR234 and NGRΩexoZ fixed nitrogen. (D) Pseudonodules of L. leucocephala induced by NGRΩexoK expressing the gusA gene (42 days postinoculation; Fix⁻ phenotype). Bacteria colonized the surface of pseudonodules, whereas GUS staining was not detected within pseudonodules. (E) Nitrogen-fixing nodule induced by a NGR234 derivative expressing the gusA gene (59 days postinoculation; Fix⁺ phenotype). Nodulated roots were stained for GUS activity. (F) Typical nodule of A. lebbek containing nitrogen-fixing bacteroids of NGR234 (87 days postinoculation; Fix⁺ phenotype). (G) Pseudonodules of A. lebbek induced by NGRΩexoK (87 days postinoculation; Fix⁻ phenotype). Bars = 1 mm.

FIG. 3.

Representative GC-MS analysis of purified EPS (HMW forms). (A) Glycosyl composition analysis: total-ion chromatogram of the TMS-α/β-methyl glycosides derived from EPS (NGR234). Peak 1, glucuronic acid; peak 2, galactose; peak 3, glucose; peak 4, glucose and 4,6-pyruvate acetal of galactose coelute; peak 5, 4,6-pyruvate acetal of galactose; peak S, internal standard. Unlabeled peaks are components arising from the culture medium. (B) Glycosyl linkage analysis: total-ion chromatogram of the partially methylated alditol acetate derivatives prepared from NGR234. For an explanation of abbreviations see Table 4.

FIG. 4.

Characterization of EOSs. (A) Purification steps and yield of HMW forms of EPS and EOSs of NGR234. Bacterial supernatants were precipitated with ethanol in a stepwise fashion. EOSs were applied to a DEAE-Sephadex A-25 column in 25 mM Tris-HCl (pH 7.6) and eluted with 100 mM NaCl added to the same buffer. The final purification step was SEC on a Sephadex G-75 column. (B) SEC (Sephadex G-75) chromatography of EOSs. Fractions (1.5 ml) were collected, and the carbohydrates were analyzed using the anthrone method (absorbance at 620 nm). EOSs produced by NGR234 eluted in fractions 40 to 44. (C) Effect of NaBH₄ reduction. HMW forms of EPS and EOSs were subjected to NaBH₄ reduction and complete acid hydrolysis. Glc, Gal, and the resulting galactitol were analyzed by Dionex chromatography. Uronic acids were not analyzed. Chromatogram a shows the results for Glc and Gal standards. As shown in chromatogram b, purified HMW forms of EPS from NGR234 resulted in formation of Glc, PvGal, and Gal. PvGal and Gal coeluted [peak (Pv)Gal]. The ratio of Glc to (Pv)Gal was 5:2, as determined by integration of peak areas. As shown in chromatogram c, the Gal standard treated with NaBH₄ was completely converted to galactitol. Chromatogram d shows that NaBH₄ reduction of EOSs from NGR234 resulted in the formation of Glc, PvGal, and galactitol (PvGal/galactitol ratio, 1:1).

FIG. 5.

Positive-ion MALDI-TOF mass spectrum of the purified EOS fraction derived from NGR234. Two major ion families were observed, corresponding to the nonasaccharide and octasaccharide subunits. All ions were sodiated and are listed in Table 3 along with their proposed formulas and the predicted ions. For an explanation of abbreviations see Table 3.

FIG. 6.

Plant growth and nodulation of various legumes inoculated with NGR234 or NGRΩexoK (ΩexoK). At the time of harvest, the following data were acquired for each plant: total plant biomass (dry weight) (A), nodule dry weight (biomass of nodules and pseudonodules) (B), number of fully developed nodules (C), and number of pseudonodules (D). The data are means ± standard errors (n = 5 for V. unguiculata; n = 6 for A. procera and K. rubicunda; n = 7 for A. lebbeck, C. fasciculata, L. leucocephala, and S. tomentosa). The asterisks indicate statistically significant differences (P < 0.05, as determined by the Kruskal-Wallis test) between plants inoculated with NGR234 and NGRΩexoK. Abbreviations: Al, A. lebbeck 69 days postinoculation (Fig. 2F and 2G); Ap, A. procera 77 days postinoculation; Cf, C. fasciculata 69 days postinoculation; Kr, K. rubicunda 77 days postinoculation; Ll, L. leucocephala cv. Cunninghami 80 days postinoculation; St, S. tomentosa 101 days postinoculation; Vu, V. unguiculata 34 days postinoculation; DW, dry weight.