Further studies of the role of cyclic beta-glucans in symbiosis. An NdvC mutant of Bradyrhizobium japonicum synthesizes cyclodecakis-(1-->3)-beta-glucosyl

Abstract

The cyclic beta-(1-->3),beta-(1-->6)-D-glucan synthesis locus of Bradyrhizobium japonicum is composed of at least two genes, ndvB and ndvC. Mutation in either gene affects glucan synthesis, as well as the ability of the bacterium to establish a successful symbiotic interaction with the legume host soybean (Glycine max). B. japonicum strain AB-14 (ndvB::Tn5) does not synthesize beta-glucans, and strain AB-1 (ndvC::Tn5) synthesizes a cyclic beta-glucan lacking beta-(1-->6)-glycosidic bonds. We determined that the structure of the glucan synthesized by strain AB-1 is cyclodecakis-(1-->3)-beta-D-glucosyl, a cyclic beta-(1-->3)-linked decasaccharide in which one of the residues is substituted in the 6 position with beta-laminaribiose. Cyclodecakis-(1-->3)-beta-D-glucosyl did not suppress the fungal beta-glucan-induced plant defense response in soybean cotyledons and had much lower affinity for the putative membrane receptor protein than cyclic beta-(1-->3),beta-(1-->6)-glucans produced by wild-type B. japonicum. This is consistent with the hypothesis presented previously that the wild-type cyclic beta-glucans may function as suppressors of a host defense response.

Figure 1

Fast-atom bombardment mass ionization spectrum of glucans from B. japonicum ndvC strain AB-1. The m/z values are reported as the nominal masses of the molecular ions plus Na (M+23). degree of polymerization₁₁ = 1805; degree of polymerization₁₂ = 1968.

Figure 2

¹³C-NMR spectra (100 MHz) of glucans from B. japonicum ndvC strain AB-1 and from S. meliloti TY7 (ndvB) carrying the glucan locus from B. japonicum on cosmid clone p5D3. The assignment of each carbon resonance is indicated.

Figure 3

Proposed structure of the β-(1→3) cyclic glucan, cyclodecakis-(1→3)-β-d-glucosyl, from B. japonicum ndvC mutant AB-1.

Figure 4

Profile of LPS after SDS-PAGE visualized by silver staining. B. japonicum strains AB-1 (lane 1), AB-14 (lane 2), and USDA 110 (lane 3).

Figure 5

Displacement of ¹²⁵I-labeled HG-APEA by increasing concentrations of B. japonicum cyclic glucans from solubilized soybean glucan-binding proteins. Maximal HG-APEA binding was set to 100%, representing an average value of 0.36 pmol mg⁻¹ protein. Independent experiments are denoted by different symbols. Cyclic β-(1→3),(1→6)-glucans from B. japonicum USDA 110 (•, ○) or cyclodecakis-(1→3)-β-d-glucosyl from strain AB-1 (ndvC) (▪, □). Values for half-maximal displacement were calculated by nonlinear regression using the Marquardt-Levenberg algorithm (see Methods).

Figure 6

Effect of increasing concentrations of cyclic glucans of B. japonicum on phytoalexin accumulation in soybean cotyledons induced by a fixed concentration of fungal β-(1→3),(1→6)-branched glucans (2 μg mL⁻¹, set to 100%). The average value of A/A_max at this concentration of fungal glucan alone was 0.81. Cyclic β-(1→3),(1→6)-glucans from B. japonicum USDA 110 (•, ○) or cyclodecakis-(1→3)-β-d-glucosyl from strain AB-1 (ndvC) (▪). Independent experiments are denoted by different symbols.