The effect of interspecies transfer of Rhizobium host-specific nodulation genes on acidic polysaccharide structure and in situ binding by host lectin

Abstract

Host specificity in the Rhizobium-legume symbiosis is controlled in the bacterium by host specific nodulation (hsn) genes residing on its symbiotic plasmid. We have examined the structure of the major acidic heteropolysaccharide produced by recombinant hybrid strains of Rhizobium leguminosarum carrying cloned R. trifolii hsn genes with those produced by the parent donor and recipient strains. Alteration of the nod gene composition of R. leguminosarum strain 300 by introduction of an 8-kilobase set of hsn genes (nodFERL and nodMN) from R. trifolii strain ANU843, resulted in a hybrid strain which conferred efficient white clover infection and nodulation, production of the R. trifolii-type acidic polysaccharide, and an increased proportion of bacterial cells which bound to the white clover lectin, trifoliin A, in the external root environment. 1H NMR studies indicated that the structure of the polysaccharide from the hybrid recombinant differed from that of the R. leguminosarum strain 300 recipient in site and stoichiometry of acetate and stoichiometry of 3-hydroxybutyrate substituents. In contrast, the polysaccharide from a different hybrid recombinant strain containing only R. trifolii nodFERL genes had the acetylation pattern of the R. leguminosarum recipient but was substituted with 3-hydroxybutyrate at a level between that made by R. trifolii and R. leguminosarum. This latter recombinant strain displays sparse infection and nodulation of white clover roots. Immunofluorescence studies indicated that the R. leguminosarum recombinant strain containing the full complement of R. trifolii hsn genes (nodFERL and nodMN) gained the ability to interact with the excreted lectin, trifoliin A, in the white clover root environment, whereas the recombinant strain containing R. trifolii nodFERL only, was significantly attenuated in this cell-lectin interaction. These results indicate that the acylation pattern of the acidic polysaccharide synthesized by these hybrid recombinants of R. leguminosarum is influenced by the introduced hsn genes of R. trifolii and suggest that the acidic polysaccharide of R. trifolii and the interaction of these bacteria with the host lectin may contribute to host specificity in the white clover-R. trifolii symbiosis.