Role of Bradyrhizobium japonicum exopolysaccharide galactosides in symbiosis with soybean

J.I. QUELAS; A. CASABUONO; M.J. ALTHABEGOITI; E. MONGIARDINI; J. PEREZ-GIMENEZ; S. LOPEZ-GARCIA; A. COUTO; A. LODEIRO

Cairns, Australia

Simposio; 12th International Symposium on Microbial Ecology; 2008

International Society of Microbial Ecology

Background and aims: Bacterial surface polysaccharides play important roles in symbiotic interactions. In particular, exopolysaccharides (EPS) from Bradyrhizobium japonicum are required for rhizobial association to soybean roots. However, detailed knowledge on EPS structure-function relationship is scarce. Here we addressed the study of the role of EPS galactosides in symbiosis.Methods: DNA regions blr2382-blr2383 (UDP-glucuronic acid epimerase-UDP-glucose-6-dehydrogenase) and blr7578 (UDP glucose-4-epimerase) were replaced by double crossing-over with antibiotic-resistance cassettes in B. japonicum USDA 110, giving strains LP 3010 and LP 3013 respectively, or the double mutant LP 3017.Polysaccharides were quantified with anthrone, and compositions analyzed by HPAEC-PAD.Lipopolysaccharides were extracted with phenol-water 55:45 and observed in polyacrylamide geles.Nodules ultrastructure was analyzed by electron microscopy.Results and conclusions Deletions were checked by PCR employing primer pairs that hybridized internally and externally to the inserted cassettes. Absences of galacturonic acid (GalA) in LP 3010 EPS, galactose (Gal) in LP 3013 EPS, or both in LP 3017 EPS were corroborated. All three strains presented complete lipopolysaccharide molecules. EPS contents ranged 6-14% wild type in all mutants.Both single mutants were defective in nodulation. The GalA-lacking LP 3010, gave red and macroscopically normal nodules, but with diffuse peribacteroidal membrane. Soybean plants co-inoculated with LP 3010 and USDA 110 at equal concentration gave 10 % nodules occupied by LP 3010. By contrast, both the Gal-lacking LP 3013 and the double mutant LP 3017 gave white, empty nodules.Thus, each galactoside has a different symbiotic role in B. japonicum EPS