EXOB MUTANTS OF BRADYRHIZOBIUM-JAPONICUM WITH REDUCED COMPETITIVENESS FOR NODULATION OF GLYCINE-MAX

Exopolysaccharide (EPS) mutants of Bradyrhizobium japonicum defective within a DNA region homologous to the Rhizobium meliloti exoB gene were constructed. Using an interspecies complementation approach, two overlapping cosmid clones of B. japonicum DNA were isolated. A 9.1-kb EcoRI subclone common to both cosmids was found to restore the ability of the exoB mutant R. meliloti H36, to induce effective nodules on alfalfa plants, and to form fluorescent colonies on agar media containing cellufluor white. Km(r) deletion as well as insertion derivatives of this fragment were introduced into B. japonicum 110spc4 by marker exchange. The resulting deletion mutants DELTAP5, DELTAP6 (4.5 kb each), and DELTAP22 (2.1 kb) were designated exoB mutants, because they lacked UDP-glucose 4'-epimerase activity, and the deleted regions hybridized with an exoB DNA probe of R. meliloti. The mutants had a nonmucoid colony morphology. In contrast to the wild-type EPS, no galactose could be detected in the residual EPS produced by the mutant strains, indicating an altered EPS composition. The mutant strains exhibited a wild-type lipopolysaccharide pattern on polyacrylamide gels. Although the mutants induced effective nodules on soybean, the early stages of the symbiotic interaction were disturbed. Nodulation was delayed by about 5 days, and the mutants exhibited a greatly reduced competitiveness. When the mutants DELTAP5 or DELTAP22 were coinoculated together with the parent strain 110 at similar titers, almost all of the nodules were occupied by the wild type. An at least 100-fold excess of DELTAP5 or DELTAP22 cells was necessary to obtain half of the nodules occupied by the mutant strains. This effect is not due to the presence of the aph gene in these mutants, as revealed by the unaffected competitiveness of the insertion mutant P29. The reduced competitiveness of B. japonicum exo mutants for nodulation of soybean clearly demonstrates that exopolysaccharides of B. japonicum carry out important functions during the early stages of the symbiotic interaction.