EXOB MUTANTS OF BRADYRHIZOBIUM-JAPONICUM WITH REDUCED COMPETITIVENESSFOR NODULATION OF GLYCINE-MAX

Exopolysaccharide (EPS) mutants of Bradyrhizobium japonicum defective within a DNA region homologous to the Rhizobium meliloti exoB gene wer e constructed. Using an interspecies complementation approach, two ove rlapping cosmid clones of B. japonicum DNA were isolated. A 9.1-kb Eco RI subclone common to both cosmids was found to restore the ability of the exoB mutant R. meliloti H36, to induce effective nodules on alfal fa plants, and to form fluorescent colonies on agar media containing c ellufluor white. Km(r) deletion as well as insertion derivatives of th is fragment were introduced into B. japonicum 110spc4 by marker exchan ge. The resulting deletion mutants DELTAP5, DELTAP6 (4.5 kb each), and DELTAP22 (2.1 kb) were designated exoB mutants, because they lacked U DP-glucose 4'-epimerase activity, and the deleted regions hybridized w ith an exoB DNA probe of R. meliloti. The mutants had a nonmucoid colo ny morphology. In contrast to the wild-type EPS, no galactose could be detected in the residual EPS produced by the mutant strains, indicati ng an altered EPS composition. The mutant strains exhibited a wild-typ e lipopolysaccharide pattern on polyacrylamide gels. Although the muta nts induced effective nodules on soybean, the early stages of the symb iotic interaction were disturbed. Nodulation was delayed by about 5 da ys, and the mutants exhibited a greatly reduced competitiveness. When the mutants DELTAP5 or DELTAP22 were coinoculated together with the pa rent strain 110 at similar titers, almost all of the nodules were occu pied by the wild type. An at least 100-fold excess of DELTAP5 or DELTA P22 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 th e insertion mutant P29. The reduced competitiveness of B. japonicum ex o mutants for nodulation of soybean clearly demonstrates that exopolys accharides of B. japonicum carry out important functions during the ea rly stages of the symbiotic interaction.