Rhizobium meliloti mutants with decreased DAHP synthase activity are sensitive to exogenous tryptophan and phenylalanine and form ineffective nodules.

Abstract

We isolated two Tn5-generated mutants of Rhizobium meliloti whose growth was inhibited by rich medium or by exogenous tryptophan or phenylalanine. These mutants, Rm7479 and Rm7480, belonged to the same genetic complementation group. The mutant locus could not be found on either indigenous megaplasmid but was localized on the chromosome. The mutants formed ineffective nodules on alfalfa plants. They invaded nodules within infection threads and were released into plant cells enclosed within peribacteroid membranes, but once released into the plant cells they failed to differentiate into mature bacteroids. The mutants demonstrated a decrease in total 2-keto-3-deoxy-D-arabino-heptonic acid 7-phosphate synthase (DAHP synthase) activity, which is the first committed step in aromatic biosynthesis. Wild-type genes were isolated that complemented in one case or suppressed in another case, all three mutant phenotypes: growth on rich medium, symbiotic effectiveness, and DAHP synthase activity. Each mutant strain gave rise to linked second-site suppressor mutations that restored growth on rich medium. The suppressor mutants showed restoration of near wild-type DAHP synthase levels. One of the suppressor strains restored effective symbiosis while the other did not. Genetic complementation experiments showed that growth on rich medium, DAHP synthase activity, and effective symbiosis were all affected by the same genetic lesion. These results suggest that normal flux of metabolites through the aromatic biosynthesis pathway is essential for bacteroid development.