RHIZOBIUM-MELILOTI MUTANTS WITH DECREASED DAHP SYNTHASE ACTIVITY ARE SENSITIVE TO EXOGENOUS TRYPTOPHAN AND PHENYLALANINE AND FORM INEFFECTIVE NODULES

We isolated two Tn5-generated mutants of Rhizobium meliloti whose grow th was inhibited by rich medium or by exogenous tryptophan or phenylal anine. These mutants, Rm7479 and Rm7480, belonged to the same genetic complementation group. The mutant locus could not be found on either i ndigenous megaplasmid but was localized on the chromosome. The mutants formed ineffective nodules on alfalfa plants. They invaded nodules wi thin infection threads and were released into plant cells enclosed wit hin peribacteroid membranes, but once released into the plant cells th ey failed to differentiate into mature bacteroids. The mutants demonst rated a decrease in total 2-keto-3-deoxy-D-arabino-heptonic acid 7-pho sphate synthase (DAHP synthase) activity, which is the first committed step in aromatic biosynthesis. Wild-type genes were isolated that com plemented in one case or suppressed in another case, all three mutant phenotypes: growth on rich medium, symbiotic effectiveness, and DAHP s ynthase activity. Each mutant strain gave rise to linked second-site s uppressor mutations that restored growth on rich medium. The suppresso r mutants showed restoration of near wild-type DAHP synthase levels. O ne of the suppressor strains restored effective symbiosis while the ot her 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 norma l flux of metabolites through the aromatic biosynthesis pathway is ess ential for bacteroid development.