Symbiotic ineffectiveness of trpCD deletion mutants of Bradyrhizobium japonicum

Genes responsible for tryptophan biosynthesis are thought to be essential for symbiotic competence of Bradyrhizobium japonicum. The B. japonicum trpCD region, cloned as a 2.8-kb HindIII-BamHI fragment, had an internal 1.2-kb SmaI fragment that was deleted and replaced in vitro with interposon omega carrying a gene specifying streptomycin and spectinomycin resistance. Site-directed marker exchange mutagenesis of the B. japonicum genome was performed in vivo and the construction of deletion mutants was confirmed by Southern blot analysis of the DNA of the exconjugants. Since the trpCD deletion mutants grew on indole they evidently were unaffected in expression of trpAB and we interpret these data as indicating that the organization of the genes for tryptophan biosynthesis in B. japonicum differs from that of enterics. The new Trp− deletion mutants lacked activities for the trpC- and trpD-encoded enzymes, indole glycerol phosphate synthase (EC 4.1.1.48) and phosphoribosyl anthranilate transferase (EC 2.4.2.18), respectively, but had a higher specific activity for the trpEG-encoded enzyme, anthranilate synthase (EC 4.1.3.27). The new trpCD deletion mutants failed to effectively nodulate soybean. Thus, we conclusively demonstrate the essential role of trpCD in symbiosis.