Phenotypic characterization and regulation of the nolA gene of Bradyrhizobium japonicum

Two Bradyrhizobium japonicum nolA mutants were constructed and used to test the functional role of NolA in nodulation, Contrary to the previous hypothesis that NolA acts as a repressor of nod gene transcription, the expression of a nodD1-lacZ or nodY-lacZ fusion in the nolA mutant strains was similar to that found in the wild type, However NolA does appear to act as a transcriptional regulatory protein since it is required for its own expression, as well as that of nodD2. Expression of NodD2 from a constitutive promoter led to a significant reduction in nodC-lacZ activity, Therefore, the repression of nod gene expression by NolA is likely an indirect effect, perhaps mediated by other genes (e.g., nodD2) that are regulated by NolA, When inoculated onto soybean roots, the nolA mutant strains showed only a slight delay in nodulation as compared to the wild type, However, the mutant strains were grossly defective in nodulation and nitrogen fixation on cowpea plants, Microscopic examination of soybean nodules induced by the nolA mutant strains showed developmental and morphological characteristics similar to nodules formed by the wild type with only a slight delay in bacteroid maturation, In contrast, cowpea nodules induced by the nolA mutant strains contained fewer infected cells and bacteroids were not found in a typical symbiosome structure, These results indicate that NolA is a transcriptional activator required for the expression of genes that play a role not only in the early stages of infection, but also during the later stages of bacteroid development and maintenance.