Bradyrhizobium (Arachis) sp strain NC92 contains two nodD genes involved in the repression of rodA and a nolA gene required for the efficient nodulation of host plants

The common nodulation locus and closely linked nodulation genes of Bradyrhizobium (Arachis) sp, strain NC92 have been isolated on an 11.0-kb EcoRI restriction fragment. The nucleotide sequence of a 7.0-kb EcoRV-EcoRI subclone was determined and found to contain open reading frames (ORFs) homologous to the nodA, nodB, nodD(1), nodD(2), and nolA genes of Bradyrhizobium japonicum and Bradyrhizobium elkanii. Nodulation assays of nodD(1), nodD(2), or noLA deletion mutants on the host plants Macroptilium atropurpureum (siratro) and Vigna unguiculata (cowpea) indicate that nolA is required for efficient nodulation, as nolA mutants exhibit a 6-day nodulation delay and reduced nodule numbers. The nolA phenotype was complemented by providing the nolA ORF in trans, indicating that the phenotype is due to the lack of the nolA ORF, nodD, mutants displayed a 2-day nodulation delay, whereas nodD(2) strains were indistinguishable from the wild type, Translational nodA-lacZ, nodD(1)-lacZ, nodD(2)-lacZ, and nolA-lacZ fusions were created. Expression of the nodA-lacZ fusion was induced by the addition of peanut, cowpea, and siratro seed exudates and by the addition of the isoflavonoids genistein and daidzein. In a nodD(1) or nodD(2) background, basal expression of the nodA-lacZ fusion increased two- to threefold. The level of expression of the nodD(2)-lacZ and nolA-lacZ fusions was low in the wild type but increased in nodD(1), nodD(2), and nodD(1) nodD(2) backgrounds independently of the addition of the inducer genistein, nolA was required for increased expression of the nodD(2)-lacZ fusion. These data suggest that a common factor is involved in the regulation of nodD(2) and nolA, and they are also consistent with a model of nod gene expression in Bradyrhizobium (Arachis) sp, strain NC92 in which negative regulation is mediated by the products of the nodD(1) and nodD(2) genes.