Phylogeny of nodulation and nitrogen-fixation genes in Bradyrhizobium: supporting evidence for the theory of monophyletic origin, and spread and maintenance by both horizontal and vertical transfer

Bacteria belonging to the genus Bradyrhizobium are capable of establishing symbiotic relationships with a broad range of plants belonging to the three subfamilies of the family Leguminosae (=Fabaceae), with the formation of specialized structures on the roots called nodules, where fixation of atmospheric nitrogen takes place. Symbiosis is under the control of finely tuned expression of common and host-specific nodulation genes and also of genes related to the assembly and activity of the nitrogenase, which, in Bradyrhizobium strains investigated so far, are clustered in a symbiotic island. Information about the diversity of these genes is essential to improve our current poor understanding of their origin, spread and maintenance and, in this study, we provide information on 40 Bradyrhizobium strains, mostly of tropical origin. For the nodulation trait, common (nodA), Bradyrhizobium-specific (nodY/K) and host-specific (nodZ) nodulation genes were studied, whereas for fixation ability, the diversity of nifH was investigated. In general, clustering of strains in all nod and nifH trees was similar and the Bradyrhizobium group could be clearly separated from other rhizobial genera. However, the congruence of nod and nif genes with ribosomal and housekeeping genes was low. nodA and nodY/K were not detected in three strains by amplification or hybridization with probes using Bradyrhizobium japonicum and Bradyrhizobium elkanii type strains, indicating the high diversity of these genes or that strains other than photosynthetic Bradyrhizobium must have alternative mechanisms to initiate the process of nodulation. For a large group of strains, the high diversity of nod genes (with an emphasis on nodZ), the low relationship between nod genes and the host legume, and some evidence of horizontal gene transfer might indicate strategies to increase host range. On the other hand, in a group of five symbionts of Acacia mearnsii, the high congruence between nod and ribosomal/housekeeping genes, in addition to shorter nodY/K sequences and the absence of nodZ, highlights a co-evolution process. Additionally, in a group of B. japonicum strains that were symbionts of soybean, vertical transfer seemed to represent the main genetic event. In conclusion, clustering of nodA and nifH gives additional support to the theory of monophyletic origin of the symbiotic genes in Bradyrhizobium and, in addition to the analysis of nodY/K and nodZ, indicates spread and maintenance of nod and nif genes through both vertical and horizontal transmission, apparently with the dominance of one or other of these events in some groups of strains.