Identification and structure of the Rhizobium galegae common nodulation genes: evidence for horizontal gene transfer.

Rhizobia are soil bacteria able to fix atmospheric nitrogen in symbiosis with leguminous plants. In response to a signal cascade coded by genes of both symbiotic partners, a specific plant organ, the nodule, is formed. Rhizobial nodulation (nod) genes trigger nodule formation through the synthesis of Nod factors, a family of chitolipooligosaccharides that are specifically recognized by the host plant at the first stages of the nodulation process. Here, we present the organization and sequence of the common nod genes from Rhizobium galegae, a symbiotic member of the RHIZOBIACEAE: This species has an intriguing phylogenetic position, being symbiotic among pathogenic agrobacteria, which induce tumors instead of nodules in plant shoots or roots. This apparent incongruence raises special interest in the origin of the symbiotic apparatus of R. galegae. Our analysis of DNA sequence data indicated that the organization of the common nod gene region of R. galegae was similar to that of Sinorhizobium meliloti and Rhizobium leguminosarum, with nodIJ downstream of nodABC and the regulatory nodD gene closely linked to the common nod operon. Moreover, phylogenetic analyses of the nod gene sequences showed a close relationship especially between the common nodA sequences of R. galegae, S. meliloti, and R. leguminosarum biovars viciae and trifolii. This relationship in structure and sequence contrasts with the phylogeny based on 16S rRNA, which groups R. galegae close to agrobacteria and separate from most other rhizobia. The topology of the nodA tree was similar to that of the corresponding host plant tree. Taken together, these observations indicate that lateral nod gene transfer occurred from fast-growing rhizobia toward agrobacteria, after which the symbiotic apparatus evolved under host plant constraint.

Sinorhizobium teranga bv. acaciae ORS1073 and Rhizobium sp. strain ORS1001, two distantly related Acacia-nodulating strains, produce similar Nod factors that are O carbamoylated, N methylated, and mainly sulfated.

We have determined the structures of Nod factors produced by strains representative of Sinorhizobium teranga bv. acaciae and the so-called cluster U from the Rhizobium loti branch, two genetically different symbionts of particular Acacia species. Compounds from both strains were found to be similar, i.e., mainly sulfated, O carbamoylated, and N methylated, indicating a close relationship between host specificity and Nod factor structure, regardless of the taxonomy of the bacterial symbiont.

The Sesbania Root Symbionts Sinorhizobium saheli and S. teranga bv. sesbaniae Can Form Stem Nodules on Sesbania rostrata, although They Are Less Adapted to Stem Nodulation than Azorhizobium caulinodans.

Sesbania species can establish symbiotic interactions with rhizobia from two taxonomically distant genera, including the Sesbania rostrata stem-nodulating Azorhizobium sp. and Azorhizobium caulinodans and the newly described Sinorhizobium saheli and Sinorhizobium teranga bv. sesbaniae, isolated from the roots of various Sesbania species. A collection of strains from both groups were analyzed for their symbiotic properties with different Sesbania species. S. saheli and S. teranga bv. sesbaniae strains were found to effectively stem nodulate Sesbania rostrata, showing that stem nodulation is not restricted to Azorhizobium. Sinorhizobia and azorhizobia, however, exhibited clear differences in other aspects of symbiosis. Unlike Azorhizobium, S. teranga bv. sesbaniae and S. saheli did not induce effective stem nodules on plants previously inoculated on the roots, although stem nodulation was arrested at different stages. For Sesbania rostrata root nodulation, Sinorhizobium appeared more sensitive than Azorhizobium to the presence of combined nitrogen. S. saheli and S. teranga bv. sesbaniae were effective symbionts with all Sesbania species tested, while Azorhizobium strains fixed nitrogen only in symbiosis with Sesbania rostrata. In a simple screening test, S. saheli and S. teranga bv. sesbaniae were incapable of asymbiotic nitrogenase activity. Thus, Azorhizobium can easily be distinguished from Sinorhizobium among Sesbania symbionts on the basis of symbiotic and free-living nitrogen fixation. The ability of Azorhizobium to overcome the systemic plant control appears to be a stem adaptation function. This last property, together with its host-specific symbiotic nitrogen fixation, makes Azorhizobium highly specialized for stem nodulation of the aquatic legume Sesbania rostrata.

In Rhizobium meliloti, the operon associated with the nod box n5 comprises nodL, noeA and noeB, three host-range genes specifically required for the nodulation of particular Medicago species.

In Rhizobium meliloti, the genes required for nodulation of legume hosts are under the control of DNA regulatory sequences called nod boxes. In this paper, we have characterized three host-specific nodulation genes, which form a flavonoid-inducible operon down-stream of the nod box n5. The first gene of this operon is identical to the nodL gene identified by Baev and Kondorosi (1992) in R. meliloti strain AK631. The product of the second gene, NoeA, presents some homology with a methyltransferase. nodL mutants synthesize Nod factors lacking the O-acetate substituent. In contrast, in strains carrying a mutation in either noeA or noeB, no modification in Nod-factor structure or production could be detected. On particular hosts, such as Medicago littoralis, mutants of the n5 operon showed a very weak nodule-forming ability, associated with a drastic decrease in the number of infection threads, while nodulation of Medicago truncatula or Melilotus alba was not affected. Thus, nodL noeA and noeB are host-specific nodulation genes. By using a gain-of-function approach, we showed that the presence of nodL, and hence of O-acetylated Nod factors, is a major prerequisite for confering the ability to nodulate alfalfa upon the heterologous bacterium Rhizobium tropici.