Rhizobia described so far belong to three distinct phylogenetic branches within the ␣-2 subclass of Proteobacteria. Here we report the discovery of a fourth rhizobial branch involving bacteria of the Methylobacterium genus. Rhizobia isolated from Crotalaria legumes were assigned to a new species, "Methylobacterium nodulans," within the Methylobacterium genus on the basis of 16S ribosomal DNA analyses. We demonstrated that these rhizobia facultatively grow on methanol, which is a characteristic of Methylobacterium spp. but a unique feature among rhizobia. Genes encoding two key enzymes of methylotrophy and nodulation, the mxaF gene, encoding the ␣ subunit of the methanol dehydrogenase, and the nodA gene, encoding an acyltransferase involved in Nod factor biosynthesis, were sequenced for the type strain, ORS2060. Plant tests and nodA amplification assays showed that "M. nodulans" is the only nodulating Methylobacterium sp. identified so far. Phylogenetic sequence analysis showed that "M. nodulans" NodA is closely related to Bradyrhizobium NodA, suggesting that this gene was acquired by horizontal gene transfer.Symbioses between leguminous plants and soil bacteria commonly referred to as rhizobia are of considerable environmental and agricultural importance since they are responsible for most of the atmospheric nitrogen fixed on land. Rhizobia are able to elicit on most of the 18,000 species of the Leguminosae family the formation of specialized organs, called nodules, in which they reduce atmospheric nitrogen to ammonia to the benefit of the plant. Nodule formation is controlled by extracellular bacterial signal molecules, called Nod factors, which are recognized by the host plant (21, 34). The rhizobial species described so far are very diverse and do not form an evolutionary homogenous clade. They belong to three distinct branches within the ␣-2 subclass of Proteobacteria and are phylogenetically intertwined with non-symbiotic bacteria (40) (Fig. 1). A first large branch groups the genera Rhizobium, Sinorhizobium, Mesorhizobium, and Allorhizobium with Agrobacterium, a pathogenic bacterium of plants. A second branch contains the genus Bradyrhizobium together with photosynthetic free-living Rhodopseudomonas, whereas the third branch includes the genus Azorhizobium as well as the chemiautotroph Xanthobacter. Each rhizobial species has a defined host range, varying from very narrow, as in the case of Azorhizobium caulinodans (6), to very broad, as in the case of Sinorhizobium sp. strain NGR234 (30). Symbionts of legumes exhibiting ecological and agronomic potential should be characterized prior to their use in sustainable agriculture and environment management.Crotalaria spp. are herbs and shrubs of the subfamily Papilionoideae; it is the largest plant genus in Africa. More than 500 species commonly occur in diverse climatological situations, from semidesert to rain forests and high mountains (1, 29). Some Crotalaria spp. are of great agronomic interest since they are used as green manure to improve soil fertility o...

Data on 72 non-pigmented bacterial strains that specifically induce nitrogen-fixing root nodules on the legume species Crotalaria glaucoides, Crotalaria perrottetii and Crotalaria podocarpa are reviewed. By SDS-PAGE analysis of total protein patterns and by 16S rRNA PCR-RFLP, these strains form a homogeneous group that is separate from other legume root-nodule-forming bacteria. The 16S rRNA gene-based phylogeny indicates that these bacteria belong to the genus Methylobacterium. They can grow on C(1) compounds such as methanol, formate and formaldehyde but not methylamine as sole carbon source, and carry an mxaF gene, encoding methanol dehydrogenase, which supports their methylotrophic metabolism. Presence of a nodA nodulation gene, and ability to nodulate plants of Crotalaria species and to fix nitrogen are features that separate the strains currently included in this group from other members of the genus Methylobacterium. The present study includes additional genotypic and phenotypic characterization of this novel Methylobacterium species, i.e. nifH gene sequence, morphology, physiology, enzymic and carbon source assimilation tests and antibiotic resistance. The name Methylobacterium nodulans sp. nov. (type strain, ORS 2060(T)=CNCM I 2342(T)=LMG 21967(T)) is proposed for this group of root-nodule-forming bacteria.