Methylotrophic Methylobacterium bacteria nodulate and fix nitrogen in symbiosis with legumes

Abstract

Rhizobia described so far belong to three distinct phylogenetic branches within the alpha-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 alpha 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.

FIG. 1

Unrooted phylogenetic tree showing the different rhizobial branches, including the new “M. nodulans” in the α subdivision of Proteobacteria. The tree was constructed by using the neighbor-joining method from almost full-length 16S rDNA sequences. The GenBank/EMBL accession numbers are as follows (the first letters of the genus and species are given in parentheses): D 12790 (Pr), D12797 (Mh), X67229 (Ml), L38825 (Mmed), X67224 (Ar), X67234 (Rt), U29386 (Rl), U28916 (Re), Y17047 (Au), X67225 (Av), X67223 (At), X67226 (Rg), X67222 (Sm), X68390 (Ss), X68387 (St), X67231 (Sf), X94198 (Xag), X94201 (Xau), X94199 (Xf), D11342 (Ac), U35000 (Be), M65248 (Af), L11661 (Nw), S46917 (Bd), D25312 (Rp), D12781 (Bj), U69637 (Bl), D32226 (Mo), D32225 (Mmes), D32227 (Mrad), D32229 (Mrhodi), D32230 (Mz), D32228 (Mrhode), D32224 (Me), D32236 (Msp), and AF220763 (Mn).

FIG. 2

Methanol utilization by “M. nodulans” ORS2060. Bradyrhizobium sp. strain ORS1810 (33) was used as a negative control. Shown is growth of ORS2060 (black circles) and ORS1810 (black squares) on minimum mineral medium 72 containing MeOH as the sole carbon source. MeOH concentrations in supernatants of cultures of ORS2060 (empty circles) and ORS1810 (empty squares) are also shown.

FIG. 3

Nodules of C. perrottetii inoculated with “M. nodulans” ORS2060. (a) Multilobed fresh nodule; (b) unstained longitudinal section displaying the classical structure of indeterminate nodules with an apical meristematic zone (m), an infection zone (iz), a senescent zone (sz), and peripheral vascular bundles (vb).

FIG. 4

Phylogenetic tree based on full-length NodA sequences construced by using the neighbor-joining method. Bootstrap values (from 1,000 replications) are indicated. The GenBank/EMBL accession numbers are as follows (the first letters of the genus and species are given in parentheses): L18897 (Ac), 106241 (Ml), M73699 (Sf), M58625 (Re), X98514 (Rt), AF266748 (Mn), U33192 (BspNC92), U04609 (BspANU289), U04609 (Be), AJ249353 (Mh), U53327 (MspN33), M11268 (Sm),X87578 (Rg), and X01650 (Rl). The NodA sequence of Bradyrhizobium sp. strain ORS1810 was kindly provided by T. Stepkowski.

FIG. 5

Phylogenetic tree based on about 140-amino-acid MxaF-homologous sequences constructed by using the neighbor-joining method. Bootstrap values (from 1,000 replications) are indicated. Cluster 1 groups only MxaF sequences with the following GenBank/EMBL accession numbers (the first letters of the genus and species are given in parentheses): M17339 (Pd), AB004097 (Hm), M22629 (Mo), M31108 (Me), AF220764 (Mn), U41040 (Mm), and AF184915 (Msp). Cluster 2 groups the rhizobial sequences (accession numbers given in Materials and Methods) together with XoxF from P. denitrificans (U34346) and MxaF′ from M. extorquens (U72662). MxaF from Bacillus sp. (M65004) was used as the root. The topology of the tree constructed with the full-length MxaF-homologous sequences (thus excluding the rhizobial sequences) is similar. (α), member of α-Proteobacteria; (β), member of β-Proteobacteria.