Legume symbiotic nitrogen fixation by beta-proteobacteria is widespread in nature

Abstract

Following the initial discovery of two legume-nodulating Burkholderia strains (L. Moulin, A. Munive, B. Dreyfus, and C. Boivin-Masson, Nature 411:948-950, 2001), we identified as nitrogen-fixing legume symbionts at least 50 different strains of Burkholderia caribensis and Ralstonia taiwanensis, all belonging to the beta-subclass of proteobacteria, thus extending the phylogenetic diversity of the rhizobia. R. taiwanensis was found to represent 93% of the Mimosa isolates in Taiwan, indicating that beta-proteobacteria can be the specific symbionts of a legume. The nod genes of rhizobial beta-proteobacteria (beta-rhizobia) are very similar to those of rhizobia from the alpha-subclass (alpha-rhizobia), strongly supporting the hypothesis of the unique origin of common nod genes. The beta-rhizobial nod genes are located on a 0.5-Mb plasmid, together with the nifH gene, in R. taiwanensis and Burkholderia phymatum. Phylogenetic analysis of available nodA gene sequences clustered beta-rhizobial sequences in two nodA lineages intertwined with alpha-rhizobial sequences. On the other hand, the beta-rhizobia were grouped with free-living nitrogen-fixing beta-proteobacteria on the basis of the nifH phylogenetic tree. These findings suggest that beta-rhizobia evolved from diazotrophs through multiple lateral nod gene transfers.

FIG. 1.

Sampling sites for M. pudica and M. diplotricha in Taiwan.

FIG. 2.

16S rDNA tree showing phylogenetic positions of legume-nodulating Ralstonia and Burkholderia species within the β-proteobacteria. The ML tree (base frequencies estimated, mutation rates drawn from an γ + INVdistribution, four classes of mutations) was reconstructed by using PAUP. Xanthomonas campestris was used as an outgroup. Legume symbionts are shown in bold type. Nodulating Burkholderia strains are named according to Vandamme et al. (24). GenBank/EMBL accession numbers for the 16S rDNA sequences were AF175314 (B. cepacia genomovar VI), AF148556 (B. cepacia genomovar III), U96928 (B. vietnamensis), U91839 (B. pseudomallei), AF110188 (B. mallei), AB021423 (B. carophylli), AJ302312 (B. phymatum), AJ505301 (B. caribensis), Y17009 (B. caribensis), AF215705 (B. fungorum), AB021394 (B. phenazinium), U96939 (B. graminis), AB024310 (B. kuruiensis), AJ302311 (B. tuberum), AF139176 (P. sputorum), AF139176 (G. gigasporum), AJ238359 (H. frisingense), AL646072 (R. solanacearum), AB004790 (R. pickettii),AF085226 (R. paucula), AF300324 (R. taiwanensis), AF076645 (R. gilardii), M32021 (R. eutropha), and AF188831 (X. campestris).

FIG. 3.

Nodules of M. pudica 4 weeks after inoculation with R. taiwanensis LMG 19424. (a) Nodulated roots. (b) Root segment with pink nodules. (c) Longitudinal section showing the structure of a nodule. Plant tissue was cleared with sodium hypochlorite and stained with methylene blue as described by Truchet et al. (21). ic, infected cells; vb, vascular bundles.

FIG. 4.

Locations of nodA and nifH genes on replicons of R. taiwanensis LMG 19424 (A) and B. phymatum STM815 (B). Lane 1, PFGE of undigested genomic DNA stained with ethidium bromide; lanes 2 and 3, autoradiographs of blotted PFGE gels hybridized with nodA and nifH probes, respectively. Sizes of replicons are indicated on the left.

FIG. 5.

Unrooted nodA phylogenetic tree of rhizobia. β-Proteobacterial strains are in shown in bold type and underlined. The tree was reconstructed by using an ML approach based on a 597-bp alignment (excluding the additional segment at the N-terminal part). Sequence lengths included ranged from 558 bp (B. phymatum STM815; partial sequence) to 597 bp (most strains). Values along branches indicate bootstrap percentages higher than 50%, based on 100 replicates. nodA sequences for published bacteria are available from GenBank. EMBL accession numbers and nodA sequences for unpublished bacteria were AJ505318 (B. phymatum), AJ505311 (R. taiwanensis), AJ505309 (B. caribensis), AJ300229 (S. terangae bv. acaciae), AJ300249 (M. plurifarium), AJ505307 (TJ172), AJ300234 (BR816), AJ505305 (TJ167), AJ300242 (A. undicola), AJ302321 (B. tuberum), AJ303088 (STM259), AJ430707 (WU425), AJ430730 (CBP70), AJ430715 (ORS938), AJ430712 (USDA3139), AJ430728 (CCT6220), AJ300260 (STM270), AJ300247 (M. ciceri), AJ300228 (S. terangae bv. sesbaniae), and J300235 (R. huautlense).

FIG. 6.

nifH phylogenetic tree. The tree was reconstructed by using an ML approach based on an 800-bp alignment matrix (partial and full sequence lengths ranged from 336 to 797 bp). Values along branches indicate bootstrap percentages higher than 50%. The tree was rooted by using sequences from V. diazotrophicus, K. pneumoniae, and A. vinelandii. Rhizobia are shown in bold type, and the α-, β-, or γ-proteobacterial classification is indicated in parentheses. Clusters 1 and 3 contain α-rhizobia only, while cluster 2 includes both symbiotic and nonsymbiotic diazotrophic β-proteobacteria. nifH sequences for published bacteria are available from GenBank EMBL. EMBL accession numbers and nifH sequences for unpublished bacteria were AJ302315 (B. tuberum), AJ505320 (R. taiwanensis), AJ512206 (B. vietnamensis), AJ512207 (B. caryophylli), AJ505317 (B. caribensis), AJ505319 (B. phymatum), and AJ512205 (M. nodulans). nifH sequences from B. fungorum, R. palustris, and R. leguminosarum were from partially completed genome Web sites.