Microarray-based detection and typing of the Rhizobium nodulation gene nodC: potential of DNA arrays to diagnose biological functions of interest.

Environmental screening of bacteria for the presence of genes of interest is a challenging problem, due to the high variability of the nucleotide sequence of a given gene between species. Here, we tackle this general issue using a particularly well-suited model system that consists of the nodulation gene nodC, which is shared by phylogenetically distant rhizobia. 41mer and 50mer oligonucleotides featuring the nucleotide diversity of two highly conserved regions of the NodC protein were spotted on glass slides and cross hybridized with the radioactive-labeled target genomic DNA under low-stringency conditions. Statistical analysis of the hybridization patterns allowed the detection of known, as well as new, nodC sequences and classified the rhizobial strains accordingly. The microarray was successfully used to type the nodC gene directly from legume nodules, thus eliminating the need of cultivation of the endosymbiont. This approach could be extended to a panel of diagnostic genes and constitute a powerful tool for studying the distribution of genes of interest in the environment, as well as for bacteria identification.

Phylogenetic analyses of symbiotic nodulation genes support vertical and lateral gene co-transfer within the Bradyrhizobium genus.

Symbiotic nitrogen fixing bacteria-known as rhizobia-harbour a set of nodulation (nod) genes that control the synthesis of modified lipo-chitooligosaccharides, called Nod factors that are required for legume nodulation. The nodA gene, which is essential for symbiosis, is responsible for the attachment of the fatty acid group to the oligosaccharide backbone. The nodZ, nolL, and noeI genes are involved in specific modifications of Nod factors common to bradyrhizobia, i.e., the transfer of a fucosyl group on the Nod factor core, fucose acetylation and fucose methylation, respectively. PCR amplification, sequencing and phylogenetic analysis of nodA gene sequences from a collection of diverse Bradyrhizobium strains revealed the monophyletic character with the possible exception of photosynthetic Bradyrhizobium, despite high sequence diversity. The distribution of the nodZ, nolL, and noeI genes in the studied strains, as assessed by gene amplification, hybridization or sequencing, was found to correlate with the nodA tree topology. Moreover, the nodA, nodZ, and noeI phylogenies were largely congruent, but did not closely follow the taxonomy of the strains shown by the housekeeping 16S rRNA and dnaK genes. Additionally, the distribution of nodZ, noeI, and nolL genes suggested that their presence may be related to the requirements of their legume hosts. These data indicated that the spread and maintenance of nodulation genes within the Bradyrhizobium genus occurred through vertical transmission, although lateral gene transfer also played a significant role.

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

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.

Symbiotic and taxonomic diversity of rhizobia isolated from Acacia tortilis subsp. raddiana in Africa.

A collection of rhizobia isolated from Acacia tortilis subsp. raddiana from various sites in the North and South of Sahara was analyzed for their diversity at both taxonomic and symbiotic levels. On the basis of whole cell protein (SDS-PAGE) and 16S rDNA sequence analysis, most of the strains were found to belong to the Sinorhizobium and Mesorhizobium genera where they may represent several different genospecies. Despite their chromosomal diversity, most A. tortilis Mesorhizobium and Sinorhizobium symbionts exhibited very similar symbiotic characters. Nodulation tests showed that the strains belong to the Acacia-Leucaena-Prosopis nodulation group, although mainly forming non-fixing nodules on species other than A. tortilis. Most of the strains tested responded similarly to flavonoid nod gene inducers, as estimated by using heterologous nodA-lacZ fusions. Thin layer chromatography analysis of the Nod factors synthesized by overproducing strains showed that most of the strains exhibited similar profiles. The structures of Nod factors produced by four different Sinorhizobium sp. strains were determined and found to be similar to other Acacia-Prosopis-Leucaena nodulating rhizobia of the Sinorhizobium-Mesorhizobium-Rhizobium branch. They are chitopentamers, N-methylated and N-acylated by common fatty acids at the terminal non reducing sugar. The molecules can also be 6-O sulfated at the reducing end and carbamoylated at the non reducing end. The phylogenetic analysis of available NodA sequences, including new sequences from A. tortilis strains, confirmed the clustering of the NodA sequences of members of the Acacia-Prosopis-Leucaena nodulation group.