The Common Nodulation Genes of Astragalus sinicus Rhizobia Are Conserved despite Chromosomal Diversity

Abstract

The nodulation genes of Mesorhizobium sp. (Astragalus sinicus) strain 7653R were cloned by functional complementation of Sinorhizobium meliloti nod mutants. The common nod genes, nodD, nodA, and nodBC, were identified by heterologous hybridization and sequence analysis. The nodA gene was found to be separated from nodBC by approximately 22 kb and was divergently transcribed. The 2.0-kb nodDBC region was amplified by PCR from 24 rhizobial strains nodulating A. sinicus, which represented different chromosomal genotypes and geographic origins. No polymorphism was found in the size of PCR products, suggesting that the separation of nodA from nodBC is a common feature of A. sinicus rhizobia. Sequence analysis of the PCR-amplified nodA gene indicated that seven strains representing different 16S and 23S ribosomal DNA genotypes had identical nodA sequences. These data indicate that, whereas microsymbionts of A. sinicus exhibit chromosomal diversity, their nodulation genes are conserved, supporting the hypothesis of horizontal transfer of nod genes among diverse recipient bacteria.Rhizobia are soil bacteria that can form nodules, in which they fix nitrogen, on leguminous plants in a host-specific manner. Nodulation (nod) genes have been identified that control the specific infection and nodulation of the plant hosts. The initial infection event is regulated by a NodD protein or proteins which activate the transcription of other nod genes in the presence of host-produced flavonoids (12, 25, 37). The nod-ABC genes are called common nod genes because they are present in all rhizobia. Other nod genes, such as nodFE, nodH, nodSU, and nodZ (12,25,37), are present in various combinations in rhizobial species and are called host-specific nod genes.Expression of common and host-specific nod genes results in the production of lipochitooligosaccharides (Nod factors) that act as morphogenic signal molecules on specific legume hosts (12, 37). All Nod factors have a ␤-1,4-linked N-acetyl glucosamine oligosaccharide backbone ranging in length from 3 to 5 residues and substituted for by an N-acyl chain at the nonreducing end and other chemical groups on the glucosamine residues. The common nodABC gene products are involved in the synthesis of the N-acylated oligosaccharide core, while the host-specific nod gene products are involved in the decoration of this backbone with substitutions that confer plant specificity. The nodABC genes encode an acyltransferase, a chitin oligosaccharide deacetylase, and a chitin oligosaccharide synthase, respectively (3, 33). The common nod genes are also involved in determining host range specificity to some extent. For example, different NodA proteins recognize and transfer different fatty acid chains to the chitooligosaccharide chain, the length of which is determined by NodC (11, 27, 32). The common nodABC genes are essential for nodule formation. Mutation in any of them abolishes the ability to produce Nod factors and results in a nonnodulating (Nod Ϫ ) phenotype (12). Astragalus sinicus L. (Chinese ...

Mesorhizobium septentrionale sp. nov. and Mesorhizobium temperatum sp. nov., isolated from Astragalus adsurgens growing in the northern regions of China

“…Internal gene fragments (495 bp) of glutamine synthetase I (glnA) were amplified and sequenced directly using the PCR primers and methods described by . Internal nodA gene sequences (470-476 bp) were amplified and sequenced directly using the PCR primers and methods of Zhang et al (2000). The newly acquired sequences were aligned with other rhizobial sequences in the databases for the 16S rRNA, glnA or nodA gene using CLUSTAL_X (Thompson et al, 1997), which was also used to construct and bootstrap (1000 replicates) the corresponding phylogenetic trees.…”

“…Different rhizobial groups have been identified among the nodule isolates from Astragalus species grown in China and other countries (Guo et al, 1999;Laguerre et al, 1997;Wang & Chen, 1996;Wdowiak & Malek, 2000). This lack of specificity is not universal: Astragalus sinicus, an important green manure in the southern regions of China, has been reported to nodulate almost exclusively with Mesorhizobium huakuii (Chen et al, 1991;Zhang et al, 2000). Among the Astragalus species used in China, Astragalus adsurgens has been planted over a vast area in desert and very dry regions to protect soils from wind erosion.…”

The Structure and Evolution of Beta-Rhizobial Symbiotic Genes Deduced from Their Complete Genomes

“…Therefore, we could suggest that the symbotic genes evolved from gene duplication, and gene transfer occurred later within two or one clades resulting from the interaction between Rhizobia, legumes, and their environment. In support of this, a 410 kb symbiosis-relevant region of the Bradyrhizobium japonicum chromosome was suggested to be comprised of DNA fragments from different origins by comparing it with other free-living bacteria [76,[84][85][86][87][88]. On the other hand, there are rhizobial strains where the chromosome-borne symbiosis genes are conserved and stable.…”

“…These Beta-rhizobia were all very distant from the Alpharhizobia with low similarities of 50.6%-70.6%, in contrast to the Mimosa-nodulators, the nodA sequences of the other group of legumenodulating Beta-rhizobia which comprises papilionoid-nodulating Burkholderia strains, were very close to Alpha-rhizobia, with 86.4% similarity to Methylobacterium nodulans ORS 2060. They were also close to Mesorhizobium, Bradyrhizobium and Rhizobium, with more than 73.5% similarities.…”

“…Phylogenies using partial sequences (338 bp) of nodA ( Figure 2) and entire sequences (822 bp) of nifH ( Figure 3) from complete genomes of Alpha-and Beta-rhizobia were constructed ( Table 1 86.4% similar to each other. These Beta-rhizobia were all very distant from the Alpharhizobia with low similarities of 50.6%-70.6%, in contrast to the Mimosa-nodulators, the nodA sequences of the other group of legumenodulating Beta-rhizobia which comprises papilionoid-nodulating Burkholderia strains, were very close to Alpha-rhizobia, with 86.4% similarity to Methylobacterium nodulans ORS 2060.…”