Genomic features separating ten strains of Neorhizobium galegae with different symbiotic phenotypes.

BACKGROUND: The symbiotic phenotype of Neorhizobium galegae, with strains specifically fixing nitrogen with either Galega orientalis or G. officinalis, has made it a target in research on determinants of host specificity in nitrogen fixation. The genomic differences between representative strains of the two symbiovars are, however, relatively small. This introduced a need for a dataset representing a larger bacterial population in order to make better conclusions on characteristics typical for a subset of the species. In this study, we produced draft genomes of eight strains of N. galegae having different symbiotic phenotypes, both with regard to host specificity and nitrogen fixation efficiency. These genomes were analysed together with the previously published complete genomes of N. galegae strains HAMBI 540T and HAMBI 1141. RESULTS: The results showed that the presence of an additional rpoN sigma factor gene in the symbiosis gene region is a characteristic specific to symbiovar orientalis, required for nitrogen fixation. Also the nifQ gene was shown to be crucial for functional symbiosis in both symbiovars. Genome-wide analyses identified additional genes characteristic of strains of the same symbiovar and of strains having similar plant growth promoting properties on Galega orientalis. Many of these genes are involved in transcriptional regulation or in metabolic functions. CONCLUSIONS: The results of this study confirm that the only symbiosis-related gene that is present in one symbiovar of N. galegae but not in the other is an rpoN gene. The specific function of this gene remains to be determined, however. New genes that were identified as specific for strains of one symbiovar may be involved in determining host specificity, while others are defined as potential determinant genes for differences in efficiency of nitrogen fixation.

Galega orientalis is more diverse than Galega officinalis in Caucasus--whole-genome AFLP analysis and phylogenetics of symbiosis-related genes.

Legume plants can obtain combined nitrogen for their growth in an efficient way through symbiosis with specific bacteria. The symbiosis between Rhizobium galegae and its host plant Galega is an interesting case where the plant species G. orientalis and G. officinalis form effective, nitrogen-fixing, symbioses only with the appropriate rhizobial counterpart, R. galegae bv. orientalis and R. galegae bv. officinalis, respectively. The symbiotic properties of nitrogen-fixing rhizobia are well studied, but more information is needed on the properties of the host plants. The Caucasus region in Eurasia has been identified as the gene centre (centre of origin) of G. orientalis, although both G. orientalis and G. officinalis can be found in this region. In this study, the diversity of these two Galega species in Caucasus was investigated to test the hypothesis that in this region G. orientalis is more diverse than G. officinalis. The amplified fragment length polymorphism fingerprinting performed here showed that the populations of G. orientalis and R. galegae bv. orientalis are more diverse than those of G. officinalis and R. galegae bv. officinalis, respectively. These results support the centre of origin status of Caucasus for G. orientalis at a genetic level. Analysis of the symbiosis-related plant genes NORK and Nfr5 reveals remarkable diversity within the Nfr5 sequence, although no evidence of adaptive evolution could be found.

[Effects of natural and hybrid lectins on the legume-rhizobium interactions].

The effects of hybrid lectins--full-sized pea Pisum sativum lectin (PSL) with the carbohydrate-binding region of white melilot Melilotus albus lectin or wild licorice Astragalus glycyphyllos lectin substituted for the corresponding PSL region (PSL/MAL and PSL/AGL, correspondingly)--on the legume-rhizobium symbiosis were studied. The treatment of the Rhizobium leguminosarum bv. viciae in the alfalfa (Medicago sativa) rhizosphere with PSL induced formation of uninfected pseudonodules on its roots, whereas the treatment of the bacteria from Astragalus cicer nodules with PSL/AGL rendered these bacteria able to form infective nodules on alfalfa roots. This ability is associated with expanded and unusual carbohydrate-binding properties (combined specificity for Gal and Glc) of this hybrid protein as compared with the natural legume lectins.

[The symbiosis between oriental goat's rue and the root nodule bacteria Rhizobium galegae: specificity and competitiveness].

Competitiveness and genetic variation of the Rhizobium galegae strains from the collection of the All-Russia Institute of Agricultural Microbiology, Russian Academy of Agricultural Sciences, causing nodulation of oriental goat's rue under conditions of Bashkortostan soils (lacking this rhizobial species) were studied. It was demonstrated that of all the tested strains, the strains CIAM 0702 and CIAM 0704, each carrying two megaplasmids of 1500 and 2000 MDa, were the most competitive. RAPD (random amplified polymorphic DNA) analysis showed that R. galegae strains were able to intensively exchange the genetic material in the host plant rhizosphere. We did not succeed in detecting the local root nodule bacteria that were either initially able to infect oriental goat's rue or had adapted to infecting this species due to various genetic rearrangements.

Genetic diversity of culturable bacteria in oil-contaminated rhizosphere of Galega orientalis.

A collection of 50 indigenous meta-toluate tolerating bacteria isolated from oil-contaminated rhizosphere of Galega orientalis on selective medium was characterized and identified by classical and molecular methods. 16S rDNA partial sequencing showed the presence of five major lineages of the Bacteria domain. Gram-positive Rhodococcus, Bacillus and Arthrobacter and gram-negative Pseudomonas were the most abundant genera. Only one-fifth of the strains that tolerated m-toluate also degraded m-toluate. The inoculum Pseudomonas putida PaW85 was not found in the rhizosphere samples. The ability to degrade m-toluate by the TOL plasmid was detected only in species of the genus Pseudomonas. However, a few Rhodococcus erythropolis strains were found which were able to degrade m-toluate. A new finding was that Pseudomonas migulae strains and a few P. oryzihabitans strains were able to grow on m-toluate and most likely contained the TOL plasmid. Because strain specific differences in degradation abilities were found for P. oryzihabitans, separation at the strain level was important. For strain specific separation (GTG)5 fingerprinting was the best method. A combination of the single locus ribotyping and the whole genomic fingerprinting techniques with the selective partial sequencing formed a practical molecular toolbox for studying genetic diversity of culturable bacteria in oil-contaminated rhizosphere.

[Contribution of lectin sugar-binding peptides structure determines specificity of rhizobium-legume symbiosis in Galega orientalis and G. officinalis].

A significant heterogeneity between bacteria Rhizobium galegae bv. officinalis and R. galegae bv. orientalis forming the nitrogen-fixing symbiosis with Galega officinalis and G. orientalis, respectively, and not forming any single cross-inoculation group, was found by means of RAPD and RFEL methods. The high level of sequence similiraty between lectins of these plants indicates at their close relationship. However the sequences of lectin sugar binding peptides (SBP) of G. orientalis (TYCNPGWDPRDR) and G. officinalis (TFYNEEWDLVIKDEH) were highly diverged. Amino acids of SBP which are involved in linkage of Ca2+ and Mn2+ ions responsible for stabilization of spatial structure of carbohydrate-binding "pocket", keep their position in peptide. It suggests that lectins participate in Rhizobium-legume symbiosis and that carbohydrate-binding site plays a key role in this process.

Symbiotic and genetic diversity of Rhizobium galegae isolates collected from the Galega orientalis gene center in the Caucasus.

This paper explores the relationship between the genetic diversity of rhizobia and the morphological diversity of their plant hosts. Rhizobium galegae strains were isolated from nodules of wild Galega orientalis and Galega officinalis in the Caucasus, the center of origin for G. orientalis. All 101 isolates were characterized by genomic amplified fragment length polymorphism fingerprinting and by PCR-restriction fragment length polymorphism (RFLP) of the rRNA intergenic spacer and of five parts of the symbiotic region adjacent to nod box sequences. By all criteria, the R. galegae bv. officinalis and R. galegae bv. orientalis strains form distinct clusters. The nod box regions are highly conserved among strains belonging to each of the two biovars but differ structurally to various degrees between the biovars. The findings suggest varying evolutionary pressures in different parts of the symbiotic genome of closely related R. galegae biovars. Sixteen R. galegae bv. orientalis strains harbored copies of the same insertion sequence element; all were isolated from a particular site and belonged to a limited range of chromosomal genotypes. In all analyses, the Caucasian R. galegae bv. orientalis strains were more diverse than R. galegae bv. officinalis strains, in accordance with the gene center theory.

AFLP fingerprinting as a tool to study the genetic diversity of Rhizobium galegae isolated from Galega orientalis and Galega officinalis.

AFLP fingerprints of Rhizobium galegae strains that infect Galega orientalis and Galega officinalis obtained from different geographical sources, and of taxonomically diverse rhizobia representing the recognized species, were generated. Comparisons of the fingerprints from fluorescent labeled AFLP products using capillary electrophoresis on ABI prism 310, slab gel electrophoresis on ABI prism 377 genetic analyzers and silver staining were in good agreement. All methods delineated the G. orientalis strains from G. officinalis strains, the G. orientalis strains formed a tight cluster whereas the G. officinalis strains seem to show a greater level of genetic diversity. Comparison of fluorescent AFLP with other detection methods revealed that fluorescent labeling is more sensitive and practical, in addition, the deleterious effect of radioactivity associated with 32P-labeling, the delicate process of blotting polyacrylamide gels or the tedious procedure of silver staining can be avoided. The automated system facilitated a large number of runs at a time and the subsequent analysis of the data by generating exportable raw data. The congruency of the experiments was analyzed using the Bionumerics software.

Description of two biovars in the Rhizobium galegae species: biovar orientalis and biovar officinalis.

Twenty-six Rhizobium galegae strains, representing the center of origin of the host plants Galega orientalis and G. officinalis as well as other geographic regions, were used in a polyphasic analysis of the relationships of R. galegae strains. Phage typing, lipopolysaccharide (LPS) profiling, pulsed field gel electrophoresis (PFGE) profiling and rep-PCR (use of repetitive sequences as PCR primers for genomic fingerprinting) with REP and ERIC primers investigated nonsymbiotic properties, whereas plasmid profiling and hybridisation with a nif gene probe, and with nodB, nodD, nod box and an IS sequence from the symbiotic region as probes, were used to reveal the relationships of symbiotic genes. The results were used in pairwise calculations of distances between the strains, and the distances were visualised as a dendrogram. Indexes of association were compared for all tests pooled, and for chromosomal tests and symbiotic markers separately, to display the input of the different categories of tests on the grouping of the strains. Our study shows that symbiosis related genetic traits in R. galegae divide strains belonging to the species into two groups, which correspond to strains forming an effective symbioses with G. orientalis and G. officinalis respectively. We therefore propose that Rhizobium galegae strains forming an effective symbiosis with Galega orientalis are called R. galegae bv. orientalis and strains forming an effective symbiosis with Galega officinalis are called R. galegae bv. officinalis.