Genomic basis of symbiovar mimosae in Rhizobium etli.

BACKGROUND: Symbiosis genes (nod and nif) involved in nodulation and nitrogen fixation in legumes are plasmid-borne in Rhizobium. Rhizobial symbiotic variants (symbiovars) with distinct host specificity would depend on the type of symbiosis plasmid. In Rhizobium etli or in Rhizobium phaseoli, symbiovar phaseoli strains have the capacity to form nodules in Phaseolus vulgaris while symbiovar mimosae confers a broad host range including different mimosa trees. RESULTS: We report on the genome of R. etli symbiovar mimosae strain Mim1 and its comparison to that from R. etli symbiovar phaseoli strain CFN42. Differences were found in plasmids especially in the symbiosis plasmid, not only in nod gene sequences but in nod gene content. Differences in Nod factors deduced from the presence of nod genes, in secretion systems or ACC-deaminase could help explain the distinct host specificity. Genes involved in P. vulgaris exudate uptake were not found in symbiovar mimosae but hup genes (involved in hydrogen uptake) were found. Plasmid pRetCFN42a was partially contained in Mim1 and a plasmid (pRetMim1c) was found only in Mim1. Chromids were well conserved. CONCLUSIONS: The genomic differences between the two symbiovars, mimosae and phaseoli may explain different host specificity. With the genomic analysis presented, the term symbiovar is validated. Furthermore, our data support that the generalist symbiovar mimosae may be older than the specialist symbiovar phaseoli.

Near-full length sequencing of 16S rDNA and RFLP indicates that Rhizobium etli is the dominant species nodulating Egyptian winter Berseem clover (Trifolium alexandrinum L.).

Egyptian winter Berseem clover (EWBC) is one of the main important forage legume crops in Egypt that is used for animal feeding in winter and it occupies about 2.5 million feddans (Feddan=4200m(2)) in winter agricultural rotation systems. Forty-eight rhizobial isolates that nodulated this legume host from different geographical regions within Egypt were isolated. RFLP analyses of 16S rDNA (1.5kb) and whole ribosomal DNA (5kb), the sequencing of 16S rDNA, and the sequencing of nodC, nifH and house keeping genes were used to identify these isolates. The RFLP analysis of 16S rDNA (1.5kb) among 15 representative strains with three enzymes generated two genotypes. The largest genotype was similar to Rhizobium etli CFN42T (93.33%) except for strain 902 that failed to re-nodulate EWBC. RFLP analysis of complete ribosomal DNA (5kb) produced five genotypes. The majority of tested strains shared the genotype with R. etli CFN42T (53.33%). Only one strain (1002) shared the genotype with Rhizobium leguminosarum sv. trifolii 3023. The other four strains were comprised of two unique genotypes. Phylogenetic analysis of 16S rDNA sequences revealed that seven representative strains could be divided into two genetic clusters sharing the ancestral clad with R. etli CFN42T. A phylogenetic tree based on nodC gene sequence confirmed that all the examined strains shared the genetic lineage with R. leguminosarum sv. trifolii WSM1325. The phylogenetic trees of house keeping genes are supported strongly the identification of majority of strains as a novel symbiovar of R. etli with new lineages.

Rhizobium etli taxonomy revised with novel genomic data and analyses.

The taxonomic position of Phaseolus vulgaris rhizobial strains with available sequenced genomes was examined. Phylogenetic analyses with concatenated conserved genomic fragments accounting for over half of each genome showed that Rhizobium strains CIAT 652, Ch24-10 (newly reported genome) and CNPAF 512 constituted a well-supported group independent from Rhizobium etli CFN 42(T). DNA-DNA hybridization results indicated that CIAT 652, Ch24-10 and CNPAF 512 could correspond to R. etli, although the hybridization values were at the borderline that distinguishes different species. In contrast, experimental hybridization results were higher (over 80%) with Rhizobium phaseoli type strain ATCC 14482(T) in congruence to phylogenetic and ANIm analyses. The latter criterion allowed the reclassification of R. etli strains 8C-3 and Brasil5 as R. phaseoli. It was therefore concluded, based on all the evidence, that the CIAT 652, Ch24-10, and CNPAF 512 strains should be reclassified as R. phaseoli in spite of several common features linking them to R. etli. The R. phaseoli and R. etli speciation process seems to be a more recent event than the speciation that has occurred among other sister species, such as R. leguminosarum-R. etli or R. rhizogenes-R. tropici.

Biochemical characterization of recombinant L-asparaginase (AnsA) from Rhizobium etli, a member of an increasing rhizobial-type family of L-asparaginases.

We report the expression, purification, and characterization of L-asparaginase (AnsA) from Rhizobium etli. The enzyme was purified to homogeneity in a single-step procedure involving affinity chromatography, and the kinetic parameters K(m), V(max), and k(cat) for L-asparagine were determined. The enzymatic activity in the presence of a number of substrates and metal ions was investigated. The molecular mass of the enzyme was 47 kDa by SDS-PAGE. The enzyme showed a maximal activity at 50 degrees C, but the optimal temperature of activity was 37 degrees C. It also showed maximal and optimal activities at pH 9.0. The values of K(m), V(max), k(cat), and k(cat)/K(m) were 8.9 +/- 0.967 × 10⁻³ M, 128 +/- 2.8 U/mg protein, 106 +/- 2 s⁻¹, and 1.2 +/- 0.105 × 104 M⁻¹s⁻¹, respectively. The L-asparaginase activity was reduced in the presence of Mn²âº, Zn²âº, Ca²âº, and Mg²âº metal ions for about 52% to 31%. In addition, we found that NH4⁺, L-Asp, D-Asn, and beta-aspartyl-hydroxamate in the reaction buffer reduced the activity of the enzyme, whereas L-Gln did not modify its enzymatic activity. This is the first report on the expression and characterization of the L-asparaginase (AnsA) from R. etli. Phylogenetic analysis of asparaginases reveals an increasing group of known sequences of the Rhizobialtype asparaginase II family.

Change in land use alters the diversity and composition of Bradyrhizobium communities and led to the introduction of Rhizobium etli into the tropical rain forest of Los Tuxtlas (Mexico).

Nitrogen-fixing bacteria of the Bradyrhizobium genus are major symbionts of legume plants in American tropical forests, but little is known about the effects of deforestation and change in land use on their diversity and community structure. Forest clearing is followed by cropping of bean (Phaseolus vulgaris) and maize as intercropped plants in Los Tuxtlas tropical forest of Mexico. The identity of bean-nodulating rhizobia in this area is not known. Using promiscuous trap plants, bradyrhizobia were isolated from soil samples collected in Los Tuxtlas undisturbed forest, and in areas where forest was cleared and land was used as crop fields or as pastures, or where secondary forests were established. Rhizobia were also trapped by using bean plants. Bradyrhizobium strains were classified into genospecies by dnaK sequence analysis supported by recA, glnII and 16S-23S rDNA IGS loci analyses. A total of 29 genospecies were identified, 24 of which did not correspond to any described taxa. A reduction in Bradyrhizobium diversity was observed when forest was turned to crop fields or pastures. Diversity seemed to recover to primary forest levels in secondary forests that derived from abandoned crop fields or pastures. The shifts in diversity were not related to soil characteristics but seemingly to the density of nodulating legumes present at each land use system (LUS). Bradyrhizobium community composition in soils was dependent on land use; however, similarities were observed between crop fields and pastures but not among forest and secondary forest. Most Bradyrhizobium genospecies present in forest were not recovered or become rare in the other LUS. Rhizobium etli was found as the dominant bean-nodulating rhizobia present in crop fields and pastures, and evidence was found that this species was introduced in Los Tuxtlas forest.

Phaseolus vulgaris is nodulated in northern Spain by Rhizobium leguminosarum strains harboring two nodC alleles present in American Rhizobium etli strains: biogeographical and evolutionary implications.

In this study a collection of rhizobial strains were isolated from effective nodules of Phaseolus vulgaris in a wide region of northern Spain, which is the major producer region of this legume in Spain. The analysis of their core genes, rrs, atpD, and recA, and the 16S-23S intergenic spacer showed that all isolates belong to the phylogenetic group of Rhizobium leguminosarum and some of them were identical to those of strains nodulating Vicia or Trifolium. None of the isolates was identified as Rhizobium etli; however, all of them carry the nodC alleles alpha and gamma harboured by American strains of this species. These alleles were also found in strains nodulating P. vulgaris in southern Spain identified as R. etli. These results suggest that R. etli was carried from America to Spain with common bean seeds, but that they could have found difficulties persisting in the soils of northern Spain, probably because of the climatic conditions. The symbiotic genes of this species could have been transferred, after the arrival of P. vulgaris, to strains of R. leguminosarum already present in northern Spanish soils.

The genetic diversity of rhizobia associated with Dalea purpurea Vent. in fragmented grasslands of west-central Minnesota.

The increase in human population and the spread of agriculture over the past 150 years have transformed the landscape in west-central Minnesota into a mosaic of agricultural fields and urban land, leaving only remnants of the once dominant prairie ecosystem. Limited natural habitat in this fragmented landscape threatens the diversity and abundance of native legumes and could impact the size and function of associated belowground microbial populations. In this study, BOXA1R PCR and 16S rRNA gene sequence analyses were used to assess the genetic diversity of rhizobia associated with Dalea purpurea (Vent.) in nine prairie remnants ranging in size from 0.04 to 3.5 ha. The variation in soil properties was also determined. While 53 different genotypes of rhizobia were identified, four of these accounted for 84% of the 1029 rhizobia characterized using BOXA1R PCR. Representatives from three of the four dominant genotypes had a 16S rRNA gene sequence similar to that of Rhizobium gallicum, with two of these genotypes recovered at all sites. The fourth genotype was similar to that of Rhizobium etli and occurred with frequency at only two sites. Rhizobium genotype richness and site area were positively correlated. The implications of these results are discussed.

Analysis of Rhizobium etli and of its symbiosis with wild Phaseolus vulgaris supports coevolution in centers of host diversification.

Common beans (Phaseolus vulgaris) comprise three major geographic genetic pools, one in Mexico, Central America, and Colombia, another in the southern Andes, and a third in Ecuador and northern Peru. Species Rhizobium etli is the predominant rhizobia found symbiotically associated with beans in the Americas. We have found polymorphism in the common nodulation gene nodC among R. etli strains from a wide range of geographical origins, which disclosed three nodC types. The different nodC alleles in American strains show varying predominance in their regional distributions in correlation with the centers of bean genetic diversification (BD centers). By cross-inoculating wild common beans from the three BD centers with soils from Mexico, Ecuador, Bolivia, and Northwestern Argentina, the R. etli populations from nodules originated from Mexican soil again showed allele predominance that was opposite to those originated from Bolivian and Argentinean soil, whereas populations from Ecuadorian soil were intermediate. These results also indicated that the preferential nodulation of beans by geographically related R. etli lineages was independent of the nodulating environment. Coinoculation of wild common beans from each of the three BD centers with an equicellular mixture of R. etli strains representative of the Mesoamerican and southern Andean lineages revealed a host-dependent distinct competitiveness: beans from the Mesoamerican genetic pool were almost exclusively nodulated by strains from their host region, whereas nodules of beans from the southern Andes were largely occupied by the geographically cognate R. etli lineages. These results suggest coevolution in the centers of host genetic diversification.

Characteristics of rhizobia nodulating beans in the central region of Minnesota.

Until recently, beans (Phaseolus vulgaris L.) grown in Minnesota were rarely inoculated. Because of this, we hypothesized that bean rhizobia collected in Minnesota would either share characteristics identifiable with Rhizobium etli of Mesoamerican or Andean origin, introduced into the region as seed-borne contaminants, or be indigenous rhizobia from prairie species, such as Dalea spp. The latter organisms have been shown to nodulate and fix N2 with Phaseolus vulgaris. Rhizobia recovered from the Staples, Verndale, and Park Rapids areas of Minnesota were grouped according to the results of BOXA1R-PCR fingerprint analysis into 5 groups, with only one of these having banding patterns similar to 2 of 4 R. etli reference strains. When representative isolates were subject to fatty acid - methyl ester analysis and 16S rRNA gene sequence analysis, the results obtained differed. 16S rRNA gene sequences of half the organisms tested were most similar to Rhizobium leguminosarum. Rhizobia from Dalea spp., an important legume in the prairie ecosystem, did not play a significant role as the microsymbiont of beans in this area. This appears to be due to the longer time needed for them to initiate infection in Phaseolus vulgaris. Strains of Rhizobium tropici IIB, including UMR1899, proved tolerant to streptomycin and captan, which are commonly applied as seed treatments for beans. Local rhizobia appeared to have very limited tolerance to these compounds.

Anions effects on biosorption of Mn(II) by extracellular polymeric substance (EPS) from Rhizobium etli.

Microbial extracellular polymeric substances (EPS) are potential biosorbents for metal remediation and recovery. The Langmuir and Freundlich kinetics of Mn(II) binding by the EPS from a novel Mn(II) oxidising strain of Rhizobium etli were determined. Maximum manganese specific adsorptions (q(max)) decreased in the sequence: sulphate (62 mg Mn per g EPS) > nitrate (53 mg g(-1)) > chloride (21 mg g(-1)). Consideration of the anion during kinetic studies is usually neglected but is important in providing more practical and comparable data between different biosorbent systems.