Phylogenetic position of Rhizobium sp. strain Or 191, a symbiont of both Medicago sativa and Phaseolus vulgaris, based on partial sequences of the 16S rRNA and nifH genes.

Phenotypic and DNA sequence comparisons are presented for eight Rhizobium isolates that were cultured from field-grown alfalfa (Medicago sativa L.) in Oregon. These isolates were previously shown to nodulate both alfalfa and common bean (Phaseolus vulgaris (L.) Savi.). The objective of the present study was to determine their phylogenetic relationships to the normal symbionts of these plants, Rhizobium meliloti and Rhizobium leguminosarum biovar phaseoli, respectively. Phenotypically, the Oregon isolates more nearly resemble strains from P. vulgaris than those from M. sativa. For example, even though nitrogen fixation levels were low with both host species, the symbiotic efficiency of a representative Rhizobium isolate (Or 191) with common bean was twice that observed with alfalfa. Comparative sequencing of a 260-bp segment of the 16S rRNA gene (directly sequenced after amplification by the polymerase chain reaction) demonstrated that Or 191 is not closely related to the type strain of R. meliloti (ATCC 9930), R. leguminosarum (ATCC 10004), or Rhizobium tropici (CIAT 899). Instead, sequence comparisons of the 16S gene indicated that Or 191 belongs to a distinct and previously unrecognized taxonomic group that includes strains that have previously been called R. leguminosarum bv. phaseoli type I. Unlike type I strains, however, Or 191 has only a single copy of the nifH gene (type I strains have three), and the nucleotide sequence of this gene is substantially different from those of other rhizobial and nonrhizobial nifH genes examined thus far.     

Genetic structure of a soil population of nonsymbiotic Rhizobium leguminosarum.

The genetic structure of a population of nonsymbiotic Rhizobium leguminosarum strains was determined by the electrophoretic mobilities of eight metabolic enzymes. Nonsymbiotic strains were isolated from the rhizosphere of bean plants and characterized by growth on differential media and at different temperatures, intrinsic antibiotic resistance, the lack of homology to a nifH probe, and their inability to form nodules on bean roots. All the isolates clustered with R. leguminosarum bv. phaseoli reference strains and did not encompass any other Rhizobium taxa. Their rRNA operon restriction fragment length polymorphisms and the nucleotide sequence of a fragment of the 16S rRNA gene were also found to be identical to those of R. leguminosarum bv. phaseoli reference strains. When complemented with an R. leguminosarum bv. phaseoli symbiotic plasmid (p42d), the nonsymbiotic isolates were able to fix nitrogen in symbiosis with bean roots at levels similar to those of the parental strain. The symbiotic isolates were found at a relative frequency of 1 in 40 nonsymbiotic R. leguminosarum strains.     

Genetic structure of a soil population of nonsymbiotic Rhizobium leguminosarum.

The genetic structure of a population of nonsymbiotic Rhizobium leguminosarum strains was determined by the electrophoretic mobilities of eight metabolic enzymes. Nonsymbiotic strains were isolated from the rhizosphere of bean plants and characterized by growth on differential media and at different temperatures, intrinsic antibiotic resistance, the lack of homology to a nifH probe, and their inability to form nodules on bean roots. All the isolates clustered with R. leguminosarum bv. phaseoli reference strains and did not encompass any other Rhizobium taxa. Their rRNA operon restriction fragment length polymorphisms and the nucleotide sequence of a fragment of the 16S rRNA gene were also found to be identical to those of R. leguminosarum bv. phaseoli reference strains. When complemented with an R. leguminosarum bv. phaseoli symbiotic plasmid (p42d), the nonsymbiotic isolates were able to fix nitrogen in symbiosis with bean roots at levels similar to those of the parental strain. The symbiotic isolates were found at a relative frequency of 1 in 40 nonsymbiotic R. leguminosarum strains.     

Distribution of insertion sequence ISRm1 in Rhizobium meliloti and other gram-negative bacteria

An internal 0.9 kb segment of Rhizobium meliloti insertion sequence ISRm1 was used as a probe to determine the distribution of ISRm1 in strains of R. meliloti and other Gram-negative bacteria. The insertion sequence was detected in 80% (12/15) of R. meliloti strains from different parts of the world. Its copy number ranged from one to at least eleven. The ISRm1 copies detected showed variation in their internal restriction sites and their degree of homology to the probe. ISRm1 was found in a variety of genomic restriction fragments, and was detected in plasmids, including the nod and exo megaplasmids of R. meliloti. Other rhizobia found to contain ISRm1 were a strain of R. leguminosarum biovar phaseoli and two Rhizobium isolates capable of nodulating both Medicago sativa and Phaseolus vulgaris. It was also found in a diazotrophic soil bacterium isolated from the roots of wetland rice.     

Rhizobium tropici, a novel species nodulating Phaseolus vulgaris L. beans and Leucaena sp. trees.

A new Rhizobium species that nodulates Phaseolus vulgaris L. and Leucaena spp. is proposed on the basis of the results of multilocus enzyme electrophoresis, DNA-DNA hybridization, an analysis of ribosomal DNA organization, a sequence analysis of 16S rDNA, and an analysis of phenotypic characteristics. This taxon, Rhizobium tropici sp. nov., was previously named Rhizobium leguminosarum biovar phaseoli (type II strains) and was recognized by its host range (which includes Leucaena spp.) and nif gene organization. In contrast to R. leguminosarum biovar phaseoli, R. tropici strains tolerate high temperatures and high levels of acidity in culture and are symbiotically more stable. We identified two subgroups within R. tropici and describe them in this paper.     

Phylogeny and genetic diversity of native rhizobia nodulating common bean (Phaseolus vulgaris L.) in Ethiopia

The diversity and phylogeny of 32 rhizobial strains isolated from nodules of common bean plants grown on 30 sites in Ethiopia were examined using AFLP fingerprinting and MLSA. Based on cluster analysis of AFLP fingerprints, test strains were grouped into six genomic clusters and six single positions. In a tree built from concatenated sequences of recA, glnII, rpoB and partial 16S rRNA genes, the strains were distributed into seven monophyletic groups. The strains in the groups B, D, E, G1 and G2 could be classified as Rhizobium phaseoli, R. etli, R. giardinii, Agrobacterium tumefaciens complex and A. radiobacter, respectively, whereas the strains in group C appeared to represent a novel species. R. phaseoli, R. etli, and the novel group were the major bean nodulating rhizobia in Ethiopia. The strains in group A were linked to R. leguminosarum species lineages but not resolved. Based on recA, rpoB and 16S rRNA genes sequences analysis, a single test strain was assigned as R. leucaenae. In the nodC tree the strains belonging to the major nodulating groups were clustered into two closely linked clades. They also had almost identical nifH gene sequences. The phylogenies of nodC and nifH genes of the strains belonging to R. leguminosarum, R. phaseoli, R. etli and the putative new species (collectively called R. leguminosarum species complex) were not consistent with the housekeeping genes, suggesting symbiotic genes have a common origin which is different from the core genome of the species and indicative of horizontal gene transfer among these rhizobia.     

Identification and characterization of a nodH ortholog from the alfalfa-nodulating Or191-like rhizobia

Nodulation of Medicago sativa (alfalfa) is known to be restricted to Sinorhizobium meliloti and a few other rhizobia that include the poorly characterized isolates related to Rhizobium sp. strain Or191. Distinctive features of the symbiosis between alfalfa and S. meliloti are the marked specificity from the plant to the bacteria and the strict requirement for the presence of sulfated lipochitooligosaccharides (Nod factors [NFs]) at its reducing end. Here, we present evidence of the presence of a functional nodH-encoded NF sulfotransferase in the Or191-like rhizobia. The nodH gene, present in single copy, maps to a high molecular weight megaplasmid. As in S. meliloti, a nodF homolog was identified immediately upstream of nodH that was transcribed in the opposite direction (local synteny). This novel nodH ortholog was cloned and shown to restore both NF sulfation and the Nif+Fix+ phenotypes when introduced into an S. meliloti nodH mutant. Unexpectedly, however, nodH disruption in the Or191-like bacteria did not abolish their ability to nodulate alfalfa, resulting instead in a severely delayed nodulation. In agreement with evidence from other authors, the nodH sequence analysis strongly supports the idea that the Or191-like rhizobia most likely represent a genetic mosaic resulting from the horizontal transfer of symbiotic genes from a sinorhizobial megaplasmid to a not yet clearly identified ancestor.     

Large plasmids of fast-growing rhizobia: homology studies and location of structural nitrogen fixation (nif) genes.

A single large plasmid was isolated from multiplasmid-harboring strains Rhizobium leguminosarum 1001 and R. trifolii 5. These single plasmids, as well as the largest plasmid detectable in R. phaseoli 3622, hybridized with part of the nif structural genes of Klebsiella pneumoniae. In contrast, the plasmids of R. meliloti strains V7 and L5-30 did not show hybridization with the nif genes of K. pneumoniae, indicating that these genes might be located either on the chromosome or on a much larger plasmid which as yet has not been isolated. Studies of the homology between plasmids of fast-growing Rhizobium species showed that a specific deoxyribonucleic acid sequence, which carries the structural genes for nitrogenase, is highly conserved on a plasmid in R. leguminosarum, R. trifolii, and R. phaseoli. Furthermore, it was found that this type of plasmid in the different species shares extensive deoxyribonucleic acid homology, suggesting that strains in the R. leguminosarum cluster have preserved a nif plasmid.     

The Rhizobium meliloti host range nodQ gene encodes a protein which shares homology with translation elongation and initiation factors.

The Rhizobium meliloti nod region IIb is involved in host-range determination: (i) the presence of region IIb is necessary for transfer of alfalfa root hair curling ability to Rhizobium leguminosarum biovar trifolii; (ii) a mutation in region IIb extends the R. meliloti infection host range to Vicia sativa nigra; (iii) dominance of R. meliloti nod genes over R. leguminosarum biovar viciae nod genes is abolished by mutations in region IIb. The nucleotide sequence of this region has been determined. Genes corresponding to the two open reading frames identified are designated nodP and nodQ. The predicted amino acid sequence of the NodQ protein shows homology with translation initiation and elongation factors. The consensus sequence involved in the GTP-binding domain is conserved.     

Recognition of individual strains of fast-growing rhizobia by using profiles of membrane proteins and lipopolysaccharides.

Membrane protein and lipopolysaccharide profiles of Rhizobium leguminosarum (biovars viciae, trifolii, and phaseoli), R. meliloti, and Agrobacterium tumefaciens strains were analyzed and compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Differences in one or both profiles allowed us to distinguish all 18 R. leguminosarum strains tested in this study from each other.     

Two genes that regulate exopolysaccharide production in Rhizobium meliloti.

We describe a new Rhizobium meliloti gene, exoX, that regulates the synthesis of the exopolysaccharide, succinoglycan, exoX resembled the psi gene of R. leguminosarum bv. phaseoli and the exoX gene of Rhizobium sp. strain NGR234 in its ability to inhibit exopolysaccharide synthesis when present in multiple copies, exoX did not appear to regulate the expression of exoP. The effect of exoX was counterbalanced by another R. meliloti gene, exoF. exoF is equivalent to Rhizobium sp. strain NGR234 exoY and resembles R. leguminosarum bv. phaseoli pss2 in its mutant phenotype and in portions of its deduced amino acid sequence. The effect of exoF on the succinoglycan-inhibiting activity of exoX depended on the relative copy numbers of the two genes. exoX-lacZ fusions manifested threefold-higher beta-galactosidase activities in exoF backgrounds than in the wild-type background. exoX mutants produced increased levels of succinoglycan. However, the exoF gene was required for succinoglycan synthesis even in an exoX mutant background. exoF did not affect the expression of exoP. Strains containing multicopy exoX formed non-nitrogen-fixing nodules on alfalfa that resembled nodules formed by exo mutants defective in succinoglycan synthesis. exoX mutants formed nitrogen-fixing nodules, indicating that, if the inhibition of succinoglycan synthesis within the nodule is necessary for nitrogen fixation, then exoX is not required for this inhibition. We present indirect evidence that succinoglycan synthesis within the nodule is not necessary for bacteroid function.     

Phylogeny of fast-growing soybean-nodulating rhizobia support synonymy of Sinorhizobium and Rhizobium and assignment to Rhizobium fredii.

We determined the sequences for a 260-base segment amplified by the polymerase chain reaction (corresponding to positions 44 to 337 in the Escherichia coli 16S rRNA sequence) from seven strains of fast-growing soybean-nodulating rhizobia (including the type strains of Rhizobium fredii chemovar fredii, Rhizobium fredii chemovar siensis, Sinorhizobium fredii, and Sinorhizobium xinjiangensis) and broad-host-range Rhizobium sp. strain NGR 234. These sequences were compared with the corresponding previously published sequences of Rhizobium leguminosarum, Rhizobium meliloti, Agrobacterium tumefaciens, Azorhizobium caulinodans, and Bradyrhizobium japonicum. All of the sequences of the fast-growing soybean rhizobia, including strain NGR 234, were identical to the sequence of R. meliloti and similar to the sequence of R. leguminosarum. These results are discussed in relation to previous findings; we concluded that the fast-growing soybean-nodulating rhizobia belong in the genus Rhizobium and should be called Rhizobium fredii.     

Transconjugants of Agrobacterium radiobacter harbouring sym genes of Rhizobium galegae can form an effective symbiosis with Medicago sativa

It is known that the Rhizobium galegae genomes contain megaplasmids. The suicide vector pSUP2111 with nifH gene of R. meliloti was introduced into the strains CIAM 0703 and CIAM 0711 of R. galegae inducing effective nodules on Galega orientalis plants. The formation of self-transmissible megaplasmids was observed. The megaplasmid transfer into non-nodulating R. meliloti mutants resulted in partial complementation of the nodulation defect in recipient strains though only one transconjugant showed the nitrogen-fixing activity in symbiosis with alfalfa and another one in symbiosis with G. orientalis plants. Among the Agrobacterium strains harbouring R. galegae megaplasmids there were four classes of transconjugants: (1) Nod+ Fix- in symbiosis with goat's rue plants (three strains); (2) Nod+ Fix- on Medicago sativa (two strains); (3) Nod+ Fix+ on M. sativa (five strains); (4) Nod- with both plant hosts (11 strains).     

The diversity of Phaseolus-nodulating rhizobial populations is altered by liming of acid soils planted with Phaseolus vulgaris L. in Brazil

PCR-mediated restriction fragment length polymorphism (RFLP) analysis of the 16S-23S rRNA internally transcribed spacer (ITS) region and the 16S rRNA gene indicated that the rhizobial populations isolated from common bean (Phaseolus vulgaris L.) nodules in the unlimed soil from a series of five lime rates applied 6 years previously to plots of an acidic oxisol had less diversity than those from plots with higher rates of liming. Isolates affiliated with Rhizobium tropici IIB and Rhizobium leguminosarum bv. phaseoli were predominant independent of lime application. An index of richness based on the number of ITS groups increased from 2.2 to 5.7 along the soil liming gradient, and the richness index based on "species" types determined by RFLP analysis of the 16S rRNA gene varied from 0.5 to 1.4. The Shannon index of diversity, based on the number of ITS groups, increased from 1.8 in unlimed soil to 2.8 in limed soil, and, based on RFLP analysis of the 16S rRNA gene, ranged from 0.9 to 1.4. In the limed soil, the subpopulation of R. tropici IIB pattern types contained the largest number of ITS groups. In contrast, there were more R. leguminosarum bv. phaseoli types in the unlimed soil with the lowest pH than in soils with the highest pH. The number of ITS ("strain") groups within R. leguminosarum bv. phaseoli did not change with increased abundance of rhizobia in the soil, while with R. tropici IIB, the number of strain groups increased significantly. Some cultural and biochemical characteristics of Phaseolus-nodulating isolates were significantly related to changes in soil properties caused by liming, largely due to changes in the predominance of the rhizobial species groups.     

Comparison of geographically distant populations of Rhizobium isolated from root nodules of Phaseolus vulgaris

Seventy-two rhizobial strains were isolated from the root nodules of french beans ( Phaseolus vulgaris ). They were sampled from two geographically distant field populations and 18 additional different sites in France. They were characterized by a) plasmid profiles, (b) RFLP analysis of total cellular DNA using various chromosomal and symbiotic gene probes (including nif H from Rhizobium etli bv. phaseoli ) and c) their ability to nodulate a potential alternative host, L. leucocephala. Over half of the isolates were ascribed to Rhizobium leguminosarum bv. phaseoli on the basis of the hybridization analysis, the possession of multiple copies of nif H and their inability to nodulate L. leucocephala. The remaining isolates belonged to 2 groups which were shown to be genomically distinct from R. leguminosarum bv. phaseoli, R. etli bv. phaseoli and R. tropici. Most members of these two groups shared with R. tropici the ability to nodulate L. leucocephala and, for isolates of only one of these groups, the presence of one copy of nif H. Members of each of the 3 taxa were widely distributed in France and circumstantial evidence of pSym transfer between them was shown. R. leguminosarum bv. phaseoli and one of the two novel groups co-occurred within the two geographically distant populations. Individual genotypes were conserved between them. The finding of a third taxon at various other locations indicated additional diversity among rhizobia nodulating beans.     

Application of subtraction hybridization for the development of a Rhizobium leguminosarum biovar phaseoli and Rhizobium tropici group-specific DNA probe

Abstract A combined subtraction hybridization and polymerase chain reaction/amplification technique was used to develop a DNA probe which was specific for the Rhizobium leguminosarum biovar phaseoli and the Rhizobium tropici group. Total genomic DNA preparations from Rhizobium leguminosarum biovar viciae, Rhizobium leguminosarum biovar trifolii, Rhizobium sp., Agrobacterium tumefaciens, Rhizobium fredii, Bradyrhizobium japonicum, Bradyrhizobium ssp. and Rhizobium meliloti were pooled and used as subtracter DNA against total genomic DNA from the Rhizobium leguminosarum biovar phaseolo strain KIM5s. Only one round of subtraction hybridization at 65°C was necessary to remove all cross-hybridizing sequences. Dot blot hybridizations with total genomic DNA of the eight subtracter organisms and 29 bacteria of different groups confirmed the high specificity of the isolated DNA sequences. Dot blot hybridizations and total genomic DNA from ten different R. Leguminosarum biovar phaseoli and R. tropici strains resulted in strong hybridization signals for all strains tested. The DNA probe for the R. tropici and R. leguminosarum biovar phaseoli group was used for dot blot hybridization with DNA extracts from three tropical and one boreal soil. When correlated with data from Most Probable Number analyses the probe was capable of detecting as low as 3 × 10⁴ homologous indigenous rhizobia per g soil. The technique offers great benefits for the development of DNA probes for monitoring bacterial populations in environmental samples.     

Characterization, distribution, and localization of ISRl2, and insertion sequence element isolated from Rhizobium leguminosarum bv. viciae.

An insertion sequence (IS) element, ISR12, from Rhizobium leguminosarum bv. viciae strain MSDJ4184 was isolated by insertional inactivation of the sacRB gene of pSUP104-sac, which allows positive selection. ISRl2 is 932 bp long, is flanked by 17-bp imperfect terminal inverted repeats, and generated a 3-bp target site duplication. ISRl2 was found to be 63 to 77% homologous to insertion elements of the IS5 group of the IS4 superfamily. A probe incorporating a full-length copy of ISRl2 was used to screen genomic DNAs from a collection of strains and from two field populations of R. leguminosarum to detect and estimate the copy numbers of homologous sequences. Among the collection of 63 strains representing the different species and genera of members of the family Rhizobiaceae, homology to ISRl2 was found within strains belonging to Sinorhizobium meliloti and S. fredii; within four of the six recognized Rhizobium species. R. leguminosarum, R. tropici, R. etli, and R. galegae; and within Rhizobium sp. (Phaseolus) genomic species 2. The apparent copy numbers of ISRl2 varied from one to eight. Among 139 isolates of R. leguminosarum from two field populations, homology to ISRl2 was detected in 91% of the isolates from one site and in 17% from the other. Analysis of the 95 isolates that hybridize to ISRl2 revealed a total of 20 distinct hybridization patterns composed of one to three bands. Probing blots of Eckhardt gels showed that sequences with homology to ISRl2 may be found on plasmids or the chromosome. Analysis of their genomic distribution demonstrated relationships and diversity among the R. leguminosarum isolates tested.