Phylogenetic analysis of 23S rRNA gene sequences of. Agrobacterium, Rhizobium and Sinorhizobium strains

The phylogenetic relationship among twelve Agrobacterium, four Rhizobium, and two Sinorhizobium strains originating from various host plants and geographical regions was studied by analysis of the 23S rDNA sequences. The study included Agrobacterium strains belonging to biovars 1, 2 (with tumor- or hairy-root inducing and non-pathogenic strains), A. vitis, A. rubi; representative species of the Rhizobium genus: R. galegae, R. leguminosarum and R. tropici and Sinorhizobium meliloti strains. The phylogenetic analysis showed that within Agrobacterium, the biovar designation was reflected in the 23S rDNA similarity and that strains of Agrobacterium and Rhizobium are closely related to each other. The results suggest that the taxonomic definition of Agrobacterium and Rhizobium should be considered for revision and that the Agrobacterium-biovar identity is probably a reliable taxonomic trait.     

Effect of protein additives on acetylene reduction (nitrogen fixation) by Rhizobium in the presence and absence of soybean cells

The effect of protein additives on acetylene reduction (N(2) fixation) by Rhizobium associated with soybean cells (Glycine max [L.] Merr.) in vitro was studied. Acetylene reduction was promoted on the basal medium supplemented with 1.4 mg of N/ml supplied as aqueous extracts of hexane-extracted soybean, red kidney beans (Phaseolus vulgaris L.), or peas (Pisum sativum L.). Commercial samples of alpha-casein, or bovine serum albumin also promoted acetylene reduction at a concentration of 1.4 mg of N/ml of basal medium, but egg albumin supplying an equal amount of nitrogen to the basal medium completely suppressed acetylene reduction. Autoclaving the aqueous extract of hexane-extracted soybean meal had no effect on its ability to promote acetylene reduction. The presence of 40 mm succinate decreased acetylene reduction with leguminous proteins supplying 1.4 mg of N/ml but promoted acetylene reduction by Rhizobium 32H1-soybean cell associations on media containing alpha-casein, bovine serum albumin, or egg albumin suppling 1.4 mg of N/ml. Similar results were obtained with both cowpea Rhizobium 32H1 and Rhizobium japonicum 61A96. Pure cultures of Rhizobium 32H1 developed acetylene-reducing activity in the presence of soybean extract on basal agar medium and in vermiculite supplied with N-free mineral salts plus crude soybean meal. The results suggest that in certain situations, free living Rhizobium may reduce N(2) under field conditions.     

Identification of Rhizobium-specific intergenic mosaic elements within an essential two-component regulatory system of Rhizobium species.

Analysis of the DNA regions upstream of the phosphoenolpyruvate carboxykinase gene (pckA) in Rhizobium meliloti and Rhizobium sp. strain NGR234 identified an open reading frame which was highly homologous to the Agrobacterium tumefaciens chromosomal virulence gene product ChvI. A second gene product, 500 bp downstream of the chvI-like gene in R. meliloti, was homologous to the A. tumefaciens ChvG protein. The homology between the R. meliloti and A. tumefaciens genes was confirmed, because the R. meliloti chvI and chvG genes complemented A. tumefaciens chvI and chvG mutants for growth on complex media. We were unable to construct chvI or chvG insertion mutants of R. meliloti, whereas mutants carrying insertions outside of these genes were readily obtained. A 108-bp repeat element characterized by two large palindromes was identified in the chvI and chvG intergenic regions of both Rhizobium species. This element was duplicated in Rhizobium sp. strain NGR234. Another structurally similar element with a size of 109 bp was present in R. meliloti but not in Rhizobium sp. strain NGR234. These elements were named rhizobium-specific intergenic mosaic elements (RIMEs), because their distribution seems to be limited to members of the family Rhizobiaceae. A homology search in GenBank detected six more copies of the first element (RIME1), all in Rhizobium species, and three extra copies of the second element (RIME2), only in R. meliloti. Southern blot analysis with a probe specific to RIME1 showed the presence of several copies of the element in the genome of R. meliloti, Rhizobium sp. strain NGR234, Rhizobium leguminosarum, and Agrobacterium rhizogenes, but none was present in A. tumefaciens and Bradyrhizobium japonicum.     

Host genes inPisum sativum L. conferring resistance to EuropeanRhizobium leguminosarum strains

Summary Using primitive and wild pea plants from Afghanistan, Iran and Turkey, three host genes were detected, which confer resistance to nodulation by Rhizobium strains of cultivated peas from Europe. A dominant gene Sym 1 controls temperature-sensitive nodulation in pea cv. Iran. Another gene Sym 2 confers general resistance to a large number of European Rhizobium strains at all temperatures used. The degree of dominance of the latter gene is dependent on the Rhizobium strain used. A third gene Sym 4 is responsible for specific resistance to a single Rhizobium strain.     

Isolation and characterization of the DNA region encoding nodulation functions in Bradyrhizobium japonicum.

The DNA region encoding early nodulation functions of Bradyrhizobium japonicum 3I1b110 (I110) was isolated by its homology to the functionally similar region from Rhizobium meliloti. Isolation of a number of overlapping recombinant clones from this region allowed the construction of a restriction map of the region. The identified nodulation region of B. japonicum shows homology exclusively to those regions of R. meliloti and Rhizobium leguminosarum DNA known to encode early nodulation functions. The region of homology with these two fast-growing Rhizobium species was narrowed to an 11.7-kilobase segment. A nodulation-defective mutant of Rhizobium fredii USDA 201, strain A05B-2, was isolated and found to be defective in the ability to curl soybean root hairs. Some of the isolated recombinant DNA clones of B. japonicum were found to restore wild-type nodulation function to this mutant. Analysis of the complementation results allows the identification of a 1.8-kilobase region as essential for restoration of Hac function.     

Isolation of unique nucleic acid sequences from rhizobia by genomic subtraction: Applications in microbial ecology and symbiotic gene analysis

A subtraction hybridization and PCR amplification procedure was used to isolate two Rhizobium DNA probes which exhibited high degrees of specificity at different levels of taxonomic organization and which could be used as tools for detection of rhizobia in ecological studies. First, a probe was isolated from Rhizobium leguminosarum bv. trifolii strain P3 by removing those Sau3A restriction fragments from a P3 DNA digest which cross hybridized with pooled DNA from seven other strains of the same biovar. The remaining restriction fragments hybridized to DNA from strain P3 but not to DNA from any of the seven other strains. In a similar experiment another DNA probe, specific for the Rhizobium leguminosarum bv. phaseoli and Rhizobium tropici group, was generated by removing sequences from R. leguminosarum bv phaseoli strain Kim 5s with pooled subtracter DNA from eight other Rhizobium, Bradyrhizobium and Agrobacterium species. The same subtraction hybridization technique was also used to isolate symbiotic genes from a Rhizobium species. Results from a 1:1 subtractive DNA hybridization of the broad host range Rhizobium sp NGR234 against highly homologous S. fredii USDA257, combined with those from competitive RNA hybridizations to cosmid digests of the NGR234 symbiotic plasmid, allowed the identification of several NGR234 loci which were flavonoid-inducible and not present in S. fredii USDA257. One of these, ORF-1, was highly homologous to the leucine responsive regulatory protein of E. coli.     

AMF和饭豆根瘤菌对饭豆、玉米套种促生作用的研究

将从饭且根瘤中分离的饭豆根瘤菌(Rhizobium sp.CYY3302,Rhizobium sp.HCY9101,Rhizobium SP.JMC1402)与ANF(Arbuscular Mycorrltizal Fungi)共同接种于饭豆,进行饭豆、玉米田间小区各种试验。结果表明,接种饭豆根瘤菌和AMF的处理与未接种的处理相比,饭豆的结瘸率比对照提高52％-134％;饭豆及玉米的菌根感染率比对照分别增加43．1％-80％和46．8％-97．6％;饭豆的产量提高了54％-67％,而玉米的产量提高了2．4％-19．5％。研究结果还表明：豆科作物接种根瘸菌,即使在种过豆科作物的老区,也是有效的。     

Comparative properties of glutamine synthetases I and II in Rhizobium and Agrobacterium spp

Some properties of glutamine synthetase I (GSI) and GSII are described for a fast-growing Rhizobium sp. (Rhizobium trifolii T1), a slow-growing Rhizobium sp. (Rhizobium japonicum USDA 83), and Agrobacterium tumefaciens C58. GSII of the fast-growing Rhizobium sp. and GSII of the Agrobacterium sp. were considerably more heat labile than GSII of the slow-growing Rhizobium sp. As previously shown in R. japonicum 61A76, GSI became adenylylated rapidly in all species tested in response to ammonium. GSII activity disappeared within one generation of growth in two of the strains, but the disappearance of GSII activity required two generations in another. Isoactivity points for transferase assay, which were derived from the pH curves of adenylylated GSI and deadenylylated GSI, were approximately pH 7.8 for both R. trifolii and A. tumefaciens. No isoactivity point was found for R. japonicum under the standard assay conditions used. When the feedback inhibitor glycine was used to inhibit differentially the adenylylated GSI and deadenylylated GSI of R. japonicum, an isoactivity point was observed at pH 7.3. Thus, the transferase activity of GSI could be determined independent of the state of adenylation. A survey of 23 strains of bacteria representing 11 genera indicated that only Rhizobium spp. and Agrobacterium spp. contained GSII. Thus, this enzyme appears to be unique for the Rhizobiaceae.     

Isolation of the replication DNA region from a Rhizobium plasmid and examination of its potential as a replicon for Rhizobiaceae cloning vectors

The DNA region essential for replication and stability of a native plasmid (pTM5) from Rhizobium sp. (Hedysarum) has been identified and isolated within a 5.4-kb PstI restriction fragment. The isolation of this region was accomplished by cloning endonuclease-restricted pTM5 DNA into a ColE1-type replicon and selecting the recombinant plasmids containing the pTM5 replicator (pTM5 derivative plasmids) by their ability to replicate in Rhizobium. DNA homology studies revealed that pTM5-like replicons are present in cryptic plasmids from some Rhizobium sp. (Hedysarum) strains but not in plasmids from strains of other Rhizobium species or Agrobacterium tumefaciens. The pTM5 derivative plasmids were able to replicate in Escherichia coli and A. tumefaciens and in a wide range of Rhizobium species. On the basis of stability assays in the absence of antibiotic selective pressure, the pTM5 derivative plasmids were shown to be highly stable in both free-living and symbiotic cells of Rhizobium sp. (Hedysarum). The stability of these plasmids in other species of Rhizobium and in A. tumefaciens varied depending on the host and on the plasmid. Most pTM5 derivative plasmids tested showed significantly higher symbiotic stability than RK2 derivative plasmids pRK290 and pAL618 in Rhizobium sp. (Hedysarum), R. meliloti, and R. leguminosarum by. phaseoli. Consequently, we consider that the constructed pTM5 derivative plasmids are potentially useful as cloning vectors for Rhizobiaceae.     

Suicide plasmid vehicles for insertion mutagenesis in Rhizobium meliloti and related bacteria.

We describe the construction and use of a set of plasmid vectors of the transposons Tn1, Tn5, and Tn9 that are suicidal in Rhizobium species and therefore suitable for mutagenesis with these three transposons. The vectors are composed of the p15A replicon which functions in Escherichia coli but not in Rhizobium species and a region encoding the N type of bacterial conjugation system which is very efficient in matings between E. coli and Rhizobium species. The usefulness of the vectors has been most extensively assessed in Rhizobium meliloti. It is likely that they will be useful for mutagenesis and genome manipulation in other bacteria as well.     

Adsorption and selection of rhizobia with ion-exchange papers

Ion exchange papers were used to study the adsorption of 32P-labelled rhizobia on defined surfaces. Two strains of Rhizobium japonicum and one each of R. leguminosarum and R. lupini were compared with Escherichia coli and Bacillus subtilis. The ratio of adsorption to strong and to weak acid papers/strong and weak basic papers was consistantly higher for all rhizobial strains compared to the other bacteria. The process of desorption by increasing the ion-concentration causes about 35% desorption between 0.02 and 0.1 M MgCl2, however, an increase to 1 M does not desorb more labelled Rhizobium japonicum or E. coli cells. The ratio of adsorbed cpm to colony formers, desorbed by 0.1 M NaCl was similar with Rhizobium japonicum for all six ion exchange papers. For E. coli this ratio varied widely for the different papers. The selection of Rhizobium against a more closely related bacterium by this adsorption/desorption procedure was demonstrated with mixed cultures of Rhizobium japonicum and Chromobacterium violaceum giving a more than 80 fold enrichment of the former. Rhizobium japonicum cells, ad/desorbed from all ion exchange papers kept their infectivity and formed nodules on Glycine max with an activity of 20-40 nM C2H4-hr(-1)-mg nodule(-1). A desorption of Rhizobium japonicum from soybean roots also occurred by increasing the ion concentration. 2-3 times as many cells were removed in this way compared to washing with water.     

Enzymic degradation of chemically modified extracellular polysaccharides from Rhizobia

Extracellular polysaccharides from Rhizobium trifolii, U226, Coryn and Bart A; Rhizobium phaseoli, U453; Rhizobium leguminosarum, U331; and Rhizobium meliloti, U27, after chemical modification, become substrates for certain β-d-glucan hydrolases. The Streptomyces (1 → 4)-β-d-glucan endohydrolase (EC 3.2.1.4) hydrolyses reduced and deacetylated rhizobial polysaccharides, both before and after removal of carboxyethylidene substituents, to produce a series of oligosaccharides. The Rhizopus arrhizus (1 → 3)-β-d-glucan endohydrolase (EC 3.2.1.6) hydrolyses only fully modified polysaccharides to yield, in the case of R. meliloti U27, laminarabiose, and, in all other instances, a disaccharide identified β-d-Gal-(1 → 3)-D-Glc. The same disaccharides are released by the Rhizopus enzyme from oligosaccharides produced by the action of the Streptomyces enzyme on fully modified polysaccharides. The results are discussed in relation to the available data for the structure of the polysaccharides and the specificity of the enzymes.     

Control of ammonium assimilation in Rhizobium 32H1.

The symbiotic, nitrogen-fixing bacterium Rhizobium sp. 32H1 is a specialized ammonium producer during symbiosis. However, during free-living growth, Rhizobium 32H1 assimilates ammonium very poorly. Two pathways of ammonium assimilation exist in enteric bacteria. One is mediated by glutamate dehydrogenase, and the other is mediated by glutamine synthetase-glutamate synthase. The former pathway is altogether inoperative in Rhizobium 32H1; the latter pathway operates at a slow rate and is under strict negative control by ammonium itself. Rhizobium 32H1 glutamine synthetase activity is modulated by both repression-derepression and reversible adenylylation. For a biochemical process lacking an alternative pathway, such a regulatory pattern exacerbates the very process. This suggests that Rhizobium 32H1 restricts its own ammonium assimilation to maximize the contribution of fixed nitrogen to the host plant during symbiosis.     

Identification of 3-deoxy-lyxo-2-heptulosaric acid in the core region of lipopolysaccharides from Rhizobiaceae

A 3-deoxy-2-heptulosaric acid (DHA), very probably with the lyxo-configuration, was identified in the R-core region of lipopolysaccharides from nodulating strains of Rhizobium leguminosarum, Rhizobium meliloti and from all three biovars of the phytopathogenic Agrobacterium tumefaciens. Its structure could be deduced from the fragmentation pattern of the corresponding alditol acetates obtained after reduction of the 2-keto and the 1.7-carboxy groups by sodium borohydride or sodium borodeuteride. DHA in lipopolysaccharide was not destroyed by periodate and is therefore not in a terminal position. Two DHA-containing oligosaccharides, namely glucosyl (1----4)-3-deoxy-2-heptulosaric acid and rhamnosyl-rhamnosyl-(1----5)-3-deoxy-2-heptulosaric acid could be tentatively identified by mass spectrometric methods amongst the products of mild acidic hydrolysis of lipopolysaccharides of Rhizobium leguminosarum strain 24. The two types of non-nodulating mutants of Rhizobium leguminosarum included in this study did not contain 3-deoxy-2-heptulosaric acid.     

Conservation of plasmid-encoded traits among bean-nodulating Rhizobium species

Rhizobium etli type strain CFN42 contains six plasmids. We analyzed the distribution of genetic markers from some of these plasmids in bean-nodulating strains belonging to different species (Rhizobium etli, Rhizobium gallicum, Rhizobium giardinii, Rhizobium leguminosarum, and Sinorhizobium fredii). Our results indicate that independent of geographic origin, R. etli strains usually share not only the pSym plasmid but also other plasmids containing symbiosis-related genes, with a similar organization. In contrast, strains belonging to other bean-nodulating species seem to have acquired only the pSym plasmid from R. etli.     

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.     

Co-evolution of the legume-Rhizobium association

Summary A number of examples is given demonstrating the co-existence of pea genotypes and their specific Rhizobium, strains isolated within the same region.R. leguminosarum strains compatible with the cultivated pea have a narrow symbiotic range and they are widely distributed in European soils. This is presumably due to the narrow genetic base of the cultivated pea and its wide-spread cultivation in European soils. Rhizobium strains capable of nodulating a primitive pea line from Afghanistan were only found in soils of the Middle East and Central Asia. A more restricted distribution of specific Rhizobium strains was found for fulvum peas from Israel. Rhizobium strains effective with the fulvum pea were found in Israeli soils. A good example of co-evolution due to geographical isolation was found in south Turkey. Here a pea line was found which can form an effective symbiosis with local Rhizobium strains but not with strains from other parts of Turkey.