Legume Symbiont Research Articles

A Positive Role for Rhizobitoxine in Rhizobium-legume Symbiosis

Although Bradyrhizobium elkanii is a mutualistic symbiont of legumes, it synthesizes a phytotoxin, rhizobitoxine, that causes chlorosis on a variety of legume hosts, giving a pathogenic character to these interactions. No positive role for rhizobitoxine has been previously demonstrated. Interestingly, rhizobitoxine inhibits the rate-limiting step for ethylene biosynthesis, a plant hormone known to inhibit or down-regulate nodule development. We hypothesized that rhizobitoxine plays a positive role in nodule development through its inhibition of ethylene biosynthesis. To test this hypothesis, host plants of B. elkanii were screened for a differential nodulation response to the wild-type and rhizobitoxine mutant strains. In Vigna radiata (mungbean), the rhizobitoxine mutant strains induced many aborted nodules arrested at all stages of pre-emergent and post-emergent development and formed significantly fewer mature nodules than the wild type. Experiments revealed that nodulation of mungbean plants is sensitive to exogenous ethylene, and that the ethylene inhibitors aminoethoxyvinylglycine and Co2+ were able to partially restore a wild-type nodulation pattern to the rhizobitoxine mutants. This is the first demonstration of a nodulation phenotype of the rhizobitoxine mutants and suggests that rhizobitoxine plays a positive and necessary role in Rhizobium-legume symbiosis through its inhibition of ethylene biosynthesis.

Comparison of the evolutionary dynamics of symbiotic and housekeeping loci: a case for the genetic coherence of rhizobial lineages.

In prokaryotes, lateral gene transfer across chromosomal lineages may be mediated by plasmids, phages, transposable elements, and other accessory DNA elements. However, the importance of such transfer and the evolutionary forces that may restrict gene exchange remain largely unexplored in native settings. In this study, tests of phylogenetic congruence are employed to explore the range of horizontal transfer of symbiotic (sym) loci among distinct chromosomal lineages of native rhizobia, the nitrogen-fixing symbiont of legumes. Rhizobial strains isolated from nodules of several host plant genera were sequenced at three loci: symbiotic nodulation genes (nodB and nodC), the chromosomal housekeeping locus glutamine synthetase II (GSII), and a portion of the 16S rRNA gene. Molecular phylogenetic analysis shows that each locus generally subdivides strains into the same major groups, which correspond to the genera Rhizobium, Sinorhizobium, and Mesorhizobium. This broad phylogenetic congruence indicates a lack of lateral transfer across major chromosomal subdivisions, and it contrasts with previous studies of agricultural populations showing broad transfer of sym loci across divergent chromosomal lineages. A general correspondence of the three rhizobial genera with major legume groups suggests that host plant associations may be important in the differentiation of rhizobial nod and chromosomal loci and may restrict lateral transfer among strains. The second major result is a significant incongruence of nod and GSII phylogenies within rhizobial subdivisions, which strongly suggests horizontal transfer of nod genes among congenerics. This combined evidence for lateral gene transfer within, but not between, genetic subdivisions supports the view that rhizobial genera are "reproductively isolated" and diverge independently. Differences across rhizobial genera in the specificity of host associations imply that the evolutionary dynamics of the symbiosis vary considerably across lineages in native settings.

Genetic diversity of rhizobial symbionts isolated from legume species within the genera Astragalus, Oxytropis, and Onobrychis.

The genetic diversity of 44 rhizobial isolates from Astragalus, Oxytropis, and Onobrychis spp. originating from different geographic locations was evaluated by mapped restriction site polymorphism (MRSP) analysis of 16S rRNA genes and by PCR DNA fingerprinting with repetitive sequences (REP-PCR). A comparison of tree topologies of reference strains constructed with data obtained by MRSP and by 16S rRNA gene sequence analyses showed that the topologies were in good agreement, indicating that the MSRP approach results in reasonable estimates of rhizobial phylogeny. The isolates were distributed into 14 distinct 16S rRNA gene types clustering into three major groups which corresponded with three of the genera within the legume symbionts. Most of the isolates were within the genus Mesorhizobium. Five were identified with different genomic species nodulating Lotus spp. and Cicer arietinum. Three Astragalus isolates were classified as Bradyrhizobium, one being similar to Bradyrhizobium elkanii and another being similar to Bradyrhizobium japonicum. Six of the isolates were related to species within the genus Rhizobium. Two were similar to Rhizobium leguminosarum, and the remainder were identified as Rhizobium gallicum. DNA fingerprinting by REP-PCR revealed a high level of diversity within single 16S ribosomal DNA types. The 44 isolates were distributed into 34 REP groups. Rhizobial classification at the genus and probably also the species levels was independent of geographic origin and host plant affinity.

Isolation and characterization of the UDP-glucose 4′-epimerase-encoding gene, galE, from Brucella abortus 2308

The UDP-glucose 4′-epimerase-encoding gene, galE or exoB, was isolated from Brucella abortus 2308 by complementation of an exoB mutant of Sinorhizobium meliloti. Confirmation of the identity was done by constructing an in-frame deletion of 660 bp with galE of the B. abortus genome by marker exchange. The resulting galE mutant lacked UDP-glucose 4′-epimerase activity. This activity was restored by in trans complementation with the intact gene. The B. abortus gal E mutant is not altered in colony morphology compared to wt 2308. The lack of UDP-glucose 4′-epimerase activity in the mutant and PCR analysis strongly suggest that only one copy of galE exists in B. abortus 2308. The galE sequence of B. abortus 2308 is more similar to galE from other animal-inhabiting bacteria than it is to exoB from the Sinorhizobium legume symbionts. We propose that galE in B. abortus evolved by lateral transfer from other animal-inhabiting bacteria rather than from a common ancestor of Brucella and Sinorhizobium.

Rhizobium gone native: unexpected plasmid stability of indigenous Rhizobium leguminosarum.

Lateral transfer of bacterial plasmids is thought to play an important role in microbial evolution and population dynamics. However, this assumption is based primarily on investigations of medically or agriculturally important bacterial species. To explore the role of lateral transfer in the evolution of bacterial systems not under intensive, human-mediated selection, we examined the association of genotypes at plasmid-encoded and chromosomal loci of native Rhizobium, the nitrogen-fixing symbiont of legumes. To this end, Rhizobium leguminosarum strains nodulating sympatric species of native Trifolium were characterized genetically at plasmid-encoded symbiotic (sym) regions (nodulation AB and nodulation CIJT loci) and a repeated chromosomal locus not involved in the symbiosis with legumes. Restriction fragment length polymorphism analysis was used to distinguish genetic groups at plasmid and chromosomal loci. The correlation between major sym and chromosomal genotypes and the distribution of genotypes across host plant species and sampling location were determined using chi2 analysis. In contrast to findings of previous studies, a strict association existed between major sym plasmid and chromosomal genetic groups, suggesting a lack of successful sym plasmid transfer between major Rhizobium chromosomal types. These data indicate that previous observations of sym plasmid transfer in agricultural settings may seriously overestimate the rates of successful conjugation in systems not impacted by human activities. In addition, a nonrandom distribution of Rhizobium genotypes across host plant species and sampling site demonstrates the importance of both factors in shaping Rhizobium population dynamics.

Sequencing the 500-kb GC-rich symbiotic replicon of Rhizobium sp. NGR234 using dye terminators and a thermostable "sequenase": a beginning.

Genomes of the soil-borne nitrogen-fixing symbionts of legumes [Azo(Brady)Rhizobium species] typically have GC contents of 59-65 mol%. As a consequence, compressions (up to 400 per cosmid) are common using automated dye primer shotgun sequencing methods. To overcome this difficulty, we have exclusively applied dye terminators in combination with a thermostable "sequenase" for shotgun sequencing GC-rich cosmids from pNGR234a, the 500-kbp symbiotic replicon of Rhizobium sp. NGR234. A thermostable sequenase incorporates dye terminators into DNA more efficiently than Taq DNA polymerase, thus reducing the concentrations needed (20- to 250-fold). Unincorporated dye terminators can simply be removed by ethanol precipitation. Here, we present data of pXB296, one of 23 overlapping cosmids representing pNGR234a. We demonstrate that the greatly reduced number of compressions results in a much faster assembly of cosmid sequence data by comparing assembly of the shotgun data from pXB296 and the data from another pNGR234a cosmid (pXB110) sequenced using dye primer methods. Within the 34,010-bp sequence from pXB296, 28 coding regions were predicted. All of them showed significant homologies to known proteins, including oligopeptide permeases, an essential cluster for nitrogen fixation, and the C4-dicarboxylate transporter DctA.

Perception of Rhizobium nodulation factors by tomato cells and inactivation by root chitinases.

The bacterial genera Rhizobium and Bradyrhizobium, nitrogen-fixing symbionts of legumes, secrete specific lipo-chitooligosaccharides that induce the formation of nodules on their host plants. When preparations of such nodulation-inducing factors (Nod factors) were added to suspension-cultured tomato cells, a rapid and transient alkalinization of the culture medium occurred. Lipo-oligosaccharide preparations from Rhizobium or Bradyrhizobium treated with flavonoids, known inducers of Nod factor synthesis, were up to 100 times more potent in inducing alkalinization than the ones from untreated bacteria. The activity was absent from preparations of the mutant strain Rhizobium sp. NGR234 delta nodABC, unable to produce any Nod factors. Preparations of Nod factors from various bacteria as well as individual, highly purified Nod factors from Rhizobium sp. NGR(pA28) induced alkalinization in the tomato cell cultures at nanomolar concentrations. This demonstrates that Nod factors can be perceived by tomato, a nonhost of rhizobia. Using the alkalinization response as a sensitive bioassay, Nod factors were found to be inactivated by plant chitinases. Root chitinases purified from different legumes differed in their potential to inactivate differently substituted Nod factors produced by Rhizobium sp. NGR(pA28). This indicates that the specificity of the bacterium-host plant interaction may be due, at least in part, to differential inactivation of Nod factors by root chitinases.

Rapid and sensitive assay for the phytotoxin rhizobitoxine

Rhizobitoxine is a phytotoxin synthesized by some strains of the legume symbiont genus Bradyrhizobium and the plant pathogen Pseudomonas andropogonis. We demonstrate here a new enzymatic assay which is 100-fold more sensitive than previous assays and can detect as little as 1.0 pmol of rhizobitoxine. The assay is based on the inhibition of Salmonella typhimurium beta-cystathionase by rhizobitoxine. Interestingly, beta-cystathionase from Bradyrhizobium japonicum is insensitive to rhizobitoxine at concentrations lower than 75 microM.

Rhizobia in tropical legumes: Cultural characteristics of Bradyrhizobium and Rhizobium sp.

We studied the cultural characteristics (carbohydrate utilization and associated pH changes in the media, temperature and pH optima, as well as flagellation patterns) of 51 Rhizobium sp. and 27 Bradyrhizobium sp. symbionts of tropical legumes. Both fast- and slow-growing rhizobia were isolated from nodules of Desmodium heterophyllum and D. uncinatum. Contrary to previous data, two-thirds of the bradyrhizobia could use disaccharides as the sole carbon-source. Twice as many Rhizobium sp. as Bradyrhizobium sp. utilized inositol and trehalose, suggesting that they might have some use in differentiating between the genera. The extent to which bradyrhizobia raised the pH of media was inversely correlated with the initial pH (no change at pH 7). A number of bradyrhizobia could, in combination with various carbohydrates, lower the pH, suggesting that some Bradyrhizobium sp. are able to modify their excretion products in response to environmental signals. Some Rhizobium sp. possessed only a single, subpolar flagellum. Our data thus demonstrate that Rhizobium sp. associated with tropical legumes are as heterogeneous as bradyrhizobia of the “cowpea miscellany”.

Conserved nodulation genes from the non-legume symbiont Bradyrhizobium sp. (Parasponia).

A nodulation locus from the broad-host-range, non-legume symbiont Bradyrhizobium sp. (Parasponia) strain ANU289, has been identified by hybridisation to cloned Rhizobium trifolii nodulation (nod) genes. Transfer of cloned ANU289 nod genes to R.trifolii nodulation-deficient mutants showed that the locus contains a functional homologue of the R. trifolii nodD gene. DNA sequence analysis revealed the presence of three additional genes nodA, nodB and nodC clustered adjacent to nodD. The four genes from ANU289 share substantial sequence homology with those characterised from narrow-host-range Rhizobium strains. A novel 700-bp sequence inserted between the nodD and nodABC genes encodes an open reading frame designated nodK and is oriented in the same direction as nodABC. nodKABC appear to be organized in a single transcriptional unit and nodD is oriented divergently to nodKABC. A 35-bp sequence containing the ribosome binding site for the nodD gene and an AT-rich core sequence has been identified by comparison with sequences from other Rhizobium strains and is likely to be implicated in the plant-mediated induction of nodulation gene expression.

Biological nitrogen fixation: primary structure of the Rhizobium trifolii iron protein gene.

Biological nitrogen fixation in the Rhizobium-legume symbiosis is dependent on the induction of a bacterially-encoded enzyme complex, nitrogenase. To examine the organization and expression of the genes encoding the components of nitrogenase in this complex system, these genes have been isolated from the legume symbiont Rhizobium trifolii by molecular cloning. DNA sequence analysis of the entire nifH gene (encoding the Fe-protein component of nitrogenase) and of the amino-terminal 141 codons of the nifD gene (encoding the alpha-subunit of the Mo-Fe protein) indicates that these genes are linked on a single operon in this strain. The Fe-protein amino acid sequence shares considerable homology with the sequence from other organisms, in particular the related organism Rhizobium meliloti (90% homology). The nif structural genes are preceded by a DNA sequence which is repeated at least three times in the Rhizobium trifolii genome.

Plants interact with microbial polysaccharides.

AbstractPlants are resistant to almost all of the microorganisms with which they come in contact. In response to invasion by a fungus, bacterium, or a virus, many plants produce low molecular weight compounds, phytoalexins, which inhibit the growth of microorganisms. Phytoalexins are produced whether or not the invading microorganism is a pathogen. The production of phytoalexins appears to be a widespread mechanism by which plants attempt to defend themselves against pests. Molecules of microbial origin which trigger phytoalexin accumulation in plants are called elicitors. Structural polysaccharides from the mycelial walls of several fungi elicit phytoalexin accumlation in plants. Approximately 10 ng of the polysaccharide elicits the accumulation in plants of more than sufficient amounts of phytoalexin to stop the growth of microorganisms in vitro. The best characterized elicitors have been demonstrated to be β‐1,3‐glucans with branches to the 6 position of some of the glucosyl residues. Oligosaccharides, produced by partial acid hydrolysis of the mycelial wall glucans, are exceptionally active elicitors. The smallest oligosaccharide which is still an effective elicitor is composed of about 8 sugar residues.Bacteria also elicit phytoalexin accumulation in plants, but the Rhizobium symbionts of legumes presumably have a mechanism which allows them to avoid either eliciting phytoalexin accumulation or the effects of the phytoalexins if they are accumulated. The lectins of legumes bind to the lipopolysaccharides of their symbiont, but not of their non‐symbiont, Rhizobium. It is not known whether the lectin‐lipopolysaccharide interaction is involved with the establishment of symbiosis. However, evidence will be presented that suggests that lectins are, in fact, enzymes capable of modifying the structurs of the lipopolysaccharides of their symbiont, but not of their non‐symbiont, Rhizobium. It will also be shown that the lipopolysaccharides isolated from different Rhizobium species and from different strains of individual Rhizobium species have different sugar compositions. Thus, the different strains of a single Rhizobium species are as different from one another as the different species of Salmonella and other gram‐negative bacteria. This conclusion is substantiated by experiments demonstrating that antibodies to the lipopolysaccharide from a single Rhizobium strain can differentiate that strain from other strains of the same species as well as from other Rhizobium species. The role in symbiosis of the strain‐specific O‐antigens is unknown.