Expression by Soil Bacteria of Nodulation Genes from Rhizobium leguminosarum biovar trifolii

Gram-negative, rod-shaped bacteria from the soil of white clover-ryegrass pastures were screened for their ability to nodulate white clover (Trifolium repens) cultivar Grasslands Huia and for DNA homology with genomic DNA from Rhizobium leguminosarum biovar trifolii ICMP2668 (NZP582). Of these strains, 3.2% were able to hybridize with strain ICMP2668 and nodulate white clover and approximately 19% hybridized but were unable to nodulate. Strains which nodulated but did not hybridize with strain ICMP2668 were not detected. DNA from R. leguminosarum biovar trifolii (strain PN165) cured of its symbiotic (Sym) plasmid and a specific nod probe were used to show that the relationship observed was usually due to chromosomal homology. Plasmid pPN1, a cointegrate of the broad-host-range plasmid R68.45 and a symbiotic plasmid pRtr514a, was transferred by conjugation to representative strains of nonnodulating, gram-negative, rod-shaped soil bacteria. Transconjugants which formed nodules were obtained from 6 of 18 (33%) strains whose DNA hybridized with that of PN165 and 1 of 9 (11%) strains containing DNA which did not hybridize with that of PN165. The presence and location of R68.45 and nod genes was confirmed in transconjugants from three of the strains which formed nodules. Similarly, a pLAFR1 cosmid containing nod genes from a derivative of R. leguminosarum biovar trifolii NZP514 formed nodules when transferred to soil bacteria.     

Alfalfa Root Exudates and Compounds which Promote or Inhibit Induction of Rhizobium meliloti Nodulation Genes

Using a plate induction assay, we demonstrate that alfalfa exudes inducer of Rhizobium meliloti nodulation genes. The inducer is exuded from the infectible zone of the root, accumulates to at least 1 micromolar, and is not affected by 10 millimolar nitrate. No zones of inhibition are observed. A nodulation minus mutant line of alfalfa, MN-1008, exudes normal levels of inducer. R. meliloti grown in rich medium requires ten-fold higher concentrations of luteolin to achieve half-maximal induction as compared to cells grown in a minimal medium. Flavonoids other than luteolin are found to have activity in R. meliloti nodulation gene induction assays. The compounds apigenin and eriodictyol have activities two-fifths and one-seventh that of luteolin, respectively. Several of the flavonoids tested (morin = naringenin > kaempferol = chrysin > quercetin = fisetin = hesperitin) demonstrate antagonistic activity toward induction by luteolin. The most effective antagonist is the coumarin, umbelliferone.     

Influence of Soil and Nonsoil Environments on Nodulation by Rhizobium trifolii

Indigenous serotypes 1-01 and 2-02 of Rhizobium trifolii occupied similar percentages (18 to 23%) of root nodules on soil-grown subclover (Trifolium subterraneum L.) and were virtually absent (4.5%) from nodules of soil-grown white clover (Trifolium repens L.). In contrast (with the exception of one dilution [10⁻⁴]), serotype 1-01 occupied a substantial portion of nodules (16 to 40%) on white clover seedlings grown on mineral salts agar and exposed to samples of the same soil in the form of a 10-fold dilution series (10⁻¹ to 10⁻⁵). Under the latter conditions, occupancy of subclover nodules by 1-01 and of nodules of both plant species by 2-02 was consistent with the results obtained with soil-grown plants.     

Interference between Rhizobium meliloti and Rhizobium trifolii nodulation genes: genetic basis of R. meliloti dominance.

Transfer of an IncP plasmid carrying the Rhizobium meliloti nodFE, nodG, and nodH genes to Rhizobium trifolii enabled R. trifolii to nodulate alfalfa (Medicago sativa), the normal host of R. meliloti. Using transposon Tn5-linked mutations and in vitro-constructed deletions of the R. meliloti nodFE, nodG, and nodH genes, we showed that R. meliloti nodH was required for R. trifolii to elicit both root hair curling and nodule initiation on alfalfa and that nodH, nodFE, and nodG were required for R. trifolii to elicit infection threads in alfalfa root hairs. Interestingly, the transfer of the R. meliloti nodFE, nodG, and nodH genes to R. trifolii prevented R. trifolii from infecting and nodulating its normal host, white clover (Trifolium repens). Experiments with the mutated R. meliloti nodH, nodF, nodE, and nodG genes demonstrated that nodH, nodF, nodE, and possibly nodG have an additive effect in blocking infection and nodulation of clover.     

Expression of Nodulation Genes of Rhizobium in Azotobacter

Rhizobium meliloti nif nod gene cluster was transferred conjugally to Azotobacter, and the modified Azotobacter showed nodulation and nitrogen fixation on alfalfa plants.     

Role of Motility and Chemotaxis in Efficiency of Nodulation by Rhizobium meliloti

Spontaneous mutants of Rhizobium meliloti L5-30 defective in motility or chemotaxis were isolated and compared against the parent with respect to symbiotic competence. Each of the mutants was able to generate normal nodules on the host plant alfalfa (Medicago sativa), but had slightly delayed nodule formation, diminished nodulation in the initially susceptible region of the host root, and relatively low representation in nodules following co-inoculation with equal numbers of the parent. When inoculated in growth pouches with increasing dosages of the parental strain, the number of nodules formed in the initially susceptible region of the root increased sigmoidally, with an optimum concentration of about 10⁵ to 10⁶ bacteria/plant. The dose-response behavior of the nonmotile and nonchemotactic mutants was similar, but they required 10- to 30-fold higher concentrations of bacteria to generate the same number of nodules. The distribution frequencies of nodules at different positions along the primary root were very similar for the mutants and parent, indicating that reduced nodulation by the mutants in dose-response experiments probably reflects reduced efficiency of nodule initiation rather than developmentally delayed nodule initiation. The number of bacteria that firmly adsorbed to the host root surface during several hours of incubation was 5- to 20-fold greater for the parent than the mutants. The mutants were also somewhat less effective than their parent as competitors in root adsorption assays. It appears that motility and chemotaxis are quantitatively important traits that facilitate the initial contact and adsorption of symbiotic rhizobia to the host root surface, increase the efficiency of nodule initiation, and increase the rate of infection development.     

Morphogenetic Rescue of Rhizobium meliloti Nodulation Mutants by trans-Zeatin Secretion

The development of nitrogen-fixing nodules is induced on the roots of legume host plants by Rhizobium bacteria. We employed a novel strategy to probe the underlying mechanism of nodule morphogenesis in alfalfa roots using pTZS, a broad host range plasmid carrying a constitutive trans-zeatin secretion (tzs) gene from Agrobacterium tumefaciens T37. This plasmid suppressed the Nod- phenotype of Rhizobium nodulation mutants such that mutants harboring pTZS stimulated the formation of nodulelike structures. Alfalfa roots formed more or fewer of these nodules according to both the nitrogen content of the environment and the position along the root at which the pTZS+ bacteria were applied, which parallels the physiological and developmental regulation of true Rhizobium nodule formation. This plasmid also conferred on Escherichia coli cells the ability to induce root cortical cell mitoses. Both the pattern of induced cell divisions and the spatially restricted expression of an alfalfa nodule-specific marker gene (MsENOD2) in pTZS-induced nodules support the conclusion that localized cytokinin production produces a phenocopy of nodule morphogenesis.     

Extended Region of Nodulation Genes in Rhizobium meliloti 1021. I. Phenotypes of Tn5 Insertion Mutants

Rhizobium meliloti Nod^- mutant WL131, a derivative of wild-type strain 102F51, was complemented by a clone bank of wild-type R. meliloti 1021 DNA, and clone pRmJT5 was recovered. Transfer of pRmJT5 conferred alfalfa nodulation on other Rhizobium species, indicating a role in host range determination for pRmJT5. Mutagenesis of pRmJT5 revealed several segments in which transposon insertion causes delay in nodulation, and/or marked reduction of the number of nodules formed on host alfalfa plants. The set of mutants indicated five regions in which nod genes are located; one mutant, nod-216, is located in a region not previously reported to encode a nodulation gene. Other mutant phenotypes correlated with the positions of open reading frames for nodH, nodF and nodE , and with a 2.2-kb EcoRI fragment. A mutant in nodG had no altered phenotype in this strain. One nodulation mutant was shown to be a large deletion of the common nod gene region. We present a discussion comparing the various studies made on this extended nod gene region.     

Influence of Phosphate on the Growth and Nodulation Characteristics of Rhizobium trifolii

The growth and nodulating characteristics of Rhizobium trifolii 6 and 36 differed under different external phosphate conditions. Under growth conditions designed to deplete the internal phosphate content of the rhizobia, strain 6 maintained a generation time of 5 h during the exponential phase over two cycles of growth in phosphate-depleted medium. In contrast, the generation time of strain 36 was extended from 3.5 to 9.8 h over two cycles of phosphate-depleted growth, although the organism eventually achieved the same cell density and cellular phosphate content as that of strain 6 at stationary phase. Phosphate-depleted strain 6 required 0.51 ± 0.08 μM phosphate to commence proliferation, whereas phosphate-depleted strain 36 required 0.89 ± 0.04 μM phosphate under the same conditions. Phosphate-depleted strain 6 maintained viability when exposed to external phosphate concentrations subcritical for growth to occur, whereas phosphate-depleted strain 36 lost viability within 48 h when exposed to medium containing phosphate at concentrations subcritical for growth. Phosphate-depleted strain 36 was inferior to phosphate-depleted strain 6 at nodulating subterranean clover (Trifolium subterraneum L. cv. Mt. Barker) by taking 2 to 4 days longer to develop nodules in phosphatedepleted plant grown medium at pH 5.5. Nodulation by phosphate-depleted strain 36 was accelerated either by including phosphate in the plant growth medium at pH 5.5 or by raising the solution pH of phosphate-depleted plant growth medium to pH 6.5. External phosphate and pH effects were not observed on the nodulating capabilities of phosphate-depleted strain 6 or on luxury phosphate-grown cells of either strain. Phosphatedepleted strains 6 and 36 proliferated to a similar extent on the rhizoplanes even under stringently low external P_i concentrations. The phosphatase activities of both phosphate-depleted strains were significantly (P = 0.05) higher at pH 6.5 than at pH 5.5, and the activity of strain 6 was significantly higher (P = 0.05) than that of strain 36 at pH 5.5 and 5.0.     

Chemotaxis of Rhizobium meliloti towards Nodulation Gene-Inducing Compounds from Alfalfa Roots

Luteolin, a flavone present in seed exudates of alfalfa, induces nodulation genes (nod) in Rhizobium meliloti and also serves as a biochemically specific chemoattractant for the bacterium. The present work shows that R. meliloti RCR2011 is capable of very similar chemotactic responses towards 4′,7-dihydroxyflavone, 4′,7-Dihydroxyflavanone, and 4,4′-dihydroxy-2-methoxychalcone, the three principal nod gene inducers secreted by alfalfa roots. Chemotactic responses to the root-secreted nod inducers in capillary assays were usually two- to four-fold above background and, for the flavone and flavonone, occurred at concentrations lower than those required for half-maximal induction of the nodABC genes. Complementation experiments indicated that the lack of chemotactic responsiveness to luteolin seen in nodD1 and nodA mutants of R. meliloti was not due to mutations in the nod genes, as previously thought. Thus, while nod gene induction and flavonoid chemotaxis have the same biochemical specificity, these two functions appear to have independent receptors or transduction pathways. The wild-type strain was found to suffer selective, spontaneous loss of chemotaxis towards flavonoids during laboratory subculture.     

Nodulation of specific legumes is controlled by several distinct loci in Rhizobium trifolii

Summary Three distinct loci (designated regions III, IV and V) were identified in the 14 kb Nod region of Rhizobium trifolii strain ANU843 and were found to determine the host range characteristics of this strain. Deletion of region III or region V only from the 14 kb Nod region affected clover nodulation capacity. The introduction to R. Leguminosarum of DNA fragments on multicopy vectors carrying regions III, IV and V (but not smaller fragments) extended the host range of R. leguminosarum so that infection threads and nodules occurred on white clover plants. The same DNA fragments were introduced to the Sym plasmid-cured strain (ANU845) carrying the R. meliloti recombinant nodulation plasmid pRmSL26. Plasmid pRmSL26 alone does not confer root hair curling or nodulation on clover plants. However, the introduction to ANU845 (pRmSL26) of a 1.4 kb fragment carrying R. trifolii region IV only, resulted in the phenotypic activation of marked root hair curling ability to this strain on clovers but no infection events or nodules resulted. Only the transfer of regions III, IV and V to strain ANU845 (pRmSL26) conferred normal nodulation and host range ability of the original wild type R. trifolii strain. These results indicate that the host range genes determine the outcome of early plant-bacterial interactions primarily at the stage of root hair curling and infection.     

Bacterial Growth Rates and Competition Affect Nodulation and Root Colonization by Rhizobium meliloti

The addition of streptomycin to nonsterile soil suppressed the numbers of bacterial cells in the rhizosphere of alfalfa (Medicago sativa L.) for several days, resulted in the enhanced growth of a streptomycin-resistant strain of Rhizobium meliloti, and increased the numbers of nodules on the alfalfa roots. A bacterial mixture inoculated into sterile soil inhibited the colonization of alfalfa roots by R. meliloti, caused a diminution in the number of nodules, and reduced plant growth. Enterobacter aerogenes, Pseudomonas marginalis, Acinetobacter sp., and Klebsiella pneumoniae suppressed the colonization by R. meliloti of roots grown on agar and reduced nodulation by R. meliloti, the suppression of nodulation being statistically significant for the first three species. Bradyrhizobium sp. and “Sarcina lutea” did not suppress root colonization nor nodulation by R. meliloti. The doubling times in the rhizosphere for E. aerogenes, P. marginalis, Acinetobacter sp., and K. pneumoniae were less and the doubling times for Bradyrhizobium sp. and “S. lutea” were greater than the doubling time of R. meliloti. Under the same conditions, Arthrobacter citreus injured alfalfa roots. We suggest that competition by soil bacteria reduces nodulation by rhizobia in soil and that the extent of inhibition is related to the growth rates of the rhizosphere bacteria.     

根瘤菌共生结瘤基因的分子遗传学研究进展

根瘤菌共生结瘤基因的分子遗传学研究进展①樊妙姬陈丽梅马庆生（广西大学生物技术与糖业工程学院,南宁５３０００５）ＴｈｅＡｄｖａｎｃｅｉｎＭｏｌｅｃｕｌａｒＧｅｎｅｔｉｃｏｆＲｈｉｚｏｂｉｕｍＳｙｍｂｏｔｉｃＮｏｄｕｌａｔｉｏｎＧｅｎｅｓＦＡＮＭｉａｏｊ...     

Growth and Nodulation Responses of Rhizobium meliloti to Water Stress Induced by Permeating and Nonpermeating Solutes

Isolates of Rhizobium meliloti, representing antigenically distinct indigenous serogroups 31 and 17, were grown in yeast extract-mannitol broth (YEM) containing NaCl or polyethylene glycol (PEG) to provide external water potentials ranging from −0.15 to −1.5 MPa. Several differences were found between representatives of the two groups in their abilities to adapt to water stress induced by the nonpermeating solute PEG. At potentials below −0.5 MPa, strain 31 had a lower specific growth rate than strain 17 and an irregular cell morphology. In contrast, neither growth nor cell morphology of either strain was affected significantly over the same range of water potentials created by a permeating solute, NaCl. Despite the superior growth of strain 17 at the low water potentials imposed by PEG, upshock of water-stressed cells (−1.0 MPa; PEG) into normal YEM (−0.15 MPa) resulted in a faster recovery of growth by strain 31 than by strain 17. Different responses of the two strains to a water potential increase were also revealed in nodulation studies. Strain 31 required significantly fewer days to nodulate alfalfa than strain 17 did when the strains were transferred from YEM with PEG at −1.0 MPa onto the roots of alfalfa seedlings in plant growth medium (−0.1 MPa). The addition of supplemental calcium (0.1 mM) to growth medium with PEG (−1.0 MPa) reduced the differences between strains in their responses to water stress. The severe growth restriction and morphological abnormalities shown by strain 31 were corrected, and the prolonged recovery time shown by water-stressed cells (−1.0 MPa; PEG) of strain 17 upon transfer to normal YEM was shortened. The latter strain also nodulated earlier and more rapidly after growth in PEG medium at −1.0 MPa in the presence of supplemental calcium ions. These results indicate that the efficacy of osmoregulation can vary among strains of the same species and that the mechanism of osmoregulation may differ depending on the nature of the water stress.