Nodulation Competitiveness Research Articles

Isolation and characterization of the Rhizobium meliloti Tn5-mutants with impaired nodulation competitiveness

Two Tn5-mutants, T37 and T107, with decreased nodulation competitiveness (Cmp−) were isolated in Rhizobium meliloti strain CXM1-105. Their Cmp− phenotype was repeatedly displayed in sterile plant tests, where competitiveness was measured against a Fix− reference strain CXM1-48 and the parental strain CXM1-105. In addition mutant T37 showed a decreased nodulation rate (Nra−) at the early stages of alfalfa development (9–18 days after germination). Its mutant phenotype was demonstrated also under greenhouse conditions when it was evaluated against reference strain CXM1-48, and against indigenous rhizobial strains persisting in the soil. Tn5 insertions from mutants T37 and T107 were transduced using phage ϕM12 to the parental strain CXM1-105. All tested transductants showed Cmp− and Nra− phenotype in the case of T37 and Cmp− phenotype in the case of T107. By means of conjugations with avirulent R. meliloti mutant ZB121 and plasmidless Agrobacterium tumefaciens UBAPF2 we located both Tn5-insertions on the R. meliloti Sym-plasmid.

A Hydrophobic Mutant of Rhizobium etli Altered in Nodulation Competitiveness and Growth in the Rhizosphere

We isolated and characterized CE3003, a Tn5-induced mutant with altered colony morphology derived from Rhizobium etli CE3. CE3003 produced domed colonies and was highly hydrophobic as indicated by its ability to partition into hexadecane, whereas its parent produced flat colonies and was hydrophilic. On bean plants, CE3003 induced nodules and reduced acetylene. CE3003 and CE3 grew at similar rates when they were grown separately or together in culture medium or inoculated singly onto bean seeds. However, when they were mixed at a 1:1 ratio and applied to seeds, CE3003 achieved significantly lower populations than CE3 in the rhizosphere. Five days after coinoculation of CE3 and CE3003, the population of the mutant was less than 10% of the population of CE3 in the bean rhizosphere. To determine the nodulation competitiveness of the mutant, it was coinoculated with CE3 at various ratios at planting, and the ratio of the nodules occupied by each strain was determined 21 days later. A 17,000-fold excess of CE3003 in mixed inocula was required to obtain equal nodule occupancy by the two strains. A genomic library of strain CE3 was mobilized into CE3003, and we identified a cosmid, pRA3003, that restored the parental colony morphology and hydrophilicity to the mutant. Restoration of the parental colony morphology was accompanied by recovery of the ability to grow competitively in the rhizosphere and to compete for nodulation of beans. The data show an association between cell surface hydrophobicity, nodulation competitiveness, and competitive growth in the rhizosphere in mutant CE3003.

Symbiotic Characteristics of Rhizobium leguminosarum bv. trifolii Isolates Which Represent Major and Minor Nodule-Occupying Chromosomal Types of Field-Grown Subclover ( Trifolium subterraneum L.)

The symbiotic effectiveness and nodulation competitiveness of Rhizobium leguminosarum bv. trifolii soil isolates were evaluated under nonsoil greenhouse conditions. The isolates which we used represented both major and minor nodule-occupying chromosomal types (electrophoretic types [ETs]) recovered from field-grown subclover (Trifolium subterraneum L.). Isolates representing four ETs (ETs 2, 3, 7, and 8) that were highly successful field nodule occupants fixed between 2- and 10-fold less nitrogen and produced lower herbage dry weights and first-harvest herbage protein concentrations than isolates that were minor nodule occupants of field-grown plants. Despite their equivalent levels of abundance in nodules on field-grown subclover plants, ET 2 and 3 isolates exhibited different competitive nodulation potentials under nonsoil greenhouse conditions. ET 3 isolates generally occupied more subclover nodules than isolates belonging to other ETs when the isolates were mixed in 1:1 inoculant ratios and inoculated onto seedlings. In contrast, ET 2 isolates were less successful at nodulating under these conditions. In many cases, ET 2 isolates required a numerical advantage of at least 6:1 to 11:1 to occupy significantly more nodules than their competitors. We identified highly effective isolates that were as competitive as the ET 3 isolates despite representing serotypes that were rarely recovered from nodules of field-grown plants. When one of the suboptimally effective isolates (ET2-1) competed with an effective and competitive isolate (ET31-5) at several different inoculant ratios, the percentages of nodules occupied by the former increased as its numerical advantage increased. Although subclover yields declined as nodule occupancy by ET2-1 increased, surprisingly, this occurred at inoculant ratios at which large percentages of nodules were still occupied by ET31-5.

A Bradyrhizobium japonicum gene essential for nodulation competitiveness is differentially regulated from two promoters.

We report the identification and nucleotide sequence of a new symbiotic gene (nfeC) from the soybean root nodule bacterium, Bradyrhizobium japonicum. A Tn5 insertion (NAD14) in this gene did not affect nitrogen fixation but caused a significant delay in soybean nodulation. In addition, this mutant exhibited a reduction in its competitive ability to nodulate soybean when coinoculated with the wild type. Sequence analysis of the mutated region revealed that the NAD14 Tn5 insertion mapped within an open reading frame of 825 bp. Primer extension using B. japonicum mRNA from three different growth conditions, aerobic, anaerobic, and bacteroids (i.e., symbiotic form) indicated that the upstream region of the gene contained two promoters, which were differentially regulated in response to the growth conditions. One promoter was expressed in bacteroids, but not under aerobic or anaerobic free-living conditions. The other promoter was expressed only under aerobic conditions.

Enhanced competitiveness for nodulation of Medicago sativa by Rhizobium meliloti transconjugants harbouring the root-inducing plasmids of Agrobacterium rhizogenes strain 15834

Abstract The root-inducing plasmid of Agrobacterium rhizogenes strain 15834 marked with transposon Tn5-mob with a helper plasmid RP4-4 was mobilized into nitrogen-fixing Rhizobium meliloti strain CIAM 1759. The resulting transconjugants did not induce ‘hairy’ root syndrome but developed nitrogen-fixing nodules on alfalfa. Among the 9 transconjugants tested, 6 strains had increased nodulation rates. The competitiveness of 2 of these R. meliloti (pRi) strains was significantly enhanced as compared with the parent strain CIAM 1759; this was confirmed both in tube and in pot tests.

Enhanced competitiveness for nodulation of Medicago sativa by Rhizobium meliloti transconjugants harbouring the root-inducing plasmids of Agrobacterium rhizogenes strain 15834 FEMS Microbiology Ecology 12 (1993) 61–68

Enhanced competitiveness for nodulation of Medicago sativa by Rhizobium meliloti transconjugants harbouring the root-inducing plasmids of Agrobacterium rhizogenes strain 15834 FEMS Microbiology Ecology 12 (1993) 61–68

Molecular cloning and characterization of a sym plasmid locus that regulates cultivar‐specific nodulation of soybean by Rhizobium fredii USDA257

Rhizobium fredii strain USDA257 produces nitrogen-fixing nodules on primitive soybean cultivars such as Peking but fails to nodulate agronomically improved cultivars such as McCall. Transposon-mutant 257DH4 has two new phenotypes: it nodulates McCall, and its ability to do so is sensitive to the presence of parental strain USDA257, i.e. it is subject to competitive nodulation blocking. We have isolated a cosmid containing DNA that corresponds to the site of transposon insertion in 257DH4 and have localized Tn5 on an 8.0 kb EcoRI fragment. The 5596 bp DNA sequence that surrounds the insertion site contains seven open reading frames. Five of these, designated nolBTU, ORF4, and nolV, are closely spaced and of the same polarity. nolW and nolX are of the opposite polarity. The initiation codon for nolW lies 155 bp upstream from that of nolB, and its is separated from nolX by 281 bp. The predicted NolT and NolW proteins have putative membrane-spanning regions. The N-terminus of the hypothetical NolW protein also has limited homology to NodH of Rhizobium meliloti, but none of the deduced protein sequences has significant homology to known nodulation gene products. Site-directed mutagenesis with mudII1734 confirms that inactivation of nolB, nolT, nolU, nolV, nolW, or nolX extends host range for nodulation to McCall soybean. This phenotype could not be genetically dissected from sensitivity to competitive nodulation blocking. Expression of nolBTU and nolX is induced as much as 30-fold by flavonoid signal molecules, even though these genes lack nod-box promoters. Histochemical staining of McCall roots inoculated with nolB-, nolU-, or nolX-lacZ fusions verifies that these genes are expressed continuously from preinfection to the stage of the functional nodule. Although a nolU-ORF4-nolV clone hybridizes to a single 8.0 kb EcoRI fragment from 10 strains of R. fredii and broad-host-range Rhizobium sp. NGR234, hybridizing sequences are not detectable in other rhizobia.

Enhanced competitiveness for nodulation of Medicago sativa by Rhizobium meliloti transconjugants harbouring the root-inducing plasmids of Agrobacterium rhizogenes strain 15834

The root-inducing plasmid of Agrobacterium rhizogenes strain 15834 marked with transposon Tn5-mob RP4-4 with a helper plasmid was mobilized into nitrogen-fixing Rhizobium meliloti strain CIAM 1759. The resulting transconjugants did not induce ‘hairy’ root syndrome but developed nitrogen-fixing nodules on alfalfa. Among the 9 transconjugants tested, 6 strains had increased modulation rates. The competitiveness of 2 of these R. meliloti (pRi) strains was significantly enhanced as compared with the parent strain CIAM 1759; this was confirmed both in tube and in pot tests.

Enhanced competitiveness for nodulation of Medicago sativa by Rhizobium meliloti transconjugants harbouring the root-inducing plasmids of Agrobacterium rhizogenes strain 15834

The root-inducing plasmid of Agrobacterium rhizogenes strain 15834 marked with transposon Tn5-mob RP4-4 with a helper plasmid was mobilized into nitrogen-fixing Rhizobium meliloti strain CIAM 1759. The resulting transconjugants did not induce ‘hairy’ root syndrome but developed nitrogen-fixing nodules on alfalfa. Among the 9 transconjugants tested, 6 strains had increased modulation rates. The competitiveness of 2 of these R. meliloti (pRi) strains was significantly enhanced as compared with the parent strain CIAM 1759; this was confirmed both in tube and in pot tests.

Evaluation of a Strategy for Identifying Nodulation Competitiveness Genes in Rhizobium Leguminosarum Biovar Phaseoli

Rhizobium leguminosarum biovar phaseoli strain KIM5s is consistently much more competitive than strain CE3 in nodulation of beans (Phaseolus vulgaris L.) in the laboratory and in the field. To identify genes that contribute to the competitiveness of KIM5s, we transferred a cosmid library containing KIM5s DNA into CE3 and applied the transconjugants to bean plants to allow the plants to enrich for those with enhanced nodulation competitiveness. The nodule isolates were then applied to plants for further enrichment. Of 75 isolates from nodules sampled after the two enrichments, 9 were more competitive than CE3. For example, when outnumbered in the inocula 40-fold by a reference strain, these nine strains typically occupied 15-40% of the nodules compared with 0-3% for CE3. However, when these strains were cured of the cosmids, they remained highly competitive, demonstrating that the enhanced competitiveness of the strains was not associated with the cosmids. We found no evidence for cosmid insertion into the chromosome or for cosmid-induced genetic changes in these cured strains. We found some evidence suggesting that their altered competitiveness was due to spontaneous genetic changes that did not involve the cosmids. Although these highly competitive variants remain genetically uncharacterized, they may provide insight into bacterial traits that contribute to, or detract from, successful nodulation competitiveness.

Nucleotide Sequence and Characterization of Rhizobium meliloti Nodulation Competitiveness Genes nfe

Rhizobium meliloti large plasmid pRmeGR4b carries the nodulation competitiveness locus nfe responsible for the nodulation efficiency and competitive ability of strain GR4 on alfalfa roots. We report here the nucleotide sequence and characterization of a 3345 base-pair DNA section of the nfe region. Sequence analysis revealed four open reading frames (ORFs), two of them with rightward polarity, termed nfe1 and nfe2, are preceded by functional nif consensus sequences and NifA-binding motifs. An additional, NifA-independent, transcriptional start site for nfe1 was also found. Two other ORFs with leftward polarity, designated ORFA and ORFB, were indentified upstream from nfe1 and nfe2 but no nif consensus sequences were found. However, expression of ORFA might be indirectly coupled to the NifA-NtrA regulatory network. The gene products of nfe1 and nfe2 were identified using in vitro transcription/translation and bacteriophage T7 RNA polymerase/promoter system, respectively. A high degree of homology between the amino terminal domain of Nfe1 and the nifH gene product was found. In addition, nfe1 shows homology with the upstream non-coding DNA region of the fixABCX operon. Furthermore, the putative ORFB encoded protein contains a helix-turn-helix motif that resembles the DNA-binding consensus sequence proposed for many prokaryotic regulatory proteins.

Nodulation competitiveness of Rhizobium leguminosarum bv. phaseoli and Rhizobium tropici strains measured by glucuronidase (gus) gene fusion

The nodulation competitiveness of 17 Rhizobium leguminosarum bv. phaseoli and 3 R. tropici strains was analysed in growth pouches, at pH 5.2 and 6.4. All 20 strains were coinoculated with a gus+ strain of R. leguminosarum bv. phaseoli strain KIM5s. The gus+ phenotype, carrying the glucuronidase gene, was used to type nodules directly in the growth pouches. Nodule occupancy ranged from 4% for the least competitive to 96% for the most competitive R. leguminosarum bv. phaseoli strain. The R. tropici strains showed low rates of nodule occupancy at pH 6.4 but their competitiveness improved significantly under acid conditions. CIAT 895 was the only R. leguminosarum bv. phaseoli strain that was less competitive (P<0.05) at the lower pH. The competitiveness of all the other R. leguminosarum bv. phaseoli strains was unaffected by pH. Various physiological and genetic properties of the strains were analysed in search of correlations with nodulation competitiveness. Hybridisation patterns with three different DNA probes (nif KDH, common nod genes, and hup genes) and the metabolism of 53 different C sources were compared. No general correlations were found between hybridisation or growth pattern and competitiveness. The less competitive R. tropici strains had a unique DNA hybridisation pattern and were not able to use shikimate, ferulate, coumarate, or asparagine as C sources. Most of the less competitive R. leguminosarum bv. phaseoli strains could not metabolize either ferulate or coumarate. This might indicate a relationship between nodulation competitiveness and the ability to degrade aromatic compounds.

Genetics of competition for nodulation of legumes.

An economically important problem in microbial ecology concerns the efficacy of rhizobial inoculants for the formation of nitrogen-fixing root nodules on legume crop plants such as soybean, alfalfa, and clover. Some strains of rhizobia can increase symbiotic nitrogen fixation under controlled conditions. However, attempts to improve nitrogen fixation under agricultural conditions with such strains often fail, usually as a result of the presence of indigenous rhizobia limiting nodulation by the inoculum strains. This problem is referred to as the Rhizobium competition problem, and molecular genetics is being used to address the problem from two perspectives. First, the host specificity of rhizobia is being characterized with the long term goal of developing strains that can nodulate a very strain-specific host-legume genotype. Second, the genetic basis of competitiveness in several strains is being examined. Genetic determinants of nodulation competitiveness have been isolated and mechanisms for their stable integration into the genome of superior nitrogen-fixing strains have been developed. Several phenotypes have been identified as playing an important role in nodulation competitiveness including antibiosis, motility, speed of nodulation, cell-surface characteristics, and nodulation efficiency. Several solutions to this problem are likely to result from these strategies and will be useful for certain legumes in specific locations.

Nucleotide sequence of Rhizobium meliloti GR4 insertion sequence ISRm3 linked to the nodulation competitiveness locus nfe.

Nucleotide sequence of Rhizobium meliloti GR4 insertion sequence ISRm3 linked to the nodulation competitiveness locus nfe.

A Rhizobium meliloti lipopolysaccharide mutant altered in competitiveness for nodulation of alfalfa.

A transposon Tn5-induced mutant of Rhizobium meliloti Rm2011, designated Rm6963, showed a rough colony morphology on rich and minimal media and an altered lipopolysaccharide (LPS). Major differences from the wild-type LPS were observed in (i) hexose and 2-keto-3-deoxyoctonate elution profiles of crude phenol extracts chromatographed in Sepharose CL-4B, (ii) silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis patterns of crude and purified LPS fractions, and (iii) immunoreactivities otherwise present in purified LPS of the parental strain Rm2011. In addition, Rm6963 lost the ability to grow in Luria-Bertani medium containing the hydrophobic compounds sodium deoxycholate or SDS and showed a decrease in survival in TY medium supplemented with high calcium concentrations. The mutant also had altered symbiotic properties. Rm6963 formed nodules that fixed nitrogen but showed a delayed or even reduced ability to nodulate the primary root of alfalfa without showing changes in the position of nodule distribution profiles along the roots. Furthermore, 2 to 3 weeks after inoculation, plants nodulated by Rm6963 were smaller than control plants inoculated with wild-type bacteria in correlation with a transient decrease in nitrogen fixation. In most experiments, the plants recovered later by expressing a full nitrogen-fixing phenotype and developing an abnormally high number of small nodules in lateral roots after 1 month. Rm6963 was also deficient in the ability to compete for nodulation. In coinoculation experiments with equal bacterial numbers of both mutant and wild-type rhizobia, only the parent was recovered from the uppermost root nodules. A strain ratio of approximately 100 to 1 favoring the mutant was necessary to obtain an equal ratio (1:1) of nodule occupancy. These results show that alterations in Rm6963 which include LPS changes lead to an altered symbiotic phenotype during the association with alfalfa that affects the timing of nodule emergence, the progress of nitrogen fixation, and the strain competitiveness for nodulation.

Isolation of transposon Tn5-induced hydrophobic mutants of aBradyrhizohium japonicumstrain with improved competitive nodulation abilities

Abstract Hydrophobic mutants of the Bradyrhizohium japonicum strain 138NR were obtained by transposon Tn5 mutagenesis followed by replica-plating on polystyrene plates. Fifteen mutants were isolated at a frequency of 106. Gel-filtration analysis of the exopolysaccharides revealed that the hydrophobic mutants produced a significantly smaller amount of low-molecular-weight polysaccharides than the parent. Four of the isolated mutants formed a larger number of nodules on soybean (Glycine max L. Merr. cv. Tamahomare) than the parent, two were superior in symbiotic nitrogen fixation, and two were symbiotically defective on soybean. Competitive nodulation abilities of the mutants were examined by inoculating them to soybean with B. japonicum strain 123ET as a competitor and determining the nodule occupancy based on the antibiotic resistance. All the mutants tested except for the symbiotically defective ones were superior in their competitive nodulation ability to the parent strain. When inoculated 24 h before t...

A nolC-lacZ gene fusion in Rhizobium fredii facilitates direct assessment of competition for nodulation of soybean

A constitutively expressed chromosomal gene, nolC, regulates the ability of Rhizobium fredii strain USDA257 to nodulate certain agronomically improved soybean cultivars. Other advanced cultivars, as well as unimproved varieties such as 'Peking', form nitrogen-fixing nodules with parental strain USDA257 and with nolC mutants. We have used 257DH512, a derivative of USDA257 that contains a nolC-lacZ fusion, to directly measure strain competition for nodule occupancy. Seedlings of the soybean cultivar 'Peking' were inoculated with mixtures containing various ratios of 257DH512 and R. fredii USDA208; nodules were harvested 14 days later and stained histochemically with X-gal, a chromogenic substrate for the β-galactosidase product of lacZ. We were able to stain and evaluate batches of nearly 500 nodules in just 4 h, without the use of expensive equipment. Under the conditions of these experiments, USDA208 was considerably more competitive than 257DH512, even when it had been present in the inoculum at a numerical disadvantage. We suggest that gene fusions can be used to greatly simplify the assessment of nodulation competitiveness in rhizobia. Key words: competition, nolC-lacZ fusion, soybean – Rhizobium fredii, nodule occupancy.

Identification and cloning of Bradyrhizobium japonicum genes expressed strain selectively in soil and rhizosphere.

The growth of Bradyrhizobium japonicum USDA 110 and USDA 438 in soil extract-supplemented medium led to transcription of a large amount of DNA not expressed in basal medium. Strain USDA 438 was more competitive for the nodulation of soybean than strain USDA 110. To identify and isolate DNA regions which were expressed specifically in strain USDA 438 but not in strain USDA 110 in response to soil extract or soybean root exudate, we developed a subtractive RNA hybridization procedure. Several cosmid clones which showed strain-specific gene expression were isolated from a USDA 438 gene library. Two clones enhanced competitive nodulation when mobilized to USDA 110. The method described may be useful for identifying genes expressed in response to environmental stimuli or genes expressed differently in related microbial strains.

Genetics of Competition for Nodulation of Legumes

Genetics of Competition for Nodulation of Legumes

Isolation and Characterization of a Competition-Defective Bradyrhizobium japonicum Mutant

Tn5 mutagenesis was coupled with a competition assay to isolate mutants of Bradyrhizobium japonicum defective in competitive nodulation. A double selection procedure was used, screening first for altered extracellular polysaccharide production (nonmucoid colony morphology) and then for decreased competitive ability. One mutant, which was examined in detail, was deficient in acidic polysaccharide and lipopolysaccharide production. The wild-type DNA region corresponding to the Tn5 insertion was isolated, mapped, and cloned. A 3.6-kb region, not identified previously as functioning in symbiosis, contained the gene(s) necessary for complementation of the mutation. The mutant was motile, grew normally on minimal medium, and formed nodules on soybean plants which fixed almost as much nitrogen as the wild type during symbiosis.