Rhizobium Etli Strains Research Articles

Regulation of pyruvate carboxylase in Rhizobium etli.

Pyruvate carboxylase (PYC) is a biotin-dependent enzyme catalyzing the anaplerotic conversion of pyruvate to oxaloacetate in Rhizobium etli strain CE3. A pyc::Tn5 mutant had severely reduced growth, or failed to grow on sugars, three-carbon organic acids or glycerol, consistent with these substrates being metabolized via pyruvate. Transconjugants expressing a pyc::beta-glucuronidase gene fusion had slightly increased apparent pyc transcription during growth on pyruvate as compared to succinate, similar to the modest carbon source dependent changes in PYC activity reported previously. Biotin supplementation of cultures growing on pyruvate dramatically increased PYC activity but not apparent pyc transcription. Bacteroids isolated from bean nodules did not contain detectable PYC activity while apparent pyc transcription occurred at a moderate level.

Persistence of twoRhizobium etli inoculant strains in clay and silty loam soils

The persistence of Rhizobium etli strains CE3 and Ph 163 was studied in two soil types representing major French bean growing areas in Egypt. Clay soil from MENOUFIA and silty loam soil from ISMAILIA were planted by bean Cultivars; Bronco and Giza 6. The inoculation with strain Ph 163 in the first bean cultivation was significantly higher in nodule biomass and number; whereas, the strain CE3 was significantly higher in plant biomass accumulation (Moawad et al. 2004). The persisting inocula strains seem to perform differently in the two soils in terms of nodulation, biomass accumulation and N-uptake by the two cultivars as compared with their performance with the first inoculation. CE3 strain persisting in the soil performed better than Ph.163 strain. The nodule occupancy by the persisting inoculant rhizobial was determined by two approaches; fluorescent antibody (FA) technique and other Rep-PCR fingerprinting. Both techniques were close in the evaluation of persisting inoculant strains which nodulated beans in the second planting season without inoculation. The results obtained showed that both strains are good survivors in the two soils.

High Salt and High pH Tolerance of New Isolated Rhizobium etli Strains from Egyptian Soils

Saline and alkaline soils are major problems contributing to the low productivity of common bean (Phaseolus vulgaris) in arid and semi-arid regions such as Egypt. Therefore our study was directed toward selecting strains more tolerant to these environmental stresses. Among seven Rhizobium etli strains isolated from Egyptian soils, we found a high degree of diversity. Strains EBRI 21 and EBRI 26 are highly tolerant to a salt concentration up to 4% NaCl. A positive correlation was found between the salt tolerance and the adaptation to alkaline pH (9). Strains EBRI 2 and EBRI 26 were adapted to elevated temperatures (42 degrees C). The minimum level of low pH for the majority of Rhizobium etli strains from Egypt was pH 4.7 while the Colombian strain Rhizobium tropici CIAT 899 survived well at pH 4. At 0.4% NaCl, the symbiotic efficiency of the salt-tolerant strain EBRI 26 was superior in cultivar Giza 6 compared with the salt-sensitive strain EBRI 2 (18.2 compared with 13.9 nM: C2H4 h(-1) mg(-1) nodule fresh weight). In the bean cultivar Saxa, nitrogen fixation was much more affected by high salt concentration (0.4% NaCl) than in the cultivar Giza 6 with both strains (3.9 and 3.8 nM: C2H4 h(-1) mg(-1) nodule fresh weight, respectively). In general, stress of alkalinity had a less detrimental effect on nodulation and N2 fixation than stress of salinity.

Selection of Competitive Strains of Rhizobium Nodulating Phaseolus Vulgaris and Adapted to Environmental Conditions in Egypt, Using the Gus-Reporter Gene Technique

Two Rhizobium etli strains, EBRI 2 and EBRI 26, isolated from Egypt were tested for nodulation competitiveness on beans using Rhizobium tropici CIAT 899G as the competing strain. The insertion of the gus-reporter transposon mTn5ssgusA30 did not alter the nodulation or nitrogen fixation capacity of mutant strain CIAT 899G compared to the wild type. At neutral pH, R. etli strains EBRI 2 and EBRI 26 were more competitive than CIAT 899G with the bean cultivar Saxa. These two strains gave nodule occupancies of 52.1 and 61.1% competing with equal cell numbers of CIAT 899G. Nodule occupancies from these two native strains increased with the bean cultivar Giza 6 from Egypt to 66 and 67.5%. Based on these results, cultivar Giza 6 was used to select the most competitive strains under stress of salinity or alkalinity as a major problem for a large part of Egyptian soils. Under stress of salinity (0.2% NaCl or 34.2 mM NaCl), the salt-sensitive strain EBRI 2 was more competitive than the salt-resistant strain EBRI 26. Strain EBRI 2 gave 87.4% but strain EBRI 26 gave 63.7% nodule occupancy against CIAT 899G. The same trend of results was observed under stress of alkalinity (pH 8). Strain EBRI 2 occupied 83% while Strain EBRI 26 occupied 53.2%.

Importance of the O-antigen, core-region and lipid A of rhizobial lipopolysaccharides for the induction of systemic resistance in potato to Globodera pallida

AbstractLipopolysaccharides (LPS) of the rhizobacterium Rhizobium etli strain G12 are involved in the induction of systemic resistance in potato roots towards the potato cyst nematode Globodera pallida. In the present study, the wild type strain G12 and three LPS-mutant strains with truncated O-antigens all reduced nematode infection of potato plants to about 30% that of untreated controls. Therefore, the O-antigen of the bacterial LPS cannot be the sole inducer of the resistance mechanism. In further search of the mode of action, the isolated core-region and lipid A-fraction of the LPS of one O-antigen-mutant strain R. etli #4-153 were tested for elicitor activity. Using a split-root system, which provides spatial separation of the inducing agent from the parasite, it was shown that both the entire LPS and its core-region systemically reduced G. pallida infection to 45% that of the untreated control. In contrast, the lipid A-fraction was less effective; the 20% reduction in nematode infection was not different from the control nor from the LPS and core region treatments. It is concluded that the oligosaccharides of the core-region are the main trigger of systemic resistance in potato roots towards G. pallida infection.

Lipopolysaccharides of Rhizobium etli strain G12 act in potato roots as an inducing agent of systemic resistance to infection by the cyst nematode Globodera pallida.

Recent studies have shown that living and heat-killed cells of the rhizobacterium Rhizobium etli strain G12 induce in potato roots systemic resistance to infection by the potato cyst nematode Globodera pallida. To better understand the mechanisms of induced resistance, we focused on identifying the inducing agent. Since heat-stable bacterial surface carbohydrates such as exopolysaccharides (EPS) and lipopolysaccharides (LPS) are essential for recognition in the symbiotic interaction between Rhizobium and legumes, their role in the R. etli-potato interaction was studied. EPS and LPS were extracted from bacterial cultures, applied to potato roots, and tested for activity as an inducer of plant resistance to the plant-parasitic nematode. Whereas EPS did not affect G. pallida infection, LPS reduced nematode infection significantly in concentrations as low as 1 and 0.1 mg ml(-1). Split-root experiments, guaranteeing a spatial separation of inducing agent and challenging pathogen, showed that soil treatments of one half of the root system with LPS resulted in a highly significant (up to 37%) systemic induced reduction of G. pallida infection of potato roots in the other half. The results clearly showed that LPS of R. etli G12 act as the inducing agent of systemic resistance in potato roots.

Structural Characterization of the O-antigenic Polysaccharide of the Lipopolysaccharide from Rhizobium etli Strain CE3: A UNIQUE O-ACETYLATED GLYCAN OF DISCRETE SIZE, CONTAINING 3-O-METHYL-6-DEOXY-l-TALOSE AND 2,3,4-TRI-O-METHYL-l-FUCOSE

The O-antigenic polysaccharide of the Rhizobium etli CE3 lipopolysaccharide (LPS) was structurally characterized using chemical degradations (Smith degradation and beta-elimination of uronosyl residues) in combination with alkylation analysis, electrospray, and matrix-assisted laser desorption ionization-time of flight mass spectrometry, tandem mass spectrometry, and (1)H COSY and TOCSY nuclear magnetic resonance spectroscopy analyses of the native polysaccharide and the derived oligosaccharides. The polysaccharide was found to be a unique, relatively low molecular weight glycan having a fairly discrete size, with surprisingly little variation in the number of repeating units (degree of polymerization = 5). The polysaccharide is O-acetylated and contains a variety of O-methylated glycosyl residues, rendering the native glycan somewhat hydrophobic. The molecular mass of the major de-O-acetylated species, including the reducing end 3-deoxy-d-manno-2-octulosonic acid (Kdo) residue, is 3330 Da. The polysaccharide is comprised of a trisaccharide repeating unit having the structure -->4)-alpha-d-GlcpA-(1-->4)-[alpha-3-O-Me-6-deoxy-Talp-(1--> 3)]-alpha -l-Fucp-(1-->. The nonreducing end of the glycan is terminated with the capping sequence alpha-2,3, 4-tri-O-Me-Fucp-(1-->4)-alpha-d-GlcpA-(1-->, and the reducing end of the molecule consists of the non-repeating sequence -->3)-alpha-l-Fucp-(1-->3)-beta-d-Manp-(1-->3)-beta-QuiNA cp-(1-->4)-a lpha-Kdop-(2-->, where QuiNAc is N-acetylquinovosamine (2-N-acetamido-2,6-dideoxyglucose). The reducing end Kdo residue links the O-chain polysaccharide to the core region oligosaccharide, resulting in a unique location for a Kdo residue in LPS, removed four residues distally from the lipid A moiety. Structural heterogeneity in the O-chain arises mainly from the O-acetyl and O-methyl substitution. Methylation analysis using trideuteriomethyl iodide indicates that a portion of the 2,3,4-tri-O-methylfucosyl capping residues, typically 15%, are replaced with 2-O-methyl- and/or 2,3-di-O-methylfucosyl residues. In addition, approximately 25% of the 3,4-linked branching fucosyl residues and 10% of the 3-linked fucosyl residues are 2-O-methylated. A majority of the glucuronosyl residues are methyl-esterified at C-6. These unique structural features may be significant in the infection process.

A method for screening Phaseolus vulgaris L. germplasm for preferential nodulation with a selected Rhizobium etli strain

As part of a breeding program to improve the nitrogen-fixing symbiosis between common bean (Phaseolus vulgaris) and Rhizobium etli, we developed a rapid screen for common bean accessions that preferentially nodulate with KIM5s, a high nitrogen fixing strain of R. etli. We constructed a mutant of KIM5s that did not fix nitrogen (Fix-) but was otherwise indistinguishable from KIM5s. We screened plants for symptoms of nitrogen deficiency when grown in a Honduran soil containing indigenous common bean-nodulating rhizobia (104 per gram) and KM6001, the Fix- mutant of KIM5s (104/seedling added 7 days after planting). Leaf color was scored on a scale of 1 to 5, in which 1 was dark green and 5 was bright yellow. Of 820 genetically diverse accessions of P. vulgaris screened, 51 were scored 1, 626 were scored 2 or 3, and 143 were scored 4 or 5. Selfed seed was produced from common bean plants of the accessions scored 1, 4 or 5. Twenty-four accessions that scored 1, and 58 that scored 4 or 5 were screened in soil containing indigenous rhizobia and the wild type KIM5s (Fix+), and nodule occupancy was determined by antibiotic resistance. On the 24 common bean accessions that were scored 1, KIM5s occupied 0-6% of the nodules, on 26 of the accessions that were scored 4 or 5, KIM5s occupied 90%-100% of the nodules, and on the remaining 34 that scored 4 or 5, there was a distribution of nodule occupancy. Foliar color was highly correlated with nodule occupancy (r = 0.786,p = 0.01). The results indicate that the rapid visual screen using the Fix- mutant accurately identified common bean accessions that preferentially nodulate with the wild-type KIM5s (Fix+) strain in soil containing indigenous rhizobia. This screen will facilitate introduction of the preferential nodulation trait into superior cultivars and provides the foundation for studies of the genetic basis of preferential nodulation.

Regulation of pyruvate carboxylase in Rhizobium etli

Pyruvate carboxylase (PYC) is a biotin-dependent enzyme catalyzing the anaplerotic conversion of pyruvate to oxaloacetate in Rhizobium etli strain CE3. A pyc::Tn 5 mutant had severely reduced growth, or failed to grow on sugars, three-carbon organic acids or glycerol, consistent with these substrates being metabolized via pyruvate. Transconjugants expressing a pyc::β-glucuronidase gene fusion had slightly increased apparent pyc transcription during growth on pyruvate as compared to succinate, similar to the modest carbon source dependent changes in PYC activity reported previously. Biotin supplementation of cultures growing on pyruvate dramatically increased PYC activity but not apparent pyc transcription. Bacteroids isolated from bean nodules did not contain detectable PYC activity while apparent pyc transcription occurred at a moderate level.

RosR, a determinant of nodulation competitiveness in Rhizobium etli.

We previously described a Tn5 mutant of Rhizobium etli strain CE3, designated CE3003, that is decreased in nodulation competitiveness, reduced in competitive growth in the rhizosphere, and has a hydrophobic cell surface (R. S. Araujo, E. A. Robleto, and J. Handelsman, Appl. Environ. Microbiol., 60:1430-1436, 1994). To determine the molecular basis for the mutant phenotypes, we identified a 1.2-kb fragment of DNA derived from the parent that restored the wild-type phenotypes to the mutant. DNA sequence analysis indicated that this 1.2-kb fragment contained a single open reading frame that we designated rosR. The Tn5 insertion in CE3003 was within rosR. We constructed a derivative of CE3 that contained a deletion in rosR, and this mutant was phenotypically indistinguishable from CE3003 in cell surface and competitive characteristics. Based on the nucleotide sequence, the deduced RosR amino acid sequence is 80% identical to that of the Ros protein from Agrobacterium tumefaciens and the MucR protein from Rhizobium meliloti. Both Ros and MucR are transcriptional repressors that contain a putative zinc-finger DNA-binding domain. This study defines a gene, rosR, that is homologous to a family of transcriptional regulators and contributes to nodulation competitiveness of R. etli. Moreover, we established that a single gene affects nodulation competitiveness, competitive growth in the rhizosphere, and cell surface hydrophobicity.

Glutamine Synthetase II Constitutes a Novel Taxonomic Marker in Rhizobium etli and Other Rhizobium Species

Different Rhizobium species may be identified by using polymorphisms in their glutamine synthetases (GSII) but not by their GSI profiles. We analyzed the GSs of various Rhizobium tropici and Rhizobium etli strains (which are capable of nodulating and fixing nitrogen in Phaseolus vulgaris beans), as well as strains of other species included for comparison. The GS polymorphisms were determined by identifying variations in native enzyme mobility (revealed by GS activity staining) and in the isoelectric points of the monomers (revealed by immunodetection with antibodies against the GS proteins) by using gel electrophoresis. Restriction fragment length polymorphism patterns obtained by hybridizing an internal fragment of the GSII gene obtained from R. etli with total fragmented DNAs from different strains clearly distinguished the different groups. GSII is a novel and useful marker for Rhizobium groups and species, and GSII data support R. tropici and R. etli as bona fide species.

Discrete differences between strains of different Rhizobium spp. for competitive nodule occupancy on beans

Rhizobium etli strain TAL182 and R. leguminosarum bv phaseoli strain 8002, both of which produce melanin pigment, were tested for their nodulation competitiveness on beans by paired inoculation with two strains which do not produce melanin: R. tropici strain CIAT899 and Rhizobium sp. strain TAL1145. An assay was developed to distinguish nodules formed by the melanin-producing and non-producing strains. Strain TAL182 had discrete competitive superiority over CIAT899 and TAL1145 for nodulation of beans. Nodulation competitiveness was not correlated with the ability to produce melanin pigment or the host range of the Rhizobium strains tested.

Preference in the nodulation of Phaseolus vulgaris cultivar RAB39

The low nodule occupancy achieved by superior nitrogen-fixing inoculant strains is a problem in the production of many traditional legume species, including Phaseolus vulgaris. Cultivars that select for inoculant strains, rather than nodulate with ineffective indigenous rhizobia, offer one approach to the resolution of this problem. In this study we identify a bean cultivar, RAB39, that nodulates preferentially with Rhizobium tropici, including the inoculant-quality strain UMR1899. This preference in nodulation was not affected by temperature or pH, though strain preference by a second, alkaline-producing Rhizobium etli strain did vary markedly with temperature. When RAB39 was inoculated with 1:1 mixtures of UMR1899 and seven serologically distinct strains of Rhizobium etli, more than 75% of the nodules formed by each strain pair contained UMR1899. A number of studies have reported close correlations between the percent nodule occupancy and speed of nodulation, measured using the root-tip-marking procedure. Exceptions to this correlation were evident in the present study, suggesting that host preference in nodulation was not related to differences in the parameters normally used to estimate speed of nodulation. The preference of RAB39 for R. tropici, a species of Rhizobium that occurs at low frequency in most bean soils, and especially for the inoculant-quality strain UMR1899, provides a tool to overcome the lack of response to inoculation in common bean.Key words: competition, nodulation efficiency, strain preference, common bean.

Competition for nodule occupancy on Phaseolus vulgaris by Rhizobium etli and Rhizobium tropici strains can be efficiently monitored in an ultisol during the early stages of growth using a constitutive GUS gene fusion

The GUS gene fusion (pKW107) was transferred into 4 Rhizobium phaseoli strains (KIM5s, CIAT 895, CIAT 7202, CIAT 151) and 1 Rhizobium tropici strain (CIAT 899) in order to determine the nodulation competitiveness of these strains on common bean ( Phaseolus vulgaris) in soil. DNA was fingerprinted with the different mutants to verify correct integration of the glucuronidase gene in the strains' genomes. Co-inoculation trials in growth pouches proved that the GUS + mutant strains were unaltered in their nodulation capabilities. Plant studies in a non-sterile ultisol examined the value of GUS-marked Rhizobium strains for monitoring nodule occupancy under tropical conditions. At 14 and 21 days after planting (DAP), blue nodule color allowed consistently clear and reliable differentiation between nodules formed by GUS + rhizobia and indigenous soil rhizobia. At 30 DAP, however, expression of the GUS trait was inconsistent. Older nodules formed by the GUS + rhizobia were only a faint green after incubation in detection buffer, probably because of the restriction of gusA gene expression in the construct to free-living rhizobia. Nodules formed by GUS-harboring inocula strains were differentiated from ineffective green nodules formed by native rhizobia in 30 DAP nodules in microplates by crushing nodules before adding detection buffer. Data obtained by use of the GUS gene fusion were confirmed by data derived using the ELISA technique and antibiotic resistance markers. The GUS gene fusion was found to be a powerful tool to examine competitiveness of inocula strains in a tropical soil.

Ability of selected accessions of Phaseolus vulgaris L. to restrict nodulation by particular rhizobia

Plant genotypes that limit nodulation by indigenous rhizobia while nodulating normally with inoculant-strain nodule occupancy in Phaseolus vulgaris. In this study, eight of nine Rhizobium tropici strains and six of 15 Rhizobium etli strains examined, showed limited ability to nodulate and fix nitrogen with the two wild P. vulgaris genotypes G21117 and G10002, but were effective in symbiosis with the cultivated bean genotypes Jamapa and Amarillo Gigante. Five of the R. etli strains restricted in nodulation by G21117 and G10002 produced an alkaline reaction in yeast mannitol medium. In a competition experiment in which restricted strains were tested in 1:1 mixtures with the highly effective R. etli strain CIAT632, the restricted strains produced a low percentage of the nodules formed on G2117, but produced over 40% of the nodules formed on Jamapa. The interaction of the four Rhizobium strains with the two bean genotypes, based on the percentage of nodules formed, was highly significant (P<0.001).

Characterization of a Rhizobium etli chromosomal gene required for nodule development on Phaseolus vulgaris L.

A chromosomal gene, required for nodule development on Phaseolus bean, was characterized from Rhizobium etli strain TAL182. MLC640 is a Tn5 insertion mutant of TAL182 which shows decreased motility in soft TY agar and is defective in nodule development. The site of Tn5 insertion in MLC640 mapped to a 3.6-kb EcoRI chromosomal fragment. The 3.6-kb fragment was subcloned from the cosmid pUHR80 which complemented MLC640. Further subcloning and site-directed Tn5 mutagenesis localized the gene for nodule development to a 1.7-kb region within the 3.6-kb EcoRI fragment. Southern hybridization using the 3.6-kb EcoRI fragment as the probe against genomic DNA of several Rhizobium spp. indicated that this gene is conserved in different rhizobia.