Characterization of recA genes and recA mutants of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae

Summary DNA fragments carrying the recA genes of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae were isolated by complementing a UV-sensitive recA ^– Escherichia coli strain. Sequence analysis revealed that the coding region of the R. meliloti recA gene consists of 1044 by coding for 348 amino acids whereas the coding region of the R. leguminosarum bv. viciae recA gene has 1053 bp specifying 351 amino acids. The R. meliloti and R. leguminosarum bv. viciae recA genes show 84.8% homology at the DNA sequence level and of 90.1% at the amino acid sequence level. recA ^– mutant strains of both Rhizobium species were constructed by inserting a gentamicin resistance cassette into the respective recA gene. The resulting recA mutants exhibited an increased sensitivity to UV irradiation, were impaired in their ability to perform homologous recombination and showed a slightly reduced growth rate when compared with the respective wild-type strains. The Rhizobium recA strains did not have altered symbiotic nitrogen fixation capacity. Therefore, they represent ideal candidates for release experiments with impaired strains.The accession numbers: X59956 R. LEGUMINOSARUM REC A ALAS-DNA; X59957 R. MELITOTI REC A ALAS-DNA     

Ensifer meliloti bv. lancerottense establishes nitrogen-fixing symbiosis with Lotus endemic to the Canary Islands and shows distinctive symbiotic genotypes and host range

Eleven strains were isolated from root nodules of Lotus endemic to the Canary Islands and they belonged to the genus Ensifer, a genus never previously described as a symbiont of Lotus. According to their 16S rRNA and atpD gene sequences, two isolates represented minority genotypes that could belong to previously undescribed Ensifer species, but most of the isolates were classified within the species Ensifer meliloti. These isolates nodulated Lotus lancerottensis, Lotus corniculatus and Lotus japonicus, whereas Lotus tenuis and Lotus uliginosus were more restrictive hosts. However, effective nitrogen fixation only occurred with the endemic L. lancerottensis. The E. meliloti strains did not nodulate Medicago sativa, Medicago laciniata Glycine max or Glycine soja, but induced non-fixing nodules on Phaseolus vulgaris roots. nodC and nifH symbiotic gene phylogenies showed that the E. meliloti symbionts of Lotus markedly diverged from strains of Mesorhizobium loti, the usual symbionts of Lotus, as well as from the three biovars (bv. meliloti, bv. medicaginis, and bv. mediterranense) so far described within E. meliloti. Indeed, the nodC and nifH genes from the E. meliloti isolates from Lotus represented unique symbiotic genotypes. According to their symbiotic gene sequences and host range, the Lotus symbionts would represent a new biovar of E. meliloti for which bv. lancerottense is proposed.     

Structural and functional analysis of the fixLJ genes of Rhizobium leguminosarum biovar phaseoli CNPAF512

The fixLJ genes of Rhizobium leguminosarum biovar phaseoli CNPAF512 were identified by DNA hybridization of a genomic library with an internal fragment of the Rhizobium meliloti fixJ gene. The nucleotide sequence was determined and the corresponding amino acid sequence was aligned with the amino acid sequences of the FixL proteins of R. meliloti, Bradyrhizobium japonicum and Azorhizobium caulinodans. While the FixJ protein and the carboxy-terminal part of the FixL protein are highly homologous to the other FixL and FixJ proteins, the homology in the central heme-binding, oxygen-sensing domain and in the amino-terminal domain of FixL is very low. The R. leguminosarum bv. phaseoli FixL protein does not contain the heme-binding motif defined for the previously described FixL proteins. R. leguminosarum bv. phaseoli fixLJ and fixJ mutants were constructed. These mutants can still fix nitrogen, albeit at a reduced level. Expression analysis of nifA-gusA and nifH-gusA fusions in the constructed mutants revealed that the R. leguminosarum bv. phaseoli fixLJ genes are involved in microaerobic nifH expression but not in nifA expression.The nucleotide sequence data reported will appear in the EMBL, Genbank and DDBJ Nucleotide Sequence Databases under the accession number U27314     

The new class 11 transposon Tn163 is plasmid-borne in two unrelated Rhizobium leguminosarum biovar viciae strains

Tn163 is a transposable element identified in Rhizobium leguminosarum bv. viciae by its high insertion rate into positive selection vectors. The 4.6 kb element was found in only one further R. leguminosarum bv. viciae strain out of 70 strains investigated. Both unrelated R. leguminosarum bv. viciae strains contained one copy of the transposable element, which was localized in plasmids native to these strains. DNA sequence analysis revealed three large open reading frames (ORFs) and 38 bp terminal inverted repeats. ORF1 encodes a putative protein of 990 amino acids displaying strong homologies to transposases of class 11 transposons. ORF2, transcribed in the opposite direction, codes for a protein of 213 amino acids which is highly homologous to DNA invertases and resolvases of class II transposons. Homology of ORF1 and ORF2 and the genetic structure of the element indicate that Tn163 can be classified as a class II transposon. It is the first example of a native transposon in the genus Rhizobium. ORF3, which was found not to be involved in the transposition process, encodes a putative protein (256 amino acids) of unknown function. During transposition Tn163 produced direct repeats of 5 bp, which is typical for transposons of the Tn3 family. However, one out of the ten insertion sites sequenced showed a 6 by duplication of the target DNA; all duplicated sequences were A/T rich. Insertion of Tn163 into the sacB gene revealed two hot spots. Chromosomes of different R. leguminosarum bv. viciae strains were found to be highly refractory to the insertion of Tn163.     

Tolerance ofRhizobium leguminosarum bv.phaseoli to acidity and drought

Ten strains ofRhizobium leguminosarum bv.phaseoli isolated from soils of Morocco were more tolerant than three culture collection strains to acid conditions in culture media or in sterile soil. The survival rate of a tolerant strain in a sandy acid soil was greater than a sensitive strain at different humidity levels. These properties should give locally selected strains an advantage in nodulatingPhaseolus vulgaris roots in soils similar to those used here.     

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%.     

Rhizobium etli and Rhizobium gallicum Nodulate Common Bean (Phaseolus vulgaris) in a Traditionally Managed Milpa Plot in Mexico: Population Genetics and Biogeographic Implications

The stability of the genetic structure of rhizobial populations nodulating Phaseolus vulgaris cultivated in a traditionally managed milpa plot in Mexico was studied over three consecutive years. The set of molecular markers analyzed (including partial rrs, glnII, nifH, and nodB sequences), along with host range experiments, placed the isolates examined in Rhizobium etli bv. phaseoli and Rhizobium gallicum bv. gallicum. Cluster analysis of multilocus enzyme electrophoresis and plasmid profile data separated the two species and identified numerically dominant clones within each of them. Population genetic analyses showed that there was high genetic differentiation between the two species and that there was low intrapopulation differentiation of the species over the 3 years. The results of linkage disequilibrium analyses are consistent with an epidemic genetic structure for both species, with frequent genetic exchange taking place within conspecific populations but not between the R. etli and R. gallicum populations. A subsample of isolates was selected and used for 16S ribosomal DNA PCR-restriction fragment length polymorphism analysis, nifH copy number determination, and host range experiments. Plasmid profiles and nifH hybridization patterns also revealed the occurrence of lateral plasmid transfer among distinct multilocus genotypes within species but not between species. Both species were recovered from nodules of the same plants, indicating that mechanisms other than host, spatial, or temporal isolation may account for the genetic barrier between the species. The biogeographic implications of finding an R. gallicum bv. gallicum population nodulating common bean in America are discussed.     

Different species and symbiotic genotypes of field rhizobia can nodulate Phaseolus vulgaris in Tunisian soils

A collection of 160 isolates of rhizobia nodulating Phaseolus vulgaris in three geographical regions in Tunisia was characterized by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified 16S rDNA, nifH and nodC genes. Nine groups of rhizobia were delineated: Rhizobium gallicum biovar (bv.) gallicum, Rhizobium leguminosarum bv. phaseoli and bv. viciae, Rhizobium etli bv. phaseoli, Rhizobium giardinii bv. giardinii, and four groups related to species of the genus Sinorhizobium, Sinorhizobium meliloti, Sinorhizobium medicae and Sinorhizobium fredii. The most abundant rhizobial species were R. gallicum, R. etli, and R. leguminosarum encompassing 29–20% of the isolates each. Among the isolates assigned to R. leguminosarum, two-thirds were ineffective in nitrogen fixation with P. vulgaris and harbored a symbiotic gene typical of the biovar viciae. The S. fredii-like isolates did not nodulate soybean plants but formed numerous effective nodules on P. vulgaris. Comparison of nodC gene sequences showed that their symbiotic genotype was not related to that of S. fredii, but to that of the S. fredii-like reference strain GR-06, which was isolated from a bean plant grown in a Spanish soil. An additional genotype including 16% of isolates was found to be closely related to species of the genus Agrobacterium. However, when re-examined, these isolates did not nodulate their original host.     

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).     

Tn5 insertion mutants of Pseudomonas sp. 267 defective in siderophore production and their effect on clover (Trifolium pratense) nodulated with Rhizobium leguminosarum bv. trifolii

Pseudomonas sp. strain 267 isolated from soil promoted growth of different plants under field conditions and enhanced symbiotic nitrogen fixation in clover under gnotobiotic conditions. This strain produced pyoverdine-like compound under low-iron conditions and secreted vitamins of the B group. The role of fluorescent siderophore production in the beneficial effect of strain 267 on nodulated clover plants was investigated. Several non-fluorescent (Pvd^-) Tn5 insertion mutants of Pseudomonas sp. strain 267 were isolated and characterized. The presence of Tn5 insertions was confirmed by Southern analysis of EcoRI digested genomic DNA of each derivative strain. The siderophore-negative mutants were compared to the parental strain with respect to their growth promotion of nodulated clover infected with Rhizobium leguminosarum bv. trifolii 24.1. We found that all isolated Pvd^- mutants stimulated growth of nodulated clover plants in a similar manner to the parental strain. No consistent differences were observed between strain 267 and Pvd^- derivatives strains with respect to their plant growth promotion activity under gnotobiotic conditions.Dr Deryto died in august 1994     

Effect of pH on competition for nodule occupancy by type I and type II strains ofRhizobium leguminosarum bv.phaseoli

The effect of soil pH on the competitive abilities of twoRhizobium leuminosarum bv.phaseoli type I and one type II strains was examined in a nonsterile soil system.Phaseolus vulgaris seedlings, grown in unlimed (pH 5.2) or limed (pH 7.6) soil, were inoculated with a single-strain inoculum containing 1 × 10⁶ cells mL^–1 of one of the three test strains or with a mixed inoculum (1:1, type I vs. type II) containing the type II strain CIAT 899 plus one type I strain (TAL 182 or CIAT 895). At harvest, nodule occupants were determined. In a separate experiment, a mixed suspension (1:1, type I vs. type II) of CIAT 899 paired with either TAL 182 or CIAT 895 was used to inoculateP. vulgaris seedlings grown in sterile, limed or unlimed soil. The numbers of each strain in the rhizosphere were monitored for 10 days following inoculation. The majority of nodules (> 60%) formed on plants grown in acidic soil were occupied by CIAT 899, the type II strain. This pattern of nodule occupancy changed in limed soil. When CIAT 899 was paired with TAL 182, the type I strain formed 78% of the nodules. The number of nodules formed by CIAT 899 and CIAT 895 (56% and 44%, respectively) were not significantly different. The observed patterns of nodule occupancy were not related to the relative numbers or specific growth rates of competing strains in the host rhizosphere prior to nodulation. The results indicate that soil pH can influence which symbiotype ofR. leguminosarum bv.phaseoli will competitively nodulateP. vulgaris.     

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°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.     

Use of DNA marker to select well-adapted <Emphasis Type="Italic">Phaseolus</Emphasis>-symbionts strains under acid conditions and high temperature

Soil acidity and high temperature contribute to the failure of nodulation in the common bean. It is therefore urgent to select strains with a high competitive ability under these stress conditions. Two Egyptian Rhizobium etli strains, EBRI 2 and EBRI 26, were examined against Rhizobium tropici CIAT 899G labeled with the gus (β-glucuronidase) reporter gene. EBRI 2 and EBRI 26 were less competitive than CIAT 899G under acid conditions with both the Egyptian cultivar Giza 3 and the Colombian cultivar Rab 39. However, EBRI 2 and EBRI 26 gave higher nodule occupancy (78% and 62.5, respectively) than the nodule occupancy (18.5% and 35%) obtained by CIAT 899G at 35°C with cultivar Giza 3. Soil acidity (pH 5.8) was less detrimental to the nodule occupancy of EBRI 2 than EBRI 26 when they tested in competition with CIAT 899G.     

Evaluation of strain competitiveness in Rhizobium leguminosarum bv phaseoli using a nod + fix− natural mutant

Competition from native soil rhizobia is likely to be an important factor limiting Phaseolus vulgaris L. inoculant response in Latin America. We used UMR 1116, a nod ⁺ fix^– natural mutant of Rhizobium leguminosarum bv phaseoli strain CC511, as a reference strain to study competition for nodulation sites in this species. When P. vulgaris cv Carioca was planted in soils containing different proportions of UMR 1116 and the effective and competitive strain UMR 1899, UMR 1116 occupied more than 50% of the nodules at all inoculant ratios tested, though increasing the proportion of UMR 1899 in the inoculant did enhance the number and percentage of effective nodules and plant dry weight. Sixty two strains of bean rhizobia were tested in competition with UMR 1116. An inoculant ratio of 1:1 was used, with all strains applied to the soil rather than to seeds. Strains varied in the number and percentage of effective nodules produced in competition with UMR 1116, and in plant dry weight, and there was a strong correlation between variation in each of these traits and plant N accumulation. Seven of the strains (UMR 1073, 1084, 1102, 1125, 1165, 1378 and 1384) were identified as both superior in competitive ability and active in N₂ fixation. Site of placement of the inoculant and ambient temperature influenced strain response.Journal paper 16736, Agricultural Experiment Station, University of Minnesota, St. Paul, MN 55108, USA     

Molecular cloning and nucleotide sequence of the Rhizobium phaseoli recA gene

Summary A recombinant phage carrying the recA gene of Rhizobium phaseoli was isolated from a R. phaseoli genomic library by complementation of the Fec^– phenotype of the recombinant phage in Escherichia coli. When expressed in E. coli, the cloned recA gene was shown to restore resistance to both UV irradiation and the DNA alkylating agent methyl methanesulphonate (MMS). The R. phaseoli recA gene also promoted homologous recombination in E. coli. The cloned recA gene was only weakly inducible in E. coli recA strains by DNA damaging agents. The DNA sequence of the R. phaseoli recA gene was determined and compared with published recA sequences. No LexA-binding site was detected in the R. phaseoli recA upstream region.     

Rhizobium tropici nodulates field-grown Phaseolus vulgaris in France

Two hundred and eighty seven isolates of Rhizobium nodulating Phaseolus vulgaris L. were sampled in France from four geographically distant field populations. They were characterized by their colony morphology and by plasmid profiles. A representative sample was further characterized: a) by the ability of each isolate to nodulate a potential alternative host Leucaena leucocephala and to grow on specific media, and b) by RFLP analysis of PCR amplified 16S rRNA genes. On the basis of their phenotypic and genetic characteristics the isolates could be assigned either to Rhizobium leguminosarum bv phaseoli, or to R. tropici. The two species co-occurred at three sites. R. leguminosarum bv phaseoli represented 2%, 4%, 72% and 100% of the population at the four different sites. Eighteen and 22 different plasmid profiles were identified within R. tropici and R. leguminosarum bv phaseoli, respectively. Some of them were conserved between distant geographical regions. The fact that R. tropici was found in France shows that this species is not limited to tropical regions and gives additional evidence of the multi-specific nature of the Phaseolus microsymbiont, even over a geographically limited area.     

Nitrogen assimilation and partitioning in two nitrogen-fixing cultivars of Phaseolus vulgaris L.

Two cultivars of Phaseolus vulgaris L., one responsive (Mexico 309) and one less-responsive (Rio Tibagi) to nodulation with Rhizobium were grown in Leonard jars in a greenhouse. Bean plants were either inoculated with a strain of Rhizobium leguminosarum bv. phaseoli (UMR-1899), a vesicular-arbuscular mycorrhizal (VAM) fungus (Glomus etunicatum) or were left non-inoculated (controls). At two harvests (21 and 28 days post-emergence), extracts containing soluble proteins and free amino acids were prepared from leaves, roots and nodules of field beans. Nodulated plants contained a significantly higher concentration of protein and amino acids in all plant parts. Nitrogen-fixing beans invested a significantly greater proportion of total N as protein-N and amino acid-N as compared to VAM or control beans. Abundant nodule-specific proteins (nodulins) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), identified and quantified using scanning densitometry. Rio Tibagi nodules contained a significantly lower concentration of glutamine synthetase (GS) subunits than did Mexico 309 nodules. Glutamate synthase (GOGAT) and GS activities were low relative to other legumes. The transferase/synthetase ratio for GS was also low indicating that the synthetase activity was caturated and was operating at maximal level in these young N₂-fixing associations. Specific nodule activity (SNA) and the level of GS were correlated (r=0.90, p<0.05) for both cultivars at both harvests. GS activity was only 8 or 24% higher than SNA in nodules of Mexico 309 or Rio Tibagi cultivars, respectively, under conditions where substrate was not limiting. This suggests that early in the functioning of this symbiosis N assimilation by GS is the rate-limiting step in N₂ fixation by these two bean cultivars, each with a different symbiotic efficiency. Phaseolus breeding programs that attempt to improve N₂ fixation in beans should identify germplasm that expresses elevated levels of nodule-specific GS or GOGAT, and this material should be used along with effective R. leguminosarum bv. phaseoli strains that have already been selected, to determine superior host-microsymciont associations.     

Nodulation and nitrogen fixation by fast- and slow-growing rhizobia strains of soybean on several temperate and tropical legumes

Three slow-growingBradyrhizobium japonicum (G₃, USDA-110 and KUL-150) of diverse origins and two fast-growing strains ofRhizobium fredii (USDA-192 and USDA-193) were tested with a cropped soybean (Glycine max L. Merrill) cultivar, two cowpeas (Vigna unguiculata), one mung-bean (Phaseolus radiata), one winged-bean (Psophocarpus tetragonolobus) and one field bean (Phaseolus vulgaris) varieties.TheR. fredii strains nodulated and fixed Nitrogen as effectively as the strains ofB. japonicum in a modern european soybean cultivar, namely Fiskeby V. The other western bred soybeans tested were not nodulated by theseR. fredii strains. All of the soybean rhizobia produced nodules in both cowpeas and in mung-bean; theR. fredii strains showed effective N₂-fixation in the cowpeas, particularly USDA-193, yielding shoot dry weights greater than those from theB. japonicum. The symbiotic performance of theR. fredii strains with soybean and other legumes indicated that they should be placed in an intermediate group between the slow-growingB. japonicum and cowpearhizobium sp.The hydrogen uptake activites suggested a possible host effect on the expression of such genes in one out of theB. japonicum strains tested. Furthermore, the slow-growing rhizobia showed significantly higher nitrate-reduction than theR. fredii in the nodules.     

Some properties of carbohydrate and C4-dicarboxylic acid utilization negative mutants ofRhizobium leguminosarum biovarphaseoli strain P121

After NTG treatment of the very effective wild type strain P121 ofRhizobium leguminosarum biovarphaseoli, mutants defective in the utilization of sugars or organic acids were obtained. All the mutants nodulated the cultivar Goldie ofPhaseolus vulgaris. The arabinose, fructose, glucose and pyruvate utilization mutants formed nodules similar in shape and size to the nodules formed by the wild type strain. These mutants exhibited an acetylene reduction activity significantly lower than the activity observed with the wild type strain. All the C₄-dicarboxylic acid utilization mutatns, formed ineffective nodules that did not show a significant acetylene reduction activity. The C₄-dicarboxylic acids uptake system is apparently inducible in the free-living bacteria of strain P121. When P121 cells were grown on glucose in the presence of 2.5 mM malate, the rate of glucose-dependent O₂ consumption significantly decreased suggesting the presence of a catabolite repression-like phenomenon. Isolated bacteroids of strain P121, under the experimental conditions used, were able to oxidize succinate, fumarate or malate but did not oxidize pyruvate, glucose, fructose or sucrose.