Genetic Analysis of Rhizobium leguminosarum bv. Phaseoli Mutants Defective in Nodulation and Nodulation Suppression

Nodulation-defective rhizobia and their nodule-forming derivatives containing cloned DNA from the wild type were used to study nodulation suppression in Phaseolus vulgaris L. Non-nitrogen-fixing derivatives which formed rhizobia-containing white nodules induced partial suppression. Comparison of this with the complete suppression by Fix⁺ derivatives and a Fix^- mutant which formed rhizobia-containing pink nodules suggests that the extent of suppression may be related to successive stages of nodule development.     

High-frequency rearrangements in Rhizobium leguminosarum bv. phaseoli plasmids.

High-frequency genomic rearrangements affecting the plasmids of Rhizobium leguminosarum bv. phaseoli CFN42 were analyzed. This strain contains six large plasmids ranging in size from 200 to 600 kb. In the absence of any selective pressure, we found 11 strains from 320 analyzed colonies that presented different kinds of plasmid-borne rearrangements, including sequence amplification, deletion, cointegration, and loss of plasmids. These data support the concept that the R. leguminosarum bv. phaseoli genome is a dynamic structure and imply that strains are mixtures of similar but not identical cells.     

Response of Rhizobium leguminosarum bv phaseoli to acidity

Soil acidity constraints grain legume production in tropical soils, both limiting Rhizobium survival and reducing nodulation. Strains of rhizobia with greater tolerance to hydrogen-ion concentration have been identified, but the basis for strain differences in pH tolerance has yet to be determined. In this study, strains of Rhizobium leguminosarum by phaseoli which differed in their tolerance to acidity were exposed to acid pH, then cell levels of potassium and calcium determined, and specific ‘acid-shock’ proteins identified. Lowering the external pH to 4.6–4.7 resulted in an immediate efflux of calcium from the cell of both acid tolerant and sensitive bean strains. Change in cell potassium levels on exposure to acidity varied with the strain. Strain UMR 1899 and an acid-sensitive mutant derived from it maintained high cytoplasmic potassium at acid pH, whereas an acid-sensitive strain UMR 1632 underwent a marked decline in cell potassium at pH 4.6. Exposure of these strains to pH 4.5 in the presence of [³⁵S]-labeled methionine enhanced production of a number of proteins, while synthesis of other proteins at this pH was significantly reduced. Differences in banding pattern were also evident between UMR1899 and the Tn5-induced pH-sensitive mutant UMR5005 derived from it, and between cells grown in the presence and absence of calcium and phosphorus.     

Multiple copies of nodD in Rhizobium tropici CIAT899 and BR816.

Rhizobium tropici strains are able to nodulate a wide range of host plants: Phaseolus vulgaris, Leucaena spp., and Macroptilium atropurpureum. We studied the nodD regulatory gene for nodulation of two R. tropici strains: CIAT899, the reference R. tropici type IIb strain, and BR816, a heat-tolerant strain isolated from Leucaena leucocephala. A survey revealed several nodD-hybridizing DNA regions in both strains: five distinct regions in CIAT899 and four distinct regions in BR816. Induction experiments of a nodABC-uidA fusion in combination with different nodD-hybridizing fragments in the presence of root exudates of the different hosts indicate that one particular nodD copy contributes to nodulation gene induction far more than any other nodD copy present. The nucleotide sequences of both nodD genes are reported here and show significant homology to those of the nodD genes of other rhizobia and a Bradyrhizobium strain. A dendrogram based on the protein sequences of 15 different NodD proteins shows that the R. tropici NodD proteins are linked most closely to each other and then to the NodD of Rhizobium phaseoli 8002.     

Identification and cloning of nodulation genes and host specificity determinants of the broad host-range Rhizobium leguminosarum biovar phaseoli strain CIAT899

Rhizobium Ieguminosarum biovar phaseoli type II strain CIAT899 nodulates a wide range of hosts: Phaseolus vulgaris (beans), Leucaena esculenta (leucaena) and Macroptilium atropurpureum (siratro). A nodulation region from the symbiotic plasmid has been isolated and characterized. This region, which is contained in the overlapping cosmid clones pCV38 and pCV117, is able to induce nodutes in beans, leucaena and siratro roots when introduced in strains cured for the symbiotic plasmid, pSym. In addition, this cloned region extends the host range of Rhizobium meliloti and R. leguminosarum biovar (bv.) trifolii wild-type strains to nodulate beans. Analysis of constructed subclones indicates that a 6.4 kb Hin dlll fragment contains the essential genes required for nodule induction on all three hosts. Rhizobium leguminosarum bv. phaseoli type I strain CE3 nodulates only beans. However, CE3 transconjugants harbouring plasmid pCV3802 (which hybridized to a nodD heterologous probe), were capable of eliciting nodules on leucaena and siratro roots. Our results suggest that the CIAT899 DNA region hybridizing with the R. meliloti nodD detector is involved in the extension of host specificity to promote nodule formation in P. vulgaris, L. esculenta and M. atropurpureum.     

Comparative strain typing of Rhizobium leguminosarum bv. viciae natural populations

372 natural isolates of Rhizobium leguminosarum bv. viciae, rescued from nodules of pea plants grown in an agricultural field in northern Italy, were analyzed by different methods. Three DNA-based fingerprinting techniques were lined up to compare their relative degree of resolution and possible advantages of each approach. The methods included (i) Eckhardt gel plasmid profiles, (ii) pulsed-field gel electrophoresis (PFGE) of genomic large fragment digests, and (iii) random amplified polymorphic DNA (RAPD) profiles, generated with arbitrary primers. The scheme also involved the isolation of a number of different isolates per nodule to estimate the level of intra-nodular variability. It was therefore possible to evaluate the frequency of double and multiple occupancies, and the proportion of the alternative profiles sharing the same nodule, generally resulting in a numerically dominant, main representative accompanied by a secondary one with a slightly different fingerprint. This finding revealed that the different profiles within a nodule are normally due to bacteria derived from the same single invader following genetic alterations possibly occurred during infection, e.g., by plasmid loss. The analysis of 31 nodules revealed 16 different patterns, representing the most frequently occurring nodulation-proficient isolates of the natural soil examined, five of which were found with frequencies around 15%. The sensitivity of the methods in differentiating isolates was compared. The relatedness of the different natural rhizobial isolates was investigated by densitometrical gel analysis of the fingerprints, allowing a comparison of the results. One of the most interesting conclusions was that the degree of information yielded by the plasmid gel profiling alone, carried out by simple visual inspection without software-aided analyses, was surprisingly high, as it enabled a placement of the isolates, whose accuracy, in terms of relatedness, was subsequently confirmed by each of the two genomic methods.     

青海蚕豆根瘤菌共生固氮效应的研究

蚕豆根瘤菌属于快生型根瘤菌 ,2 4h形成菌落。在盆栽试验中 ,蚕豆根瘤菌Qx -2与蚕豆具有良好共生效应 ,其株高、茎叶干重及含氮量 ,株瘤数 ,根瘤重量以及固氮酶活性等 ,均明显高于不接种对照 ,固氮率提高 69.4 9%。接种根瘤菌是提高蚕豆共生固氮效应的技术措施。     

Competition among Rhizobium leguminosarum bv. phaseoli strains for nodulation of common bean.

Six effective Rhizobium leguminosarum bv. phaseoli strains were examined for nodulation competitiveness on common bean (Phaseolus vulgaris L.), using all possible two-strain combinations of inoculum. Nodule occupancy was determined with strain-specific fluorescent antibodies. The strains were divided into three groups according to their overall competitive abilities on pole bean cv. Kentucky Wonder and bush bean cv. Bountiful. Strains TAL 182 and TAL 1472 were highly competitive (greater than 70% nodule occupancy); strains KIM-5, Viking 1, and CIAT 899 were moderately competitive (approximately 50% nodule occupancy); and strain CIAT 632 was poorly competitive (less than 5% nodule occupancy). The competitiveness of the six strains was similar on the two host cultivars. The proportion of competing strains in the inoculum influenced the nodule occupancy of the highly competitive and moderately competitive strains, but not that of the poorly competitive strain. Two outstanding strains (TAL 182 and TAL 1472) were identified as ideal model strains for molecular and genetic studies on nodulation competitiveness.     

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.     

Competition in Kenyan soils between Rhizobium leguminosarum biovar phaseoli strain Kim5 and R. tropici strain CIAT899 using the gusA marker gene

The contribution of appropriate inoculum strains to more efficient nitrogen fixation by legumes has been difficult to assess due to the laborious nature of the assays involved in assessing establishment of inoculum strains in the field. The use of marker genes, in particular the GUS system, changes this, making it possible to assess occupancy by the inoculum strain in large numbers of nodules on whole root systems. Here we used the GUS system to evaluate the competitive ability of two rhizobial strains, Rhizobium leguminosarum bv. phaseoli strain Kim5 and R. tropici strain CIAT899 in two soil types from Kenya. The results confirm that Kim5 is a highly competitive strain, forming 86% of the nodules in a near-neutral pH soil. Although the competitiveness of CIAT899 is enhanced in an acid (pH 4.5) soil it still only formed 35% of the nodules. There were no differences between inoculum strains in their efficiency of nitrogen fixation in either soil type, and virtually no N2-fixation occurred in the acid soil due to the lack of tolerance of the Phaseolus genotype to soil acidity.     

Structural complexity of the symbiotic plasmid of Rhizobium leguminosarum bv. phaseoli.

The complete physical map of the symbiotic plasmid of Rhizobium leguminosarum bv. phaseoli strain CFN42 was established. The data support the concept that Rhizobium symbiotic genes are part of a complex genomic structure which contains a large amount of reiterated DNA sequences. This plasmid is a circular structure of 390 kb with approximately 10 families of internally reiterated DNA sequences of two to three elements each. One family includes two directly oriented nitrogenase operons situated 120 kb apart. We also found several stretches of pSym that are reiterated in other replicons of the cell. Localization of symbiotic gene sequences by heterologous hybridization revealed that nodABC sequences are separated in two regions, each of which contains a nod boxlike element, and it also suggested the presence of two copies of the nifA and nodD gene sequences. We propose that the complex structure of the symbiotic plasmid allows interactions between repeated DNA sequences which, in turn, might result in frequent rearrangements.     

Symbiotic effects of deltamatB Rhizobium leguminosarum bv. trifolii mutant on clovers

The role of malonate in symbiotic nitrogen metabolism has long been controversial, although it is known to occur in legume roots, especially in the nodules. Here we report that malonate metabolism plays a key role in the differentiation of bacteroids Rhizobium leguminosarum bv. trifolii in clover nodules. An operon, mat, that consists of three consecutive genes (matABC) has been discovered. Mat encodes enzymes that catalyze the uptake and conversion of malonate to acetyl-CoA through malonyl-CoA. A mutant bacteria, which replaced matB that encodes malonyl-CoA synthetase with a kanamycin resistant gene, was generated and infected with white clover. Clover growth was considerably reduced, even though nodules were formed. However, the nodules were filled with vacuoles, but not with bacteroids. This indicates that malonate metabolism is an important requirement for the formation of mature nodules that are filled with bacteroids.     

Molecular and symbiotic characterization of exopolysaccharide-deficient mutants of Rhizobium tropici strain CIAT899

We studied the symbiotic behaviour of 20 independent Tn5 mutants of Rhizobium tropici strain CIAT899 that were deficient in exopolysaccharide (EPS) production. The mutants produced non-mucoid colonies, were motile, grew in broth cultures at rates similar to those of the parent, and produced significantly less EPS than did CIAT899 in broth culture. A genomic library of strain CIAT899, constructed in pLA2917, was mobilized into all of the mutants, and cosmids that restored EPS production were identified. EcoRI restriction digests of the cosmids revealed nine unique inserts. Mutant complementation and hybridization analysis showed that the mutations affecting EPS production fell into six functional and physical linkage groups. On bean, the mutants were as efficient in nodulation and as effective in acetylene reduction as strain CIAT899, induced a severe interveinal chlorosis, and all but one were less competitive than CIAT899. On siratro, CIAT899 induced nodules that were ineffective in acetylene reduction, whereas the EPS-deficient mutants induced effective nodules. Microscopic examination of thin sections showed that nodules from both siratro and bean plants inoculated with either CIAT899 or an EPS-deficient mutant contained infected cells. These data indicate that EPS is not required for normal nodulation of bean by R. tropici, that it may contribute to competitiveness of R. tropici on bean, and that the loss of EPS production is accompanied by acquisition of the ability to reduce acetylene on siratro.     

Selection of Portuguese Rhizobium leguminosarum bv. trifolii strains for production of legume inoculants

From several native clover species, growing in six different soil types, 170 Rhizobium leguminosarum biovar trifolii strains were isolated, covering the central and southern regions of Portugal. The effectiveness of the strains varied from ineffective to highly effective on T. subterraneum cv. Clare and on T. fragiferum cv. Palestine, with a predominance of medium and high effectiveness on both host plants. The effectiveness was not influenced by provenence (soil or plant), except for the strains from the rankers soils and for the strains isolated from T. pratense, that were ineffective or medium effective on T. subterraneum.Selected strains were evaluated for effectiveness on T. subterraneum cv. Clare, using the commercial strain TA1 as reference. Several of the isolated strains were more effective than TA1, indicating that local strains may be used to produce better inoculants.     

Different plasmids of Rhizobium leguminosarum bv. phaseoli are required for optimal symbiotic performance.

Rhizobium leguminosarum bv. phaseoli CFN42 contains six plasmids (pa to pf), and pd has been shown to be the symbiotic plasmid. To determine the participation of the other plasmids in cellular functions, we used a positive selection scheme to isolate derivatives cured of each plasmid. These were obtained for all except one (pe), of which only deleted derivatives were recovered. In regard to symbiosis, we found that in addition to pd, pb is also indispensable for nodulation, partly owing to the presence of genes involved in lipopolysaccharide synthesis. The positive contribution of pb, pc, pe, and pf to the symbiotic capacity of the strain was revealed in competition experiments. The strains that were cured (or deleted for pe) were significantly less competitive than the wild type. Analysis of the growth capacity of the cured strains showed the participation of the plasmids in free-living conditions: the pf- strain was unable to grow on minimal medium, while strains cured of any other plasmid had significantly reduced growth capacity in this medium. Even on rich medium, strains lacking pb or pc or deleted for pe had a diminished growth rate compared with the wild type. Complementation of the cured strains with the corresponding wild-type plasmid restored their original phenotypes, thus confirming that the effects seen were due only to loss of plasmids. The results indicate global participation of the Rhizobium genome in symbiotic and free-living functions.     

Symbiotic plasmid rearrangement in Rhizobium leguminosarum bv. viciae VF39SM

A rearrangement between the symbiotic plasmid (pRleVF39d) and a nonsymbiotic plasmid (pRleVF39b) in Rhizobium leguminosarum bv. viciae VF39 was observed. The rearranged derivative showed the same plasmid profile as its parent strain, but hybridization to nod, fix, and nif genes indicated that most of the symbiotic genes were now present on a plasmid corresponding in size to pRleVF39b instead of pRleVF39d. On the other hand, some DNA fragments originating from pRleVF39b now hybridized to the plasmid band at the position of pRleVF39d. These results suggest that a reciprocal but unequal DNA exchange between the two plasmids had occurred.     

Characterization of the urease gene cluster from Rhizobium leguminosarum bv. viciae

Moderate levels of urease activity (ca. 300 mU mg(-1)) were detected in Rhizobium leguminosarum bv. viciae UPM791 vegetative cells. This activity did not require urea for induction and was partially repressed by the addition of ammonium into the medium. Lower levels of urease activity (ca. 100 mU mg(-1)) were detected also in pea bacteroids. A DNA region of ca. 9 kb containing the urease structural genes ( ureA, ureB and ureC), accessory genes ( ureD, ureE, ureF, and ureG), and five additional ORFs ( orf83, orf135, orf207, orf223, and orf287) encoding proteins of unknown function was sequenced. Three of these ORFs ( orf83, orf135 and orf207) have a homologous counterpart in a gene cluster from Sinorhizobium meliloti, reported to be involved in urease and hydrogenase activities. R. leguminosarum mutant strains carrying Tn 5 insertions within this region exhibited a urease-negative phenotype, but induced wild-type levels of hydrogenase and nitrogenase activities in bacteroids. orf287 encodes a potential transmembrane protein with a C-terminal GGDEF domain. A mutant affected in orf287 exhibited normal levels of urease activity in culture cells. Experiments aimed at cross-complementing Ni-binding proteins required for urease and hydrogenase synthesis (UreE and HypB, respectively) indicated that these two proteins are not functionally interchangeable in R. leguminosarum.