Nitrogen-Fixing Nodules with Ensifer adhaerens Harboring Rhizobium tropici Symbiotic Plasmids

Ensifer adhaerens is a soil bacterium that attaches to other bacteria and may cause lysis of these other bacteria. Based on the sequence of its small-subunit rRNA gene, E. adhaerens is related to Sinorhizobium spp. E. adhaerens ATCC 33499 did not nodulate Phaseolus vulgaris (bean) or Leucaena leucocephala, but with symbiotic plasmids from Rhizobium tropici CFN299 it formed nitrogen-fixing nodules on both hosts. The nodule isolates were identified as E. adhaerens isolates by growth on selective media.     

Nitrogen-fixing nodules induced by Agrobacterium tumefaciens harboring Rhizobium phaseoli plasmids.

Rhizobium phaseoli CFN299 forms nitrogen-fixing nodules in Phaseolus vulgaris (bean) and in Leucaena esculenta. It has three plasmids of 185, 225, and 410 kilobases. The 410-kilobase plasmid contains the nitrogenase structural genes. We have transferred these plasmids to the plasmid-free strain Agrobacterium tumefaciens GMI9023. Transconjugants containing different combinations of the R. phaseoli plasmids were obtained, and they were exhaustively purified before nodulation was assayed. Only transconjugants harboring the 410-kilobase plasmid nodulate P. vulgaris and L. esculenta. Nodules formed by all such transconjugants are able to reduce acetylene. Transconjugants containing the whole set of plasmids from CFN299 nodulate better and fix more nitrogen than the transconjugants carrying only the Sym plasmid. Microscopic analysis of nodules induced by A. tumefaciens transconjugants reveals infected cells and vascular bundles. None of the A. tumefaciens transconjugants, not even the one with the whole set of plasmids from CFN299, behaves in symbiosis like the original R. phaseoli strain; the transconjugants produce fewer nodules and have lower acetylene reduction (25% as compared to the original R. phaseoli strain) and more amyloplasts per nodule. More than 2,000 bacterial isolates from nodules of P. vulgaris and L. esculenta formed by the transconjugants were analyzed by different criteria. Not a single rhizobium could be detected. Our results show that R. phaseoli plasmids may be expressed in the A. tumefaciens background and direct the formation of effective, differentiated nodules.     

Enhanced symbiotic performance by Rhizobium tropici glycogen synthase mutants

We isolated a Tn5-induced Rhizobium tropici mutant that has enhanced capacity to oxidize N,N-dimethyl-p-phenylendiamine (DMPD) and therefore has enhanced respiration via cytochrome oxidase. The mutant had increased levels of the cytochromes c(1) and CycM and a small increase in the amount of cytochrome aa(3). In plant tests, the mutant increased the dry weight of Phaseolus vulgaris plants by 20 to 38% compared with the control strain, thus showing significantly enhanced symbiotic performance. The predicted product of the mutated gene is homologous to glycogen synthases from several bacteria, and the mutant lacked glycogen. The DNA sequence of the adjacent gene region revealed six genes predicted to encode products homologous to the following gene products from Escherichia coli: glycogen phosphorylase (glgP), glycogen branching enzyme (glgB), ADP glucose pyrophosphorylase (glgC), glycogen synthase (glgA), phosphoglucomutase (pgm), and glycogen debranching enzyme (glgX). All six genes are transcribed in the same direction, and analysis with lacZ gene fusions suggests that the first five genes are organized in one operon, although pgm appears to have an additional promoter; glgX is transcribed independently. Surprisingly, the glgA mutant had decreased levels of high-molecular-weight exopolysaccharide after growth on glucose, but levels were normal after growth on galactose. A deletion mutant was constructed in order to generate a nonpolar mutation in glgA. This mutant had a phenotype similar to that of the Tn5 mutant, indicating that the enhanced respiration and symbiotic nitrogen fixation and decreased exopolysaccharide were due to mutation of glgA and not to a polar effect on a downstream gene.     

Formation of Rhizobium phaseoli symbiotic plasmids by genetic recombination

We report here the formation of symbiotic plasmids (pSyms), by genetic recombination between rearranged pSyms, which lack symbiotic information, and resistance plasmids carrying parts of different symbiotic plasmids (R's). This recombination was found to occur both between plasmids derived from different Rhizobium phaseoli isolates, and between plasmids derived from strains obtained from the same original isolate. We also present evidence on the formation of a functional symbiotic plasmid by recombination of an R', carrying nif and nod genes from strain CFN42, and an indigenous plasmid present in this strain (pCFN42e), which was thought to be unrelated to its symbiotic plasmid (pCFN42d). These data are discussed with respect to the stability and transfer of Rhizobium symbiotic information.     

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.     

Rhizobium tropici chromosomal citrate synthase gene.

Two genes encoding citrate synthase, a key enzyme in the Krebs cycle, have been found in Rhizobium tropici. One of them is in the bacterial chromosome, while the other is in the symbiotic plasmid. We sequenced the chromosomal gene and found that it is very similar to the previously reported plasmidic gene sequence in its structural region but not in its regulatory region. The chromosomal gene is able to complement an Escherichia coli citrate synthase mutant. In R. tropici, a mutant in the chromosomal citrate synthase gene has a diminished citrate synthase activity (in free-living bacteria), a diminished nodulation capacity, and forms nitrogen-fixing nodules. In contrast, the citrate synthase double mutant forms ineffective nodules devoid of bacteroids and forms less nodules than the single chromosomal mutant. It is inferred that both genes are functional and required during the nodulation process in R. tropici.     

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.     

Two-dimensional proteome reference map of Rhizobium tropici PRF 81 reveals several symbiotic determinants and strong resemblance with agrobacteria

Rhizobium tropici strain PRF 81 is used in commercial inoculants for common-bean crops in Brazil because of its high efficiency in nitrogen fixation and, as in other strains belonging to this species, its tolerance of environmental stresses, representing a useful biological alternative to chemical nitrogen fertilizers. In this study, a proteomic reference map of PRF 81 was obtained by two-dimensional gel electrophoresis and MALDI-TOF/TOF-TOF mass spectrometry. In total, 115 spots representing 109 different proteins were successfully identified, contributing to a better understanding of the rhizobia-legume symbiosis and supporting, at proteomics level, a strong resemblance with agrobacteria.     

Nitrogen-fixing root nodules inDatisca Cannabina L.

Summary The occurrence of root nodules inDatisca cannabina is reported. The nodules are typically of the Alnus type, forming dichotomously-branched coralloid clusters. The enlarged cortical cells contain vesicle clusters of the endophyte. The nodules reduced acetylene to ethylene at a rate of 5.5 moles ethylene per g fresh wt of nodules per hour.     

GuaB activity is required in Rhizobium tropici during the early stages of nodulation of determinate nodules but is dispensable for the Sinorhizobium meliloti-alfalfa symbiotic interaction

The guaB mutant strain Rhizobium tropici CIAT8999-10T is defective in symbiosis with common bean, forming nodules that lack rhizobial content. In order to investigate the timing of the guaB requirement during the nodule formation on the host common bean by the strain CIAT899-10.T, we constructed gene fusions in which the guaB gene is expressed under the control of the symbiotic promoters nodA, bacA, and nifH. Our data indicated that the guaB is required from the early stages of nodulation because full recovery of the wild-type phenotype was accomplished by the nodA-guaB fusion. In addition, we have constructed a guaB mutant derived from Sinorhizobium meliloti 1021, and shown that, unlike R. tropici, the guaB S. meliloti mutant is auxotrophic for guanine and induces wild-type nodules on alfalfa and Medicago truncatula. The guaB R. tropici mutant also is defective in its symbiosis with Macroptilium atropurpureum and Vigna unguiculata but normal with Leucaena leucocephala. These results show that the requirement of the rhizobial guaB for symbiosis is found to be associated with host plants that form determinate type of nodules.     

Glutathione produced by Rhizobium tropici is important to prevent early senescence in common bean nodules

In this paper, we examine the importance of glutathione in symbiosis, using a glutathione biosynthetic gshB mutant derived from Rhizobium tropici CIAT899, a common bean (Phaseolus vulgaris) endosymbiont. Plants infected with the mutant strain presented a delayed nodulation phenotype and a reduction in the dry weight of aerial part of plants, suggesting diminished nitrogen-fixation activity. In addition, bacterial gshB expression was assayed in wild-type infected nodules, during the different steps of nodulation, and found to increase in mature and early senescent nodules. Conspicuously, nodules induced by gshB mutant bacteria presented an early senescent pattern, which was associated with increased levels of superoxide accumulation. These results provide a direct evidence of the role of bacterial glutathione in protecting nodules from reactive oxygen species, which may determine nodule senescence.     

Studies of symbiotic plasmids in Rhizobium trifolii and fast-growing bacteria that nodulate soybeans

The Rhizobium trifolii symbiotic plasmid pRt5a was transferred to the fast-growing soybean strain USDA 194. Transconjugants carrying pRt5a were not able to nodulate clovers and one of the transconjugants had lost its smallest resident plasmid and did not fix nitrogen in soybean. Transconjugants of USDA 194 carrying pRt5a were able to transfer pRt5a back to a non-nodulating R. trifolii which inherited the symbiotic properties of the R. trifolii strain from which the plasmid was derived.     

Requirement of succinate dehydrogenase activity for symbiotic bacteroid differentiation of Rhizobium meliloti in alfalfa nodules.

Transmission electron microscopy was used to study the cellular morphologies of a wild-type Rhizobium meliloti strain (L5-30), a nitrogen fixation-ineffective (Fix-) succinate dehydrogenase mutant (Sdh-) strain, and a Fix+ Sdh+ revertant strain within alfalfa nodules and after free-living growth in a minimal medium containing 27 mM mannitol plus 20 mM succinate. The results showed a requirement of succinate dehydrogenase activity for symbiotic differentiation and maintenance of R. meliloti bacteroids within alfalfa nodules and for succinate-induced cellular pleomorphism in free-living cultures. Also, the Sdh- strain had a 3.5-fold lower rate of oxygen consumption in the defined medium than did the wild type.     

Evidence for genetic exchange and recombination of Rhizobium symbiotic plasmids in a soil population

A soil population of 16 Rhizobium leguminosarum bv. trifolii isolates was characterized by using three Sym (for symbiotic) plasmid-specific DNA hybridization probes: (i) an R. leguminosarum bv. trifolii-specific, repeated-sequence probe; (ii) a nifHDK gene probe, and (iii) a nod gene probe. A predominant Sym plasmid family was identified among the isolates. Three other unrelated Sym plasmid families were also identified. The isolates were also classified either by using a chromosomal DNA hybridization probe or by serological relatedness to 25 different R. leguminosarum bv. trifolii antisera. With either method, it was possible to group the 16 soil isolates into identical or related families. However, the correlation between the two techniques was not high. Irrespective of the means used to classify the bacterial host strain, it was possible to identify the same Sym plasmids in unrelated strains, as well as unrelated Sym plasmids in identical host strains. These data indicate that, within this soil population, there has been genetic exchange of Sym plasmids, and in one instance the hybridization pattern indicates that in vivo recombination of two different Sym plasmids may have occurred. Symbiotic effectiveness tests on red, strawberry, and subterranean clovers clearly differentiated the isolates. In general, the pattern of response was similar within groupings on the basis of Sym plasmid and chromosomal profiles but different between such groups.     

Negative effects of agrocin 84-encoding Agrobacterium plasmids on symbiotic properties of Rhizobium meliloti

Two plasmids, pAgK84::Tn5-Mob from Agrobacterium radiobacter carrying genes for the production of agrocin 84, and RP4-4 from E. coli were inserted either separately or together into a strain of Rhizobium meliloti. Each of these plasmid-containing R. meliloti transconjugants was less effective than the wild type strain in their ability to fix nitrogen in Medicago tornata. The pAgK84::Tn5-Mob-containing transconjugant was significantly less effective than that containing RP4-4. The transconjugant strains were inferior to the wild type strain in their ability to nodulate seedlings and to compete for nodulation.     

Functional relationships between plasmids and their significance for metabolism and symbiotic performance of Rhizobium leguminosarum bv. trifolii

Rhizobium leguminosarum bv. trifolii TA1 (RtTA1) is a soil bacterium establishing a highly specific symbiotic relationship with clover, which is based on the exchange of molecular signals between the host plant and the microsymbiont. The RtTA1 genome is large and multipartite, composed of a chromosome and four plasmids, which comprise approximately 65 % and 35 % of the total genome, respectively. Extrachromosomal replicons were previously shown to confer significant metabolic versatility to bacteria, which is important for their adaptation in the soil and nodulation competitiveness. To investigate the contribution of individual RtTA1 plasmids to the overall cell phenotype, metabolic properties and symbiotic performance, a transposon-based elimination strategy was employed. RtTA1 derivatives cured of pRleTA1b or pRleTA1d and deleted in pRleTA1a were obtained. In contrast to the in silico predictions of pRleTA1b and pRleTA1d, which were described as chromid-like replicons, both appeared to be completely curable. On the other hand, for pRleTA1a (symbiotic plasmid) and pRleTA1c, which were proposed to be unessential for RtTA1 viability, it was not possible to eliminate them at all (pRleTA1c) or entirely (pRleTA1a). Analyses of the phenotypic traits of the RtTA1 derivatives obtained revealed the functional significance of individual plasmids and their indispensability for growth, certain metabolic pathways, production of surface polysaccharides, autoaggregation, biofilm formation, motility and symbiotic performance. Moreover, the results allow us to suggest broad functional cooperation among the plasmids in shaping the phenotypic properties and symbiotic capabilities of rhizobia.     

Rhizobium phaseoli symbiotic mutants with transposon Tn5 insertions.

Rhizobium phaseoli CFN42 DNA was mutated by random insertion of Tn5 from suicide plasmid pJB4JI to obtain independently arising strains that were defective in symbiosis with Phaseolus vulgaris but grew normally outside the plant. When these mutants were incubated with the plant, one did not initiate visible nodule tissue (Nod-), seven led to slow nodule development (Ndv), and two led to superficially normal early nodule development but lacked symbiotic nitrogenase activity (Sna-). The Nod- mutant lacked the large transmissible indigenous plasmid pCFN42d that has homology to Klebsiella pneumoniae nitrogenase (nif) genes. The other mutants had normal plasmid content. In the two Sna- mutants and one Ndv mutant, Tn5 had inserted into plasmid pCFN42d outside the region of nif homology. The insertions of the other Ndv mutants were apparently in the chromosome. They were not in plasmids detected on agarose gels, and, in contrast to insertions on indigenous plasmids, they were transmitted in crosses to wild-type strain CFN42 at the same frequency as auxotrophic markers and with the same enhancement of transmission by conjugation plasmid R68.45. In these Ndv mutants the Tn5 insertions were the same as or very closely linked to mutations causing the Ndv phenotype. However, in two mutants with Tn5 insertions on plasmid pCFN42d, an additional mutation on the same plasmid, rather than Tn5, was responsible for the Sna- or Ndv phenotype. When plasmid pJB4JI was transferred to two other R. phaseoli strains, analysis of symbiotic mutants was complicated by Tn5-containing deleted forms of pJB4JI that were stably maintained.     

Nitrogenase and antioxidant enzyme activities in Phaseolus vulgaris nodules formed by Rhizobium tropici isogenic strains with varying tolerance to salt stress

Common bean plants inoculated with salt-tolerant Rhizobium tropici wild-type strain CIAT899 formed a more active symbiosis than did its decreased salt-tolerance (DST) mutant derivatives (HB8, HB10, HB12 and HB13). The mutants formed partially effective (HB10, HB12) or almost ineffective (HB8, HB13) nodules (Fix(d)) under non-saline conditions. The DST mutant formed nodules that accumulated more proline than did the wild-type nodules, while soluble sugars were accumulated mainly in ineffective nodules. Under salt stress, plant growth, nitrogen fixation, and the activities of the antioxidant defense enzymes of nodules were affected in all symbioses tested. Overall, mutant nodules showed lower antioxidant enzyme activities than wild-type nodules. Levels of nodule catalase appeared to correlate with symbiotic nitrogen-fixing efficiency. Superoxide dismutase and dehydroascorbate reductase seem to function in the molecular mechanisms underlying the tolerance of nodules to salinity.