Genetic and functional analysis of the nodulation region of the Rhizobium leguminosarum Sym plasmid pRL1JI

Thirty Tn5- or Tn1831-induced nodulation (nod) mutants of Rhizobium leguminosarum were examined for their genetic and symbiotic properties. Thirteen mutants contained a deletion in Sym plasmid pRL1JI. These deletions cover the whole nod region and are 50 kb in size. All remaining seventeen mutations are located in a 6.6 kb EcoRI nod fragment of the Sym plasmid. Mutations in a 3.5 kb part on the right hand side of this 6.6 kb fragment completely prevent nodulation on Vicia sativa. All mutants in this 3.5 kb area are unable to induce marked root hair curling and thick and short roots.Mutations in a 1.5 kb area on the left hand side of the 6.6 kb nod fragment generate other symbiotic defects in that nodules are only rarely formed and only so after a delay of several days. Moreover, infection thread formation is delayed and root hair curling is more excessive than that caused by the parental strain. Their ability to induce thick and short roots is unaltered.Mutations in this 1.5 kb region are not complemented by pRmSL26, which carries nod genes of R. meliloti, whereas mutations in the 3.5 kb region are all complemented by pRmSL26.Abbreviations Rps repression of production of small bacteriocin - Mep medium bacteriocin production - Nod nodulation - Fix fixation - Tsr thick and short roots - Flac root hair curling - Hsp host specificity - Flad root hair deformation - Tc tetracycline - Km kanamycin - Cm chloramphenicol - Sp spectinomycin - Sm streptomycin - R resistant     

Analysis of R-primes demonstrates that genes for broad host range nodulation of Rhizobium strain NGR234 are dispersed on the Sym plasmid

Summary R-prime plasmids carrying regions of the symbiotic (Sym) plasmid of the broad host range Rhizobium strain NGR234 were isolated in intergeneric matings with Escherichia coli K12. Three R-primes carrying approximately 180 kb (pMN23), 220 kb (pMN31) and 330 kb (pMN49) of Sym DNA were characterized in more detail. Restriction enzyme analysis and hybridization studies showed that these R-primes carried large overlapping regions of the Sym plasmid, and had the symbiotic genes (two copies of nifH, D and K; nodA, B, C and D; region II; host specific nodulation (hsn) genes) located over half of the 470 kb Sym plasmid. Only the largest of these R-primes (pMN49) contained the complete nodulation host range of the original parent strain NGR234. This broad host range was shown to be present on plasmid pMN49 by being expressed in Agrobacterium tumefaciens strain A136. Furthermore the R-prime plasmids were shown to contain different regions of distinctive host specific nodulation (hsn) for tropical legume infection and for the nodulation of the non-legume Parasponia. Nodulation of soybeans, however, required an additional region that was not essential for the nodulation of other tropical legumes. Strain NGR234 was also found to nodulate the stem and roots of the tropical legume Sesbania rostrata at a very low efficiency. However, the R-prime mini Sym plasmid constructions enabled a greater efficiency of nodulation of Sesbania rostrata to occur.     

Identification of host range determinants in the Rhizobium species MPIK3030

Summary R. meliloti primarily nodulates Medicago sativa but cannot nodulate Macroptilium atropurpureum. By introducing an 11.4 kb region into R. meliloti from the Symplasmid of Rhizobium strain MPIK3030, the host range of the R. meliloti transconjugants were shown to be extended to M. atropurpureum, one of the hosts of MPIK3030 but not normally nodulated by R. meliloti. The region responsible for host range extension was isolated by mass conjugating a clone bank from MPIK3030 into the R. meliloti wild type, and subsequent screening for nodulation on M. atropurpureum. Using deleted derivatives of a plasmid reisolated from endosymbiotic bacteria, the host range region was further narrowed down to three EcoRI fragments. Tn5 mutagenesis allowed the isolation of three discrete regions on an 11.4 kb section, which are involved in the extension of host range to M. atropurpureum. Finally, complementation experiments performed with R. meliloti common nod and hsn mutants indicated that none of the genes involved in the early steps of nodulation, including host-range functions, can be complemented by genes carried on the 11.4 kb fragment derived from MPIK3030.     

Host-specific nodulation is encoded on a 14kb DNA fragment in Rhizobium trifolii

Summary The Rhizobium trifolii genes necessary for nodule induction and development have been isolated on a 14.0kb fragment of symbiotic (Sym) plasmid DNA. When cloned into a broad-host-range plasmid vector, these sequences confer a clover nodulation phenotype on a derivative of R. trifolii which has been cured of its endogenous Sym plasmid. Furthermore, these sequences encode both host specificity and nodulation functions since they confer the ability to recognize and nodulate clover plants on Agrobacterium and a fast-growing cowpea Rhizobium. This indicates that the bacterial genes essential for the initial, highly-specific interaction with plants are closely linked.     

Cloned nodulation genes of Rhizobium leguminosarum determine host-range specificity

Summary Three nodulation-deficient (nod) mutants of Rhizobium leguminosarum were isolated following insertion of the transposon Tn5 into pRL1JI, the R. leguminosarum plasmid known to carry the nodulation genes. DNA adjacent to the nod: Tn5 alleles was subcloned and used to probe a cosmid clone bank containing DNA from a Rhizobium strain carrying pRL1JI. Two cosmid clones which showed homology with the probe contained about 10 kb of DNA in common. The R. leguminosarum host-range determinants were found to be present within this 10 kb common region since either of the cosmid clones could enable a cured R. phaseoli strain to nodulate peas instead of Phaseolus beans, its normal host. Electron microscopy of nodules induced by Rhizobium strains cured of their normal symbiotic plasmid but containing either of the two cosmid clones showed bacteroid-forms surrounded by a peri-bacteroid membrane, indicating that normal infection had occurred. Thus it is clear that this 10 kb region of nodDNA carries the genes that determine host range and that relatively few bacterial genes may be involved in nodule and bacteroid development.     

Plasmid-mediated control of nodulation in Rhizobium trifolii

The nodulation ability was effectively eliminated from different Rhizobium trifolii strains incubated at elevated temperature (urkowski and Lorkiewicz, 1978). Non-nodulating (Nod^-) mutants were stable and no reversion of Nod^- to Nod⁺ phenotype was observed. Strains R. trifolii 24 and T12 which showed a high percentage of elimination of nodulation ability were examined in detail. Two plasmids were detected in strain 24 using neutral and alkaline sucrose gradient centrifugation of plasmid preparations. Molecular weights of the plasmids pWZ1 and pWZ2 were 460 Mdal and 190 Mdal, respectively. Rhizobium lysates labeled with ³H-thymidine and ultracentrifuged in caesium chloride — ethidium bromide gradients demonstrated a 40% reduction of the plasmid DNA content in R. trifolii 24 Nod^- mutants in comparison with the nodulating wild type strain 24. It was found further that non-nodulation of mutants 24 Nod^- was due to the absence of plasmid pWZ2. Sucrose gradient data also demonstrated that strain T12 contained two plasmids with molecular weights corresponding to those of pWZ1 and pWZ2, respectively. In Nod^- mutant clones derived from strain T12, pWZ2 plasmid was missing.Non Standard Abbreviations CCC covalently closed circular - OC open cirucular - Sarkosyl sodium N-lauroylsarcosinate     

Nodulation of specific legumes is controlled by several distinct loci in Rhizobium trifolii

Summary Three distinct loci (designated regions III, IV and V) were identified in the 14 kb Nod region of Rhizobium trifolii strain ANU843 and were found to determine the host range characteristics of this strain. Deletion of region III or region V only from the 14 kb Nod region affected clover nodulation capacity. The introduction to R. Leguminosarum of DNA fragments on multicopy vectors carrying regions III, IV and V (but not smaller fragments) extended the host range of R. leguminosarum so that infection threads and nodules occurred on white clover plants. The same DNA fragments were introduced to the Sym plasmid-cured strain (ANU845) carrying the R. meliloti recombinant nodulation plasmid pRmSL26. Plasmid pRmSL26 alone does not confer root hair curling or nodulation on clover plants. However, the introduction to ANU845 (pRmSL26) of a 1.4 kb fragment carrying R. trifolii region IV only, resulted in the phenotypic activation of marked root hair curling ability to this strain on clovers but no infection events or nodules resulted. Only the transfer of regions III, IV and V to strain ANU845 (pRmSL26) conferred normal nodulation and host range ability of the original wild type R. trifolii strain. These results indicate that the host range genes determine the outcome of early plant-bacterial interactions primarily at the stage of root hair curling and infection.     

In vitro andin vivo nitrogenase activity ofRhizobium mutants and their symbiotic effectivity

A slow growing nitrogen-fixing strain ofVigna radiata var.aureus (mung bean)Rhizobium which expressed nitrogenase activity in a synthetic medium was isolated from its native population. Mutants with decreased and increased nitrogenase activity were derived from this strain by treatment with acridine orange and ethidium bromide. These mutants were tested for symbiotic effectivity invivo. The effectivity of mutants with decreased nitrogenase activity in the culture medium was lower than the parent strain; however, the effectivity of mutants with higher nitrogenase activity did not increase above that of the parent. This suggests that the plant is perhaps a limiting factor in the full expression of rhizobial nitrogenase in the nodules.     

Identification and cloning of nodulation genes from the stem-nodulating bacterium ORS571

Summary After random Tn5 mutagenesis of the stem-nodulating Sesbania rostrata symbiont strain ORS571, Nif^-, Fix^- and Nod^- mutants were isolated. The Nif^- mutants had lost both free-living and symbiotic N₂ fixation capacity. The Fix^- mutants normally fixed N₂ in the free-living state but induced ineffective nodules on S. rostrata. They were defective in functions exclusively required for symbiotic N₂ fixation. A further analysis of the Nod^- mutants allowed the identification of two nod loci. A Tn5 insertion in nod locus 1 completely abolished both root and stem nodulation capacity. Root hair curling, which is an initial event in S. rostrata root nodulation, was no longer observed. A 400 bp region showing weak homology to the nodC gene of Rhizobium meliloti was located 1.5 kb away from this nod Tn5 insertion. A Tn5 insertion in nod locus 2 caused the loss of stem and root nodulation capacity but root hair curling still occurred. The physical maps of a 20.5 kb DNA region of nod locus 1 and of a 40 kb DNA region of nod locus 2 showed no overlaps. The two nod loci are not closely linked to nif locus 1, containing the structural genes for the nitrogenase complex (Elmerich et al. 1982).     

A kinetic study on the plasmid stability of three <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strains

Absract The plasmid stability of three wild type Lactococcus lactis strains and their mutants was investigated at different incubation time and temperatures in two different media [M17 broth and reconstituted skim milk (RSM)]. The results showed that both incubation times and temperature are effective on plasmid loss. The plasmid profiles of wild type strains exhibited 8 to 9 distinct plasmid species with molecular weights from 2.1 to 24.0 kb. Lactose fermentation ability was found to be encoded by 22.2 (strain U70), 23.6 (strain U29) and 24.0 (strain U52) kb plasmids in the wild type strains, respectively. The stabilities of the plasmids were explained by applying a second-order polynomial modeling system. Reasonable fittings were obtained for the model and the adjusted regression coefficients (R ² _adj) were between 0.76 and 0.99 for the overall data. Overall, it was found that incubation time had the most profound effect on plasmid stability, with plasmid loss occurring after 72 h, while temperatures in the range of 15–40°C also induced plasmid instability.     

Characterization of the ntrBC genes of Azospirillam brasilense Sp7: Their involvement in the regulation of nitrogenase synthesis and activity

A 7.1 kb EcoRI fragment from Azospirillum brasilense, that hybridized with a probe carrying the ntrBC genes from Bradyrhizobium japonicum, was cloned. The nucleotide sequence of a 3.8 kb subfragment was established. This led to the identification of two open reading frames, encoding polypeptides of 401 and 481 amino acids, that were similar to NtrB and NtrC, respectively. A broad host range plasmid containing the putative Azospirillum ntrC gene was shown to restore nitrogen fixation under free-living conditions to a ntrC-Tn5 mutant of Azorhizobium caulinodans. Several Tn5 insertion mutants were isolated in the ntrBC coding region in A. brasilense. These mutants were prototrophic and Nif⁺. However, their nitrogenase activity was slightly lower than in the wild type and they were unable to grow on nitrate as sole nitrogen source. Under microaerobiosis and in the absence of ammonia, a nifA-lacZ fusion was expressed in the mutants at about 60% of the level in the wild type. In the presence of ammonia, the fusion was similarly expressed (60% of the maximum) both in the wild type and mutants. Addition of ammonia to a nitrogen-fixing culture of ntrBC mutants did not abolish nitrogenase activity, in contrast with the wild type. It thus appears that in Azospirillum the ntrBC genes are not essential for nitrogen fixation, although NtrC controls nifA expression to some extent. They are, however, required for the switch-off of nitrogenase activity.     

Symbiotic effectiveness of Hup+ Rhizobium,VAM fungi and phosphorus levels in relation to nitrogen fixation and plant growth ofCajanus cajan

Effect of hydrogen uptake positive (Hup⁺) strain ofRhizobium sp. (pigeon pea) and VAM fungus (Glomus fasciculatum) was studied on the symbiotic parameters of pigeon pea (Cajanus cajan) cv. AL-15 at various levels of phosphorus. The Hup⁺ Rhizobium strain showed more nodulation, plant biomass and plant nitrogen content than its Hup⁻ counterpart. VAM infection in pigeon pea roots helped in translocating phosphorus from the soil and improved nitrogen fixation. Similarly, addition of phosphorus was found to play a positive role in enhancing all these parameters. Dual inoculation of Hup⁺ Rhizobium strain and VAM significantly increased nodulation, nitrogenase activity, plant nitrogen and phosphorus content and plant biomass compared to single inoculation of either organism and dual inoculation with Hup⁻ and VAM fungus.     

Conserved plasmid/chromosome sequences in fast- and slow-growing rhizobia that nodulate the same plant

Apart from the ability to nodulate legumes, fast-and slow-growing rhizobia have few bacteriological traits in common. Given that there is only one pathway to nodulation, DNA sequences conserved in fast- and slow-growing organisms that nodulate the same host should be strongly enriched in infectivity genes. We tested this hypothesis with seven fast-growing and five slow-growing strains that produced responses varying from fully effective nodulation through various ineffective associations to non-nodulation on four different hosts (Lotus pedunculatus, Lupinus nanus, Macroptilium atropurpureum, and Vigna unguiculata). When restriction enzyme digested total DNA from 10 of the strains was separately hybridized with nick-translated plasmid DNA isolated from 4 fast-growing strains, variable but significant homologies were found with all 10 strains. Part of this homology was shown to be associated with the nifKDH genes for nitrogenase and part with putative nodulation genes carried on pC2, a cosmid clone containing a 37 kbp region of the large sym plasmid present in the fast-growing broad-host range Rhizobium sp. strain NGR234. Analysis of the extent of homology between the plasmids of 3 fastgrowing strains (NGR234, TAL 996 and UMKL 19) able to effectively nodulate Vigna unguiculata showed them to have homologous DNA fragments totalling 47 kbp. This core homology represents less than 12% of the total coding capacity of the sym plasmid present in each of these strains.Abbreviations Sym symbiotic sequences/plasmids - nod genes required for nodulation - nod putative nod genes - nif genes required for the synthesis of the enzyme nitrogenase     

A molecular linkage map of nitrogenase and nodulation genes in Rhizobium trifolii

Summary A molecular map was constructed linking the nitrogenase structural genes (nif) and nodulation genes (nod) in the white clover symbiont, Rhizobium trifolii. In R. trifolii strain ANU843 these two genetic regions are located some 16 kilobases (kb) apart on the 180 kb symbiotic (Sym) plasmid. The molecular linkage of nod and nif genetic regions was established by hybridization analysis using recombinant plasmids containing overlapping cloned sequences. Nodulation genes were located by means of a Tn5-induced nodulation-defective mutant that failed to induce clover root hair curling (Hac^- phenotype). A cloned wild-type DNA fragment was shown to phenotypically correct the Hac^- mutation by complementation. The nifHDK genes were cloned by positive hybridization to another R. trifolii nif-specific probe. Location of the nif genes relative to the nod genes was established by analysis of a Sym plasmid deletion derivative.     

Rhizobium nodulation genes involved in root hair curling (Hac) are functionally conserved

Summary Five specific transposon-induced nodulation defective (Nod⁻) mutants from different fast-growing species ofRhizobium were used as the recipients for the transfer of each of several endogenous Sym(biosis) plasmids or for recombinant plasmids that encode early nodulation and host-specificity functions. The Nod⁻ mutants were derived fromR. trifolii, R. meliloti and from a broad-host-rangeRhizobium strain which is able to nodulate both cowpea (tropical) legumes and the non-legumeParasponia. These mutants had several common features (a), they were Nod⁻ on all their known plant hosts, (b), they could not induce root hair curling (Hac⁻) and (c), the mutations were all located on the endogenous Sym-plasmid of the respective strain. Transfer to these mutants of Sym plasmids (or recombinant plasmids) encoding heterologous information for clover nodulation (pBR1AN, pRt032, pRt038), for pea nodulation (pJB5JI, pRL1JI::Tn1831), for lucerne nodulation (pRmSL26), or for the nodulation of both tropical legumes and non-legumes (pNM4AN), was able to restore root hair curling capacity and in most cases, nodulation capacity of the original plant host(s). This demonstrated a functional conservation of at least some genes involved in root hair curling. Positive hybridization between Nod DNA sequences fromR. trifolii and from a broad-host-rangeRhizobium strain (ANU240) was obtained to other fast-growingRhizobium strains. These results indicate that at least some of the early nodulation functions are common in a broad spectrum ofRhizobium strains.     

Cloning and mutagenesis of nodulation genes from Rhizobium leguminosarum TOM, a strain with extended host range

Summary Some primitive pea lines, e.g. cultivar Afghanistan, are resistant to nodulation by most strains of Rhizobium leguminosarum. However the Turkish strain TOM can nodulate cv. Afghanistan in addition to commercial pea varieties, and this extended host range is a property of its symbiotic plasmid, pRL5JI. A gene bank was constructed using DNA from a strain of R. leguminosarum containing pRL5JI. Following transfer to a strain of R. leguminosarum that had been cured of its symbiotic plasmid, two derivatives were isolated that contained cloned nodulation determinants, and were able to nodulate both cv. Afghanistan and a commercial pea variety. In addition, these clones conferred the ability to nodulate peas to a strain of R. phaseoli that had been previously cured of its symbiotic plasmid. One of these clones was subjected to mutagenesis with transposon Tn5, and 11 mutants were identified that were affected in nodulation ability. The sites of Tn5 insertions were mapped using restriction endonucleases and all were found to be within a region of 5 kb. The mutants fell into three classes on the basis of their map positions and their phenotypes on the two different pea lines tested. One class of mutants was affected in gene functions that were common to the nodulation of both pea hosts; a second class was impaired specifically in the nodulation of the commercial pea variety; a third class of mutant failed to confer on a normal strain of R. leguminosarum the supplementary ability to nodulate cv. Afghanistan.     

Long term field study shows increased biomass production in tree legumes inoculated with Rhizobium

Nodulation potential, nitrogen fixation efficiency (nitrogenase activity) and biomass yield response of Leucaena leucocephala and Acacia nilotica to inoculation with 6 selected fast growing Rhizobium strains was explored in long-term (5 year) field trials. All the strains formed nodules and fixed nitrogen in L. leucocephala and A. nilotica. Seasonal effects on nitrogenase activity was observed and in winter (ambient temperature about 20 °C), nitrogenase activity could not be detected. However, with the onset of spring and a rise in temperature, fresh nodulation (renodulation) by all the inoculant rhizobial strains was observed in both the tree legumes. In L. leucocephala, maximum renodulation was exhibited by strain A1 while in A. nilotica, strain AB3 formed the maximum renodulation 24 months after transplantation. Dry matter yield of all the inoculated plants demonstrated a significant increase over that of the uninoculated plants at the end of five years after transplanting. In L. leucocephala, strain NGR8 gave the maximum response (45% more dry matter yield) in dry matter production while in A. nilotica, strain USDA 3325 showed a 25% increase in total dry matter yield five years after transplantation.     

Plant growth-promoting rhizobacteria for improving nodulation and nitrogen fixation in the common bean (Phaseolus vulgaris L.)

A greenhouse experiment was performed to evaluate the effects of plant growth-promoting rhizobacteria (PGPR) on nodulation, biological nitrogen fixation (BNF) and growth of the common bean (Phaseolus vulgaris L. cv. Tenderlake). Single and dual inoculation treatments of bean with Rhizobium and/or PGPR were administered to detect possible changes in the levels of and interactions between the phytohormones IAA and cytokinin. Bean plants cv. Tenderlake were grown in pots containing Fluvic Neosol eutrophic (pH 6.5). Fourteen kilogram aliquots of soil contained in 15-l pots were autoclaved. Bean seeds were surface sterilized and inoculated with Rhizobium tropici (CIAT 899-standard strain) alone and in combination with one of the PGPR strains: Bacillus endophyticus (DSM 13796), B. pumilus (DSM 27), B. subtilis (DSM 704), Paenibacillus lautus (DSM 13411), P. macerans (DSM 24), P. polymyxa (DSM 36), P. polymyxa (Loutit L.) or Bacillus sp.(65E180). The experimental design was randomized block design with three replications. Beans co-inoculated with Rhizobium tropici (CIAT899) and Paenibacillus polymyxa (DSM 36) had higher leghemoglobin concentrations, nitrogenase activity and N₂ fixation efficiency and thereby formed associations of greater symbiotic efficiency. Inoculation with Rhizobium and P. polymyxa strain Loutit (L) stimulated nodulation as well as nitrogen fixation. PGPR also stimulated specific-nodulation (number of nodules per gram of root dry weight) increases that translated into higher levels of accumulated nitrogen. The activities of phytohormones depended on their content and interactions with Rhizobium tropici and Paenibacillus and/or Bacillus (PGPR) strains which affect the cytokinin in content in the common bean.     

Azide-resistant mutants ofAzorhizobium caulinodans with enhanced symbiotic effectiveness

Azide-resistant mutants ofAzorhizobium caulinodans strains Sb3, S78, SrR13 and SrS8 were isolated and screened for nitrate reductase activity. Selected nitrate reductase negative mutants were inoculated onSesbania bispinosa andS. rostrata under sterile conditions in chillum jars to study their symbiotic behavior. Azide-resistant mutants exhibited either similar or higher symbiotic effectiveness than the parent strain after 30 d of plant growth. Nodule mass, nitrogenase activity and uptake hydrogenase activity of the mutants varied depending on the host as well as on the plant growth stage. In comparison to wild-type parent strains, four azide-resistant mutants, Sb3Az18, S78Az21, SrR13Az17 and SrS8Az6 showed significant increase in nodulation and nitrogen fixation as well as shoot dry mass of the inoculated plants.     

Strain selection for pigeon pea Rhizobium under greenhouse conditions

Pigeon pea obtains N for growth by N₂ fixation although yield generally is not improved by either the inoculation of Rhizobium or by the application of N fertilizer in Puerto Rico. Sixteen strains ofRhizobium spp., different in geographical origin, were tested for N-fixing effectiveness, determined from comparisons with uninoculated controls, N controls and the standard strain 176A22. Inoculated treatments showed significant differences in nodulation, plant dry weight and %N. Several strain x plant combinations had higher N content than the N treatment, reflecting the ubiquity of effective strains and the possible lack of response of pigeon pea to inoculation or N fertilization. Strains superior in N₂ fixation were selected for testing for symbiotic effectiveness under field conditions.