Fredii Strain Research Articles

Elaboration of flavonoid-induced proteins by the nitrogen-fixing soybean symbiont Rhizobium fredii is regulated by both nodD1 and nodD2, and is dependent on the cultivar-specificity locus, nolXWBTUV

Genistein and other flavonoids from host legumes are known to stimulate cells of the nitrogen-fixing soybean symbiont Rhizobium fredii to synthesize Nod factors, which function as signals during nodule initiation. Flavonoids also trigger R. fredii to secrete a set of signal-responsive (SR) proteins into the environment. By insertion mutagenesis, we showed that secretion of SR proteins by this organism has an absolute dependence on the regulatory gene nodD1. We isolated and sequenced nodD1 and nodD2 of R. fredii USDA257 and constructed strains containing additional, plasmid-borne copies of these genes. Extra copies of nodD1 had no effect on secretion of SR proteins, but extra copies of nodD2 rendered the process constitutive. Extracts from seeds of the soybean cultivars McCall and Peking can substitute for purified flavonoids as inducers of SR proteins. The nolXWBTUV locus is known to control cultivar-specific nodulation of McCall soybean in a negative, flavonoid-dependent manner. Inactivation of any of these genes prevented SR proteins from accumulating in culture fluids. Protein secretion in response to host signals was a characteristic of nine out of ten R. fredii strains tested. Immunological probes failed to detect SR3 or SR5 in mature soybean or cowpea nodules. Although the functions of these proteins remain unknown, their potential role in symbiosis is strengthened by the discovery that their accumulation depends on nodD1, nodD2 and nolXWBTUV.

Evaluation of the Symbiotic Properties of Rhizobium fredii in European Soils

Summary The symbiotic properties of different Rhizobium fredii strains were evaluated in a controlled environment growth chamber, greenhouse, and field conditions. R. fredii strains HH 102-2 and HH 103 showed to be as good soybean inoculants as Bradyrhizobium japonicum USDA 110 in three years (1987, 1988 and 1992) of field experiments in Spanish soils. Thus, it is concluded that these two R. fredii strains are valid soybean inoculants in Spanish soils which, as all European soils, are devoid of indigenous B. japonicum and R. fredii strains. Competition experiments in field conditions (1987) showed that R. fredii strains HH102-2 and HH 103-2 were more competitive than B. japonicum USDA 110-1 to nodulate soybean cv. Williams. Results of competition studies in greenhouse conditions using acid (pH 4.9), neutral (pH 6.6) and alkaline (pH 8.1) soils showed that in the acid soil B. japonicum strains USDA 110 and 3-15-b3 totally outcompeted R. fredii strain HH 103 to nodulate on cv. Williams. Conversely, in the alkaline soil the fast-growing strain occupied more than 80% of the nodules. These results indicate that the soil pH could be an important factor in determining the competitive success of R. fredii and B. japonicum for nodulating soybeans.

Host range, RFLP, and antigenic relationships betweenRhizobium fredii strains andRhizobium sp. NGR234

Rhizobium fredii is a nitrogen-fixing symbiont from China that combines broad host range for nodulation of legume species with cultivar specificity for nodulation of soybean. We have compared 10 R. fredii strains with Rhizobium sp. NGR234, a well known broad host range strain from Papua New Guinea. NGR234 nodulated 16 of 18 tested lugume species, and nodules on 14 of the 16 fixed nitrogen. The R. fredii strains were not distinguishable from one another. They nodulated 13 of the legumes, and in only nine cases were nodules effective. All legumes nodulated by R. fredii were included within the host range of NGR234. Restriction fragment length polymorphisms (RFLPs) were detected with four DNA hybridization probes: the regulatory and common nod genes, nodDABC; the soybean cultivar specificity gene, nolC; the nitrogenase structural genes, nifKDH; and RFRS1, a repetitive sequence from R. fredii USDA257. A fifth locus, corresponding to a second set of soybean cultivar specificity genes, nolBTUVWX, was monomorphic. Using antisera against whole cells of three R. fredii strains and NGR234, we separated the 11 strains into four serogroups. The anti-NGR234 sera reacted with a single R. fredii strain, USDA191. Only one serogroup, which included USDA192, USDA201, USDA217, and USDA257, lacked cross reactivity with any of the others. Although genetic and phenotypic differences among R. fredii strains were as great as those between NGR234 and R. fredii, our results confirm that NGR234 has a distinctly wider host range than R. fredii.

Genetic allelism and linkage tests of a soybean gene, Rfg1, controlling nodulation with Rhizobium fredii strain USDA 205

A soybean gene, Rfg1, controlling nodulation with strain USDA 205, the type strain for the fast-growing species Rhizobium fredii, was tested for allelism with the Rj4 gene. The Rj4 gene conditions ineffective nodulation primarily with certain strains of the slow-growing soybean microsymbiont, Bradyrhizobium elkanii. The F2 seeds of the cross of the cultivars Peking, carrying the alleles rfg1, Rj4, i (controlling inhibition of seed coat color) and W1 (controlling flower color), and Kent, carrying the alleles Rfg1, rj4, i-i and w1, were evaluated for nodulation response with strain USDA 205 by planting surface disinfested seeds in sterilized vermiculite in growth trays and inoculating with a stationary phase broth culture of strain USDA 205 at planting. Plants were classified for nodulation response visually after four weeks growth and transplanted to the field for F3 seed production. Flower color, purple (W1) vs white (w1), was determined in the field. The allele present at the i locus was determined by classification of F3 seed coat color. The F3 seeds were planted in growth trays and inoculated with strain USDA 61 of Bradyrhizobium elkanii to determine the genotype for the Rj4 locus. The Rfg1 and Rj4 genes were determined to be located at separate loci. Chi-square analysis for linkage indicated that Rfg1 segregated independently of the Rj4, I and W1 loci.

Molecular cloning and characterization of a sym plasmid locus that regulates cultivar‐specific nodulation of soybean by Rhizobium fredii USDA257

Rhizobium fredii strain USDA257 produces nitrogen-fixing nodules on primitive soybean cultivars such as Peking but fails to nodulate agronomically improved cultivars such as McCall. Transposon-mutant 257DH4 has two new phenotypes: it nodulates McCall, and its ability to do so is sensitive to the presence of parental strain USDA257, i.e. it is subject to competitive nodulation blocking. We have isolated a cosmid containing DNA that corresponds to the site of transposon insertion in 257DH4 and have localized Tn5 on an 8.0 kb EcoRI fragment. The 5596 bp DNA sequence that surrounds the insertion site contains seven open reading frames. Five of these, designated nolBTU, ORF4, and nolV, are closely spaced and of the same polarity. nolW and nolX are of the opposite polarity. The initiation codon for nolW lies 155 bp upstream from that of nolB, and its is separated from nolX by 281 bp. The predicted NolT and NolW proteins have putative membrane-spanning regions. The N-terminus of the hypothetical NolW protein also has limited homology to NodH of Rhizobium meliloti, but none of the deduced protein sequences has significant homology to known nodulation gene products. Site-directed mutagenesis with mudII1734 confirms that inactivation of nolB, nolT, nolU, nolV, nolW, or nolX extends host range for nodulation to McCall soybean. This phenotype could not be genetically dissected from sensitivity to competitive nodulation blocking. Expression of nolBTU and nolX is induced as much as 30-fold by flavonoid signal molecules, even though these genes lack nod-box promoters. Histochemical staining of McCall roots inoculated with nolB-, nolU-, or nolX-lacZ fusions verifies that these genes are expressed continuously from preinfection to the stage of the functional nodule. Although a nolU-ORF4-nolV clone hybridizes to a single 8.0 kb EcoRI fragment from 10 strains of R. fredii and broad-host-range Rhizobium sp. NGR234, hybridizing sequences are not detectable in other rhizobia.

Broad host‐range effective mutants of Rhizobium fredii strains

Rhizobium fredii strains USDA 192 and USDA 205 form nitrogen‐fixing nodules on Asiatic and different unbred soybean cultivars but are ineffective on American‐adapted soybeans. Further to our report on the isolation of spontaneous mutant derivatives of Rh. fredii strains USDA 192 and USDA 205 that were able to form nitrogen‐fixing nodules on the American soybean cultivars Wells and Fred, we show that the symbiotic properties of these mutants are very different from those of their parental strains. All the mutants effectively nodulated other American soybean cultivars different from that (Wells or Fred) used to select the mutants and they had also acquired the capacity to form nitrogen‐fixing nodules on Cajanus cajan and Macroptilium atropurpurcum. These results demonstrated that although Rh. fredii strains USDA 192 and USDA 205 are either completely ineffective or poorly effective on American soybean cultivars, C. cajan and M. atropurpureum, they have the inherent capacity to establish effective nitrogen‐fixing symbioses with these legumes. Transfer of the symbiotic plasmid of strain AB‐7 (a broad host‐range mutant derivative of USDA 192) to Rh. fredii strain SVQ‐127 (USDA192‐background) produced transconjugants that formed nitrogen‐fixing nodules on the American soybean cultivar Williams and M. atropurpureum. These results indicate that the mutation that produces the broad host‐range phenotype of mutant strain AB‐7 is located in its symbiotic plasmid. Rhizobium fredii strain USDA 192 was more competitive than its derivative AB‐7 to nodulate on the unbred soybean cultivar Malayan. Moreover, the presence of USDA 192 in the inocula severely inhibited nodulation on soybean cultivar Williams by the effective derivative AB‐7. Strain SVQ‐127 was able to nodulate effectively on the Asiatic soybean cultivar Peking although its symbiotic plasmid was no longer visible by agarose gel electrophoresis. DNA‐DNA hybridization experiments showed that the symbiotic plasmid, or part of it, was still present in the bacterial genome.

Performance of a Superior Bradyrhizobium japonicum and a Selected Sinorhizobium fredii Strain with Soybean Cultivars

AbstractStrain 532C of Bradyrhizobium japonicum has outperformed other soybean [Glycine max (L.) Merr.] rhizobial strains in Ontario trials conducted with cv. Evans. Strain HH303 of Sinorhizobium fredii has proven effective with some Ontario cultivars. The objective of this research was to compare the performance of 532C with that of USDA 110, CB 1809, a three‐strain mixture, and HH303, by 16 commonly‐grown, early‐maturing soybean cultivars. Trials were conducted from 1988 to 1990 (only five cultivars were used in 1990) on soils that had low available N and had not grown soybeans previously. There were significant (P < 0.01) strain‐by‐genotype interactions for yield in all 3 yr and for seed protein content in 1988 and 1989, but interactions were small compared to main effects. Averaged over cultivars, strain 532C caused 11 to 17% higher seed yield than USDA 110 or CB 1809 in all 3 yr. Strain 532C appeared effective with all 16 cultivars tested. Strain 532C, used alone, caused higher average yields than the three strain mixture, which included 532C, only in 1988. The S. fredii strain HH303 caused consistently lower average yields than 532C, USDA 110, and the three‐strain mixture, but average yields with HH303 and CB 1809 were not different in 1988 and 1989. Cultivars with cv. Evans in their pedigree yielded well with HH303. There were strong positive correlations (r = 0.75−0.85) between seed yield and seed protein contents. Strain 532C supported higher seed yields than the three‐strain mixture at equivalent seed protein contents. Strain 532C was an effective N2 fixer with all cultivars tested and is now used in all soybean inoculants sold in Ontario.

A nolC-lacZ gene fusion in Rhizobium fredii facilitates direct assessment of competition for nodulation of soybean

A constitutively expressed chromosomal gene, nolC, regulates the ability of Rhizobium fredii strain USDA257 to nodulate certain agronomically improved soybean cultivars. Other advanced cultivars, as well as unimproved varieties such as 'Peking', form nitrogen-fixing nodules with parental strain USDA257 and with nolC mutants. We have used 257DH512, a derivative of USDA257 that contains a nolC-lacZ fusion, to directly measure strain competition for nodule occupancy. Seedlings of the soybean cultivar 'Peking' were inoculated with mixtures containing various ratios of 257DH512 and R. fredii USDA208; nodules were harvested 14 days later and stained histochemically with X-gal, a chromogenic substrate for the β-galactosidase product of lacZ. We were able to stain and evaluate batches of nearly 500 nodules in just 4 h, without the use of expensive equipment. Under the conditions of these experiments, USDA208 was considerably more competitive than 257DH512, even when it had been present in the inoculum at a numerical disadvantage. We suggest that gene fusions can be used to greatly simplify the assessment of nodulation competitiveness in rhizobia. Key words: competition, nolC-lacZ fusion, soybean – Rhizobium fredii, nodule occupancy.

Tn [formula omitted] mutagenesis of chinese Rhizobium fredii for siderophore overproduction

In the midwestem United States, members of USDA Bradyrhizobium japonicum serogroup 135 dominate in alkaline soils for nodulation of soybean [ Glycine max (L.) Merr.], whereas members of USDA serogroup 123 dominate in non-alkaline soils. A possible explanation for the dominance of 135 in alkaline soils is that native 135 strains can produce siderophores that assist in acquiring iron. Our objectives were to test six reference strains (three slow-growing B. japonicum and three fast-growing Rhizobium fredii) for siderophore production and to evaluate mutants of R.fredii (developed by Tn 5 -insertion for overproduction of siderophores) for their competitive abilities in an alkaline soil. B. japonicum reference strains USDA 110, 123 and 135 were tested for siderophore production in liquid culture by using Neilands' CAS Assay Solution. Mutants were developed for siderophore overproduction by introducing transposon Tn 5 (Km r) into a siderophore-producing Chinese R. fredii strain. Two siderophore overproducing mutants that produced mature and pink nodules on soybean in growth pouches were examined for the presence of Tn 5 by Southern hybridization with a Tn 5 probe. The Tn 5 -carrying mutants were further tested in the greenhouse for their competitiveness against native strains present in an alkaline soil of Iowa and for their symbiotic effectivity. Among the three slow-growing bradyrhizobia tested for siderophore production, USDA 135 was the only strain producing siderophore in a liquid medium low in iron. Nodule occupancies in the greenhouse by the two siderophore overproducing mutants of R. fredii were 3 and 4%, compared with 19% for the wild-type strain. Thus, either the wild-type was acquiring all the iron needed for maximum growth and nodulation, or the insertion of Tn 5 and the overproduction of siderophore resulted in less competitive strains.

NolC, a Rhizobium fredii gene involved in cultivar-specific nodulation of soybean, shares homology with a heat-shock gene.

Rhizobium fredii strain USDA257 does not nodulate soybean (Glycine max (L.) Merr.) cultivar McCall. Mutant 257DH5, which contains a Tn5 insert in the bacterial chromosome, forms nodules on this cultivar, but acetylene-reduction activity is absent. We have sequenced the region corresponding to the site of Tn5 insertion in this mutant and find that it lies within a 1176bp open reading frame that we designate nolC. nolC encodes a protein of deduced molecular weight 43564. Nucleotide sequences homologous to nolC are present in several other Rhizobium strains, as well as Agrobacterium tumefaciens, but not in Pseudomonas syringae pathovar glycinea. nolC lacks significant sequence homology with known genes that function in nodulation, but is 61% homologous to dnaJ, an Escherichia coli gene that encodes a 41 kDa heat-shock protein. Both R. fredii USDA257 and mutant 257DH5 produce heat-shock proteins of 78, 70, 22, and 16kDa. A 4.3kb EcoRI-HindIII subclone containing nolC expresses a single 43kDa polypeptide in mini-cells. A longer, 9.4kb EcoRI fragment expresses both the 43kDa polypeptide and a 78kDa polypeptide that corresponds in size to that of the largest heat-shock protein. Thus, although nolC has strong sequence homology to dnaJ and appears to be linked to another heat-shock gene, it does not directly function in the heat-shock response.

Rhizobia and bradyrhizobia under salt stress: possible role of trehalose in osmoregulation

Seven rhizobium fredii strains and seven Bradyrhizobium japonicum strains were grown in defined medium with or without 20m~ trehalose in the presence or absence of NaCI. Trehalose had no effect on the growth rate of the strains in the absence of NaCI, but increased the growth rate of some strains in the presence of NaCl. Bradyrhizobiurn japonicum strain RCR 3827 was completely inhibited by 0.08 M NaCl in absence of trehalose, but multiplied when trehalose was added. The results indicate that trehalose may act as an osmoregulator in these strains of Rhizobium and Bradyrhizobium. 10, 127-129.

Interaction of Rhizobium fredii USDA257 and nodulation mutants derived from it with the agronomically improved soybean cultivar McCall

Rhizobium fredii USDA257 does not nodulate McCall soybean (Glycine max (L.) Merr.), but two transposon-mutants derived from it, 257DH4 and 257DH5, do. All three organisms cause curling of McCall root hairs and induce the formation of underlying cortical cell divisions. The mutants produce infection threads, and many of the meristematic foci develop into nodules. In contrast, root hairs that deform in response to USDA257 lack infection threads, and meristematic activity ceases prior to the appearance of nodule meristems. Root systems nodulated by mutant 257DH4 reduce acetylene at rates similar to those of roots nodulated by reference R. fredii strain USDA191. The presence of living cells of USDA257 in inocula leads to strong inhibition of nodulation by 257DH4 but not by 257DH5. This blocking effect depends on the ratio of USDA257 cells to 257DH4 cells in the inoculum; nodules that form contain cells of 257DH4, but not those of parental strain USDA257.

A Comparative Study of the Physiological Characteristics, Plasmid Content and Symbiotic Properties of Different Rhizobium fredii Strains

Summary Three of the new Rhizobium fredii strains (HH003, HH102 and HH103) isolated by Dowdle and Bohlool (1985) were investigated for their plasmid content, carbon nutritional patterns, salt tolerance and host-range and symbiotic efficiency with eight different legumes of the “cowpea cross-inoculation group”. The three R. fredii strains harbour nif structural genes, homologous to nif H, D, K, on large plasmids (ca. 200 Md). Their carbon nutritional patterns and salt tolerance were the same as those reported for the R. fredii strains isolated by Keyser et al. (1982a). All three strains formed nitrogen fixing nodules on Macroptilium atropurpureum, Cajanus cajan, Phaseolus aureus and Vigna unguiculata var unguiculata but failed to nodulate on Vigna unguiculata subsp. cylindrica and Vigna angularis. Vigna radiata and Psophocarpus tetragonolobus were very poorly nodulated by at least one of the three R. fredii strains.

Inter and Intraspecific Transfer of a Rhizobium fredii Symbiotic Plasmid: Expression and Incompatibility of Symbiotic Plasmids

Summary The Rhizobium fredii symbiotic plasmid pRfr193a has been marked by Tn5-Mob insertion and transferred to Rhizobium sp. ( Hedysarum coronarium ) strain IS123 and to a Fix - mutant derivative (AB-24) of R. fredii strain USDA 194. Most of the IS123 transconjugants carrying pRfr193a::Tn5-Mob (pAB1) nodulated ineffectively on Glycine max and all of them failed to nodulate on H. coronarium . These transconjugants had lost the indigenous IS123 symbiotic plasmid (pRHcRB16b) and showed an additional plasmid band. Transfer of the R. fredii symbiotic plasmid pAB1 to R. fredii strain AB-24 did not correct the Fix - phenotype of the recipient strain.

Growth of Fast- and Slow-Growing Rhizobia on Ethanol

Free-living soybean rhizobia and Bradyrhizobium spp. (lupine) have the ability to catabolize ethanol. Of the 30 strains of rhizobia examined, only the fast- and slow-growing soybean rhizobia and the slow-growing Bradyrhizobium sp. (lupine) were capable of using ethanol as a sole source of carbon and energy for growth. Two strains from each of the other Rhizobium species examined (R. meliloti, R. loti, and R. leguminosarum biovars phaseoli, trifolii, and viceae) failed to grow on ethanol. One Rhizobium fredii (fast-growing) strain, USDA 191, and one (slow-growing) Bradyrhizobium japonicum strain, USDA 110, grew in ethanol up to concentrations of 3.0 and 1.0%, respectively. While three of the R. fredii strains examined (USDA 192, USDA 194, and USDA 205) utilized 0.2% acetate, only USDA 192 utilized 0.1% n-propanol. None of the three strains utilized 0.1% methanol, formate, or n-butanol as the sole carbon source.

DNA Homologies between Rhizobium fredii, Rhizobia That Nodulate Galega sp., and Other Rhizobium and Bradyrhizobium Species

The relationships between Rhizobium fredii and the rhizobia that nodulate Galega officinalis and Galega orientalis (goat’s rue) and recognized species of Rhizobium and Bradyrhizobium were investigated by using deoxyribonucleic acid (DNA)-DNA hybridization, legume nodulation tests, and phage typing. The R. fredii strains formed a distinct DNA homology group which could be divided into two subgroups. The mean levels of relative homology at 65°C of 11 strains of R. fredii with R. fredii reference strains USDA 208 and USDA 191 were 86 and 80%, respectively. These values were significantly higher (Student’s t test, P < 0.001) than the mean levels of relative homology with DNAs from other groups of rhizobia. The Galega-nodulating rhizobia also formed a distinct DNA homology group. The mean levels of relative homology at 65°C of DNAs from 11 strains with DNAs from reference strains gall and galNW3, which effectively nodulate G. officinalis, were 79 and 85%, respectively. These values were also significantly higher (Student’s t test, P < 0.001) than the values obtained with DNAs from other groups of rhizobia. These results correlated with cross-inoculation and phage-typing results and indicated that the two groups are genetically distinct. Genomic DNA was used as a probe in a modified colony hybridization autoradiographic procedure for the identification of DNAs from Rhizobium leguminosarum, R. fredii, and Rhizobium sp. (Galega) in colonies and from nodules.

Fast‐Growing Rhizobium fredii are Poor Nitrogen‐Fixing Symbionts of Soybean1

Soybean‐Rhizobium genotype combinations with high rates of N2 fixation are useful in soybean (Glycine max L. Merr.) breeding programs. Nitrogen fixation was examined in 13 soybean cultivars and wild soybean (G. soja Sieb. & Zucc.) plant introductions inoculated with Bradyrhizobium japonicum strain 61A76 and four fast‐growing strains of Rhizobium fredii. Five‐week‐old plants were assessed for nodule morphology, acetylene reduction rate, nodule number, nodule fresh weight, and plant‐top dry weight. All fast‐growing strains formed effective nodules on cultivars ‘Peking’, ‘Virginia’, ‘Hardee’, and the G. soja plant introductions. In contrast, three of the fast‐growing strains formed ineffective nodules on the other cultivars. One strain, USDA191, formed effective nodules on all cultivars. No nodules were formed by the fast‐growing R. fredii on ‘Harosoy rj1 rj1’. Rhizobium fredii strain USDA191 fixed N, as well as the slow‐growing control with cultivars Peking, Virginia, ‘Harosoy 63’, and ‘Rampage’, and G. soja plant introductions PI 342622A and PI 101404B, but fixed less NJ than the slow‐growing control with cultivars ‘Evans’, ‘Williams’, Hardee, ‘Hill’, and G. soja PI 407217 and PI 81762.

Competition for nodulation of field-grown soybeans by strains of Rhizobium fredii

Nodulation of Glycine max (L) Merr. by six Rhizobium fredii strains was measured in two Midwestern fields containing high indigenous populations of Bradyrhizobium japonicum (3 × 105/gm soil). The soils were inoculated with antibiotic-resistant mutants using liquid inoculum at two levels on soybean cv. Peking and cv. Jacques 130. Strain establishment was measured 40 days after planting. In the first year, USDA206, USDA217, and USDA257 were the most competitive strains, occupying greater than 50% of the nodules on cv. Peking in both soils. None of the strains were competitive on Jacques 130. In the second growing season, all nodules were formed by the indigenous population on both cultivars, suggesting that these fast-growing strains do not persist in Midwestern soils.

Conservation of IS66 homologue of octopine Ti plasmid DNA in Rhizobium fredii plasmid DNA

DNA sequences homologous to the T-DNA region of the octopine Ti plasmid from Agrobacterium tumefaciens are found in various fast-growing Rhizobium fredii strains. The largest fragment (BamHI fragment 2) at the right-boundary region of the 'core' T-DNA hybridizes to more than one plasmid present in R. fredii. However, one smaller fragment (EcoRI fragment 19a) adjacent to the 'core' T-DNA shows homology only with the plasmid carrying the symbiotic nitrogen-fixation genes (pSym). Hybridization data obtained with digested R. fredii USDA193 pSym DNA suggests that the homology is mainly with two HindIII fragments, 1.7 kb and 8.8 kb in size, of the plasmid. The 1.7 kb HindIII fragment also hybridizes to two regions of the virulence plasmid of A. tumefaciens, pAL1819, a deletion plasmid derived from the octopine Ti plasmid, pTiAch5. Hybridization studies with an insertion element IS66 from A. tumefaciens indicate that the 1.7 kb HindIII fragment of R. fredii plasmid, homologous to the T-DNA and the virulence region of Ti plasmid, is itself an IS66 homologue.

The formation of R-prime deletion mutants and the identification of the symbiotic genes in Rhizobium fredii strain USDA191

R-prime plasmids were formed between the plasmid of Rhizobium fredii strain USDA191 containing nodulation and nitrogen-fixation genes, pRjaUSDA191c, and pRL180, and RP1 derivative. R. fredii USDA191 contains four HindIII fragments that hybridize with an 8.7 kb EcoRI fragment that contains nodulation genes from R. meliloti. These four fragments are on pRjaUSDA191c and are 15.5 kb, 12.5 kb, 6.8 kb, and 5.2 kb in size. A series of R-primes generated in E. coli of pRjaUSDA191c were transferred into a Nod(-) Nif(-) derivative of strain USDA191 to determine which nodulation region is necessary for nodule formation. Transconjugants containing the 12.5 kb and the 6.8 kb HindIII fragments on segments of pRjaUSDA191c produced nodules on soybean plants. However, transconjugants containing the 12.5 kb HindIII fragment alone were unable to form nodules, suggesting that the 6.8 kb HindIII fragment or the 6.8 kb and the 12.5 kb HindIII fragments together were needed for nodule formation. The 6.8 kb HindIII fragment was subcloned into the vector pVK102 and transferred into transconjugants containing no sequences homologous to R. meliloti nodulation DNA or to transconjugants containing only the 12.5 kb HindIII fragment. Nodules were formed on soybeans only when both the 12.5 kb and the 6.8 kb HindIII fragments were present in R. frediistrain USDA191.