Isolation and characterization of transposon Tn5-induced symbiotic mutants of Rhizobium loti

Rhizobium loti NZP2037 and NZP2213, each cured of its single large indigenous plasmid, formed effective nodules on Lotus spp., suggesting that the symbiotic genes are carried on the chromosome of these strains. By using pSUP1011 as a vector for introducing transposon Tn5 into R. loti NZP2037, symbiotic mutants blocked in hair curling (Hac), nodule initiation (Noi), bacterial release (Bar), and nitrogen fixation (Nif/Cof) on Lotus pedunculatus were isolated. Cosmids complementing the Hac, Noi, and Bar mutants were isolated from a pLAFR1 gene library of NZP2037 DNA by in planta complementation and found to contain EcoRI fragments of identical sizes to those into which Tn5 had inserted in the mutants. The cosmids that complemented the mutants of these phenotypic classes did not share common fragments, nor did cosmids that complemented four mutants within the Noi class, suggesting that these symbiotically important regions are not tightly linked on the R. loti chromosome.     

Symbiotic properties of Lotus pedunculitis root nodules induced by Rhizobium loti and Bradyrhizobium sp. (Lotus)

Vance, C. P., Reibach, P. H. and Pankhurst, C. E. 1987. Symbiotic properties of Lotus pedunculatus root nodules induced by Rhizobium loti and Bradyrhizobium sp. ( Lotus ).
Symbiotic properties of root nodules were evaluated in glasshouse-grown Lotus pedunculatus Cav. cv. Maku inoculated with either a fast-growing Rhizobium loti strain NZP2037 or a slow-growing Bradyrhizobium sp. ( Lotus ) strain CC814s. Although the nodule mass of plants inoculated with NZP2037 was twice that of plants inoculated with CC814s, the yield of NZP2037 shoots and roots was 50% that of CC814s shoots and roots. Nodules induced by Bradyrhizobium fixed substantially more N than nodules induced by R. loti. Glucose requirements [mol glucose (mol N₂ fixed)^-1] of nodules induced by CC814s and NZP2037 were 7.1 and 16.6, respectively. Nodule enzymes of carbon and nitrogen assimilation reflected the disparity of the two sym-bioses. Xylem sap of the symbiosis with the higher yield contained a higher concentration of asparagine [9.86 μmol (ml xylem sap)'] than did the lower yielding symbiosis [5.80 umol (ml xylem sap)"']. Nodule CO₂ fixation was directly linked to nodule N assimilation in both symbioses. The results indicate that the difference between the two symbioses extend to nodule N and C assimilation and whole plant N transport. The data support a role for host plant modulation of bacterial efficiency and assimilation of fixed N.     

Transfer of an indigenous plasmid of Rhizobium loti to other rhizobia and Agrobacterium tumefaciens

Rhizobium loti strains NZP2037 and NZP2213 were each found to contain a single large plasmid: pRlo2037a (240 MDal) and pRlo2213a (120 MDal), respectively. Plasmid DNA present in crude cell lysates of each strain and purified pRlo2037a DNA did not hybridize with pID1, a recombinant plasmid containing part of the nitrogen fixation (nif) region of R. meliloti, indicating that nif genes were not present on these plasmids. The transposon Tn5 was inserted into pRlo2037a and this plasmid was then transferred into R. leguminosarum, R. meliloti and Agrobacterium tumefaciens. All transconjugants failed to nodulate Lotus pedunculatus, suggesting that the ability to nodulate this legume was also not carried on pRlo2037a. Transfer of pRlo2037a to R. loti strain NZP2213 did not alter the Nod+ Fix- phenotype of this strain for L. pedunculatus. Determinants for flavolan resistance, believed to be necessary for effective nodulation of L. pedunculatus, were not carried on pRlo2037a. These data suggest that nodulation, nitrogen fixation and flavolan resistance genes are not present on the large plasmid in R. loti strain NZP2037.     

Exopolysaccharide mutants of Rhizobium loti are fully effective on a determinate nodulating host but are ineffective on an indeterminate nodulating host.

By Tn5 mutagenesis of Rhizobium loti PN184 (NZP2037 str-1) and selection for nonfluorescence of colonies on Calcofluor agar, eight independently generated expolysaccharide (EPS) mutants (three smooth and five rough) were isolated. The parent strain, PN184, was found to produce an acidic EPS. This EPS was produced. with reduced O acetylation, by the smooth EPS mutants but not by the rough EPS mutants. Lipopolysaccharide was isolated from all mutants and was identical to that of PN184 as defined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All mutants were resistant to lysis by R. loti bacteriophage phi 2037/1. Cosmids that complemented the mutations in the rough EPS mutants were isolated from a pLAFR1 gene library of NZP2037 by complementation of the nonfluorescent phenotype. The genes identified were shown to be unlinked and located on the chromosome. All mutants were fully effective when inoculated onto Lotus pedunculatus, a determinate nodulating host, but were ineffective, inducing the formation of very small nodules or tumorlike growths, when inoculated onto Leucaena leucocephala, an indeterminate nodulating host. These results, obtained in an isogenic Rhizobium background, support suggestions that acidic EPS is required for effective nodulation of indeterminate nodulating legumes but is not required for effective nodulation of determinate nodulating legumes.     

Isolation and characterization of a Rhizobium loti gene required for effective nodulation of Lotus pedunculatus

A Rhizobium loti gene required for effective invasion of the host Lotus pedunculatus has been identified by transposon Tn5 mutagenesis. Cosmids that complemented a previously isolated mutation (239) at this invasion (inv) locus were identified by in planta complementation and used to construct a physical map of the gene region. The insertion site of Tn5 in PN239 was mapped to a 7.5-kb EcoRI fragment, which complemented the mutation when subcloned into pLAFR1. Further Tn5 mutagenesis of the 7.5-kb fragment was carried out in Escherichia coli using bacteriophage lambda 467, and the mutations homogenotized into R. loti NZP2037. Three additional Fix- mutations were isolated, and these were found to map adjacent to the position of the original mutation in strain PN239. All the other Tn5 insertions isolated in the 7.5-kb fragment gave a Fix+ phenotype on L. pedunculatus. Electron microscopic examination of the L. pedunculatus nodules induced by the isolated Fix- mutants showed that bacteria were either blocked in release from the infection threads or were unable to undergo normal bacteroid development. The inv locus as defined by the Tn5 insertions was sequenced, and a single open-reading frame (ORF) of 576 bp, corresponding to a polypeptide of 21.3 kDa, was identified. The position and orientation of this ORF were consistent with those of the isolated Tn5 Fix- insertions.     

Mutations conferring azide resistance enhance symbiotic nitrogen fixation in Rhizobium loti

Azide-resistant (AzR) mutants of Rhizobium loti strain NZP2037 were isolated. Mutations conferring azide resistance (azi) appeared at a frequency of 0.5 × 10-7. Nine AzR mutants of R. loti were characterised for their symbiotic behaviour with Lotus pedunculatus plants. In comparison to the wild type parent strain, AzR mutants exhibited either similar or higher symbiotic effectiveness. The azi mutations which enhanced nitrogen fixation as well as improving shoot dry weight of the inoculated plants also increased nodulation. Unlike several azi mutations in Escherichia coli, these azi mutations did not alter sensitivity of R. loti to phenethyl alcohol. One of the AzR mutants exhibited higher micro-aerobic, N, N, N, N-tetramethyl-p-phenylenediamine (TMPD) oxidase activity.     

Molecular cloning of a nodulation gene from fast- and slow-growing strains of Lotus rhizobia

Summary Cosmids containing a nodulation gene from Rhizobium loti NZP2037 were isolated using a 12.8 kb nod:: Tn5 EcoRI fragment from the Nod^- mutant strain PN233, as a hybridisation probe. A physical map of the nod region was established using the enzymes EcoRI and HindIII and the site of insertion of Tn5 in PN233 determined. Site-specific exchange of the cloned nod:: Tn5 fragment demonstrated that Tn5, and not an indigenous insertion sequence, was responsible for the nod mutation in PN233. The nod cosmids isolated complemented the Nod^- phenotype of strain PN233 but restoration of the Fix phenotype was variable suggesting a need for marker rescue to occur before nitrogen fixation occurred.Corresponding nod cosmids were isolated from a R. loti strain, NZP2213, that forms ineffective tumour-like structures on Lotus pedunculatus and from the slow-growing strain (Bradyrhizobium sp), CC814s, by in planta complementation of PN233. Hybridisation experiments suggested that the nod gene region of R. loti NZP2037 was more homologous to Bradyrhizobium strain CC814s than with a nod gene region of R. trifolii strain PN100. However, transfer of the R. trifolii nod cosmid into the R. loti Nod mutant PN233, restored the ability of this strain to initiate nodules on Lotus pedunculatus.     

Four unnamed species of nonsymbiotic rhizobia isolated from the rhizosphere of Lotus corniculatus.

Previously, we found that genetically diverse rhizobia nodulating Lotus corniculatus at a field site devoid of naturalized rhizobia had symbiotic DNA regions identical to those of ICMP3153, the inoculant strain used at the site (J. T. Sullivan, H. N. Patrick, W. L. Lowther, D. B. Scott, and C. W. Ronson, Proc. Natl. Acad. Sci. USA 92:8985-8989, 1995). In this study, we characterized seven nonsymbiotic rhizobial isolates from the rhizosphere of L. corniculatus. These included two from plants at the field site sampled by Sullivan et al. and five from plants at a new field plot adjacent to that site. The isolates did not nodulate Lotus species or hybridize to symbiotic gene probes but did hybridize to genomic DNA probes from Rhizobium loti. Their genetic relationships with symbiotic isolates obtained from the same sites, with inoculant strain ICMP3153, and with R. loti NZP2213T were determined by three methods. Genetic distance estimates based on genomic DNA-DNA hybridization and multilocus enzyme electrophoresis were correlated but were not consistently reflected by 16S rRNA nucleotide sequence divergence. The nonsymbiotic isolates represented four genomic species that were related to R. loti; the diverse symbiotic isolates from the site belonged to one of these species. The inoculant strain ICMP3153 belonged to a fifth genomic species that was more closely related to Rhizobium huakuii. These results support the proposal that nonsymbiotic rhizobia persist in soils in the absence of legumes and acquire symbiotic genes from inoculant strains upon introduction of host legumes.     

Evaluation and improvement of the energy efficiency of nitrogen fixation in Lotus corniculatus nodules induced by Rhizobium loti strains indigenous to Uruguay

In order to evaluate energy efficiency of nitrogen fixation by the Lotus corniculatus/Rhizobium loti symbiosis, Uruguayan R. loti strains were tested for hydrogen-uptake (Hup) status. Nodules induced in L. corniculatus by all eight R. loti strains tested evolved high amounts of hydrogen (2.0–8.7 mol H2/h.g nodule fresh weight). This production of hydrogen corresponds to 38–69% of total nitrogenase activity estimated as acetylene reduction, suggesting that hydrogen is not recycled within these nodules. This was confirmed by the lack of hydrogenase activity in bacteroid suspensions. Additionally, no hybridization signals were observed in total DNA restriction digests from these strains when a DNA fragment containing part of hydrogenase structural genes from Rhizobium leguminosarum bv. viciae was used as probe. Cosmid pHU52, containing the complete gene cluster required for hydrogen oxidation in Bradyrhizobium japonicum, was introduced into two R. loti strains. Transconjugants from only one of the strains were able to express hydrogenase activity in vegetative cells incubated under the derepression conditions described for B. japonicum. Bacteroids induced by both transconjugant strains in L. corniculatus and Lotus tenuis expressed hydrogenase activity in nodules. The level of hydrogenase activity induced in L. tenuis nodules was two-fold higher than those induced in L. corniculatus. This implies the existence of a strong host effect on hydrogenase expression in this symbiotic system.     

Effect of plant nutrient supply on nodule effectiveness and rhizobium strain competition for nodulation ofLotus pedunculatus

Summary The effect of nutrient supply on nodule formation and competition between Rhizobium strains for nodulation ofLotus pedunculatus was studied. Limiting plant growth by decreasing the supply of nutrients in an otherwise nitrogen-free medium, increased the size but decreased the number and the nitrogenase activity of nodules formed by a fast-growing strain of Lotus Rhizobium (NZP2037). In contrast decreasing nutrient supply caused only a small decline in the size, number and nitrogenase activity of nodules formed by a slow-growing strain (CC814s). Providing small quantities of NH₄NO₃ (50 to 250 g N) to plants grown with a normal supply of other nutrients stimulated nodule development by both Rhizobium strains and increased the nitrogenase activity of the NZP2037 nodules. Differences in the level of effectiveness (nitrogen-fixing ability) of nodules formed by different Rhizobium strains on plants grown with a normal supply of nutrients were less apparent when the plants were grown with decreased nutrient supply or when the plants were supplied with low levels of inorganic N.Inter-strain competition for nodulation ofL. pedunculatus between the highly effective slow-growing strain CC814s and 7 other fast- and slow-growing strains, showed CC814s to form 42 to 100% of the nodules in all associations. The greater nodulating competitiveness of strain CC814s prevailed despite changes in the nutrient supply to the host plant. A tendency was observed for partially effective Lotus Rhizobium strains to become more competitive in nodule formation when plant growth was supplemented with low levels of inorganic nitrogen.     

Genetic locus in Rhizobium japonicum (fredii) affecting soybean root nodule differentiation.

A genetic locus in fast-growing Rhizobium japonicum (fredii) USDA 191 (Fix+ on several contemporary soybean cultivars) was identified by random Tn5 mutagenesis as affecting the development and differentiation of root nodules. This mutant (MU042) is prototrophic and shows no apparent alterations in its surface properties. It induces aberrant nodules, arrested at the same early level of differentiation, on all its host plants. An 8.1-kilobase EcoRI fragment containing Tn5 was cloned from MU042. In USDA 191 as well as another fast-growing strain, USDA 201, the affected locus was found to be unlinked to the large symbiotic plasmid and appears to be chromosomal. An analogous sequence has been shown to be present in Bradyrhizobium japonicum (J. Stanley, G.G. Brown, and D.P.S. Verma, J. Bacteriol. 163:148-154, 1985) as well as in R. trifolii and R. meliloti. MU042 was complemented for effective nodulation of soybean by a cosmid clone from USDA 201, and the complementing locus was delimited to a 6-kilobase EcoRI subfragment. An R. trifolii strain (MU225), whose indigenous symbiotic plasmid was replaced by that of strain USDA 191, induced more highly differentiated nodules on soybean than did MU042. This suggests that the mutation in MU042 can be functionally substituted by similar loci of other fast-growing rhizobia. Leghemoglobin and nodulin-35 (uricase II) were present in the differentiated Fix- nodules induced by MU225, whereas both were absent in MU042-induced pseudonodule structures.     

Effectiveness of Lotus Root Nodules: I. MORPHOLOGY AND FLAVOLAN CONTENT OF NODULES FORMED ON LOTUS PEDUNCULATUS BY FAST-GROWING LOTUS RHIZOBIA

The morphology of root nodules formed on Lotus pedunculatusby two fast-growing strains of Rhizobium, NZP2037 which formseffective (nitrogen-fixing) nodules and NZP2213 which formsineffective (non-nitrogen-fixing) nodules, has been studied.The nodules formed by NZP2037 contained a central zone of bacteroid-filledplant cells surrounded by a cortex. In contrast the nodulesformed by NZP2213 contained no Rhizoblum-infected plant cells,but rhizobia were found in localized areas on the nodule surfaceand between the outer two or three cell layers of the nodule.Electron-dense osmiophilic deposits identified as flavolans(condensed tannins) were present in the vacuoles of many uninfectedplant cells in the nodules formed by both Rhizobium strains.This is the first time that flavolans have been positively identifiedin legume root nodules. In the NZP2037 nodule flavolans werepresent in the outer cortical and epidermal cells. In the ineffecitveNZP2213 nodule fiavolans were present in many of the centralnodule cells. The concentration of flavolan in the NZP2213 nodulewas 12 times higher than in the NZP2037 nodule.     

Symbiotic effectiveness of phage-resistant mutants of two strains of Lotus rhizobia

Summary Phage-resistant mutants were obtained from a fast-growing (NZP2037) and a slow-growing (CC814s) strain of Rhizobium nodulating Lotus. All the mutants were stable and did not differ from the original parent strain in their cultural characteristics. OnLotus pedunculatus the mutants of NZP2037 were as effective in N-fixation as the parent strain but most mutants of CC814s were less effective. These mutants of CC814s were also less effective than the parent strain on several other host plants.     

Identification of a Rhizobium trifolii plasmid coding for nitrogen fixation and nodulation genes and its interaction with pJB5JI, a Rhizobium leguminosarum plasmid

Rhizobium trifolii T37 contains at least three plasmids with sizes of greater than 250 megadaltons. Southern blots of agarose gels of these plasmids probed with Rhizobium meliloti nif DNA indicated that the smallest plasmid, pRtT37a, contains the nif genes. Transfer of the Rhizobium leguminosarum plasmid pJB5JI, which codes for pea nodulation and the nif genes and is genetically marked with Tn5, into R. trifolii T37 generated transconjugants containing a variety of plasmid profiles. The plasmid profiles and symbiotic properties of all of the transconjugants were stably maintained even after reisolation from nodules. The transconjugant strains were placed into three groups based on their plasmid profiles and symbiotic properties. The first group harbored a plasmid similar in size to pJB5JI (130 megadaltons) and lacked a plasmid corresponding to pRtT37a. These strains formed effective nodules on peas but were unable to nodulate clover and lacked the R. trifolii nif genes. This suggests that genes essential for clover nodulation as well as the R. trifolii nif genes are located on pRtT37a and have been deleted. The second group harbored hybrid plasmids formed from pRtT37a and pJB5JI which ranged in size from 140 to ca. 250 megadaltons. These transconjugants had lost the R. leguminosarum nif genes but retained the R. trifolii nif genes. Strains in this group nodulated both peas and clover but formed effective nodules only on clover. The third group of transconjugants contained a hybrid plasmid similar in size to pRtT37b. These strains contained the R. trifolii and R. leguminosarum nif genes and formed N2-fixing nodules on both peas and clover.     

Expression by Soil Bacteria of Nodulation Genes from Rhizobium leguminosarum biovar trifolii

Gram-negative, rod-shaped bacteria from the soil of white clover-ryegrass pastures were screened for their ability to nodulate white clover (Trifolium repens) cultivar Grasslands Huia and for DNA homology with genomic DNA from Rhizobium leguminosarum biovar trifolii ICMP2668 (NZP582). Of these strains, 3.2% were able to hybridize with strain ICMP2668 and nodulate white clover and approximately 19% hybridized but were unable to nodulate. Strains which nodulated but did not hybridize with strain ICMP2668 were not detected. DNA from R. leguminosarum biovar trifolii (strain PN165) cured of its symbiotic (Sym) plasmid and a specific nod probe were used to show that the relationship observed was usually due to chromosomal homology. Plasmid pPN1, a cointegrate of the broad-host-range plasmid R68.45 and a symbiotic plasmid pRtr514a, was transferred by conjugation to representative strains of nonnodulating, gram-negative, rod-shaped soil bacteria. Transconjugants which formed nodules were obtained from 6 of 18 (33%) strains whose DNA hybridized with that of PN165 and 1 of 9 (11%) strains containing DNA which did not hybridize with that of PN165. The presence and location of R68.45 and nod genes was confirmed in transconjugants from three of the strains which formed nodules. Similarly, a pLAFR1 cosmid containing nod genes from a derivative of R. leguminosarum biovar trifolii NZP514 formed nodules when transferred to soil bacteria.     

The region for exopolysaccharide synthesis in Rhizobium leguminosarum biovar trifolii is located on the non-symbiotic plasmid.

An Exo- mutant of Rhizobium leguminosarum biovar trifolii was isolated which did not produce acidic exopolysaccharide and induced defective, non-fixing nodules on clover plants. The nodules were defective at a late stage of development, they contained infection threads and bacteria were released into the host cells. Cosmid pARF136 capable of complementing the Exo- mutation was isolated from a cosmid bank made from total R. trifolii DNA. Hybridization between DNA of pARF136 and plasmids of R. trifolii strains separated by Eckhardt's technique suggested that the exo locus is located on a 300 kb megaplasmid, and nodDABC and nifKDH genes are located on another 180 kb pSym plasmid. A 5.4 kb BamH1 fragment of the recombinant cosmid pARF136 was able to restore exopolysaccharide synthesis in Exo- mutant of R. trifolii 93 but it did not complement the symbiotic defect.     

Nodules are induced on alfalfa roots by Agrobacterium tumefaciens and Rhizobium trifolii containing small segments of the Rhizobium meliloti nodulation region.

Regions of the Rhizobium meliloti nodulation genes from the symbiotic plasmid were transferred to Agrobacterium tumefaciens and Rhizobium trifolii by conjugation. The A. tumefaciens and R. trifolii transconjugants were unable to elicit curling of alfalfa root hairs, but were able to induce nodule development at a low frequency. These were judged to be genuine nodules on the basis of cytological and developmental criteria. Like genuine alfalfa nodules, the nodules were initiated from divisions of the inner root cortical cells. They developed a distally positioned meristem and several peripheral vascular bundles. An endodermis separated the inner tissues of the nodule from the surrounding cortex. No infection threads were found to penetrate either root hairs or the nodule cells. Bacteria were found only in intercellular spaces. Thus, alfalfa nodules induced by A. tumefaciens and R. trifolii transconjugants carrying small nodulation clones of R. meliloti were completely devoid of intracellular bacteria. When these strains were inoculated onto white clover roots, small nodule-like protrusions developed that, when examined cytologically, were found to more closely resemble roots than nodules. Although the meristem was broadened and lacked a root cap, the protrusions had a central vascular bundle and other rootlike features. Our results suggest that morphogenesis of alfalfa root nodules can be uncoupled from infection thread formation. The genes encoded in the 8.7-kilobase nodulation fragment are sufficient in A. tumefaciens or R. trifolii backgrounds for nodule morphogenesis.     

The requirement for exopolysaccharide precedes the requirement for flavolan-binding polysaccharide in nodulation of Leucaena leucocephala by Rhizobium loti

Rhizobium loti strain PN4115 (NZP2213 str-1) ineffectively nodulates Leucaena leucocephala, i.e., strain PN4115 induces nodulation (Nod⁺) and is able to invade these nodules (Inv⁺), but fails to fix nitrogen (Fix^–). Strain PN4115 does not synthesize a flavolan-binding polysaccharide (FBP), which is synthesized by the fully effective (Nod⁺Inv⁺Fix⁺) R. loti strain PN184 (NZP2037 str-1). The FBP may offer protection from prodelphinidin-rich flavolans synthesized by Lc. leucocephala. In this work, we show that exopolysaccharide (EPS)-negative mutants derived from strain PN4115 have a more severe ineffective phenotype (Nod⁺Inv^–Fix^–) on Lc. leucocephala than strain PN4115. This suggests that EPS from strain PN4115 is functional during invasion of Lc. leucocephala and that the requirement for EPS precedes the requirement for FBP. Received: 8 October 1996 / Accepted: 11 December 1996