Characterization and symbiotic importance of acidic extracellular polysaccharides of Rhizobium sp. strain GRH2 isolated from acacia nodules.

Rhizobium sp. wild-type strain GRH2 was originally isolated from root nodules of the leguminous tree Acacia cyanophylla and has a broad host range which includes herbaceous legumes, e.g., Trifolium spp. We examined the extracellular exopolysaccharides (EPSs) produced by strain GRH2 and found three independent glycosidic structures: a high-molecular-weight acidic heteropolysaccharide which is very similar to the acidic EPS produced by Rhizobium leguminosarum biovar trifolii ANU843, a low-molecular-weight native heterooligosaccharide resembling a dimer of the repeat unit of the high-molecular-weight EPS, and low-molecular-weight neutral beta (1,2)-glucans. A Tn5 insertion mutant derivative of GRH2 (exo-57) that fails to form acidic heteropolysaccharides was obtained. This Exo- mutant formed nitrogen-fixing nodules on Acacia plants but infected a smaller proportion of cells in the central zone of the nodules than did wild-type GRH2. In addition, the exo-57 mutant failed to nodulate several herbaceous legume hosts that are nodulated by wild-type strain GRH2.     

Induction of pathogenic-like responses in the legume Macroptilium atropurpureum by a transposon-induced mutant of the fast-growing, broad-host-range Rhizobium strain NGR234.

Mutant strain ANU2861, a transposon Tn5 mutant of the fast-growing, broad-host-range Rhizobium strain ANU280 (NGR234 Smr Rfr) overproduces polysaccharide, is an ade auxotroph, and induces poorly developed nodules on Leucaena leucocephala and Lablab purpureus (H.C. Chen, M. Batley, J.W. Redmond, and B.G. Rolfe, J. Plant Physiol. 120:331-349, 1985). Strain ANU2861 cannot form nodules on Macroptilium atropurpureum Urb. (siratro) or on Desmodium intortum and D. uncinatum and the nonlegume Parasponia. The parent strain, ANU280, effectively nodulates all these legume species except Parasponia, on which it forms ineffective nodules. Ultrastructural examination of infection sites on the legume siratro showed that mutant strain ANU2861 caused root hair curling (Hac+ phenotype), some cortical cell division (Noi+), but no infection threads (Inf-). Localized cellular responses, known to occur in phytopathological interactions, were observed in electron micrographs of the epidermal tissue at or near the infection zone after inoculation with strain ANU2861 but not the wild-type parental strain. These include (i) the rapid (within 20 h) accumulation of osmiophilic droplets attached to membranes at potential sites of strain ANU2861 penetration and (after 48 h) in the epidermal cells in the immediate region of the curled root hairs, and (ii) localized cell death of the epidermal cells. In addition, strain ANU2861 can initiate a systemic response in split-root siratro plants which prevents the successful nodulation of strain ANU280. A 6.3-kilobase fragment of wild-type genomic DNA, which includes the site of Tn5 insertion in strain ANU2861, was cloned and introduced to strain ANU2861. All the phenotypic defects of the mutant strain were corrected by the introduction of this DNA fragment. This indicates that the original Tn5 insertion is responsible for the phenotype.     

Exopolysaccharides of Rhizobium leguminosarum biovar trifolii harbouring cloned exo region.

Rhizobium leguminosarum bv. trifolii produces an acidic exopolysaccharide (EPS) which plays an important role in the development of nitrogen-fixing nodules. Tn5 mutant of R. trifolii 93 defective in EPS production (Exo-) forms ineffective (Fix-) nodules on red clover. This Exo- mutation is complemented by the pARF1368 and pARF25 cosmids isolated from gene bank of Rhizobium trifolii TA1, but the complementation is not correlated with restoration of Fix+ phenotype. Furthermore, these cosmids introduced to wild-type of R. trifolii 24 repress its ability to form nitrogen-fixing nodules. These results might suggest that bacteria with cosmids carrying the exo region form EPS of altered structure. It has been shown by 1H-n.m.r. that exopolysaccharides produced by R. trifolii 93pARF-1368 and 93pARF25 contain less non-carbohydrate residues (acetyl, pyruvyl and 3-hydroxybutanoyl) than the wild type EPS. These data suggest that the biological activity of the exopolysaccharide of R. trifolii depends on the contents of the non-carbohydrate substitutions.     

Exo-oligosaccharides of Rhizobium sp. strain NGR234 are required for symbiosis with various legumes

Rhizobia are nitrogen-fixing bacteria that establish endosymbiotic associations with legumes. Nodule formation depends on various bacterial carbohydrates, including lipopolysaccharides, K-antigens, and exopolysaccharides (EPS). An acidic EPS from Rhizobium sp. strain NGR234 consists of glucosyl (Glc), galactosyl (Gal), glucuronosyl (GlcA), and 4,6-pyruvylated galactosyl (PvGal) residues with beta-1,3, beta-1,4, beta-1,6, alpha-1,3, and alpha-1,4 glycoside linkages. Here we examined the role of NGR234 genes in the synthesis of EPS. Deletions within the exoF, exoL, exoP, exoQ, and exoY genes suppressed accumulation of EPS in bacterial supernatants, a finding that was confirmed by chemical analyses. The data suggest that the repeating subunits of EPS are assembled by an ExoQ/ExoP/ExoF-dependent mechanism, which is related to the Wzy polymerization system of group 1 capsular polysaccharides in Escherichia coli. Mutation of exoK (NGROmegaexoK), which encodes a putative glycanase, resulted in the absence of low-molecular-weight forms of EPS. Analysis of the extracellular carbohydrates revealed that NGROmegaexoK is unable to accumulate exo-oligosaccharides (EOSs), which are O-acetylated nonasaccharide subunits of EPS having the formula Gal(Glc)5(GlcA)2PvGal. When used as inoculants, both the exo-deficient mutants and NGROmegaexoK were unable to form nitrogen-fixing nodules on some hosts (e.g., Albizia lebbeck and Leucaena leucocephala), but they were able to form nitrogen-fixing nodules on other hosts (e.g., Vigna unguiculata). EOSs of the parent strain were biologically active at very low levels (yield in culture supernatants, approximately 50 microg per liter). Thus, NGR234 produces symbiotically active EOSs by enzymatic degradation of EPS, using the extracellular endo-beta-1,4-glycanase encoded by exoK (glycoside hydrolase family 16). We propose that the derived EOSs (and not EPS) are bacterial components that play a crucial role in nodule formation in various legumes.     

Identification of a nodD-dependent locus in the Rhizobium strain NGR234 activated by phenolic factors secreted by soybeans and other legumes

Transfer of the strain NGR234nodD 1 gene into the narrow host range R. trifolii strain ANU843 on either a 6.7-kb HindIII or 17-kb XhoI fragment broadens the host range of this bacterium to include the tropical legumes Vigna unguiculata, Glycine ussuriensis, Leucaena leucocephala, and siratro (Macroptilium atropurpureum). Contrary to previous data (Bassam et al. 1986), mutagenesis of the 17-kb XhoI fragment with a mini-Mu lac transposon (Mu dII1734) showed that a functional nodD 1 gene was essential for extended host range. Gene expression studies using both Mu dII1734 fusions and a promoter-cloning vector indicated that several loci, including the nodD 1 gene, are constitutively expressed. No evidence was found for regulation of the strain NGR234 nodD 1 gene by its product. Another locus nod-81, was induced only in the presence of exudates from various plant species, including soybean (Glycine max). Whereas the expression of nod-81 was dependent on the presence of a functional nodD 1 gene product, a regulatory nod-box DNA sequence was not detected 5' to this gene by using available oligonucleotide hybridization probes. The nod-81 locus was induced by genistein, daidzein, naringenin, and coumestrol from both cotyledon and root tissue of freshly germinated soybean seedlings. A broad spectrum of commercially available phenolic compounds stimulated induction of the nod-81 locus, including some that antagonize nod gene induction in other Rhizobium species. The nodD 1 gene product from strain NGR234 was shown to determine the spectrum of compounds that induce nod-81 expression.     

Heterologous exopolysaccharide production in Rhizobium sp. strain NGR234 and consequences for nodule development.

Rhizobium sp. strain NGR234 produces large amounts of acidic exopolysaccharide. Mutants that fail to synthesize this exopolysaccharide are also unable to nodulate the host plant Leucaena leucocephala. A hybrid strain of Rhizobium sp. strain NGR234 containing exo genes from Rhizobium meliloti was constructed. The background genetics and nod genes of Rhizobium sp. strain NGR234 are retained, but the cluster of genes involved in exopolysaccharide biosynthesis was deleted. These exo genes were replaced with genes required for the synthesis of succinoglycan exopolysaccharide from R. meliloti. As a result of the genetic manipulation, the ability of these hybrids to synthesize exopolysaccharide was restored, but the structure was that of succinoglycan and not that of Rhizobium sp. strain NGR234. The replacement genes were contained on a cosmid which encoded the entire known R. meliloti exo gene cluster, with the exception of exoB. Cosmids containing smaller portions of this exo gene cluster did not restore exopolysaccharide production. The presence of succinoglycan was indicated by staining with the fluorescent dye Calcofluor, proton nuclear magnetic resonance spectroscopy, and monosaccharide analysis. Although an NGR234 exoY mutant containing the R. meliloti exo genes produced multimers of the succinoglycan repeat unit, as does the wild-type R. meliloti, the deletion mutant of Rhizobium sp. strain NGR234 containing the R. meliloti exo genes produced only the monomer. The deletion mutant therefore appeared to lack a function that affects the multiplicity of succinoglycan produced in the Rhizobium sp. strain NGR234 background. Although these hybrid strains produced succinoglycan, they were still able to induce the development of an organized nodule structure on L. leucocephala. The resulting nodules did not fix nitrogen, but they did contain infection threads and bacteroids within plant cells. This clearly demonstrated that a heterologous acidic exopolysaccharide structure was sufficient to enable nodule development to proceed beyond the developmental barrier imposed on mutants of Rhizobium sp. strain NGR234 that are unable to synthesize any acidic exopolysaccharide.     

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.     

Macroptilium atropurpureum (siratro) host specificity genes are linked to a nodD-like gene in the broad host range Rhizobium strain NGR234

Summary A 6.7 kb HindIII fragment from the Sym-plasmid of strain NGR234 was found to code a nodD-like gene flanked by two loci which were required for siratro host range. Transfer of the 6.7 kb fragment from NGR234 to R. trifolii strain ANU843 conferred extended host range ability to this strain on siratro plants but not to other plants normally nodulated by strain NGR234. Tn5 mutagenesis of the 6.7 kb fragment showed that insertions located into loci flanking the nodD-like gene abolished the extended host range phenotype. A hybridization probe spanning one of the host specificity loci was shown to hybridize to three specific bands in the NGR234 genome. Complementation and DNA hybridization data showed that the nodD-like gene of strain NGR234 was functionally similar to that in R. trifolii. The introduction to R. trifolii of the 6.7 kb HindIII fragment containing Tn5 insertions located in the nodD-like gene did not abolish the ability to extend the host range of R. trifolii to siratro plants. However, transfer of the 6.7 kb HindIII to R. trifolii derivatives containing Tn5 insertions into either nodA, B or C or other R. trifolii nod genes failed to confer siratro nodulation to these recipients. Reconstruction experiments showed that the 6.7 kb fragment from strain NGR234 and the 14 kb nodulation region of R. trifolii could induce the nodulation of siratro plants when introduced together into Sym-plasmid-cured Rhizobium strains.     

Extension of host range of Rhizobium leguminosarum bv. trifolii caused by point mutations in nodD that result in alterations in regulatory function and recognition of inducer molecules

The positive activation of several nodulation genes in strain ANU843 of Rhizobium leguminosarum biovar trifolii is mediated by the product of the nodD gene and by the interaction of NodD with plant-secreted inducer and anti-inducer compounds. We have mutagenized the nodD gene of strain ANU843 with nitrosoguanidine and have found that the ability of the mutated nodD products to interact with inducer and anti-inducer compounds is affected by the amino acid sequence in at least two key regions, including a novel area between amino acids 77 and 123. Several novel classes of mutants were recognized by phenotypic and molecular analysis of the mutant nodD genes. Classes 1 and 4 mutants were able to induce nodA expression independently of the addition of inducer and anti-inducer compounds and were unable to mediate autoregulation of the nodD gene. Classes 2 and 3 mutants retained several properties of the wild-type nodD, including the ability to interact with inducer and anti-inducer compounds and the capacity to autoregulate nodD expression. In addition, class 2 mutants showed an inducer-independent ability to mediate nodA expression to 10-fold higher levels over control strains. The class 3 mutant showed reactivity to compounds that had little or no inducing ability with the wild-type nodD. An alteration in NodD function was demonstrated with classes 2 and 3 mutants, which showed greatly enhanced ability to complement a Tn5-induced mutation in the nodD1 gene of strain NGR234 and to restore nodulation ability on the tropical legume siratro. Mutants of nodD possessing inducer-independent ability to activate nod gene expression (classes 1, 2, and 4) were capable of extending the host range of R. l. bv. trifolii to the nonlegume Parasponia. DNA sequence analysis showed that single base changes were responsible for the altered phenotypic properties of five of six mutants examined. Four of the six mutations affected amino acid residues in a putative receiver domain in the N-terminal end of the nodD protein.     

Induction of nitrogen-fixing nodules on clover requires only 32 kilobase pairs of DNA from the Rhizobium trifolii symbiosis plasmid.

Overlapping subclones from the Rhizobium trifolii symbiosis plasmid pRt843a were generated by using in vivo and in vitro methods. Subclones were assayed for symbiotic phenotype by introducing them into a derivative of R. trifolii ANU843 cured of its symbiosis plasmid and testing the transconjugant strains for the ability to induce nitrogen-fixing nodules on clover. One subclone spanning 32 kilobase pairs (kb) of DNA from pRt843a was found to restore nitrogen fixation ability. This subclone included all known nodulation genes of R. trifolii ANU843 and the nitrogenase structural genes nifHDK. In addition, regions homologous to fixABC, nifA, nifB, nifE, and nifN genes of other nitrogen-fixing bacteria were identified in this 32-kb subclone by DNA-DNA hybridization. Transposon mutagenesis of this subclone confirmed that regions containing these nif and fix genes were required for induction of nitrogen-fixing nodules on clover. In addition, a region located 5 kb downstream of the nifK gene was found to be required for induction of nitrogen-fixing nodules. No homology to known nif and fix genes could be detected in this latter region.     

Rhizobium meliloti nodD genes mediate host-specific activation of nodABC.

To differentiate among the roles of the three nodD genes of Rhizobium meliloti 1021, we studied the activation of a nodC-lacZ fusion by each of the three nodD genes in response to root exudates from several R. meliloti host plants and in response to the flavone luteolin. We found (i) that the nodD1 and nodD2 products (NodD1 and NodD2) responded differently to root exudates from a variety of hosts, (ii) that NodD1 but not NodD2 responded to luteolin, (iii) that NodD2 functioned synergistically with NodD1 or NodD3, (iv) that NodD2 interfered with NodD1-mediated activation of nodC-lacZ in response to luteolin, and (v) that a region adjacent to and upstream of nodD2 was required for NodD2-mediated activation of nodC-lacZ. We also studied the ability of each of the three R. meliloti nodD genes to complement nodD mutations in R. trifolii and Rhizobium sp. strain NGR234. We found (i) that nodD1 complemented an R. trifolii nodD mutation but not a Rhizobium sp. strain NGR234 nodD1 mutation and (ii) that R. meliloti nodD2 or nodD3 plus R. meliloti syrM complemented the nodD mutations in both R. trifolii and Rhizobium sp. strain NGR234. Finally, we determined the nucleotide sequence of the R. meliloti nodD2 gene and found that R. meliloti NodD1 and NodD2 are highly homologous except in the C-terminal region. Our results support the hypothesis that R. meliloti utilizes the three copies of nodD to optimize the interaction with each of its legume hosts.     

Involvement of Genes on a Megaplasmid in the Acid-Tolerant Phenotype of Rhizobium leguminosarum Biovar Trifolii

The acid-tolerant Rhizobium leguminosarum biovar trifolii strain ANU1173 exhibited several new phenotypes when cured of its symbiotic (Sym) plasmid and the second largest megaplasmid. Strain P22, which has lost these two plasmids, had reduced exopolysaccharide production and cell mobility on TY medium. The parent strain ANU1173 was able to grow easily in laboratory media at pH 4.5, whereas the derivative strain P22 was unable to grow in media at a pH of <4.7. The intracellular pH of strain ANU1173 was 6.8 when the external pH was 4.5. In contrast, strain P22 had an acidic intracellular pH of <6.4 when the external pH was <5.5. Strain P22 had a dramatically increased membrane permeability to protons and decreased proton extrusion activity. Analysis with sodium dodecyl sulfate-polyacrylamide gels showed that strain P22 lacked a slow-migrating lipopolysaccharide (LPS) banding group which was present in the parent strain. Mobilization of the second largest megaplasmid of strain ANU1173 back into strain P22 restored the altered LPS structure and physiological characteristics of strain P22. Mobilization of the Sym plasmid of strain ANU1173 into strain P22 showed that the second largest megaplasmid of strain ANU1173 was required for the establishment of nitrogen-fixing nodules on Trifolium repens and Trifolium subterraneum. Furthermore, an examination of a large number of specific exopolysaccharide- or LPS-deficient Rhizobium mutants did not show a positive correlation between exopolysaccharide or LPS synthesis and acid tolerance.     

Split-Root Assays Using Trifolium subterraneum Show that Rhizobium Infection Induces a Systemic Response That Can Inhibit Nodulation of Another Invasive Rhizobium Strain

Subterranean clover plants possessing two equally infectible and robust lateral root systems (“split roots”) were used in conjunction with several specific mutant strains (derived from Rhizobium trifolii ANU843) to investigate a systemic plant response induced by infective Rhizobium strains. This plant response controls and inhibits subsequent nodulation on the plant. When strain ANU843 was inoculated onto both root systems simultaneously or 24, 48, 72, or 96 h apart, an inhibitory response occurred which retarded nodulation on the root exposed to the delayed inoculum but only when the delay period between inocula was greater than 24 h. Equal numbers of nodules were generated on both roots when ANU843 was inoculated simultaneously or 24 h apart. The ability to infect subterranean clover plants was required to initiate the plant inhibitory response since preexposure of one root system to non-nodulating strains did not retard the ability of the wild-type strain to nodulate the opposing root system (even when the delay period was 96 h). Moreover, the use of specific Tn5-induced mutants subtly impaired in their ability to nodulate demonstrated that the plant could effectively and rapidly discriminate between infections initiated by either the parent or the mutant strains. When inoculated alone onto clover plants, these mutant strains were able to infect the most susceptible plant cells at the time of inoculation and induce nitrogen-fixing nodules. However, the separate but simultaneous inoculation on opposing root systems of the parent and the mutant strains resulted in the almost complete inhibition of the nodulation ability of the mutant strains. We concluded that the mutants were affected in their competitive ability, and this finding was reflected by poor nodule occupancy when the mutants were coinoculated with the parent strain onto a single root system. Thus the split-root system may form the basis of a simple screening method for the ranking of competitiveness of various rhizobia on small seeded legumes.     

豇豆根瘤菌NGR234和紫云英根瘤菌109感染紫云英的比较研究

利用光学和电子显微镜对紫云英根瘤菌菌株109和广宿主的快生型根瘤菌菌株NGR234感染温带型豆科植物紫云英进行了研究,结果表明根瘤菌感染紫云英是通过在根毛中形成侵染线的途径。电子显微镜研究揭示了固氮根瘤中细胞内侵染线的存在。接种二天后,首先可观察到根毛的卷曲或分枝。接种四至五天后,在每株植物卷曲的根毛中可看到侵染线。接种八至十天后的植株出现肉眼可见的根瘤。菌株NGR234能够在紫云英上诱导根毛的卷曲,侵染线和根瘤的形成,但所形成的根瘤却未能固氮,根瘤中无明显的类菌体区,但有少数包有细菌的侵染线。NGR234抗抗菌素的衍生菌均未能使紫云英结瘤。将NGR234的共生质粒转移至三叶草、苜蓿、豌豆、快生型大豆根瘤菌和农杆菌,亦未能使这些细菌获得紫云英上结瘤的能力。     

Two genes that regulate exopolysaccharide production in Rhizobium meliloti.

We describe a new Rhizobium meliloti gene, exoX, that regulates the synthesis of the exopolysaccharide, succinoglycan, exoX resembled the psi gene of R. leguminosarum bv. phaseoli and the exoX gene of Rhizobium sp. strain NGR234 in its ability to inhibit exopolysaccharide synthesis when present in multiple copies, exoX did not appear to regulate the expression of exoP. The effect of exoX was counterbalanced by another R. meliloti gene, exoF. exoF is equivalent to Rhizobium sp. strain NGR234 exoY and resembles R. leguminosarum bv. phaseoli pss2 in its mutant phenotype and in portions of its deduced amino acid sequence. The effect of exoF on the succinoglycan-inhibiting activity of exoX depended on the relative copy numbers of the two genes. exoX-lacZ fusions manifested threefold-higher beta-galactosidase activities in exoF backgrounds than in the wild-type background. exoX mutants produced increased levels of succinoglycan. However, the exoF gene was required for succinoglycan synthesis even in an exoX mutant background. exoF did not affect the expression of exoP. Strains containing multicopy exoX formed non-nitrogen-fixing nodules on alfalfa that resembled nodules formed by exo mutants defective in succinoglycan synthesis. exoX mutants formed nitrogen-fixing nodules, indicating that, if the inhibition of succinoglycan synthesis within the nodule is necessary for nitrogen fixation, then exoX is not required for this inhibition. We present indirect evidence that succinoglycan synthesis within the nodule is not necessary for bacteroid function.     

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.     

Two C4-dicarboxylate transport systems in Rhizobium sp. NGR234: rhizobial dicarboxylate transport is essential for nitrogen fixation in tropical legume symbioses.

To investigate the role of dicarboxylate transport in nitrogen-fixing symbioses between Rhizobium and tropical legumes, we made a molecular genetic analysis of the bacterial transport system in Rhizobium sp. NGR234. This braod host range strain fixes nitrogen in association with evolutionarily divergent legumes. Two dicarboxylate transport systems were cloned from Rhizobium NGR234. One locus was chromosomally located, whereas the other was carried on the symbiotic plasmid (pSym) and contained a dctA carrier protein gene, which was analyzed in detail. Although the DNA and derived amino acid sequences of the structural gene were substantially homologous to that of R. meliloti, its promoter sequences was quite distinct, and the upstream sequence also exhibited no homology to dctB, which is found at this position in R. meliloti. A site-directed internal deletion mutant in dctA of NGR234 exhibited a (unique) exclusively symbiotic phenotype that could grow on dicarboxylates ex planta, but could not fix nitrogen in planta. This phenotype was found for tested host plants of NGR234 with either determinate- or indeterminate-type nodules, confirming for the first time that symbiosis-specific uptake of dicarboxylates is a prerequisite for nitrogen fixation in tropical legume symbioses.     

Studies on the Characterization of Root Morphology of White Clover Infected by Transconjugant of Rhizobium leguminosarum

White clover plants were inoculated with transconjugant strain' 290 which was obtained from introduction of host specific nodulation genes of wild-type Rhizobium trifolii strain ANU 843 to Rhizobium leguminosarum strain 300. The characterization of root morphology of white clover induced by the transconjugant was observed and compared to the plants induced by the parent strains. White clover started tO form a typical root hair curling inoculated with transconjugant strain 290 24h after inoculation, at 48h a part of cell wall of root hair was degradated, infection thread was observed in the infected root hair cell, cortical cell divisions occurred extensively. All these characterizations were similar to that infected by strain ANU 843. Plant inoculation test indicated that no nodule was formed when inoculated by R. leguminosarum strain 300, while plants nodulated when inoculated with transconjugant strain 290 as well as R. trifolii ANU 843. This suggests that introduction of host specific nodulation genes of R. trifolii results in conferring the nodulation ability of R. leguminosarum on white clover.     

Distribution of O-acetyl groups in the exopolysaccharide synthesized by Rhizobium leguminosarum strains is not determined by the Sym plasmid

The patterns of O-acetylation of the exopolysaccharide (EPS) from the Sym plasmid-cured derivatives of Rhizobium leguminosarum bv. trifolii strain LPR5, R. leguminosarum bv. trifolii strain ANU843 and R. leguminosarum bv. viciae strain 248 were determined by 1H and 13C NMR spectroscopy. Beside a site indicative of the chromosomal background, these strains have one site of O-acetylation in common, namely residue b of the repeating unit. The O-acetyl esterification pattern of EPS of the Sym plasmid-cured derivatives of strains LPR5, ANU843, and 248 was not altered by the introduction of a R. leguminosarum bv. viciae Sym plasmid or a R. leguminosarum bv. trifolii Sym plasmid. The induction of nod gene expression by growth of the bacteria in the presence of Vicia sativa plants or by the presence of the flavonoid naringenin, produced no significant changes in either amount or sites of O-acetyl substitution. Furthermore, no such changes were found in the EPS from a Rhizobium strain in which the nod genes are constitutively expressed. The substitution pattern of the exopolysaccharide from R. leguminosarum is, therefore, determined by the bacterial genome and is not influenced by genes present on the Sym plasmid. This conclusion is inconsistent with the suggestion of Philip-Hollingsworth et al. (Philip-Hollingsworth, S., Hollingsworth, R. I., Dazzo, F. B., Djordjevic, M. A., and Rolfe, B. G. (1989) J. Biol. Chem. 264, 5710-5714) that nod genes of R. leguminosarum bv. trifolii, by influencing the acetylation pattern of EPS, determine the host specificity of nodulation.     

Isolation and characterization of Azospirillum brasilense loci that correct Rhizobium meliloti exoB and exoC mutations.

The occurrence in Azospirillum brasilense of genes that code for exopolysaccharide (EPS) synthesis was investigated through complementation studies of Rhizobium meliloti Exo- mutants. These mutants are deficient in the synthesis of the major acidic EPS of Rhizobium species and form empty, non-nitrogen-fixing root nodules on alfalfa (J. A. Leigh, E. R. Signer, and G. C. Walker, Proc. Natl. Acad. Sci. USA 82:6231-6235, 1985). We demonstrated that the exoC mutation of R. meliloti could be corrected for EPS production by several cosmid clones of a clone bank of A. brasilense ATCC 29145. However, the EPS produced differed in structure from the wild-type R. meliloti EPS, and the symbiotic deficiency of the exoC mutation was not reversed by any of these cosmid clones. The exoB mutation could be corrected not only for EPS production but also for the ability to form nitrogen-fixing nodules on alfalfa by one particular cosmid clone of A. brasilense. Tn5 insertions in the cloned DNA were isolated and used to construct Azospirillum mutants with mutations in the corresponding loci by marker exchange. It was found that these mutants failed to produce the wild-type high-molecular-weight EPS, but instead produced EPSs of lower molecular weight.