An Azorhizobium caulinodans ORS571 locus involved in lipopolysaccharide production and nodule formation on Sesbania rostrata stems and roots.

Azorhizobium caulinodans ORS571 is able to nodulate roots and stems of the tropical legume Sesbania rostrata. An ORS571 Tn5 insertion mutant, strain ORS571-X15, had a rough colony morphology, was nonmotile, and showed clumping behavior on various media. When this pleiotropic mutant was inoculated on roots or stems of the host, no nodules developed (Nod-). Compared with the wild type, strain ORS571-X15 produced lipopolysaccharides (LPS) with an altered ladder pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, suggestive of a different O-antigen structure with a lower degree of polymerization. A cosmid clone, pRG20, that fully complemented all phenotypes of ORS571-X15 was isolated. With a 6-kb EcoRI subfragment of pRG20, clumping was relieved and nodulation was almost completely restored, but the strain was still nonmotile. LPS preparations from these complemented strains resembled the wild-type LPS, although minor quantitative and qualitative differences were evident. The sequence of the locus hit by the Tn5 in ORS571-X15 (the oac locus) revealed a striking homology with the rfb locus of Salmonella typhimurium, which is involved in O-antigen biosynthesis. The Tn5 insertion position was mapped to the oac3 gene, homologous to rfbA, encoding dTDP-D-glucose synthase. Biochemical assaying showed that ORS571-X15 is indeed defective in dTDP-D-glucose synthase activity, essential for the production of particular deoxyhexoses. Therefore, it was proposed that the O antigen of the mutant strain is devoid of such sugars.     

Identification of a new inducible nodulation gene in Azorhizobium caulinodans.

The narrow host range bacterial strain Azorhizobium caulinodans ORS571 induces the formation of nitrogen-fixing nodules on the root and stem of the tropical legume Sesbania rostrata. Here, a new flavonoid-inducible locus of ORS571 is described, locus 4. The locus was identified and isolated via the occurrence of particular sequences, the gamma and delta elements. These elements are reiterated in the ORS571 genome, linked to symbiotic loci. Sequencing of locus 4 showed the presence of an open reading frame (ORF6) that is flanked downstream by a gamma element and upstream by a delta element. The gamma element is approximately 180 bp in size, and shows homology to the insertion element ISRm3, an insertion sequence belonging to a distinct class of IS elements. The delta element is about 300 bp in size and has homology with repeated sequences found in other Rhizobiaceae. The ORF6 gene product shows a low, but significant homology to the mouse mastocytoma antigen P35B (Szikora et al., EMBO J. 9: 1041-1050, 1990) and to a class of NAD/NADP-binding sugar epimerase/dehydrogenases (Pissowotzki et al., Mol. Gen. Genet. 231: 113-213, 1991). Immediately upstream from ORF6, a nod box-related sequence is present, the arrangement of which is fully consistent with a recently presented model for the nod box structure (Goethals et al., Proc. Natl. Acad. Sci. USA 89: 1646-1650, 1992). Insertional inactivation of ORF6 did not affect the nodulation and fixation performance on S. rostrata. However, on S. formosa roots the nodulation kinetics of such a mutant was clearly affected (about 5 days delay). We propose to call this new symbiotic gene nolK.     

Rhizobial factors required for stem nodule maturation and maintenance in Sesbania rostrata-Azorhizobium caulinodans ORS571 symbiosis

The molecular and physiological mechanisms behind the maturation and maintenance of N(2)-fixing nodules during development of symbiosis between rhizobia and legumes still remain unclear, although the early events of symbiosis are relatively well understood. Azorhizobium caulinodans ORS571 is a microsymbiont of the tropical legume Sesbania rostrata, forming N(2)-fixing nodules not only on the roots but also on the stems. In this study, 10,080 transposon-inserted mutants of A. caulinodans ORS571 were individually inoculated onto the stems of S. rostrata, and those mutants that induced ineffective stem nodules, as displayed by halted development at various stages, were selected. From repeated observations on stem nodulation, 108 Tn5 mutants were selected and categorized into seven nodulation types based on size and N(2) fixation activity. Tn5 insertions of some mutants were found in the well-known nodulation, nitrogen fixation, and symbiosis-related genes, such as nod, nif, and fix, respectively, lipopolysaccharide synthesis-related genes, C(4) metabolism-related genes, and so on. However, other genes have not been reported to have roles in legume-rhizobium symbiosis. The list of newly identified symbiosis-related genes will present clues to aid in understanding the maturation and maintenance mechanisms of nodules.     

Photosynthetic Bradyrhizobium Sp. strain ORS285 synthesizes 2-O-methylfucosylated lipochitooligosaccharides for nod gene? Dependent interaction with Aeschynomene plants

Bradyrhizobium sp. strain ORS285 is a photosynthetic bacterium that forms nitrogen-fixing nodules on the roots and stems of tropical aquatic legumes of the Aeschynomene genus. The symbiotic interaction of Bradyrhizobium sp. strain ORS285 with certain Aeschynomene spp. depends on the presence of nodulation (nod) genes whereas the interaction with other species is nod gene independent. To study the nod gene-dependent molecular dialogue between Bradyrhizobium sp. strain ORS285 and Aeschynomene spp., we used a nodB-lacZ reporter strain to monitor the nod gene expression with various flavonoids. The flavanones liquiritigenin and naringenin were found to be the strongest inducers of nod gene expression. Chemical analysis of the culture supernatant of cells grown in the presence of naringenin showed that the major Nod factor produced by Bradyrhizobium sp. strain ORS285 is a modified chitin pentasaccharide molecule with a terminal N-C(18:1)-glucosamine and with a 2-O-methyl fucose linked to C-6 of the reducing glucosamine. In this respect, the Bradyrhizobium sp. strain ORS285 Nod factor is the same as the major Nod factor produced by the nonphotosynthetic Bradyrhizobium japonicum USDA110 that nodulates the roots of soybean. This suggests a classic nod gene-dependent molecular dialogue between Bradyrhizobium sp. strain ORS285 and certain Aeschynomene spp. This is supported by the fact that B. japonicum USDA110 is able to form N(2)-fixing nodules on both the roots and stems of Aeschynomene afraspera.     

Interactions of rhizobia with rice and wheat

Recently, evidence has been obtained that naturally occurring rhizobia, isolated from the nodules of non-legume Parasponia species and from some tropical legumes, are able to enter the roots of rice, wheat and maize at emerging lateral roots by crack entry. We have now investigated whether Azorhizobium caulinodans strain ORS571, which induces root and stem nodules on the tropical legume Sesbania rostrata as a result of crack entry invasion of emerging lateral roots, might also enter rice and wheat by a similar route. Following inoculation with ORS571 carrying a lacZ reporter gene, azorhizobia were observed microscopically within the cracks associated with emerging lateral roots of rice and wheat. A high proportion of inoculated rice and wheat plants had colonized lateral root cracks. The flavanone naringenin at 10 and 10 M stimulated significantly the colonization of lateral root cracks and also intercellular colonization of wheat roots. Naringenin does not appear to be acting as a carbon source and may act as a signal molecule for intercellular colonization of rice and wheat by ORS571 by a mechanism which is nod gene-independent, unlike nodule formation in Sesbania rostrata. The opportunity now arises to compare and to contrast the ability of Azorhizobium caulinodans with that of other rhizobia, such as Parasponia rhizobia, to intercellularly colonize the roots of non-legume crops.     

Molecular and functional characterization of the Azorhizobium caulinodans ORS571 hydrogenase gene cluster

In this work, we report the cloning and sequencing of the Azorhizobium caulinodans ORS571 hydrogenase gene cluster. Sequence analysis revealed the presence of 20 open reading frames hupTUVhypFhupSLCDFGHJK hypABhupRhypCDEhupE. The physical and genetic organization of A. caulinodans ORS571 hydrogenase system suggests a close relatedness to that of Rhodobacter capsulatus. In contrast to the latter species, a gene homologous to Rhizobium leguminosarum hupE was identified downstream of the hyp operon. A hupSL mutation drastically reduced the high levels of hydrogenase activity induced by the A. caulinodans ORS571 wild-type strain in symbiosis with Sesbania rostrata plants. However, no significant effects on dry weight and nitrogen content of S. rostrata plants inoculated with the hupSL mutant were observed in plant growth experiments.     

Cloning of an Azorhizobium caulinodans endoglucanase gene and analysis of its role in symbiosis.

Azorhizobium caulinodans ORS571, a symbiont of the tropical leguminous plant Sesbania rostrata, showed low, constitutive levels of endoglucanase (Egl) activity. A clone carrying the gene responsible for this phenotype was isolated via introduction of a genomic library into the wild-type strain and screening for transconjugants with enhanced Egl activity. By subcloning and expression in Escherichia coli, the Egl phenotype was allocated to a 3-kb EcoRI-BamHI fragment. However, sequence analysis showed the egl gene to be much larger, consisting of an open reading frame of 1,836 amino acids. Within the deduced polypeptide, three kinds of putative domains were identified: a catalytic domain, two cellulose-binding domains, and an eightfold reiterated motif. The catalytic domain belongs to the family A of cellulases. A C-terminal stretch of 100 amino acids was similar to family II cellulose-binding domains. A second copy of this domain occurred near the middle of the polypeptide, flanked by reiterated motifs. ORS571 mutants carrying a Tn5 insertion in the egl gene had lost the Egl activity. These mutants as well as Egl-overproducing strains showed a normal nodulation behavior, indistinguishable from wild-type nodulation on Sesbania rostrata under laboratory conditions.     

At least two nodD genes are necessary for efficient nodulation of alfalfa by Rhizobium meliloti

A Rhizobium meliloti DNA region (nodD1) involved in the regulation of other early nodulation genes has been delimited by directed Tn5 mutagenesis and its nucleotide sequence has been determined. The sequence data indicate a large open reading frame with opposite polarity to nodA, -B and -C, coding for a protein of 308 (or 311) amino acid residues. Tn5 insertion within the gene caused a delay in nodulation of Medicago sativa from four to seven days. Hybridization of nodD1 to total DNA of Rhizobium meliloti revealed two additional nodD sequences (nodD2 and nodD3) and both were localized on the megaplasmid pRme41b in the vicinity of the other nod genes. Genetic and DNA hybridization data, combined with nucleotide sequencing showed that nodD2 is a functional gene, while requirement of nodD3 for efficient nodulation of M. sativa could not be detected under our experimental conditions. The nodD2 gene product consists of 310 amino acid residues and shares 86.4% homology with the nodD1 protein. Single nodD2 mutants had the same nodulation phenotype as the nodD1 mutants, while a double nodD1-nodD2 mutant exhibited a more severe delay in nodulation. These results indicate that at least two functional copies of the regulatory gene nodD are necessary for the optimal expression of nodulation genes in R. meliloti.     

Competitive nodulation blocking of cv. Afghanistan pea is related to high levels of nodulation factors made by some strains of Rhizobium leguminosarum bv. viciae

Cultivar Afghanistan peas are resistant to nodulation by many strains of Rhizobium leguminosarum bv. viciae but are nodulated by strain TOM, which carries the host specificity gene nodX. Some strains that lack nodX can inhibit nodulation of cv. Afghanistan by strain TOM. We present evidence that this "competitive nodulation-blocking" (Cnb) phenotype may result from high levels of Nod factors inhibiting nodulation of cv. Afghanistan peas. The TOM nod gene region (including nodX) is cloned on pIJ1095, and strains (including TOM itself) carrying pIJ1095 nodulate cv. Afghanistan peas very poorly but can nodulate other varieties normally. The presence of pIJ1095, which causes increased levels of Nod factor production, correlates with Cnb. Nodulation of cv. Afghanistan by TOM is also inhibited by a cloned nodD gene that increases nod gene expression and Nod factor production. Nodulation of cv. Afghanistan can be stimulated if nodD on pIJ1095 is mutated, thus severely reducing the level of Nod factor produced. Repression of nod gene expression by nolR eliminates the Cnb phenotype and can stimulate nodulation of cv. Afghanistan. Addition of Nod factors to cv. Afghanistan roots strongly inhibits nodulation. The Cnb+ strains and added Nod factors inhibit infection thread initiation by strain TOM. The sym2A allele determines resistance of cv. Afghanistan to nodulation by strains of R. leguminosarum bv. viciae lacking nodX. We tested whether sym2A is involved in Cnb by using a pea line carrying the sym2A region introgressed from cv. Afghanistan; nodulation in the introgressed line was inhibited by Cnb+ strains. Therefore, the sym2A region has an effect on Cnb, although another locus (or loci) may contribute to the stronger Cnb seen in cv. Afghanistan.     

Interactions between bacterial diazotrophs and non-legume dicots: Arabidopsis thaliana as a model plant

When interactions between diazotrophic bacteria and non-legume plants are studied within the context of trying to extend biological nitrogen fixation to non-legume crops, an important first step is to establish reproducible internal colonization at high frequency of these plants. Using Azorhizobium caulinodans ORS571 (which induces stem and root nodules on the tropical legume Sesbania rostrata), tagged with a constitutively expressed lacZ reporter gene, we have studied the possibilities of internal colonization of the root system of the model dicot Arabidopsis thaliana. ORS571 was found to be able to enter A. thaliana roots after first colonizing lateral root cracks (LRCs), at the points of emergence of lateral roots. Cytological studies showed that after LRC colonization, bacteria moved into the intercellular space between the cortical and endodermal cell layers of roots. In our experimental conditions, this LRC and intercellular colonization are reproducible and occur at high frequency, although the level of colonization at each site is low. The flavonoids naringenin and daidzein, at low concentrations, were found to significantly stimulate (at the p=0.01 level) the frequency of LRC and intercellular colonization of A. thaliana roots by A. caulinodans. The role in colonization of the structural nodABC genes, as well as the regulatory gene nodD, was studied and it was found that both colonization and flavonoid stimulation of colonization are nod gene-independent. These systems should now enable the various genetic and physiological factors which are limiting both for rhizobial colonization and for endophytic nitrogen fixation in non-legumes, to be investigated. In particular, the use of A. thaliana, which has many advantages over other plants for molecular genetic studies, to study interactions between diazotrophic bacteria and non-legume dicots, should provide the means of identifying and understanding the mechanisms by which plant genes are involved in these interactions.     

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.     

Characterization of three genomic loci encoding Rhizobium sp. strain ORS571 N2 fixation genes.

Sixty-five independent, N2 fixation-defective (Nif-) vector insertion (Vi) mutants were selected, cloned, and mapped to the ORS571 genome. The recombinant Nif::Vi plasmids obtained in this way were used as DNA hybridization probes to isolate homologous phages from a genomic library of ORS571 constructed in lambda EMBL3. Genomic maps were drawn for three ORS571 Nif gene loci. Forty-five Nif::Vi mutants in genomic Nif locus 1 defined two gene clusters separated by 8 kilobase pairs (kb) of DNA. In the first cluster, 36 Nif::Vi mutants mapped to a 7-kb DNA segment that showed DNA homology with Klebsiella pneumoniae nifHDKE and encoded at least two Nif operons. In the other cluster, nine Nif::Vi mutants mapped to a 1.5-kb DNA segment that showed homology with K. pneumoniae and Rhizobium meliloti nifA; this DNA segment encoded a separate Nif operon. Fifteen Nif::Vi mutants mapped to a 3.5-kb DNA segment defined as Nif locus 2 and showed DNA homology with the R. meliloti P2 fixABC operon. Nif locus 2 carries a second nifH (nifH2) gene. Four Nif::Vi mutants mapped to a 2-kb DNA segment defined as Nif locus 3 and showed DNA homology with K. pneumoniae nifB. DNA from lambda Nif phages comprising all three genomic Nif loci was subcloned in plasmid vectors able to stably replicate in ORS571. These plasmid subclones were introduced into ORS571 strains carrying physically mapped Nif::Vi insertions, and genetic complementations were conducted. With the exception of certain mutants mapping to the nifDK genes, all mutants could be complemented to Nif+ when they carried plasmid subclones of defined genomic DNA regions. Conversely, most nifDK mutants behaved as pseudodominant alleles.