Synthesis Of Nod Factors Research Articles

A host-specific bacteria-to-plant signal molecule (Nod factor) enhances germination and early growth of diverse crop plants.

Lipo-chitooligosaccharides (LCOs), or Nod factors, are host-specific bacteria-to-plant signal molecules essential for the establishment of a successful N(2)-fixing legume-rhizobia symbiosis. At submicromolar concentrations Nod factors induce physiological changes in host and non-host plants. Here we show that the Nod factor Nod Bj V(C18:1,MeFuc) of Bradyrhizobium japonicum 532C enhances germination of a variety of economically important plants belonging to diverse botanical families: Zea mays, Oryza sativa (Poaceae), Beta vulgaris (Chenopodaceae), Glycine max, Phaseolus vulgaris (Fabaceae), and Gossypium hirsutum (Malvaceae), under laboratory, greenhouse and field conditions. Similar increases in germination were observed for filtrates of genistein-induced cultures of B. japonicum 532C, while non-induced B. japonicum, induced Bj 168 (a nodC mutant of B. japonicum deficient in Nod factor synthesis) or the pentamer of chitin did not invoke such responses, demonstrating the role of Nod factor in the observed effects. In addition, three out of four synthetic LCOs evaluated also promoted germination of corn, soybean and Arabidopsis thaliana seeds. LCO also enhanced the early growth of corn seedlings under greenhouse conditions. These findings suggest the possible use of LCOs for improved crop production.

Time-course of lipoxygenase, antioxidant enzyme activities and H2 O2 accumulation during the early stages of Rhizobium-legume symbiosis.

• The involvement of lipoxygenase and antioxidant enzyme activities as well as hydrogen peroxide (H2 O2 ) accumulation are reported during early infection steps in alfalfa (Medicago sativa) roots inoculated either with a wild type Sinorhizobium meliloti or with a mutant defective in Nod-factor synthesis (Nod C- ). • Compatibility between M. sativa and Rhizobium correlates, at least in part, with an increase in the activities of these enzymes, particularly catalase and lipoxygenase, during the preinfection period (up to 12h). The mutant strain, defective in Nod-factor biosynthesis, showed a decrease in all enzyme activities assayed, and an increase in H2 O2 accumulation. • Enhancement of scavenging activities for several reactive oxygen species correlated with compatibility of the S. meliloti-alfalfa symbiosis, whereas the Nod C- strain triggered a defence response. Nod factors were essential to suppress this response. • Increase in lipoxygenase and lipid hydroperoxide decomposing activities, observed during the first hours after inoculation with a compatible strain, could be related to tissue differentiation and/or the production of signal molecules involved in autoregulation of nodulation by the plant.

Nod genes and Nod signals and the evolution of the Rhizobium legume symbiosis.

The establishment of the nitrogen-fixing symbiosis between rhizobia and legumes requires an exchange of signals between the two partners. In response to flavonoids excreted by the host plant, rhizobia synthesize Nod factors (NFs) which elicit, at very low concentrations and in a specific manner, various symbiotic responses on the roots of the legume hosts. NFs from several rhizobial species have been characterized. They all are lipo-chitooligosaccharides, consisting of a backbone of generally four or five glucosamine residues N-acylated at the non-reducing end, and carrying various O-substituents. The N-acyl chain and the other substituents are important determinants of the rhizobial host specificity. A number of nodulation genes which specify the synthesis of NFs have been identified. All rhizobia, in spite of their diversity, possess conserved nodABC genes responsible for the synthesis of the N-acylated oligosaccharide core of NFs, which suggests that these genes are of a monophyletic origin. Other genes, the host specific nod genes, specify the substitutions of NFs. The central role of NFs and nod genes in the Rhizobium-legume symbiosis suggests that these factors could be used as molecular markers to study the evolution of this symbiosis. We have studied a number of NFs which are N-acylated by alpha,beta-unsaturated fatty acids. We found that the ability to synthesize such NFs does not correlate with taxonomic position of the rhizobia. However, all rhizobia that produce NFs such nodulate plants belonging to related tribes of legumes, the Trifolieae, Vicieae, and Galegeae, all of them being members of the so-called galegoid group. This suggests that the ability to recognize the NFs with alpha-beta-unsaturated fatty acids is limited to this group of legumes, and thus might have appeared only once in the course of legume evolution, in the galegoid phylum.

Identification and structure of the Rhizobium galegae common nodulation genes: evidence for horizontal gene transfer.

Rhizobia are soil bacteria able to fix atmospheric nitrogen in symbiosis with leguminous plants. In response to a signal cascade coded by genes of both symbiotic partners, a specific plant organ, the nodule, is formed. Rhizobial nodulation (nod) genes trigger nodule formation through the synthesis of Nod factors, a family of chitolipooligosaccharides that are specifically recognized by the host plant at the first stages of the nodulation process. Here, we present the organization and sequence of the common nod genes from Rhizobium galegae, a symbiotic member of the RHIZOBIACEAE: This species has an intriguing phylogenetic position, being symbiotic among pathogenic agrobacteria, which induce tumors instead of nodules in plant shoots or roots. This apparent incongruence raises special interest in the origin of the symbiotic apparatus of R. galegae. Our analysis of DNA sequence data indicated that the organization of the common nod gene region of R. galegae was similar to that of Sinorhizobium meliloti and Rhizobium leguminosarum, with nodIJ downstream of nodABC and the regulatory nodD gene closely linked to the common nod operon. Moreover, phylogenetic analyses of the nod gene sequences showed a close relationship especially between the common nodA sequences of R. galegae, S. meliloti, and R. leguminosarum biovars viciae and trifolii. This relationship in structure and sequence contrasts with the phylogeny based on 16S rRNA, which groups R. galegae close to agrobacteria and separate from most other rhizobia. The topology of the nodA tree was similar to that of the corresponding host plant tree. Taken together, these observations indicate that lateral nod gene transfer occurred from fast-growing rhizobia toward agrobacteria, after which the symbiotic apparatus evolved under host plant constraint.

Gene discovery through genomic sequencing of Brucella abortus.

Brucella abortus is the etiological agent of brucellosis, a disease that affects bovines and human. We generated DNA random sequences from the genome of B. abortus strain 2308 in order to characterize molecular targets that might be useful for developing immunological or chemotherapeutic strategies against this pathogen. The partial sequencing of 1,899 clones allowed the identification of 1,199 genomic sequence surveys (GSSs) with high homology (BLAST expect value < 10(-5)) to sequences deposited in the GenBank databases. Among them, 925 represent putative novel genes for the Brucella genus. Out of 925 nonredundant GSSs, 470 were classified in 15 categories based on cellular function. Seven hundred GSSs showed no significant database matches and remain available for further studies in order to identify their function. A high number of GSSs with homology to Agrobacterium tumefaciens and Rhizobium meliloti proteins were observed, thus confirming their close phylogenetic relationship. Among them, several GSSs showed high similarity with genes related to nodule nitrogen fixation, synthesis of nod factors, nodulation protein symbiotic plasmid, and nodule bacteroid differentiation. We have also identified several B. abortus homologs of virulence and pathogenesis genes from other pathogens, including a homolog to both the Shda gene from Salmonella enterica serovar Typhimurium and the AidA-1 gene from Escherichia coli. Other GSSs displayed significant homologies to genes encoding components of the type III and type IV secretion machineries, suggesting that Brucella might also have an active type III secretion machinery.

Isolation and characterization of new efficient and competitive bean ( Phaseolus vulgaris L.) rhizobia from Brazil

The common bean ( Phaseolus vulgaris L.) is widely cultivated in South and Central America and Africa, but inoculation with rhizobia often does not lead to a response in field experiments. A selection program was started in the State of Paraná, Brazil, in which three promising strains, PRF 35, PRF 54 and PRF 81, showing high rates of N 2 fixation, were competitive and tolerated high temperatures. The performance of the strains was also verified in four field experiments, where inoculation with PRF 81 allowed yield increases of up to 906 kg ha −1, compared with the non-inoculated (control) with a high population of native bean rhizobia. The high performance of PRF 81 was confirmed in several other field trials carried out in Brazil, leading to its recommendation for use in commercial Brazilian inoculants. PRF 34, PRF 54 and PRF 81 were further characterized and compared with four strains, representative of bean rhizobia species in an effort to define variables which could aid future selection programs. The Brazilian strains showed unique profiles of protein, lipopolysaccharide and PCR using specific (ERIC and REP) or arbitrary short primers. The DNA fingerprints obtained with specific or arbitrary primers showed that strains PRF 35 and PRF 54 were genetically very close, nevertheless, there were substantial differences between the strains in nodulation and N 2 fixation rates, as well as in the synthesis of Nod factors after induction with naringenin. The Brazilian strains showed Nod factor profiles similar to those of R. tropici type IIA CFN 299 and IIB CIAT 899 strains, and mixed characteristics of both types. That is, they were unable to grow in LB and PY minus Ca, as with type IIA, but were tolerant to high temperature, acidity, and had the same PCR product with Y1 and Y2 primers, as type IIB strain. The Brazilian strains showed mixed host range spectra between strain types IIA and IIB and, by the analysis of 17 fatty acids, strains PRF 35 and PRF 54 were grouped with CFN 299 and PRF 81 with CIAT 899. The performance of strain PRF 81 in field experiments indicates future potential for identification of new competitive and efficient R. tropici strains for tropical and subtropical areas.

High-resolution transcriptional analysis of the symbiotic plasmid of Rhizobium sp. NGR234.

Most of the bacterial genes involved in nodulation of legumes (nod, nol and noe ) as well as nitrogen fixation (nif and fix ) are carried on pNGR234a, the 536 kb symbiotic plasmid (pSym) of the broad-host-range Rhizobium sp. NGR234. Putative transcription regulators comprise 24 of the predicted 416 open reading frames (ORFs) contained on this replicon. Computational analyses identified 19 nod boxes and 16 conserved NifA-sigma54 regulatory sequences, which are thought to co-ordinate the expression of nodulation and nitrogen fixation genes respectively. To analyse transcription of all putative ORFs, the nucleotide sequence of pNGR234a was divided into 441 segments designed to represent all coding and intergenic regions. Each of these segments was amplified by polymerase chain reactions, transferred to filters and probed with radioactively labelled RNA. RNA was extracted from bacterial cultures grown under various experimental conditions, as well as from bacteroids of determinate and indeterminate nodules. Generally, genes involved in the synthesis of Nod factors (e.g. the three hsn loci) were induced rapidly after the addition of flavonoids, whereas others thought to act within the plant (e.g. those encoding the type III secretion system) responded more slowly. Many insertion (IS) and transposon (Tn)-like sequences were expressed strongly under all conditions tested, while a number of loci other than those known to encode nod, noe, nol, nif and fix genes were also transcribed in nodules. Many more diverse transcripts were found in bacteroids of determinate as opposed to indeterminate nodules.

Involvement of Salicylic Acid in the Establishment of the Rhizobium meliloti-Alfalfa Symbiosis

Inoculation of alfalfa plants with either incompatible Rhizobium or a Rhizobium mutant blocked in Nod factor synthesis led to an accumulation of salicylic acid in roots, in contrast to plants inoculated with a wild-type (compatible) R. meliloti strain. When salicylic acid was exogenously applied prior to inoculation of alfalfa plants with either purified Nod factor or compatible Rhizobium strains, a significant inhibition of nodule primordia formation and a reduction of the number of emerging nodules, respectively, as well as a delay in nodule visualization, were observed. These results suggest an involvement of Rhizobium-synthesized Nod factors in the inhibition of salicylic acid-mediated defense in legumes.

Isolation and sequencing of a second Rhizobium tropici CFN299 genetic locus that contains genes homologous to amino acid sulphate activation genes.

A Rhizobium tropici CFN299 DNA region, homologous to genes involved in Nod factor synthesis and amino acid sulphate activation, was isolated from a genome library. DNA sequence analysis revealed two open reading frames, orf1 and orf2. orf1 showed highest sequence similarity to the Escherichia coli cysD gene while orf2 is closely related to Rhizobium sp. N33 nodQ. However, the orf2 deduced peptide is 152 amino acids shorter than Rhizobium sp. N33 NodQ, and lacks the 3'-phosphoadenosine 5'-phosphosulphate-binding motif. A dendrogram based on the alignment of the deduced amino acid sequences of orf2/nodQ/cysN genes separated Escherichia coli cysN and orf2 from the nodQ cluster. Upstream of orf1, partial sequence analysis revealed the 3' part of an orf that is highly similar to E. coli cysH. The G + C content of orf1 and orf2 differs significantly from the G + C content of R. tropici symbiotic sulphate activation nodPQ genes. This data suggests that the isolated R. tropici CFN299 locus contains housekeeping genes for amino acid sulphate activation.

Production of nodulation factors by Rhizobium meliloti: fermentation, purification and characterization of glycolipids.

Lipooligosaccharides, synthesized by soil bacteria of the genera Rhizobium, are known to have multifunctional effects on a wide variety of plants as signal substances in symbiosis initiation, cell response elicitation and growth regulation. These so called nodulation (Nod-) factors represent interesting biotechnological products with respect to fundamental studies of symbiotic interactions as well as for potential applications. Therefore, a batch fermentation process on a scale of 30 l has been developed by means of the Rhizobium meliloti strain R.m. 1021 (pEK327) strongly overexpressing the genes for the synthesis of Nod factors. Induction by the flavone luteolin led to growth associated production of the lipooligosaccharides. Ultrafiltration was used for separating the biomass from the filtrate containing the extracellular Nod factors. Simultaneously, ultrafiltration reduced the amount of lipophilic substances, which would otherwise interfere with processes downstream. The second separation step consisted in adsorption on XAD-2, a nonspecific hydrophobic adsorptive resin. Adsorption of Nod factors was carried out by batch operation of a stirred tank. Desorption was performed by elution with methanol in a fixed bed column. A semi-preparative reversed phase HPLC (Polygoprep 100-30 C18) was chosen as the final purification step. The Nod factors were obtained after evaporation and lyophilization. Thus, about 600 mg of Nod factors were produced from 20 l of fermentation broth. The Nod factors produced by Rhizobium meliloti R.m. 1021 (pEK327) were identified by liquid secondary ion mass spectrometry and by reversed-phase HPLC as fluorescent derivatives of 2-aminobenzamide. The biological activity of the products was demonstrated by means of the root hair deformation (HAD-) assay.

NoIR controls expression of the Rhizobium meliloti nodulation genes involved in the core Nod factor synthesis

The synthesis of Rhizobium meliloti Nod signal molecules, encoded by the nod gene products, is finely regulated. A negative control of plasmid-borne nod gene expression is provided by the NolR repressor encoded by the chromosomal nolR gene. NolR was previously shown to downregulate the expression of the activator nodD1 gene and the common nodABC operon by binding to an overlapping region of the two promoters adjacent to the n1 nod-box (Kondorosi et al., 1989). We demonstrate here that NolR also controls the expression of two additional genes, nodD2 and nodM, but does not directly regulate the expression of the host-specific nod genes located downstream of the n2, n3 and n5 nod-boxes. Thus, the nod genes are differentially regulated by NolR and only those providing common nodulation functions, by determining the synthesis of the core Nod factor structure, are subjected to this negative regulation. Furthermore, NolR has a strong negative effect on the production of Nod metabolites, the level of which may serve as a fine-tuning mechanism for optimal nodulation, specific to host-plant genotypes. In addition, it elicits preferential synthesis of Nod factors carrying unsaturated C16 fatty acids. Expression of nolR was high both in the free-living bacterium and in the bacteroid and it was downregulated by its own product and by the nod gene inducer luteolin.

1995 and Beyond

We present the following hypothesis; that lipo­ oligochitin Nod-factors can act in an elicitor-Iike fash­ ion inducing, amongst other effects, a plant chitolytic enzyme, capable of hydrolysing the oligochitin chain of the Nod-factor. Decorative groups on the oligochitin chain, e.g. SUlphate, may confer partial resistance to hydrolysis upon particular Nod-factors. After entry into the plant, Nod-factor synthesis must be down­ regulated in order to avoid further, unwanted, ell· citation and the consequent abortion of the symbiosis. The plant-derived compounds Inhibiting the synthesis of bacterial Nod-factors are limiting in root tissue, leading to residual elicitation and the abortion of infec­ tion thread formation. Nod-gene anti-induction is, fur­ thermore, inactivated by both light and nitrate, thus contributing to the inhibition of nodulation under these conditions. In nitrogen-fixing nodules, the bacteroids are exposed to both nOd-gene inducing and repressing compounds. The slow accumulation of Nod-factors within the peribacteroid space eventually results in the elicitation of phytoalexin synthesis and nodule

The Rhizobium meliloti regulatory nodD3 and syrM genes control the synthesis of a particular class of nodulation factors N-acylated by (omega-1)-hydroxylated fatty acids.

Rhizobia elicit the formation of nitrogen-fixing nodules on specific legume hosts. Rhizobium meliloti nodulation (nod) genes control a signal exchange between the two symbiotic partners during infection and the early steps of nodulation. The regulatory nodD1, nodD2 and nodD3 genes are involved in the specific perception of different plant and environmental signals and activate the transcription of the nod operons. Once activated, the structural nod genes specify the synthesis of diffusible lipo-oligosaccharides, the Nod factors, which signal back to the plant. R. meliloti Nod factors are sulfated chito-oligosaccharides which are mono-N-acylated by unsaturated C16 fatty acids or by a series of C18 to C26 (omega-1)-hydroxylated fatty acids. In this paper we show that the regulatory nodD3 gene and another symbiotic regulatory gene, syrM, which mediate bacterial responses to plant signals that differ from those involving nodD1 and nodD2, determine the synthesis of Nod factors with different acyl moieties. nodD3 and syrM are required for the synthesis of Nod factors N-acylated by the (omega-1)-hydroxylated fatty acids. This regulatory mechanism makes possible the qualitative adaptation of bacterial Nod signal production to plant signals in the course of the symbiotic process.

Perception of Rhizobium nodulation factors by tomato cells and inactivation by root chitinases.

The bacterial genera Rhizobium and Bradyrhizobium, nitrogen-fixing symbionts of legumes, secrete specific lipo-chitooligosaccharides that induce the formation of nodules on their host plants. When preparations of such nodulation-inducing factors (Nod factors) were added to suspension-cultured tomato cells, a rapid and transient alkalinization of the culture medium occurred. Lipo-oligosaccharide preparations from Rhizobium or Bradyrhizobium treated with flavonoids, known inducers of Nod factor synthesis, were up to 100 times more potent in inducing alkalinization than the ones from untreated bacteria. The activity was absent from preparations of the mutant strain Rhizobium sp. NGR234 delta nodABC, unable to produce any Nod factors. Preparations of Nod factors from various bacteria as well as individual, highly purified Nod factors from Rhizobium sp. NGR(pA28) induced alkalinization in the tomato cell cultures at nanomolar concentrations. This demonstrates that Nod factors can be perceived by tomato, a nonhost of rhizobia. Using the alkalinization response as a sensitive bioassay, Nod factors were found to be inactivated by plant chitinases. Root chitinases purified from different legumes differed in their potential to inactivate differently substituted Nod factors produced by Rhizobium sp. NGR(pA28). This indicates that the specificity of the bacterium-host plant interaction may be due, at least in part, to differential inactivation of Nod factors by root chitinases.

Rhizobium NodB protein involved in nodulation signal synthesis is a chitooligosaccharide deacetylase.

The common nodulation genes nodABC are conserved in all rhizobia and are involved in synthesis of a lipooligosaccharide signal molecule. This bacterial signal consists of a chitooligosaccharide backbone, which carries at the nonreducing end a fatty acyl chain. The modified chitooligosaccharide molecule triggers development of nodules on the roots of the leguminous host plant. To elucidate the specific role of the NodB protein in nodulation factor synthesis, we have purified recombinant NodB and determined its biochemical role by direct assays. Our data show that the NodB protein of Rhizobium meliloti deacetylates the nonreducing N-acetylglucosamine residue of chitooligosaccharides. The monosaccharide N-acetylglucosamine is not deacetylated by NodB. In the pathway of Nod factor synthesis, deacetylation at the nonreducing end of the oligosaccharide backbone may be a necessary requirement for attachment of the fatty acyl chain.