Phaseolus vulgaris recognizes Azorhizobium caulinodans Nod factors with a variety of chemical substituents.

Phaseolus vulgaris is a promiscuous host plant that can be nodulated by many different rhizobia representing a wide spectrum of Nod factors. In this study, we introduced the Rhizobium tropici CFN299 Nod factor sulfation genes nodHPQ into Azorhizobium caulinodans. The A. caulinodans transconjugants produce Nod factors that are mostly if not all sulfated and often with an arabinosyl residue as the reducing end glycosylation. Using A. caulinodans mutant strains, affected in reducing end decorations, and their respective transconjugants in a bean nodulation assay, we demonstrated that bean nodule induction efficiency, in decreasing order, is modulated by the Nod factor reducing end decorations fucose, arabinose or sulfate, and hydrogen.     

The NodA proteins of Rhizobium meliloti and Rhizobium tropici specify the N-acylation of Nod factors by different fatty acids

Rhizobia synthesize mono- N -acylated chitooligosaccharide signals, called Nod factors, that are required for the specific infection and nodulation of their legume hosts. The biosynthesis of Nod factors is under the control of nodulation ( nod ) genes, including the nodABC genes present in all rhizobial species. The N -acyl substitution can vary between species and can play a role in host specificity. In Rhizobium meliloti , an alfalfa symbiont, the acyl chain is a C16 unsaturated or a (ω-1) hydroxylated fatty acid, whereas in Rhizobium tropici , a bean symbiont, it is vaccenic acid (C18:1). We constructed R. meliloti derivatives having a non-polar deletion of nodA , and carrying a plasmid with either the R. meliloti or the R. tropici nodA gene. The strain with the R. tropici nodA gene produced Nod factors acylated by vaccenic acid, instead of the C16 unsaturated or hydroxylated fatty acids characteristic of R. meliloti Nod factors, and infected and nodulated alfalfa with a significant delay. These results show that NodA proteins of R. meliloti and R. tropici specify the N -acylation of Nod factors by different fatty acids, and that allelic variation of the common nodA gene can contribute to the determination of host range.     

Multiple copies of nodD in Rhizobium tropici CIAT899 and BR816.

Rhizobium tropici strains are able to nodulate a wide range of host plants: Phaseolus vulgaris, Leucaena spp., and Macroptilium atropurpureum. We studied the nodD regulatory gene for nodulation of two R. tropici strains: CIAT899, the reference R. tropici type IIb strain, and BR816, a heat-tolerant strain isolated from Leucaena leucocephala. A survey revealed several nodD-hybridizing DNA regions in both strains: five distinct regions in CIAT899 and four distinct regions in BR816. Induction experiments of a nodABC-uidA fusion in combination with different nodD-hybridizing fragments in the presence of root exudates of the different hosts indicate that one particular nodD copy contributes to nodulation gene induction far more than any other nodD copy present. The nucleotide sequences of both nodD genes are reported here and show significant homology to those of the nodD genes of other rhizobia and a Bradyrhizobium strain. A dendrogram based on the protein sequences of 15 different NodD proteins shows that the R. tropici NodD proteins are linked most closely to each other and then to the NodD of Rhizobium phaseoli 8002.     

The nodI gene product of Rhizobium leguminosarum is closely related to ATP-binding bacterial transport proteins; nucleotide sequence analysis of the nodI and nodJ genes

The nucleotide sequence of a 2-kb fragment immediately downstream of the nodABC genes of the Rhizobium leguminosarum symbiotic plasmid pRL1JI has been determined. Genes corresponding to the two open reading frames identified are named nodI and nodJ. Tn 5 insertions into these genes result in a "nodulation-delayed" phenotype. The predicted amino acid sequence of the nodI gene shows considerable homology to inner-membrane-located gene products involved in active transport systems in Escherichia coli and Salmonella typhimurium. The predicted product of the nodJ gene is very hydrophobic, suggesting that it may be an integral membrane protein.     

Rhizobium tropici chromosomal citrate synthase gene.

Two genes encoding citrate synthase, a key enzyme in the Krebs cycle, have been found in Rhizobium tropici. One of them is in the bacterial chromosome, while the other is in the symbiotic plasmid. We sequenced the chromosomal gene and found that it is very similar to the previously reported plasmidic gene sequence in its structural region but not in its regulatory region. The chromosomal gene is able to complement an Escherichia coli citrate synthase mutant. In R. tropici, a mutant in the chromosomal citrate synthase gene has a diminished citrate synthase activity (in free-living bacteria), a diminished nodulation capacity, and forms nitrogen-fixing nodules. In contrast, the citrate synthase double mutant forms ineffective nodules devoid of bacteroids and forms less nodules than the single chromosomal mutant. It is inferred that both genes are functional and required during the nodulation process in R. tropici.     

Different and new Nod factors produced by Rhizobium tropici CIAT899 following Na+ stress

The root nodule bacterium Rhizobium tropici strain CIAT899 is highly stress resistant. It grows under acid conditions, in large amounts of salt, and at high osmotic pressure. An earlier study reported a substantial qualitative and quantitative effect of acid stress on the biosynthesis of Nod factors. The aim of the present work was to investigate the effect of high salt (NaCl) concentrations, another common stress factor, on Nod factor production. For this purpose, thin-layer chromatography, HPLC and MS analyses were carried out. The expression of nodulation genes was also studied using a nodP ∷ lacZ fusion. High concentrations of sodium enhanced nod gene expression and Nod factor biosynthesis. The effect is sodium specific because high potassium or chloride concentrations did not have this effect. Under salt stress conditions, 46 different Nod factors were identified in a CIAT899 culture, compared with 29 different Nod factors under control conditions. Only 15 Nod factor structures were common to both conditions. Under salt stress conditions, 14 different new Nod factor structures were identified that were not observed as being produced under neutral or acid conditions. The implications of our results are that stress has a great influence on Nod factor biosynthesis and that new, very interesting regulatory mechanisms, worth investigating, are involved in controlling Nod factor biosynthesis.     

Genes Essential for Nod Factor Production and Nodulation Are Located on a Symbiotic Amplicon (AMPRtrCFN299pc60) in Rhizobium tropici

Amplifiable DNA regions (amplicons) have been identified in the genome of Rhizobium etli. Here we report the isolation and molecular characterization of a symbiotic amplicon of Rhizobium tropici. To search for symbiotic amplicons, a cartridge containing a kanamycin resistance marker that responds to gene dosage and conditional origins of replication and transfer was inserted in the nodulation region of the symbiotic plasmid (pSym) of R. tropici CFN299. Derivatives harboring amplifications were selected by increasing the concentration of kanamycin in the cell culture. The amplified DNA region was mobilized into Escherichia coli and then into Agrobacterium tumefaciens. The 60-kb symbiotic amplicon, which we termed AMPRtrCFN299pc60, contains several nodulation and nitrogen fixation genes and is flanked by a novel insertion sequence ISRtr1. Amplification of AMPRtrCFN299pc60 through homologous recombination between ISRtr1 repeats increased the amount of Nod factors. Strikingly, the conjugal transfer of the amplicon into a plasmidless A. tumefaciens strain confers on the transconjugant the ability to produce R. tropici Nod factors and to nodulate Phaseolus vulgaris, indicating that R. tropici genes essential for the nodulation process are confined to an ampliable DNA region of the pSym.     

Evidence of an American origin for symbiosis-related genes in Rhizobium lusitanum

Randomly amplified polymorphic DNA (RAPD) analysis was used to investigate the diversity of 179 bean isolates recovered from six field sites in the Arcos de Valdevez region of northwestern Portugal. The isolates were divided into 6 groups based on the fingerprint patterns that were obtained. Representatives for each group were selected for sequence analysis of 4 chromosomal DNA regions. Five of the groups were placed within Rhizobium lusitanum, and the other group was placed within R. tropici type IIA. Therefore, the collection of Portuguese bean isolates was shown to include the two species R. lusitanum and R. tropici. In plant tests, the strains P1-7, P1-1, P1-2, and P1-16 of R. lusitanum nodulated and formed nitrogen-fixing symbioses both with Phaseolus vulgaris and Leucaena leucocephala. A methyltransferase-encoding nodS gene identical with the R. tropici locus that confers wide host range was detected in the strain P1-7 as well as 24 others identified as R. lusitanum. A methyltransferase-encoding nodS gene also was detected in the remaining isolates of R. lusitanum, but in this case the locus was that identified with the narrow-host-range R. etli. Representatives of isolates with the nodS of R. etli formed effective nitrogen-fixing symbioses with P. vulgaris and did not nodulate L. leucocephala. From sequence data of nodS, the R. lusitanum genes for symbiosis were placed within those of either R. tropici or R. etli. These results would support the suggestion that R. lusitanum was the recipient of the genes for symbiosis with beans from both R. tropici and R. etli.