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.     

Rhizobial NodL O-acetyl transferase and NodS N-methyl transferase functionally interfere in production of modified Nod factors

The products of the rhizobial nodulation genes are involved in the biosynthesis of lipochitin oligosaccharides (LCOs), which are host-specific signal molecules required for nodule formation. The presence of an O-acetyl group on C-6 of the nonreducing N-acetylglucosamine residue of LCOs is due to the enzymatic activity of NodL. Here we show that transfer of the nodL gene into four rhizobial species that all normally produce LCOs that are not modified on C-6 of the nonreducing terminal residue results in production of LCOs, the majority of which have an acetyl residue substituted on C-6. Surprisingly, in transconjugant strains of Mesorhizobium loti, Rhizobium etli, and Rhizobium tropici carrying nodL, such acetylation of LCOs prevents the endogenous nodS-dependent transfer of the N-methyl group that is found as a substituent of the acylated nitrogen atom. To study this interference between nodL and nodS, we have cloned the nodS gene of M. loti and used its product in in vitro experiments in combination with purified NodL protein. It has previously been shown that a chitooligosaccharide N deacetylated on the nonreducing terminus (the so-called NodBC metabolite) is the preferred substrate for NodS as well as for NodL. Here we show that the NodBC metabolite, acetylated by NodL, is not used by the NodS protein as a substrate while the NodL protein can acetylate the NodBC metabolite that has been methylated by NodS.     

Fucosylation and arabinosylation of Nod factors in Azorhizobium caulinodans: involvement of nolKnodZ as well as noeC and/or downstream genes

The DNA region downstream of the nodABCSUIJ operon of Azorhizobium caulinodans was further characterized and two new genes, nodZ and noeC were identified in the same operon. The A. caulinodans wild-type strain produces a population of Nod factors that, at the reducing end, are either unmodified or carry a D -arabinosyl and/or an L -fucosyl branch. Nod factors produced by Tn 5 -insertion mutants in nodZ noeC , and the separate nolK locus, were analysed by thin-layer chromatography and mass spectrometry. Fucosylation of Nod factors depended on both nodZ and nolK . Arabinosylation depended on noeC and/or downstream genes. Protein extracts of A. caulinodans contained an enzymatic activity for fucose transfer from GDP-fucose to chitooligosaccharides and to Nod factors. By mutant analysis and expression of nodZ in Escherichia coli , the fucosyltransferase activity was ascribed to the protein encoded by nodZ . In addition, a Nod factor fucosyltransferase activity, independent of nodZ or other known nod genes, was detected in A. caulinodans . Finally, on the basis of sequence similarity of the nolK gene product, and mass spectrometric analysis of Nod factors produced by a nolK mutant, we propose that this gene is involved in the synthesis of GDP-fucose.     

Responses of Azorhizobium caulinodans to cadmium stress

The symbiosis of Azorhizobium caulinodans and an annul legume Sesbania rostrata was recently found to be tolerant to cadmium pollution by an unknown mechanism. In this study, A. caulinodans ORS571 and ZY-20 showed much stronger tolerance to cadmium than a mutant ORS571-X15 and a common Rhizobium sp., with minimum inhibitory concentration values as high as 4 and 5 mM (versus 1 and 0.1 mM) on yeast extract mannitol agar medium, respectively. Although Cd uptake by all three strains of A. caulinodans were mostly from absorption rather than binding (both loosely or tightly) on cell surface, in resistant strains a higher portion of extractable Cd was bound on the cell surface vs. absorbed (about 1:2.5 ratio) compared to the sensitive mutant (about 1:35.1 ratio). These results suggest that certain level of metal exclusion by a permeability barrier was involved in the mechanism of resistance to Cd by A. caulinodans ORS571 and ZY-20. Over the 12-h period of cultivation in yeast extract mannitol agar medium with Cd addition, the Cd concentrations in the outer membrane and periplasm and spheroplast were the highest at the first 3 h, and declined steadily over time. The fact that Cd concentrations in spheroplast of all three strains were many folds higher than those in outer membrane and periplasm, suggests that extracellular sequestration was not the only mechanism of Cd tolerance in A. caulinodans. The decline of Cd concentrations was significantly faster and started earlier in strains ORS571 and ZY-20 than in ORS571-X15. This suggests a second, probably more substantial, mechanism involves active transport of the metal from the cell, e.g., some efflux system for maintaining homeostasis under cadmium stress.     

Review: Infection and nodulation signalling in Rhizobium-Phaseolus vulgaris symbiosis

During the Rhizobium–legume symbiosis, a mutual exchange of signalling molecules occurs. Distinct oligo- and polysaccharides are involved in nodule formation and rhizobial invasion. The common bean is a promiscuous host plant that can be nodulated by a wide range of rhizobia. Reviewing the literature on nodulation suggests that the Nod factor oligosaccharide backbone of bean-nodulating rhizobia does not require a specific attached group, except for the acyl chain at the non-reducing end. However, in Rhizobium strains that elicit nitrogen-fixing nodules on Phaseolus vulgaris and that produce methylated Nod factors, NodS mediated decorations are indispensable for invasion and/or subsequent nitrogen-fixation. Finally, we present a model that links the pathways for methylation and sulphation in nodule signalling and invasion processes.     

Regional Distribution of Methionine Adenosyltransferase in Rat Brain as Measured by a Rapid Radiochemical Method

Abstract: The distribution of methionine adenosyltransferase (MAT) in the CNS of the rat was studied by use of a rapid, sensitive and specific radiochemical method. The S -adenosyl-[methyl-¹⁴C] l -methionine ([¹⁴C]SAM) generated by adenosyl transfer from ATP to [methyl-¹⁴C] l -methionine is quantitated by use of a SAM-consuming transmethylation reaction. Catechol O -methyltransferase (COMT), prepared from rat liver, transfers the methyl-¹⁴C group of SAM to 3,4-dihydroxybenzoic acid. The ¹⁴C-labelled methylation products, vanillic acid and isovanillic acid, are separated from unreacted methionine by solvent extraction and quantitated by liquid scintillation counting. Compared to other methods of MAT determination, which include separation of generated SAM from methionine by ion-exchange chromatography, the assay described exhibited the same high degree of specificity and sensitivity but proved to be less time consuming. MAT activity was found to be uniformly distributed between various brain regions and the pituitary gland of adult male rats. In the pineal gland the enzyme activity is about tenfold higher.     

Nod factor requirements for efficient stem and root nodulation of the tropical legume Sesbania rostrata

Azorhizobium caulinodans ORS571 synthesizes mainly pentameric Nod factors with a household fatty acid, an N-methyl, and a 6-O-carbamoyl group at the nonreducing-terminal residue and with a d-arabinosyl, an l-fucosyl group, or both at the reducing-terminal residue. Nodulation on Sesbania rostrata was carried out with a set of bacterial mutants that produce well characterized Nod factor populations. Purified Nod factors were tested for their capacity to induce root hair formation and for their stability in an in vitro degradation assay with extracts of uninfected adventitious rootlets. The glycosylations increased synergistically the nodulation efficiency and the capacity to induce root hairs, and they protected the Nod factor against degradation. The d-arabinosyl group was more important than the l-fucosyl group for nodulation efficiency. Replacement of the 6-O-l-fucosyl group by a 6-O-sulfate ester did not affect Nod factor stability, but reduced nodulation efficiency, indicating that the l-fucosyl group may play a role in recognition. The 6-O-carbamoyl group contributes to nodulation efficiency, biological activity, and protection, but could be replaced by a 6-O-acetyl group for root nodulation. The results demonstrate that none of the studied substitutions is strictly required for triggering normal nodule formation. However, the nodulation efficiency was greatly determined by the synergistic presence of substitutions. Within the range tested, fluctuations of Nod factor amounts had little impact on the symbiotic phenotype.     

Methionine Recycling in Brain: A Role for Folates and Vitamin B-12

Abstract: The recycling of methionine via homocysteine was measured in vivo in brain. After constant intravenous infusions (5 h) of both [³H-methyl] methionine and [³⁵S]methionine into rats, the ratios of [³H-methyl]methionine to [³⁵S]methionine in liver, brain and plasma were determined, Similar experiments were performed in rabbits, except that the [³H-methyl]- and [³S]methionine were injected intraventricularly. If the methyl group of methionine was removed with the formation of homocysteine and then replaced by another (unlabeled) methyl group, the specific activity of the [³H-methyl]methionine would decrease more than that of [³⁵S]methionine; i.e., the ratio of [³H-methyl]- to [³⁵S]methionine in the tissue would decline. The results showed that the ratios of [³H-methyl]- to [³⁵S]methionine in liver and brain were less than the same ratio in plasma in the rats. The comparable ratios in the brain and CSF of rabbits were less than the ratio in the injectate. Since brain contains only one enzyme capable of remethylating homocysteine to methionine, the vitamin B-12–dependent methyltetrahydrofolate-homocysteine methyltransferase (EC 2.1.1.13), our results for methionine recycling via homocysteine in brain strongly support the activity of this enzyme in brain in vivo.     

Azorhizobium caulinodans ORS571 colonizes the xylem of Arabidopsis thaliana

Improved conditions were used for the aseptic growth of Arabidopsis thaliana to investigate whether xylem colonization of A. thaliana by Azorhizobium caulinodans ORS571 might occur. When seedlings were inoculated with ORS571 (pXLGD4) tagged with the lacZ reporter gene, nearly all of the plants showed blue regions of ORS571 colonization at lateral root cracks (LRC). The flavonoids naringenin and liquiritigenin significantly stimulated colonization of LRC by ORS571. Blue bands of ORS571 (pXLGD4) bacteria were observed histochemically in the xylem of intact roots of inoculated plants. Detailed microscopic analysis of sections of primary and lateral roots from inoculated A. thaliana confirmed xylem colonization. Xylem colonization also occurred with an ORS571 nodC mutant deficient in nodulation factors. There was no significant difference in the percentage of plants with xylem colonization or in the mean length of xylem colonized per plant between plants inoculated with either ORS571 (pXLGD4) or ORS571::nodC (pXLGD4), with or without naringenin.     

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.     

Biosynthesis of Polyamines in Mouse Brain: Effects of Methionine Sulfoximine and Adenosylhomocysteine

Abstract: This study examines the consequences on cerebral polyamine biosynthesis of increases and decreases in cerebral methylation. Increases were elicited by administering the convulsant agent methionine sulfoximine (MSO) and decreases by elevating in vivo the cerebral levels of the methylation inhibitor S -adenosyl-homocysteine. Following the intraventricular (i.vt.) administration of one of the two possible polyamine precursors, [1,4-¹⁴C]putrescine, the specific radioactivity (sra) of the newly formed [¹⁴C]spermidine remained unchanged. Conversely, after i.vt. l -[3,4-¹⁴C]methionine, the other polyamine precursor, significantly higher sra values for [¹⁴C]spermidine and [¹⁴C]spermine were recorded in the brains of the MSO-treated animals. [¹⁴C] S - adenosylmethionine in the brain of the MSO-treated animals was also more highly labeled following [1-¹⁴C]-methionine, indicating its accelerated formation relative to controls. We also investigated the effect of the administration of adenosine + homocysteine, a treatment that results in elevated brain adenosylhomocysteine levels, on polyamine biosynthesis from [3,4-¹⁴C]-methionine. The results of these experiments show both significantly lower sra values for [¹⁴C]spermidine and [¹⁴C]spermine and significantly higher than control endogenous methionine levels, a clear sign of the existence of a retardation in the conversion of methionine to polyamines under these conditions. In conclusion, the present study demonstrates that while interference with cerebral methylation results in significant alterations of the rate of formation of the methionine moiety of spermidine and spermine, it has no effect on the entry of the putrescine moiety into the two polyamine molecules.     

Nitrogen fixation in pure culture by rhizobia isolated from stem nodules of tropical Aeschynomene species

Abstract Asymbiotic nitrogenase activity was investigated in rhizobia strains isolated from stem and root nodules of severa Aeschynomene species. All isolated from stem-nodulating species were able to develop nitrogenase activity ex planta in the presence or in the absence of combined nitrogen, whereas root isolates from Aeschynomene species related to the cowpea group of plants showed little or no activity. Nitrogenase activity in soft-agar and in liquid cultures displayed by strains ORS310 and ORS322, isolated from stem nodules of A. indica and A. afraspera respectively, was of the same order of magnitude as that found for Azorhizobium caulinodans ORS571 and ten times higher than for Bradyrhizobium strain CB756. Furthermore, like A. caulinodans ORS571, strains ORS310 and ORS322 were able to use atmospheric nitrogen as sole nitrogen source for growth.     

The xylem of rice (Oryza sativa) is colonized by Azorhizobium caulinodans

Following inoculation with Azorhizobium caulinodans ORS571 (pXLGD4), lateral root development of rice and colonization of lateral root cracks by bacteria were shown to be stimulated by the flavonoid naringenin. Rice seedlings growing aseptically in the presence of naringenin were inoculated with ORS571 (pXLGD4), carrying the lacZ reporter gene. By microscopic analysis of sections of inoculated rice roots, it has been demonstrated that the xylem of rice roots can be colonized by Azorhizobium caulinodans. We discuss whether this colonization of the xylem of rice roots by azorhizobia could provide a suitable niche for endophytic nitrogen fixation.     

Rapid alkalinization in alfalfa root hairs in response to rhizobial lipochitooligosaccharide signals

Rhizobial lipochitooligosaccharides (Nod factors) function as symbiotic signals that trigger root hair deformations and cortical cell divisions on the roots of leguminous plants in a host-specific manner. By using pH-sensitive microelectrodes, it is shown that alfalfa root hair cells respond to Rhizobium meliloti Nod factors with a rapid intracellular alkalinization of 0.2–0.3 pH units. This alkalinization remained as long as the Nod factor was present, but slowly reversed after removal of the signal. The response was most sensitive to the sulfated tetrameric Nod factor, NodRm-IV(C16:2,S), which is morphogenic on the host plant alfalfa, suggesting a role in a signal transduction cascade. Non-sulfated Nod factor as well as chitooligosaccharides elicited a pH_c change only at elevated concentrations. The increase of PH_c in response to sulfated Nod factor was concomitant with a depolarization of the plasma membrane potential whereas the PH_c change in response to non-sulfated Nod factor occurred in the absence of membrane depolarization. In addition, whereas a first dose of sulfated Nod factor inhibited the subsequent response to a second dose of the same molecule, it did not significantly repress the activity of non-sulfated Nod factor. These results indicate that sulfated and non-sulfated Nod factors act independently and suggest the existence of two Nod signal perception systems, one transmitting the host-specific signal, the other representing an ancient reception system for a generic Nod factor structure.     

Crystal structures of NodS N-methyltransferase from Bradyrhizobium japonicum in ligand-free form and as SAH complex

NodS is an S-adenosyl-l-methionine (SAM)-dependent N-methyltransferase that is involved in the biosynthesis of Nod factor (NF) in rhizobia, which are bacterial symbionts of legume plants. NF is a modified chitooligosaccharide (COS) signal molecule that is recognized by the legume host, where it initiates symbiotic processes leading to atmospheric nitrogen fixation. We report the crystal structure of recombinant NodS protein from Bradyrhizobium japonicum, which infects lupine and serradella legumes. Two crystal forms—ligand-free NodS and NodS in complex with S-adenosyl-l-homocysteine, which is a by-product of the methylation reaction—were obtained, and their structures were refined to resolutions of 2.43 Å and 1.85 Å, respectively. Although the overall fold (consisting of a seven-stranded β-sheet flanked by layers of helices) is similar to those of other SAM-dependent methyltransferases, NodS has specific features reflecting the unique character of its oligosaccharide substrate. In particular, the N-terminal helix and its connecting loop get ordered upon SAM binding, thereby closing the methyl donor cavity and shaping a long surface canyon that is clearly the binding site for the acceptor molecule. Comparison of the two structural forms of NodS suggests that there are also other conformational changes taking place upon the binding of the donor substrate. As an enzyme that methylates a COS substrate, NodS is the first example among all SAM-dependent methyltransferases to have its three-dimensional structure elucidated. Gaining insight about how NodS binds its donor and acceptor substrates helps to better understand the mechanism of NodS activity and the basis of its functional difference in various rhizobia.     

Production of O-acetylated and sulfated chitooligosaccharides by recombinant Escherichia coli strains harboring different combinations of nod genes.

High cell density cultivation of recombinant Escherichia coli strains harboring the nodBC genes (encoding chitooligosaccharide synthase and chitooligosaccharide N-deacetylase, respectively) from Azorhizobium caulinodans has been previously described as a practical method for the preparation of gram-scale quantities of penta-N-acetyl-chitopentaose and tetra-N-acetylchitopentaose (Samain, E., Drouillard, S., Heyraud, A., Driguez, H., Geremia, R.A., 1997. Carbohydr. Res. 30, 235-242). We have now extended this method to the production of sulfated and O-acetylated derivatives of these two compounds by coexpressing nodC or nodBC with nodH and/or nodL that encode chitooligosaccharide sulfotransferase and chitooligosaccharide O-acetyltransferase, respectively. In addition, these substituted chitooligosaccharides were also obtained as tetramers by using nodC from Rhizobium meliloti instead of nodC from A. caulinodans. These compounds should be useful precursors for the preparation of Nod factor analogues by chemical modification.     

Coproporphyrin excretion by Azorhizobium caulinodans under micro-aerobic conditions

Azorhizobium caulinodans ORS571 was found to excrete moderate amounts of a fluorescent pigment into the culture medium in response to dissolved oxygen tensions below 1.0 kPa. The pigment was identified as coproporphyrin, on the basis of its optical and fluorescence spectra. FixLJ and fixK mutant derivatives of ORS571 were found to excrete 25-fold higher amounts of coproporphyrin under micro-aerobic conditions than the wild type strain. These observations suggest that A. caulinodans switches from an aerobic to an anaerobic coproporphyrinogen oxidase when the dissolved oxygen tension falls below 1.0 kPa and that the fixLJ and fixK genes are involved in the regulation of expression of the anaerobic coproporphyrinogen oxidase.     

Knockout of an azorhizobial dTDP-L-rhamnose synthase affects lipopolysaccharide and extracellular polysaccharide production and disables symbiosis with Sesbania rostrata.

A nonpolar mutation was made in the oac2 gene of Azorhizobium caulinodans. oac2 is an ortholog of the Salmonella typhimurium rfbD gene that encodes a dTDP-L-rhamnose synthase. The knockout of oac2 changed the lipopolysaccharide (LPS) pattern and affected the extracellular polysaccharide production but had no effect on bacterial hydrophobicity. Upon hot phenol extraction, the wild-type LPS partitioned in the phenol phase. The LPS fraction of ORS571-oac2 partitioned in the water phase and had a reduced rhamnose content and truncated LPS molecules on the basis of faster migration in detergent gel electrophoresis. Strain ORS571-oac2 induced ineffective nodule-like structures on Sesbania rostrata. There was no clear demarcation between central and peripheral tissues, and neither leghemoglobin nor bacteroids were present. Light and electron microscopy revealed that the mutant bacteria were retained in enlarged, thick-walled infection threads. Infection centers emitted a blue autofluorescence under UV light. The data indicate that rhamnose synthesis is important for the production of surface carbohydrates that are required to sustain the compatible interaction between A. caulinodans and S. rostrata.