Bradyrhizobium sp. Strains That Nodulate the Leguminous Tree Acacia albida Produce Fucosylated and Partially Sulfated Nod Factors

Abstract

We determined the structures of Nod factors produced by six different Bradyrhizobium sp. strains nodulating the legume tree Acacia albida (syn. Faidherbia albida). Compounds from all strains were found to be similar, i.e., O-carbamoylated and substituted by an often sulfated methyl fucose and different from compounds produced by Rhizobium-Mesorhizobium-Sinorhizobium strains nodulating other species of the Acaciae tribe.

Lipo-chitooligosaccharide Nod factors (NFs) synthesized by rhizobia act as signal molecules in the nodulation of specific legume hosts (23). NFs generally consist of four or five glucosamine residues that are N acylated at the nonreducing end and carry other substitutions on various glucosamine residues (7). Each rhizobial species (or biovar) has a defined host range and produces a set of NFs with specific structural features involved in host-range determination. Acacia species can be classified in three groups according to their ability to be nodulated in the field by fast-growing rhizobia of the Rhizobium-Sinorhizobium-Mesorhizobium branch (Acacia senegal, Acacia raddiana, and Acacia cyanophylla) (5, 6, 13, 17), by Bradyrhizobium (Acacia albida, Acacia mangium, and Acacia auriculiformis) (10, 12) or by both types of rhizobia (Acacia seyal) (8). NFs of diverse Acacia nodulating fast-growing rhizobia, Sinorhizobium terangae bv. acaciae, Mesorhizobium plurifarium, Rhizobium sp. GRH2, and Rhizobium tropici, have been characterized and shown to be structurally very close (11, 14, 15, 19). In particular, these molecules are not substituted by a glycosyl group, but they are mainly sulfated at the reducing end. NFs from Acacia bradyrhizobia have not yet been identified. However, fucosylated NF production seems to be a common feature of Bradyrhizobium (3, 22), thus raising the question as to whether rhizobia of the Rhizobium branch and Bradyrhizobium have evolved a similar or a different strategy, i.e., have developed similar or different NFs to nodulate Acacia species. Over the course of our studies on NF diversity in relation to host legume and bacterial taxonomy, we therefore examined structures produced by six genetically different Bradyrhizobium strains isolated from the leguminous tree A. albida. A. albida, recently reclassified as Faidherbia albida within the Acaciae tribe (18), is highly valuable in agroforestry for its soil improvement potential and as a source of wood and aerial forage (10).

Strains and NF production and purification.

A collection of A. albida nodulating strains was isolated in Sahelian and Sudano-Guinean areas (10) and was taxonomically characterized using both phenotypic (sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] of whole protein extracts) and genotypic approaches. The isolates were shown to mainly belong to six protein electrophoretic clusters (clusters 1, 3, 4, 6, 7, and 8) belonging to the Bradyrhizobium rRNA branch (9).

To evaluate NF diversity among A. albida bradyrhizobia and further select strains producing different molecules, 48 strains from the six SDS-PAGE clusters were analyzed by the radio thin-layer chromatography (TLC) method (24). All strains were maintained on yeast extract mannitol medium (26). TLC detection of NFs was performed as previously described (16), with the following modifications: (i) cells were induced with 2 μM daidzein (strains of cluster 4) or 2 μM genistein (other strains), since these flavonoids were identified as major inducers of Bradyrhizobium japonicum nod genes (1), and (ii) two types of TLC plates were used, silica plates developed with butanol-acetic acid-water (60:20:20) and reverse phase (RP) plates developed with acetonitrile-water (50:50). TLC analysis using silica plates showed that about 60% of the strains secreted genistein- or daidzein-induced products in sufficient amounts to be detected and that all strains produced sulfated molecules. Whereas silica plates gave similar profiles, different patterns were observed on RP-TLC plates. Strains from cluster 1 produced a continuum of labeled spots with or without induction and were not selected for structural studies. The following representative strains were selected for NF structural characterization: ORS135 and ORS166 from cluster 3 (two or three inducible spots), ORS170 from cluster 6 (one inducible spot), ORS138 from cluster 7 (five to six inducible spots), ORS130 and ORS122 from cluster 8 (five to six inducible spots).

These strains were cultured in 2-liter Erlenmeyer flasks filled with 600 ml of V medium (16) containing tetracycline (0.5 mg/liter) and 2 μM genistein as the inducer, at 30°C, until an A₆₀₀ of 0.8 to 1.0 was reached. Cells were filtered off and the cell-free medium was passed over an Amberlite XAD4 column. NFs were eluted with methanol. They were first purified on an open octadecyl-silica column that was eluted with a water-acetonitrile mixture (1:1) and then separated on a C₁₈ RP high-pressure liquid chromatography column eluted with a water-acetonitrile gradient, while UV absorbance was monitored at 206 nm (15, 19).

Molecular weight, backbone structures, and nature of the substituents.

Each UV-adsorbing peak from the high-pressure liquid chromatography separation was analyzed by positive-ion liquid secondary ion mass spectrometry (LSIMS) (15, 19), which allowed molecular mass determinations. Mass intervals of 203 U between fragment peaks due to in-source fragmentations revealed N-acetylglucosamine backbones (Fig. 1). When mixtures of chromatographically unresolved compounds were present, collision-induced spectra of each individual MH⁺ were recorded, showing the same characteristics as above. The presence of sulfate in several NFs was deduced from the loss of SO₃ (80 U) from MH⁺. Substitution of the reducing glucosaminyl end by a methyl-desoxyhexose was suggested by a mass loss of 160 U or 160 + 80 U in the spectra of nonsulfated or sulfated species, respectively.

FIG. 1.

Positive-ion LSIMS mass spectrum of the most abundant NF produced by the ORS138 strain. Fragmentations are indicated on the structure presented in the upper part of the figure. Abbreviations: Cb, CO-NH₂; Me, CH₃.

Acidic hydrolysis followed by derivatization of sugars as alditol acetates and identification by gas chromatography-mass spectrometry (GC-MS) was performed. The methyl-desoxyhexose was identified as 2-O-methyl fucose, and all NFs, except those from ORS135, liberated N-methyl glucosamine in addition to glucosamine.

Fatty acids were released by alkaline hydrolysis. They were identified by GC-MS-MS of the perfluorobenzyl esters (21). The most abundant were identified as common fatty acids, such as vaccenic, palmitic, and 9-hexadecenoic acids. NFs from ORS122, ORS130, and ORS138 strains also liberated a C-18 diunsaturated fatty acid with double bonds located at positions 11 and 15.

Taken together, the mass spectrometry and chemical analysis data indicated that the N-acylated glucosaminyl residue at the nonreducing end of NFs from ORS122, ORS130, and ORS138 strains was N methylated and O carbamoylated. No N-methyl group was detected in ORS135 NFs, and carbamoylation was incomplete. NFs from ORS170 and ORS166 were N methylated and mono- and bis-O carbamoylated. The reducing glucosaminyl residue was substituted by a partly sulfated 2-O-methyl fucose.

Location of substitutions.

Methylation analysis of borohydride-reduced NFs (4), followed by identification by GC-MS of the resulting partially methylated alditol acetates, showed that the 2-O-fucosyl moiety was sulfated on O-3. It was linked to the O-6 position of the reducing glucosaminyl residue. All glucosaminyl residues were 1,4 linked.

The carbamoyl group was located on the terminal nonreducing residue by mass spectrometry. Indeed, the collision-induced decomposition pattern of the oxonium ion of lower mass generated by in-source fragmentation (the so-called B1 ion) is characteristic of the position of substitutions (25). Abundant water loss and almost no elimination of carbamic acid or carbamic acid plus water indicated that the carbamoyl group was on O-6 in all strains exclusively producing monocarbamoylated NFs. Attempts to locate carbamoyl groups in monocarbamoylated NFs from strains synthesizing NFs with one and two carbamoyl groups suggested that these species were a mixture of O-3 and O-6 positional isomers. In addition, such spectra confirmed the presence or absence of an N-methyl group on the nonreducing residue. The results of these structural analyses are presented in Fig. 2.

FIG. 2.

Chemical structures of NFs produced by Bradyrhizobium strains isolated from Acacia albida. Components are listed in decreasing order of their molecular masses.

Plant tests.

To evaluate their symbiotic properties, a few strains were inoculated onto several legume species, and their symbiotic properties were evaluated as previously described (16) (Table 1). These strains generally nodulated wide-host-range legumes (Macroptilium atropurpureum, Aeschynomene afraspera, Aeschynomene elaphroxylon, Alysicarpus glumaceüs, Alysicarpus rugosus, Crotalaria retusa, Eriosema glomeratum, Indigofera microcarpa, and Vigna unguiculata), often forming nitrogen-fixing nodules, but failed to nodulate narrow-host-range legumes (Aeschynomene indica, Aeschynomene sensitiva, Sesbania rostrata, Sesbania pubescens, and Glycine max). They did not nodulate Acacia senegal. Nodulation of legumes known as hosts of fast-growing Acacia isolates, Leucaena leucocephala and Prosopis juliflora (16), was variable and ineffective. The strains tested nodulated Acacia mangium, but most formed nonfixing nodules. Conversely, the four A. mangium strains tested, 11C, 13C, AG3, and Bayel R (12), effectively nodulated A. albida (data not shown). No correlation between nodulation capacities and NF structural variations of the strains could be observed.

TABLE 1.

Nodulation of various legumes by Acacia albida Bradyrhizobium strains

Plant	Bradyrhizobium strainsa
	ORS122	ORS130	ORS138	ORS166	ORS170	ORS175	ORS178	ORS190	ORS191
Macroptilium atropurpureum	NT	E	NT	E	E	NT	NT	NT	E
Aeschynomene afraspera	NT	E	NT	E	E	NT	NT	NT	0
Aeschynomene elaphroxylon	NT	I	NT	E	E	NT	NT	NT	E
Alysicarpus glumaceüs	E	E	I	I	E	I	I	E	I
Alysicarpus rugosus	E	I	E	I	E	E	E	E	E
Crotalaria retusa	I	I	E	E	E	E	E	E	I
Eriosema glomeratum	E	E	I	I	E	E	E	E	I
Indigofera microcarpa	E	E	E	0	E	0	E	E	E
Vigna unguiculata	E	NT	E	I	E	NT	I	NT	I
Aeschynomene indica	NT	0	NT	0	0	NT	NT	NT	0
Aeschynomene sensitiva	NT	0	NT	0	0	NT	NT	NT	0
Sesbania rostrata	NT	0	NT	0	0	NT	NT	NT	0
Sesbania pubescens	NT	0	NT	0	0	NT	NT	NT	0
Glycine max	0	NT	0	0	0	NT	0	NT	0
Acacia senegal	0	0	0	0	0	0	0	0	0
Prosopis juliflora	I	0	I	I	I	0	0	I	I
Leucaena leucocephala	I	0	I	I	I	0	0	I	0
Acacia mangium	I	I	I	I	E	I	I	I	I

^a0, no nodulation; I, ineffective nodules; E, effective nodules, as estimated by plant visualization 45 days after inoculation; NT, not tested. 

Conclusion.

Bradyrhizobium strains nodulating A. albida produced NFs sharing the following common features. They were all chitopentamers, N acylated by common fatty acids at the nonreducing end, which is mostly carbamoylated on O-6, and N methylated. The reducing glucosamine was O-6 substituted by a 2-O-methyl fucose. The fucosyl moiety was partly sulfated on its O-3 position. Some variations were, however, observed: (i) one strain produced a mixture of noncarbamoylated species together with O-6 and O-3 mono- and bis-carbamoylated NFs, (ii) one strain produced NFs devoid of an N-methyl group, and (iii) NFs from several strains contained a new type of diunsaturated fatty acid, with double bonds located in positions 11 and 15, in addition to the common acyl chains.

Note that NF substitution by a sulfated methyl fucose is very uncommon and has been described so far only in Rhizobium sp. NGR 234 (20), a broad-host-range strain which is able to nodulate A. albida (C. Boivin, unpublished data). This structural feature could account for the relatively broad host range of A. albida strains. The presence of a fucose or a methyl fucose group in all rhizobia identified as Bradyrhizobium, e.g., B. japonicum, B. elkanii, and these six genetically different strains isolated from the wide-host-range legume A. albida, suggests that NF fucosylation is a common feature in Bradyrhizobium, even though it is also frequent in other rhizobia. The only exception possibly described so far is the slow-growing rhizobia isolated from Aspalathus (2). The three groups of Acacia defined according to their ability to be nodulated by fast-growing rhizobia, slow-growing rhizobia, or both correspond to three inoculation groups (reference 8 and our results). It may thus be hypothesized that Bradyrhizobium strains from other Acacia species produce NFs structurally close to those produced by A. albida strains. These molecules are similar to NFs produced by fast-growing Acacia strains in that they are all partly sulfated but not at the same position, and they are different in that they are fucosylated (11, 14, 15, 19). This difference between the two types of symbionts may reflect variable host-plant requirements for fucosyl substitution of NFs.