(Received for publication, December 8, 1994)
From the
Phaseolus vulgaris (common bean) can be nodulated by different Rhizobium species. A new species has been recently proposed: Rhizobium etli. Following transcriptional activation of the bacterial nodulation genes using naringenin or bean seed exudate, we have isolated, purified, and characterized R. etli extracellular nodulation factors. They are chitopentameric compounds that are N-methyl-N-vaccenoylated at their non-reducing end. At position 6 of the reducing N-acetyl-D-glucosamine, they are 4-O-acetyl-L-fucosylated. Minor compounds bear a carbamate group on the terminal non-reducing saccharidic residue.
Azorhizobium, Bradyrhizobium, and Rhizobium, collectively referred to as Rhizobia, specifically trigger stem or root nodule organogenesis on leguminous plants. In these symbiotically elicited organs, the bacteria reduce atmospheric nitrogen to ammonia(1) .
During the infection process,
signal exchange between the two symbionts controls the specificity of
this interaction(2) . Legume roots secrete flavonoids that
regulate the expression of the nodulation genes of the bacteria (the nod and nol genes)(3) . In turn, these genes
are involved in the synthesis and excretion of the so-called nodulation
factors (Nod factors) ()(2, 4, 5) .
Nod factors are lipooligosaccharides that share the same common backbone: an oligochitin core bearing a fatty acid that N-acylates the non-reducing end. These factors may also carry a variety of substitutions that complete the backbone and are involved in the specificity of the interaction between the plant and the bacteria(6) . At picomolar to nanomolar concentrations, these molecules provoke the root hair deformation effect in nodulation-competent root hairs(2, 7, 8) . They also induce rapid membrane depolarization(9) , expression of early nodulins(10) , and mitosis in the root cortex(11, 12) , which, in some cases, leads to nodule organogenesis(13, 14, 15, 16) . Thus they appear to play a key role in the establishment of the nodule symbiosis.
Recognition of Nod factors by host plants is relatively specific, and the nature and position of the Nod factor substitutions are of crucial importance. It has been proposed that a way for a bacterium to exhibit a wide host range is to synthesize a large variety of Nod factors, suggesting that each variety of plant is specifically triggered by Nod factors possessing a similar set of substitutions. For example, all Nod factors from bacteria that nodulate soybeans possess a 2-O-methylfucosyl substitution(8, 12, 16, 17) . A 6-O-sulfate group together with an N-(2E)(9Z)-hexadienoyl substitution seems specific for nodulation of alfalfa(13, 18) . Rhizobium NGR234, which nodulates more than 60 different legumes, synthesizes a very complex mixture of Nod factors bearing an N-methyl group and zero, one, or two O-carbamoyl groups at the non-reducing end together with a 2-O-methylfucose moiety, which may or may not be substituted by acetate or sulfate(19) .
To examine whether a strict relationship between the structure of Nod factors secreted by bacteria and their host plant range exists for all symbiotic associations, we examined the Nod factors from two different bacterial species that nodulate the common bean (Phaseolus vulgaris). One species is Rhizobium tropici, from which the Nod factor structure has been described earlier(20) . The species that will be dealt with in the present paper is Rhizobium etli.
R. etli(21) , previously named Rhizobium leguminosarum bv. phaseoli type I, nodulates the common bean, as does R. tropici. However, the geographical origin and breadth of the host range of these two species are different. The former, isolated from Mexican soils, seems to have a narrow host range (21) . The latter, which originated in South America, has a broad specificity as it nodulates several tropical legumes and some trees (e.g.Leucaena)(22) . In this paper we show that Nod factors from R. etli do not share identical structures with those described for R. tropici and represent a new type of Nod factor.
Mass spectra were recorded on an AutoSpec instrument (VG Analytical, Manchester, U.K.) fitted with a cesium bombardment ion source. The matrix was a mixture of m-nitrobenzyl alcohol/glycerol (1:1, v/v) spiked either with 1% trichloroacetic acid in water or with a solution of sodium iodide (1 mg/ml). The location of double bond of fatty acid was determined by remote charge fragmentation of the carboxylate anions as described (25) using a capillary gas chromatograph coupling to fractionate the fatty acids as pentafluorobenzyl esters, followed by negative ion ionization and collision-activated dissociation mass-analyzed ion kinetic energy spectrometry.
GC-MS experiments
were performed on a Hewlett-Packard 5989A mass spectrometer in
electronic impact ionization mode. One- and two-dimensional correlation
spectroscopy H NMR spectra were measured on a
Brücker AC-300 spectrometer (Karlsruhe, Germany)
using 2 mg of sample dissolved in 0.5 ml of perdeuterated dimethyl
sulfoxide (Sigma).
Deacetylation of 1 mg of NodRe
factors was achieved by 1 ml of 0.5 N sodium methanolate in
methanol at 30 °C overnight. After acidification with acetic acid,
the evaporated residue was dissolved in 1 ml of water and applied to a
Sep-Pak C cartridge. After washing with water,
deacetylated NodRe factors were eluted with methanol.
Figure 1:
Comparative analytical C
HPLC chromatograms of the butanol extracts of the culture media of the
wild type R. etli strain CFN 42 and the pSym
strain CFN 2001.
Figure 2: A, positive ion fast atom bombardment mass spectrum of the NodRe factors. Molecular ions are cationizated with the sodium ion (NaI doped matrix). B, positive ion fast atom bombardment mass-analyzed ion kinetic energy spectrum of the protonated ion at m/z 1458.
When NodRe factors were treated with 0.5 N sodium methanolate prior to analysis, the fast atom
bombardment-mass spectrum in the positive ion mode showed
(M+H) ions at m/z 1416 and
1459. However, all fragmentations remained unchanged. Thus an acetate
group was present near the reducing end. The mass difference between
the first fragment ions (m/z 1049 or 1092) and the
(M+H)
ions corresponded to the loss of both L-fucose and N-acetyl-D-glucosamine.
The fragmentation series ended at m/z 440 (or m/z 483), which was attributed to oxenium ions from the first sugar residue at the non-reducing end. m/z 440 was attributed to a N-methyl-N-vaccenoyl-D-glucosamine (20) . m/z 483, which was 43 mass units higher, indicated the presence of an additional carbamoyl substitution. Thus, NodRe factors are a mixture of carbamoylated and non-carbamoylated molecules, the former representing one-quarter to one-third of the total molecules. All NodRe factors bore a L-fucose residue linked to the terminal reducing N-acetyl-D-glucosamine together with an additional O-acetyl group.
NaBD reduction followed by permethylation produced a single compound
with a molecular mass of 1684 Da as determined by fast atom
bombardment-mass spectrometry (Fig. 3). This result indicated
that the acetyl and carbamoyl (28) groups had been lost during
the chemical process and that all the hydroxyl and amide groups had
been methylated.
Figure 3:
Positive ion fast atom bombardment mass
spectrum of the NaBD-reduced and permethylated NodRe
factors. The molecular ion is cationizated with the sodium ion (NaI
doped matrix).
The partially methylated monosaccharides were
reduced with NaBH and peracetylated. The partially
methylated alditol acetates were then analyzed by GC-MS. Four compounds
were identified:
1,5-di-O-acetyl-3,4,6-tri-O-methyl-N-acetyl-N-methylglucosaminitol
derived from the glucosamine residue at the non-reducing end of the
molecules;
1,4,5-tri-O-acetyl-3,6-di-O-methyl-N-acetyl-N-methylglucosaminitol
arising from the three internal residues;
4,6-di-O-acetyl-1,3,5-tri-O-methyl-N-acetyl-N-methylglucosaminitol
produced from the reducing glucosaminyl residue; and
1,5-di-O-acetyl 2,3,4-tri-O-methylfucositol provided
by the L-fucose residue. Therefore, this study confirmed the
(1
4) glycosidic linkages between the five N-acetyl-D-glucosamine residues of the NodRe factors
and indicated a (1
6) glycosidic linkage between the L-fucose residue and the reducing N-acetyl-D-glucosamine.
Figure 4:
Two-dimensional homonu-clear (H-
H) NMR spectrum of the NodRe
factors.
For the D-glucosamine residues, (J
= 3.5 Hz at the
reducing end) and
(J
= 9
Hz) anomeric configurations were characterized. Other signals,
classically described for the NMR spectra of nodulation factors, were
also identified (upfield signals not shown).
The wild type strain (CFN 42) of R. etli produced
enough Nod factors to enable structural studies without the need of
amplifying the Nod factors production by genetic engineering. The
structural characteristics of the R. etli nodulation factors
are as follows: (i) they have a chitopentameric core; (ii) a
4-O-acetyl--L-fucose residue is linked to carbon
6 of the reducing N-acetyl-D-glucosaminyl unit; (iii)
the glucosaminyl residue at the non-reducing end of the molecules is N-methyl-N-vaccenoyl substituted; and (iv)
one-quarter to one-third of these symbiotic signals is O-carbamoylated at their non-reducing end. According to the
nomenclature proposed by Roche and co-workers(31) , we name
these factors NodRe-V (Me, Ac-Fuc) and NodRe-V (Carb, Me, Ac-Fuc),
respectively (Fig. 5A).
Figure 5: A, structural features of the NodRe-V (Me, Ac-Fuc) and NodRe-V (Carb, Me, Ac-Fuc) factors. B, structure of the NodRt-V (Me, S) and NodRt-V (Me) factors.
The structure of NodRe
factors represents a new variation on the Nod factors structure. L-Fucose, as a substituent of chitooligomers, has been
previously found as a minor component of Nod factors from Rhizobium
fredii(8) and Bradyrhizobium
elkanii(16) . However, none of these structures bears an
additional acetyl group on L-fucose. An L-fucose ring
with an acetyl group on O-4 has been found in Rhizobium NGR234, but an O-methyl group was also present on fucosyl
O-2(19) . Carbamoyl and N-methyl groups on the
non-reducing end are more common because they have been found in Rhizobium NGR234(19) , Azorhizobium caulinodans(15) , Bradyrhizobium japonicum(17) , and B. elkanii(16) . The precise location of the carbamoyl
group needs a sufficient amount of material to perform heteronuclear H-
C bidimensional NMR. When we tried to
cultivate R. etli on a larger scale (20 liters) to purify
enough NodRe factors for this analysis, we found that the proportion of
carbamoylated molecules was lower than in the first experiments, and
thus the position of the carbamoyl group could not be reliably
assigned.
Common bean is also nodulated by R. tropici, a
bacterium with a broader host range than R. etli. When
comparing Nod factors from both bacteria, no common substituents can be
found as NodRt factors are non-fucosylated, non-carbamoylated molecules (20) . Instead, part of them possesses a sulfate group on O-6
of the reducing N-acetyl-D-glucosamine. It has been
found that these sulfated molecules are able to elicit root nodule
organogenesis on bean at 10 to 10
M(14) .
Purified Nod factors from R. etli were also found to elicit root nodules on bean in the same
concentration range as sulfate-containing NodRt factors. ()When cultivating R. etli using bean extracts as nod gene inducers, no change in the NodRe factors pattern was
found. In particular, no NodRe factors with a sulfate group could be
detected by sodium [
S]sulfate labeling.
It thus appears that bean is able to be nodulated by two rhizobial species that seem to produce Nod factors with structurally different substitutions.
However, purified fractions of Nod factors from either R. tropici or R. etli seemed to be active on bean at concentrations higher than those used to induce nodules on alfalfa by NodRm factors, for example. It remains possible that some sulfated components, being under the detection level, still contaminate the fucosylated fractions from NodRe factors, that fucosylated molecules are present in R. tropici, or that very low proportions of another common component are present in all tested fractions.
With reversed-phase HPLC, sulfated factors of R. tropici and acetylfucosylated factors of R. etli have very different retention volumes. Thus cross-contaminations with an identical component seemed unlikely. However, because reversed-phase HPLC separations are very sensitive to the hydrophilic/hydrophobic balance of the analytes, it cannot be excluded that a minor structural variation having a weak effect on the activity (such as the acyl chain length) may induce a dramatic effect on the HPLC retention volumes and thus permit the elution of analogs of cross-contaminants in the different fractions.
We are currently reinvestigating both strains to search for impurities that could be a common signal for bean nodulation.