INTRODUCTION

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Rhizobia are soil bacteria, now classified in several genera (e.g., Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, Azorhizobium), which form a symbiotic association with legume plants. The interaction between bacteria and plants results in the formation of nodules on the host plant roots, in which rhizobia fix atmospheric nitrogen. The association between rhizobia and legumes is specific: each rhizobial strain has a defined host range. For example, Mesorhizobium sp. strain N33, isolated from Oxytropis arctobia, also nodulates Astragalus alpinus and Onobrychis viciifolia (19, 26). In contrast, Sinorhizobium meliloti efficiently nodulates alfalfa (Medicago sativa) as well as Melilotus and Trigonella species. Mesorhizobium sp. strain N33 was isolated in the Canadian high arctic and is able to grow and fix nitrogen at temperatures as low as 5°C. In addition, it was shown that arctic rhizobia promoted better growth of O. viciifolia at low temperatures than did temperate strains (27). Thus, it might be valuable to extend the host range of arctic rhizobia to agronomically important legumes, in order to improve nitrogen fixation by these plants at low temperatures. It is therefore important to understand the molecular mechanisms controlling the nodulation specificity of arctic rhizobia.

Earlier work has shown that nodulation and host specificity in rhizobium-legume symbiosis are determined by signal exchanges between the bacteria and the host plant (21). Flavonoids excreted by the plant roots induce the expression of rhizobial nodulation (nod) genes. Most of these genes are involved in the biosynthesis and secretion of bacterial signals, the Nod factors, that can specifically induce symbiotic responses of the host plants. These responses include root hair deformation, division of root cortical cells and, in some instances, nodule formation (8, 32). The structures of Nod factors produced by several rhizobial species have been characterized. They are all lipochito-oligosaccharides (LCOs) consisting of -1,4-linked oligomers of three to five N-acetylglucosamine residues with an amide-linked acyl chain on the nonreducing terminal residue (8, 10, 25). In addition, the glucosamine residues can carry various substitutions. Nod factor specificity is determined by the nature of these substitutions and of the N-acyl chain.

A number of nodulation genes have been identified in Mesorhizobium sp. strain N33 (2, 3, 4). The nodABC genes, which are found in all rhizobial species, are responsible for the synthesis of the lipo-oligosaccharide core common to all Nod factors. In contrast to most rhizobial species, where nodABC belong to one operon, in Mesorhizobium sp. strain N33 nodA and nodBC belong to two different operons (2, 4). In addition to nodABC, several nod genes likely to be involved in Nod factor substitutions have been characterized (3, 4). The nodHPQ genes, which are also found in S. meliloti and Rhizobium tropici, have been shown to specify O sulfation of the Nod factor reducing end in these two species (11, 18, 29). The nodFE genes, which are also present in S. meliloti and R. leguminosarum, determine in these two species the synthesis of various polyunsaturated fatty acids with one or several double bounds conjugated to the carbonyl group (7, 34, 39). Therefore, one might expect the Mesorhizobium sp. strain N33 Nod factors to be sulfated and substituted by a fatty acid with conjugated double bonds. However, given the peculiar host range of this species, the Mesorhizobium sp. strain N33 Nod factors are likely to carry novel substitutions. In this paper, we describe the structure of the Mesorhizobium sp. strain N33 Nod factors. We show that they have original structures with substitutions never described before, and we study several features of their biosynthesis.

	MATERIALS AND METHODS

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Microbiological techniques. Bacterial strains and plasmids are described in Table 1. Mesorhizobium sp. strain N33 was grown in TY medium (33). The transfer of IncP and IncQ plasmids to Mesorhizobium sp. strain N33 was performed by triparental mating as described previously (1). Large-scale cultures were grown in Vincent medium (33) supplemented with 0.2% sodium glutamate and 0.1% sodium succinate as nitrogen and carbon sources, biotin at 0.5 µg ml¹, and other vitamins (Sigma MS vitamins) at 0.1 g liter¹. Formononetin (50 µM) or p-coumaric acid (100 µM) was used for the induction of nod gene expression.

DNA techniques. Plasmid pJC372 was constructed as follows. A lambda EMBL3 genomic bank from strain N33 (2) was screened using a N33 nodAFEG probe (4), allowing the isolation of phage 235-1. From this phage, we isolated a 4.8-kb BamHI-BglII fragment containing nodAFEG with 1.3 and 0.9 kb of 5'- and 3'-flanking sequences, respectively, including the nod box. This fragment was cloned in BamHI-BglII-digested LITMUS 28, amplified, and cloned in BamHI-digested pRK7813.

Nod factor purification. Nod factors were extracted from filtered culture supernatants by butanol extraction (30). Purification was initially performed by high-performance liquid chromatography (HPLC) with a semipreparative C₁₈ reverse-phase column (7.5 by 250 mm; Spherisorb ODS2; 5 µm) for 10 min in isocratic solvent A (water-acetonitrile, 80:20 [vol/vol]), followed by a linear gradient from solvent A to solvent B (100% acetonitrile) for 40 min at a flow rate of 1 ml min¹; the UV absorption at 206 and 220 nm was monitored. The fraction eluting between 36 and 62% acetonitrile was collected. More accurate purification was achieved with the same column and the same flow rate but with a 20-min linear gradient running from water-acetonitrile (70:30 [vol/vol]) containing 50 mM ammonium acetate (solvent A) to water-acetonitrile (50:50 [vol/vol]) containing 50 mM ammonium acetate (solvent B). Ammonium acetate and solvents from the collected fractions were removed by two successive lyophilizations.

Analytical methods. (i) Alkali hydrolysis of Nod factors and of bacteria. Nod factors were hydrolyzed with a 5 N KOH solution at 100°C for 2 h. The solution was acidified with 6 N HCl, and fatty acids were extracted with diethyl ether. Lyophilized bacteria were suspended in a 5 N KOH solution and stirred at 110°C for 4 h. After acidification, the solution was extracted with diethyl ether. The acidic components were extracted from the ether layer with a 2 N KOH solution. The aqueous phase was acidified, and the fatty acids were extracted with diethyl ether.

(ii) Derivatization of fatty acids. (a) Pentafluorobenzyl ester derivatives. Free fatty acids (less than 100 µg) were dissolved in a solution of 10 µl of anhydrous methanol and 50 µl of anhydrous acetonitrile. Two microliters of diisopropylamine and 2 µl of pentafluorobenzyl bromide were added. The reaction was complete after 1 h at room temperature. After total evaporation under nitrogen flux, the fatty acid derivatives were redissolved in dichloromethane.

(b) Methyl ester derivatives. Fatty acids dissolved in dichloromethane were methylated in situ by coinjecting them with 1 µl of trimethylsulfonium hydroxide (Macherey-Nagel, Hoerdt, Germany) into the gas chromatograph injector kept at 250°C.

(iii) Mass spectrometry (MS). An AUTOSPEC 6F sector instrument (Micromass, Altrincham, United Kingdom) with the EBE-EBE configuration was fitted with a cesium ion gun and a liquid secondary-ion MS (LSIMS) ion source. For sensitivity reasons, only the first half of the instrument was used. The energy of the Cs⁺ ions was 25 keV, and the accelerating voltage of the instrument was set to 8 kV. The 1:1 metanitrobenzyl alcohol-glycerol matrix was acidified with 1% trichloroacetic acid in water. Product ions resulting from decompositions in the first field free region were recorded in the constant B/E-linked scan mode.

(iv) NMR. Nuclear magnetic resonance (NMR) experiments were done with a Brüker AMX 500 spectrometer operating at an observation frequency of 500 MHz for ¹H. Data were recorded at 303 K. Samples were dissolved in a 3:7 D₂O-CD₃OD mixture. Methanol was used as an internal reference for chemical shift () determinations. Phase-sensitive ¹H-¹H-correlated spectroscopy (COSY) (²J, ³J, and long-range coupling) was recorded.

	RESULTS

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Mesorhizobium sp. strain N33 Nod factors are pentameric LCOs 6-O sulfated at the reducing end. Previous work had shown that the Mesorhizobium sp. strain N33 nodulation genes were induced by compounds such as formononetin, an isoflavanone, and p-coumaric acid, a phenylpropane derivative (2). In order to prepare Nod factors, 5 liters of Mesorhizobium sp. strain N33 was grown in the presence of formononetin. The culture supernatant was extracted with n-butanol, and the LCOs in the extract were purified as described in Materials and Methods. Final purification was performed by HPLC on a C₁₈ column with a water-acetonitrile gradient in the presence of ammonium acetate (Fig. 1), which allows better purification of sulfated species by reverse-phase HPLC. A total of 2.5 mg of Nod factors was obtained from a 5-liter culture. Nod factors were also extracted from a culture of Mesorhizobium sp. strain N33 induced by p-coumaric acid. Compounds identical to those obtained after induction by formononetin were obtained, as judged by the HPLC profile and MS analysis (data not shown).

View larger version (16K): [in this window] [in a new window]   FIG. 1.   HPLC profile of a Nod factor-enriched extract from a formononetin-induced N33 culture supernatant. Separation was performed in the presence of ammonium acetate as described in Materials and Methods, with monitoring of the absorption at 206 nm. See Table 2 for a description of the compounds found in fractions 1 to 4. No Nod factors were found in fraction 5.

View larger version (35K): [in this window] [in a new window]   FIG. 2.   LSIMS analysis of N33 Nod factors. (a) Spectrum of HPLC fraction 4, showing an MH+ ion at m/z 1,364.7 for the Nod factor identified as NodMN33 V (iso-C17:1, Ac, S). See Table 2 for Nod factor nomenclature. Sodium and potassium salt adducts are indicated by asterisks. (b) Product ion spectrum from dissociation of the ion at m/z 1,364 (constant B/E-linked scans). Intervals of 203 u between the fragment ions indicate N-acetylglucosamine residues, while the interval of 245 u indicates additional O acetylation of the glucosamine residue next to the nonreducing end. The section of panel b that was magnified (magnification of ×5) is indicated.

Mesorhizobium sp. strain N33 Nod factors are partly 6-O acetylated on the glucosamine residue next to the non reducing terminal glucosamine. The position of the acetyl group on the chito-oligosaccharide backbone was determined by measuring the mass intervals between consecutive ions of the B series (oxonium ions); these intervals corresponded to 203 u (N-acetylglucosamine residues), except for that between B₁ and B₂, which was equal to 245 u (Fig. 2). These results indicated that the acetyl group is located on the glucosamine residue next to the nonreducing terminal glucosamine. Location of the O-acetyl group on this residue was attempted by measuring the spontaneous decomposition of the B₂ ion. Either O-3 or O-6 acetylation is possible on an internal 1,4-linked N-acetylglucosamine residue. Studies performed previously on model compounds showed that an O-acetyl substitution at position 6 leads mostly to the elimination of water, whereas an O-acetyl substitution at position 3 is characterized by the loss of acetic acid (Fig. 3b) (38). The major decomposition product of the B₂ ion at m/z 657 was the B₁ ion at m/z 412, but the spectrum also showed the ion at m/z 639 resulting from the loss of water, whereas the elimination of acetic acid was undetectable (Fig. 3). Therefore, the acetyl group in N33 Nod factors is located at position 6. This assessment was confirmed by two-dimensional NMR (COSY) analysis (data not shown). The proton resonances were close to those observed previously for S. meliloti and R. leguminosarum bv. viciae Nod factors, which are both 6-O acetylated, although on another glucosaminyl residue (15, 26): H_6'4.43 ppm, dd, J = 12 and 2.5 Hz; H₆4.36 ppm, dd, J = 12 and 1.5 Hz; H₅4.16 ppm, m.

View larger version (25K): [in this window] [in a new window]   FIG. 3.   Location on O-6 (and not O-3) of the acetyl substituent in N33 Nod factors. (a) LSIMS of products from spontaneous dissociation (constant B/E-linked scans) of the B2 ion at m/z 657, generated in the LSIMS spectrum of the Nod factor shown in Fig. 2. Note the presence of an ion at m/z 639 resulting from the loss of water and the absence of an ion at m/z 597. The section of panel a that was magnified (magnification of ×10) is indicated. (b) Decomposition of oxonium ions (B ions) at m/z 246 from synthetic 3-O- and 6-O-acetyl-N-acetylglucosamine isomers, shown for comparison. Arrows indicate characteristic fragmentations.

Original saturated and ,-unsaturated methyl-branched acyl chain substitutions in N33 Nod factors. Calculations based on the masses of the B₁ fragment ions suggested that the terminal nonreducing glucosamine of N33 Nod factors was substituted either by an acyl chain with an uneven number of carbons (C₁₅ or C₁₇) or by a methyl group and an acyl chain with an uneven number of carbons (C₁₄ and C₁₆). To identify the acyl substituents, the Nod factor mixture was submitted to alkali hydrolysis. The liberated fatty acids were derivatized as perfluorobenzyl esters and analyzed by capillary GC-MS in the dissociative electron capture ionization mode associated with high-energy CID of the carboxylate anions (20) (Fig. 4). The major components were identified as saturated and ,-unsaturated methyl-branched fatty acids of the iso series. The presence of a methyl branch on the carbon atom next to the methyl end, characteristic of the iso series, was deduced from the greatly reduced loss of the C-2 neutral fragment (Fig. 4). The main saturated methyl-branched fatty acids were characterized as 13-methyl-tetradecanoic (iso-C_15:0) and 15-methyl-hexadecanoic (iso-C_17:0) acids. Monounsaturated methyl-branched fatty acids were also identified, the most abundant being ,-unsaturated 15-methyl-2-hexadecenoic acid (iso- C_17:1 ²). The position of the double bond in this compound was deduced from enhanced cleavage of the allylic bond, giving the ion at m/z 83 but not lower-mass fragments (20, 29). This main component was accompanied by two 15-methyl-3-hexadecenoic acids (assigned to the E and Z isomers), which probably resulted from alkali-induced isomerization of 15-methyl-2-hexadecenoic acid. Indeed, ¹H NMR studies of the whole mixture of Nod factors confirmed the above assumption, as no signal due to a double bond at position 3 could be detected. The iso-methyl group appeared as a doublet at 0.87 ppm, with J = 6.7 Hz, correlating with a CH proton at 1.53 ppm. The conjugated double bond in the E configuration induced three characteristic resonances: H₂ at 5.99 ppm, dt, J = 15 and 1.5 Hz; H₃ at 6.85 ppm, dt, J = 15 and 7 Hz; and H₄ at 2.12 ppm, m.

View larger version (29K): [in this window] [in a new window]   FIG. 4.   Characterization of fatty acids from a crude preparation of N33 Nod factors by negative-ion GC-MS of their perfluorobenzyl derivatives. (a) Total ion current (GC-MS profile). Peaks labeled by asterisks corresponded to fatty acids not characterized as Nod factor substituents by examination of the LSIMS mass spectra. This profile was recorded in the negative-ion electron capture mode. i, iso. (b) MS-MS profile (CID monitored ion kinetic energy spectrometry) of the carboxylate ion at m/z 267 from 15-methyl-2-hexadecenoic acid (iso-C17:1 2). The low abundance of the ion at m/z 237 (loss of ethane) demonstrated the presence of a methyl group on C15. The higher abundance of the ion at m/z 83 (allylic cleavage), associated with a lack of ions at lower m/z values, was characteristic of 2 unsaturations.

Synthesis of methyl-branched fatty acids by N33 is flavonoid dependent. Fatty acids of the iso series have never been described as components of Nod factors. However, they are important components of lipids in many bacteria (15, 16). We wondered whether the presence of such fatty acids in N33 Nod factors reflected the general fatty acid composition of lipids in this bacterium or whether the iso fatty acids were specific for the symbiosis signals. To address this question, we first characterized the fatty acid composition of N33 cells grown in the presence or in the absence of a flavonoid inducer. Whole N33 cell pellets were therefore submitted to alkaline hydrolysis, and the liberated fatty acids were identified by capillary GC coupled with electron impact MS (Fig. 5). For N33 cells grown in the presence of a flavonoid, vaccenic acid (C_18:1) was identified as the most abundant component by far, and palmitic (C_16:0) and stearic (C_18:0) acids were also found (Fig. 5a). These are the fatty acids most commonly found in rhizobial lipids (37). In addition, small peaks attributed to iso-C₁₅ and iso-C₁₇ fatty acids were seen.

View larger version (22K): [in this window] [in a new window]   FIG. 5.   Characterization of fatty acids released by alkali hydrolysis of N33 cells grown in the presence or in the absence of flavonoids. The GC-MS profile of methyl esters was recorded in the electron impact ionization mode. Asterisks indicate nonacidic contamination. (a) Total ion current (TIC) from formononetin-induced cells. (b) Reconstructed chromatogram of the ion at m/z 74 from the same sample. This ion is the base peak in the spectra of methyl esters of saturated fatty acids. This profile was used to improve the signal-over-noise detection of saturated fatty acids, indicating the presence of iso-C15 and iso-C17 saturated fatty acids. (c) Reconstructed chromatogram of the ion at m/z 74 ion from a similar preparation from noninduced N33 cells. (d) Reconstructed chromatogram of the ion at m/z 74 from a similar preparation of formononetin-induced cells from the N33 nodC::Tn5 mutant. i, iso.

The Mesorhizobium sp. strain N33 nodE gene is required for the synthesis of Nod factors with ,-unsaturated fatty acids. The presence of Nod factors with fatty acids bearing carbonyl-conjugated double bonds was expected, since the strain under study possesses nodFE genes. These genes are required for the synthesis of Nod factors with ,-unsaturated acyl substituents in R. leguminosarum and S. meliloti (7, 34). We thus studied Nod factors produced by an N33 strain carrying a Tn5 insertion in the nodE gene. Nod factors were purified from the culture medium of the mutant grown in the presence of flavonoids and were hydrolyzed with alkali. The released fatty acids were analyzed by capillary GC coupled with MS as methyl ester derivatives. Unsaturated fatty acids of the iso series could not be detected, whereas saturated fatty acids of the iso series were still present, with a high proportion of the iso-C₁₅ component (Fig. 6a). Fatty acids in whole-cell lipids were also characterized by the same method; again, no unsaturated fatty acids of the iso series were detected (data not shown).

View larger version (22K): [in this window] [in a new window]   FIG. 6.   Role of the nodE gene in the synthesis of N33 ,-unsaturated Nod factors. (a) GC-MS profile (electron impact ionization mode) of methyl ester derivatives of fatty acids released by hydrolysis of Nod factors secreted by the N33 nodE::Tn5 mutant. Note the absence of the iso-C17:1 fatty acid. The section of panel a that was magnified (magnification of ×3) is indicated. i, iso. (b) GC-MS profile (electron impact ionization mode) of methyl ester derivatives of fatty acids in Nod factors secreted by the S. meliloti nodAFEG (pN33nodAFEG) hybrid strain. TIC, total ion current.

	DISCUSSION

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The Nod factors of Mesorhizobium sp strain N33 (O. arctobia) are chitopentamers of N-acetylglucosamine, 6-O sulfated at the reducing end, partially 6-O acetylated at the glucosamine residue proximal to the nonreducing terminal glucosamine, and N acylated at the nonreducing end by saturated and ,-monounsaturated methyl-branched fatty acids (Fig. 7 and Table 2). In comparison with the previously described Nod factor structures, these molecules have novel features. These molecules are 6-O acetylated at the glucosamine residue proximal to the nonreducing terminal glucosamine. Recently, substitution of this residue was described for Mesorhizobium loti NZP2213, where a fucose substituent was present on O-3 (23), and for R. galegae, where an acetyl substitution was found on O-3 (40). The biological importance of these substituents is currently unknown, as are the genes encoding the substitutions. O acetylation of Nod factors on the terminal nonreducing and reducing glucosamine residues was described previously, and two different genes encoding the corresponding O-acetyltransferases were found in rhizobia: nodL and nodX, which encode 6-O-acetyltransferases specific for the nonreducing and reducing glucosaminyl ends, respectively (10, 25). Sequencing of the N33 DNA upstream of nodA revealed the presence of orfZ (4), which is homologous to bacterial O-acetyl transferase genes. However, a strain mutated in orfZ still produced acetylated Nod factors, suggesting that either this gene is not involved in the acetylation of N33 Nod factors or other N33 genes can replace it in this function.

View larger version (13K): [in this window] [in a new window]   FIG. 7.   General structure of Mesorhizobium sp. strain N33 (O. arctobia) Nod factors. i, iso.

The other substitution peculiar to N33 Nod factors is the fatty acyl chain. Methyl-branched fatty acids of the iso series, one of them , unsaturated, are substituents of N33 Nod factors. All of the Nod factor acyl chains described so far in other rhizobia are unbranched, as are the major fatty acid components of rhizobial lipids. However, in many bacteria, methyl-branched fatty acids are important components of cellular lipids. They play a role in the adaptation to cold temperatures, since they decrease the phase transition temperature of the lipids in which they are incorporated (16). We therefore wondered whether methyl-branched fatty acids could be major components of lipids in arctic strain N33 and thus could replace linear fatty acids in Nod factors. We found that in bacteria grown at 20°C in the absence of flavonoids, methyl-branched fatty acids could not be detected; in addition, they were quantitatively minor components of lipids in bacteria cultivated in the presence of flavonoids. Synthesis of methyl-branched fatty acids thus seems specific for the signal exchange between N33 and its host plant, and as such, it could contribute to the specificity of this symbiotic interaction. However, we cannot exclude the possibility that synthesis of the methyl-branched fatty acids is also induced when Mesorhizobium sp. strain N33 is grown at low temperatures. If this were the case then methyl-branched fatty acids could also be involved in a general adaptation of N33 to life at low temperatures.

In addition to LCOs, methyl-branched fatty acids could be substituents of other N33 lipids. This idea is suggested by our observation that an N33 strain mutated in nodC and therefore unable to synthesize the chito-oligosaccharide backbone of Nod factors still contained methyl-branched fatty acids. Such a substitution of lipids different from LCOs by symbiosis-specific fatty acids was described previously by Geiger et al. (12, 13). These authors showed that nodFE-dependent polyunsaturated fatty acids could be substituents of R. leguminosarum bv. viciae phospholipids. However, it is not known whether these nodFE-dependent phospholipids play a role in the symbiotic process.

The presence of methyl-branched fatty acyl chains in N33 LCOs raises the question of the biosynthesis of the corresponding fatty acids in N33. These fatty acids are important components of membrane lipids in bacteria such as Bacillus, where their biosynthetic pathways have been studied. Differences in the biosynthesis of methyl-branched fatty acids and straight-chain fatty acids lie in the primers that are used in the initial condensation reaction catalyzed by the fatty acid synthase (16). For branched-chain fatty acids of the iso series, such as iso-C_15:0 or iso-C_17:0, the primers are derivatives of the methyl-branched amino acid leucine, most likely -ketoisocaproyl- or isovaleryl-coenzyme A. Synthesis of such primers or their conversion to acyl carrier protein derivatives by a fatty acid synthase complex might be flavonoid dependent in N33. This idea would explain why flavonoids are required for the production of lipids with branched-chain fatty acids.

In addition to being methyl branched, one of the fatty acyl components of N33 Nod factors is , unsaturated. In N33, acyl groups with more than one conjugated double bond have not been detected, while in other rhizobia synthesizing Nod factors with ,-unsaturated fatty acyl groups (R. leguminosarum bv. viciae, R. leguminosarum bv. trifolii, M. huakuii, R. galegae, and S. meliloti), up to three carbonyl-conjugated double bonds have been characterized (20, 31, 34, 35, 40). In addition, in the latter four species, contrary to N33, fatty acid chains are linear and generally carry a cis double bond at the 7 position. As in R. leguminosarum and S. meliloti, the nodE gene is required in N33 for the synthesis of Nod factors with ,-unsaturated fatty acids. It was proposed earlier (12) that the acyl carrier protein NodF and the putative condensing enzyme NodE could modify normal fatty acid biosynthesis in such a way that ,-unsaturated fatty acid derivatives would not undergo the step of reduction of the enoyl intermediate. Such a model could also apply to the biosynthesis of N33 Nod factor acyl chains, if it is postulated that N33 NodFE proteins have a higher affinity for branched-chain intermediates than for unbranched ones. However, this preference would not be strict, since when the nodAFEG genes of N33 were transferred to an S. meliloti strain with a deletion of its nodAFEG genes, Nod factors possessing linear ,-unsaturated fatty acyl chains were produced. It is likely that in S. meliloti, branched precursors are not synthesized, so that N33 NodFE proteins instead use available linear precursors, such as acetyl- and malonyl-acyl carrier proteins.

In a recent work (40), it was proposed that a cluster of phylogenetically related legumes are nodulated by rhizobia producing Nod factors with ,-unsaturated fatty acids. This cluster includes the Trifolieae, Vicieae, and Galegeae tribes. Strain N33, which produces Nod factors with ,-unsaturated fatty acids, was isolated from O. arctobia, a member of the Galegeae tribe. It also nodulates O. viciifolia, a member of the Hedysareae tribe, which is closely related to the Galegeae tribe. Therefore, the present work confirms the earlier proposal and extends the cluster of related legumes nodulated by rhizobia producing Nod factors with ,-unsaturated fatty acids to members of the Hedysareae tribe. We hypothesized that in this group of plants, a new mechanism allowing the recognition of structural features of the Nod factor acyl chain evolved. This led to new possibilities for determining the specificity of rhizobium-plant interactions. Previously, two sources of variation among rhizobial LCO ,-unsaturated fatty acids were known, the length of the acyl chain and the number of conjugated double bonds. With N33, another source of variation was explored, the branching of the acyl chain; the latter might contribute to the peculiar host range of this species, together with the other substitutions. Methyl-branched fatty acids could also play a role in the adaptation of signaling mechanisms to low temperatures. It would be interesting to determine the structure of the acyl chain in Nod factors produced by other arctic rhizobia.