Symbiotic conditions induce structural modifications of Sinorhizobium sp. NGR234 surface polysaccharides

N. Fraysse2, S. Jabbouri3, M. Treilhou2, F. Couderc2 and V. Poinsot1,2

2 Laboratoire des IMRCP UMR 5623, Université Paul Sabatier, 31062 Toulouse, France, and 3 Laboratoire de biologie moléculaire, Université du Littoral, quai Masset, bassin Napoléon, BP 120, 62327 Boulogne-Sur-Mer Cedex, France

Received on March 15, 2002;; revised on April 17, 2002; accepted on June 13, 2002


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
When the rhizosphere is starved of nitrogen, the soil bacteria Rhizobium are able to infect legume roots and invade root nodules, where they can fix atmospheric nitrogen. Nod boxes, the nod gene promoters located on the rhizobial symbiotic plasmid, are activated by means of flavonoids present in the legume root exudates, leading to the synthesis of lipochitooligomers: the Nod factors. Several recent works pointed out the importance of rhizobial surface polysaccharides in establishing the highly specific symbiosis between rhizobia and legumes. Lipopolysaccharides (LPSs) exhibit specific active roles in the later stages of the nodulation processes, such as the penetration of the infection thread into the cortical cells or the setting up of the nitrogen-fixing phenotype. The study reported here concerns the structural modifications affecting surface (lipo)polysaccharides when Sinorhizobium sp. NGR234 strains are grown with nod gene induction under nitrogen starvation. In the absence of induction, NGR234 only produces fast-migrating LPSs. When cultured in the presence of flavonoids, the same strain produces large quantities of a high-molecular-weight rhamnose-rich lipopolysaccharide (RLPS). Because the synthesis of this compound seems to be coded by the symbiotic plasmid under direct or indirect gene induction by flavonoids, this RLPS is thought to be biologically relevant.

Key words: gene induction/polysaccharide/rhamnose/Rhizobium


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Soil bacteria belonging to the rhizobiaceae family interact with legume roots to form nodules in which atmospheric nitrogen can be fixed. Host plant root exudates (flavonoid-rich) activate the expression of rhizobial genes involved in the establishment of symbiosis (Long, 1996Go). Generally, the interaction between the two partners begins when rhizobia in the rhizosphere bind to the legume root hairs. In successful associations, these provoke root hair deformation (Had phenotype) and afterward curling (Hac). The following step, infection, occurs via an infection thread (Inf), and cortical cells divide to generate the nodular primordium (Nod) (Dénarié et al., 1992Go). In a later stage the bacteria differentiate into bacteroids expressing efficient nitrogenase (Nif) and synthesizing the environment in which nitrogen fixation occurs (Fix) (Fisher and Long, 1994Go). To explain such physiological changes at a molecular level, a lot of studies have focused on bacterial extracellular polysaccharides. The structural elucidation of the lipochitooligosaccharides called nod factors marked a turning point in a comprehensive study of symbiosis. Actually, these compounds, overproduced under nodD gene activation, are capable of inducing the plant phenotypes Had+, Hac+, and Nod+, even in the absence of the whole bacteria. Nevertheless, more recent studies again focused on polysaccharides suggested that they are also essential for efficient symbiosis.

Several putative roles have been considered for rhizobial exopolysaccharides (EPSs). EPSs—including apparently cyclic glucans—play an active role in the root invasion process. This function seems to be the suppression of active plant defense rather than the masking of a determinant, as coinoculation of Exo Nod+ with Exo+ Nod strains (Kapp et al., 1990Go) results in normal invasion of both bacterial types. The specificity of the EPS role is assessed by the work of Djordjevic et al. (1987)Go, where purified Sinorhizobium sp. NGR234 EPS restored nodule development to Leucena plants inoculated with Sinorhizobium sp. NGR234 exo mutants. Rolfe et al. (1996)Go even expected that EPS could moderate the ethylene production; this could explain that EPS enables the invading Exo cells to escape exclusion and to form nitrogen-fixing bacteroids. Recent studies demonstrated a specific recognition requirement (Gray et al., 1991Go; Gonzalez et al., 1996Go; Pellock et al., 2000Go). That EPSs are critical compounds only for the invasion leading to determined nodule types could be explained by the fact that suitable lipopolysaccharides (LPSs) are synthesized by the wild-type strains, which could complement the lack of EPS in the rare cortical cell penetration events.

LPSs are a very important class of polysaccharides for symbiosis. Their specific active roles appear in the later stages of symbiosis establishment. Indeed, they exhibit two apparently distinct functions. First, they are necessary for the penetration of the infection thread into the cortical cells (Gao et al., 2001Go). Second, they are keys for the Fix+ phenotype (Quandt et al., 1992Go; Jabbouri et al., 1996Go).

Only partial investigations have been done on the genetics of LPS biosynthesis in rhizobia. In contrast to other Gram-negative bacteria, where the genes involved in the O-chain biosynthesis are clustered on the chromosome (Reeves et al., 1996Go), R. etli and R. leguminosarum strains exhibit (in addition to the chromosomal loci) a ß-lps region, which is plasmidborne. This region is involved in the synthesis of the core oligosaccharide (Garcia de los Santos and Brom, 1997Go) and in O-chain biosynthesis (Vinuesa et al., 1999Go).

As extensively described by Perret et al. (1999)Go, even for such considerations, S. sp. NGR234 seems to be genetically quite different from R. etli and R. leguminosarum. First, transconjugants of A. tumefaciens harboring the symbiotic plasmid of Sinorhizobium NGR234 instead of the Ti plasmid are able to nodulate Vigna unguiculata, indicating that most of the symbiotic loci are carried on the pNGR234a plasmid (Broughton et al., 1984Go). Expression of nodulation genes is mediated by NodD proteins, which bind specifically to promoter sequences called nod boxes. Flavonoids reinforce such interactions and are required for transcriptional activation (Fischer and Long, 1993). Perret et al. (1999)Go demonstrate that addition of flavonoids to the growth medium dramatically changes the gene expression patterns. In contrast to the expression of the majority of the nod genes, which are rapidly induced, the expression of some open reading frames (ORFs) occurred in later stages.

In this article, we demonstrate that modifications occur on rhizobial surface polysaccharides, especially on LPS, when Sinorhizobium sp. NGR234 is cultured under nod gene inducers (plant flavonoids). Actually, a new rhamnose-rich LPS (RLPS) bearing 3-OH fatty acids and most of all an O-antigen, is produced. This modification confers on the LPS certain hydrophobicity, consistent with previously published works (Kannenberg and Carlson, 2001Go) on other strains. Because its synthesis seems to be coded by the symbiotic plasmid under direct or indirect gene induction by flavonoids, this RLPS is considered to be very biologically relevant.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Appearance of rhamnose is correlated with nod gene induction
The glycosyl composition of the total polysaccharides synthesized from induced and noninduced cultures has been determined by gas chromatography–mass spectrometry (GC-MS) analysis of the alditol acetate derivatives after the total hydrolysis of the polymer. These polysaccharides were isolated from the direct hot phenol-water extraction of the growth medium, including the bacteria at the end of the culture. The two showed large amounts of glucose; the presence of galactose, mannose, and glucosamine (see Table I, and Figure 1); but the polysaccharides synthesized in induced culture conditions show the presence of rhamnose (12 mol% out of glucose), which is absent under noninduced conditions. Additionally, substantial variations of the relative amounts of galactose can be observed: under noninduced conditions the molar ratio galactose/glucose is 7% and becomes 22% under nod gene-induction.


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Table I. Carbohydrate compositions of the polysaccharide purified fractions from cultured bacteria
 


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Fig. 1.GC-MS of the alditol acetate derivatives from total polysaccharide produced (I = noninduced cultures; II = induced cultures) and the purified RLPS (III). These chromatograms represent the ion current of the characteristic ion m/z 115.

 
Subtle changes in glycosyl composition of secreted polysaccharide (EPS)
The secreted polysaccharides were isolated by a simple centrifugation of the growth medium, treated with proteinase, dialyzed, and analyzed for their glycosyl composition. No detectable amounts of rhamnose were found by GC analysis of the alditol acetate derivatives for the two culture conditions (see Table I). This means that no quantitatively significant rhamnose-rich polysaccharide is excreted in the growth medium. A very intense signal of glucose was present in the two cultures, and subtle changes in the minor proportions of galactose and mannose were detected. In fact, glucose/galactose ratio was divided by four when induced, indicating at least qualitative changes in the structure of the EPS, even though large changes in their weight range were not observed by polyacrylamide gel electrophoresis (PAGE) analysis.

A slow-migrating LPS synthesized under induced conditions correlates with important changes in glycosyl composition
The aqueous phases of the hot phenol-water extracts from Sinorhizobium sp. NGR234 were compared by PAGE and glycosyl composition (see Figure 2A). For this analysis, simple silver staining was used to obtain LPS-specific coloring. The pattern obtained for the crude extract from a noninduced culture presented only one band of LPS corresponding to fast-migrating rough LPS (rLPS). The crude extract from an induced culture showed the presence of at least one additional polysaccharide: a slow-migrating, smooth type LPS (sLPS).



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Fig. 2. DOC-PAGE analysis of NGR234 surface polysaccharide. (A) Silver-stained crude extracts of, respectively, apigenine-induced and noninduced cultures. (B) Alcian blue-silver stain of induced polysaccharides separated by detoxi-gel chromatography. Left lane; LPS; right lane: KPS. (C) Alcian blue-silver stained S-300 of the gel permeation chromatography of the LPS. Eluted fractions from 15 to 35 (2.4 ml each) are enriched in RLPS. Black arrows point to the RLPS, the open arrow points to the two brown bands in the induced KPS fraction.

 
Moreover, the corresponding carbohydrate composition analysis showed the presence of rhamnose in large quantities only under induction. These proportions were about 1 for rhamnitol acetate to 3 for glucitol acetate, but became 5 to 4 when analyzed with the peracetyl aldonitrile derivatization method (data not shown). The derivatization of uronic acids into their peracetyl aldonitrile forms is not efficient unlike the alditol acetate method. Therefore, the difference between the two former ratios suggests that two-thirds of the glucitol acetate signal originates from glucuronic acid.

Analysis of capsular polysaccharide
The capsular polysaccharide (KPS) isolated by a detoxi-gel separation from the two types of cultures were also analyzed by PAGE and mass spectrometry and for glycosyl composition. The alcian blue-silver stained PAGE pattern does not show any clear difference in the banding pattern of the two molecules (see Figure 3). It is important to note that induced cultures exhibit two additional very fast-migrating intense brown bands.



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Fig. 3. Alcian blue-silver stained DOC-PAGE of different purified polysaccharides. Lane 5: The LPS produced by the bacteroids does not differ from the LPS cultured in induced conditions in terms of electrophoretic mobility or in relative abundance of the two LPSs. 1: Purified RLPS; 2: noninduced KPS; 3: induced crude extract; 4: induced KPS; 5: LPS from bacteroids; 6: noninduced KPS.

 
The GC-MS analysis of the induced culture KPS alditol acetate derivatives (see Table I) shows large quantities of glucose and little amounts of 2-keto-3-deoxy-octulosonic acid (Kdo) (10%). Note that Kdo is unstable under 2 M trifluoroacetic acid (TFA) hydrolysis, making this value an underestimated ratio. No pseudaminic acid (Pse) was detected in these GC-MS analyses, but the infrared spectrum (data not shown) revealed the presence of a band at 1648 cm–1 characteristic of an amide function, and two bands at 1705 cm–1 and 1612 cm–1 characteristic of a carboxylic acid and its conjugated salt, suggesting the presence of Pse, described previously (Reuhs et al., 1998Go). No important structural changes of the KPS occured under induction. Nevertheless, compared with the structure published by Reuhs et al. (1998)Go on NGR234 strains cultured without nod gene inducer nitrogen starvation, a nonnegligible amount of Kdo was found here. Again, it should be noted that no detectable amounts of rhamnose were detected.

Physicochemical studies of RLPS: GC-MS of sugars and fatty acids
The proportions of the alditol acetate derivatives of purified RLPS are listed in Table I. A notable amount of rhamnose (47% of the overall glycosyl content) is found in the purified slow-migrating LPS, which we call lipo-rhamno-polysaccharide as well as 4% of 4-O-Me rhamnose (Figure 1). Note, however, that the deoxycholic acid used for the chromatographies has not been completely eliminated in the final dialysis. Starting from the crude extracts, the fatty acid methyl ester (FAME) derivatives were analyzed by GC-MS. All the products present in the chromatogram shown were identified as fatty acid methyl esters, ranging from C14 up to C30 (see Table II). The comparison between the two types of culture shows a sharp increase of 3-OH FAME proportion relative to the usual fatty acids under induced conditions.


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Table II. Relative percentages of the fatty acids determined by the GC-MS analysis of the methyl ester derivatives
 
Comparison with bacteroidal LPS
The LPS isolated after polymixin affinity chromatography present a PAGE pattern similar to those from the free cultured induced cells (see Figure 3). No other polysaccharides were observed, suggesting that no plant material was retained on the polymixin phase with bacteroidal LPS. Nevertheless, the carbohydrate composition study reveals in addition to the presence of rhamnose new sugars in large quantities, for instance arabinose, known to be an important constituent of the pectic polysaccharides of the plant primary cell wall (O'Neill et al., 1990Go).


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Nod boxes, located on the rhizobial symbiotic plasmid are activated by means of flavonoids present in the legume root exudates. Their products are involved in the biosynthesis and secretion of Nod factors (Dénarié et al., 1992Go), that serve as signals to elicit the Had+, Hac+, Thr+, and Nod+ phenotypes. Nevertheless, the nitrogen-fixing phenotype (Fix+) is likely related to (lipo)polysaccharides. Indeed, numerous authors have demonstrated that LPS structure changed during the establishment of symbiosis (for review, see Carlson et al., 1999Go). Moreover, Reuhs et al. (1995)Go showed that LPS mutants of S. meliloti were inapt for nitrogen fixation. Jabbouri et al. (1996)Go made analogous observations on a Sinorhizobium sp. NGR234 Fix mutant altered in the fixF region coding for an unusual high-molecular-weight LPS. We were intrigued by structural modifications of surface polysaccharides occurring under conditions closer to symbiotic ones (nod gene induction and nitrogen starvation), and therefore started the investigation on structural modifications that can be related to nod box activation on Sinorhizobium sp. NGR234 wild-type strains.

Inducible cell-surface PS modifications seem to be specific
A first, rough investigation made on the polysaccharide extract (KPS + LPS) indicated at least one modification in the global monosaccharidic composition expressed by the appearance, following nod box induction, of large amounts of a deoxysugar: rhamnose.

To check the ubiquity of this novel sugar, the three PS classes were isolated and studied separately. Neither significant modifications nor presence of rhamnose could be demonstrated within the EPS fraction.

Ambiguous observations were made on the capsular polysaccharide KPS comparing induced and noninduced cultures. The alcian blue-silver–stained PAGE pattern does not show an evident difference in the banding pattern of the two molecules (iKPS and niKPS). Nevertheless, induced cultures exhibit two additional very-fast-migrating intense brown bands but seem not to affect the global monosaccharidic composition of KPS. No detectable amounts of rhamnose could be detected under nod gene induction. The monosaccharide composition of KPS determined here it differs from one established earlier (Reuhs et al., 1998Go) in that we observed substantial quantities of Kdo in addition to glucose and pseudaminic acid. One explanation for this could be that Sinorhizobium sp. NGR234 was cultured under nitrogen starvation (to reproduce symbiotic conditions), whereas Reuhs and colleagues used richer media. As pseudaminic acid is a diamino-sugar and Kdo a basic carbohydrate synthesized under the conditions described (see LPS composition), it might be more convenient for Sinorhizobium sp. NGR234 to replace, at least partially, Pse by Kdo when grown under nitrogen starvation.

In contrast to KPS, major modifications were observed on the LPS. Actually, a novel band appeared on the silver nitrate–stained deoxycholic (DOC)-PAGE in the high-molecular-weight domain. Interestingly, in the absence of flavonoids, Sinorhizobium sp. NGR234 produces only rLPS. Such LPSs exhibit a lipid A anchor triggering a core unit but lacking the O-antigen part. They are called rough because mutant strains producing only low-molecular-weight LPS display characteristically rough-looking colonies. Under induction, a new DOC-PAGE band is generated that displays the electrophoretic behavior of a slow-migrating smooth LPS (bearing the O-antigen polysaccharide). The sugar composition of this inducible LPS reveals that nearly half the total sugars is rhamnose, that is, this flavonoid-inducible compound is an RLPS.

Another important modification found in this compound (RLPS) concerns the fatty acid composition of its lipid A. Actually, two observations were made on these lipids. First, their length changed significantly. The usual noninduced lipids exhibit mostly C14 chains. Under induction elongated fatty acids are observed (27-OH:C28 even 29-OH:C30). Second, the 3-OH: fatty acid family (C14, C15, C16, C18, C28, C30) is overexpressed in regard to the normal ones. Notice that the minor 3-OH-C28 and 3-OH-C30 were not reported previously. The lipid A fatty acids composition here is consistent with those of R. etli exhibiting 3-OH: C14 and 27-OH: C28 chains. Because, Sinorhizobium sp. NGR234 additionally synthesize to the rLPS a smooth LPS (RLPS) in the presence of apigenine induction, we can postulate that the overexpressed 3-OH: fatty acids belong to the RLPS lipid A, even if we cannot affirm that rLPS remains unmodified. The precise structure of this RLPS is under investigation.

Freiberg et al. (1997)Go reported the presence of genes exhibiting high homology with those responsible for the synthesis of dTDP-rhamnose from glucose-1-phosphate in Escherichia coli on the Sinorhizobium sp. NGR234 Sym plasmid within loci adjacent to fixF. FixF exhibits important homology with E. coli’s kpsS and kpsD, involved in the synthesis and transport of surface polysaccharides. Some ORFs present on the same plasmid are homologous to genes coding for ABC (ATP binding cassette)-type transporters, such as y4gM (Freiberg et al., 1997Go).

Perret et al. (1999)Go demonstrated that transcription of loci y4gF to y4gI, including the genes probably involved in rhamnose synthesis, is only detected in the later stages of flavonoid gene induction. Their regulation cannot be directly associated with nod boxes. This indicates the existence of alternative or indirect regulatory pathways, which nonetheless require activation of a functional nodD1. Otherwise, downstream of hsnII, a cluster of genes unique to NGR234 seems to be involved in the transport of sugars and several proteins (even two transmembrane proteins).

Despite the structural diversity of the O-antigen, their mechanism of synthesis seems to be conserved (Whitfield, 1995Go). In general, the O-antigen is synthesized separately on a lipid carrier. This polymerization of activated sugar precursors occurs either in the cytoplasm or in the periplasm, depending on the assembly pathway. Translocation of the O-polysaccharide across the inner plasma membrane is systematically required. Three assembly pathways are known, including an ABC transporter-dependent pathway (Whitfield, 1995Go; Lerouge et al., 2001Go). Once completed, the polysaccharide is covalently bound to the suitable acceptor rLPS (lipid A + core) at the inner plasma membrane. Afterward, the complete sLPS is translocated to the cell surface by an unknown mechanism. Now, all genes required for such a synthesis pathway of the inducible RLPS are present on NGR234 Sym plasmid (y4gF-I, fixF, y4gM, and the NGR234 unique genes cluster).

LPS O-antigens are known to be affected during the symbiosis
As discussed in the review by Carlson et al. (1999)Go, several biochemical investigations with monoclonal antibodies have revealed changes in LPS expression during the symbiosis, which affect the O-chain portion. Therefore, it was concluded that rhizobial LPS expression is environment-dependent (differences between free-living bacteria and the bacteroids) (Kannenberg et al., 1994Go; Sindhu et al., 1990Go; Tao et al., 1992Go; VandenBosch et al., 1989Go). Nevertheless, such works never pointed out the structural modifications that occur and are almost exclusively based on observations made on R. leguminosarum and R. etli, and it is known that LPS composition and synthesis is at least genus specific (Kannenberg et al., 1998Go).

Nodular LPSs bear rhamnose-containing O-antigen
To make sure that such new LPS is not generated or overexpressed artifactually in our cultures, we investigated the LPS present in Vigna nodules infected with Sinorhizobium sp. NGR234. The electrophoretic pattern clearly exhibits such novel compound. Moreover, regarding the exact sugar composition, it appears that the nodular smooth LPS is also a rhamnosylated polysaccharide, even if we cannot exclude that this rhamnose could be provided by vegetal compounds, like pectic rhamno-galactouronanes.

RLPS exhibits some analogies with nodular exopolysaccharides
Streeter et al. (1992)Go published a major work concerning symbiotically induced EPS. They demonstrated that, in soybean root nodules, certain Bradyrhizobium japonicum strains produce a nodular polysaccharide (NPS), which differs from the EPS observed on the same strains in lab cultures. Moreover, this polysaccharide accumulates inside the symbiosome membrane, which encloses the bacteroid. Cultured B. japonicum secretes EPS composed of glucose, mannose, galactose, 4-O-methylgalactose, and galacturonic acid and in NPS nodules composed of rhamnose, galactose, and 2-O-methylglucuronic acid. In contrast, B. elkanii synthesize, both in cultures and in nodules, EPS only composed of rhamnose and 4-O-methylglucuronic acid.

Because the inducible LPS described here is mostly composed of rhamnose, 4-O-methylrhamnose, glucose, or glucuronic acid and galactose—not counting the typical LPS monosaccharides Kdo and glucosamine—we are tempted to make a parallel with NPS. In fact, B. japonicum’s EPSs gains in hydrophobicity when a polysaccharide rich in rhamnose and methyl-glucuronic acid is produced as a replacement for a compound containing glucose, mannose, and galacturonic acid. This is also the case when Sinorhizobium sp. NGR234 produces a rhamnose-rich smooth LPS.

Even if Sinorhizobium sp. NGR234 EPSs are apparently not as affected by the induction of nod and fix genes as the EPSs of B. japonicum, we can observe notable changes in the glucose/galactose ratio before and after induction with apigenine. The proportions of sugars determined in the EPS of noninduced cultures are consistent (with respect to the medium differences) with the structure published by Djordjevic and Rolfe (1986)Go where a nonasaccharidic repeating unit composed by seven glucose and two galactose (30% mol to Glu) derivatives was found. The important relative decrease of the galactose content under nod gene induction could be explained by the overexpression of glucose-rich polysaccharides like neutral ß-glucans.

Putative role for these RLPSs
In addition to rLPS, sLPS is synthesized only under symbiosis-like conditions. Moreover, the monosaccharide analysis demonstrated the presence of abundant rhamnose in the newly synthesized O-antigen. This indicates that when hydrophobic domains are absent or insufficient in EPS and LPS, the bacteria have to synthesize them to attain efficient symbiosis (Kannenberg and Carlson, 2001Go; present study). But the question remains open: At which step might it be necessary to present an enhanced surface hydrophobicity?

The first hypothesis proposed by Carlson et al. (1999)Go is a simple adaptation of the bacteria to important environmental changes occurring between the rhizosphere and the nodule. Such an adaptation is classically attributed to EPS. A second possibility could be to mimic a plant-like interface necessary for setting up the symbiosome where a plant membrane surrounds the bacteroid, which has lost its own bacterial cell wall, in a mechanism resembling endocytosis. Intriguing analogies could be observed between the characterized RLPS and the rhamnogalacturonane II present in the primary plant cell walls that contains rhamnose, galacturonic acid, and Kdo (York et al., 1985Go). A third possibility would be the involvement of the sLPS in the bacteroidal differentiation processes, where the bacteria have to undergo dramatic morphological changes (very great enlargement and even distortion into Y shapes). Even though recent mutant screening has revealed certain genes involved in such a developmental pathway, their function remains vague or unknown (Oke and Long, 1999Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Bacterial cultures
Sinorhizobium sp.NGR234 wild-type strains were obtained from F. Debellé (INRA Toulouse). These were precultured for 2 days at 28°C in TY broth supplemented with 10 mM CaCl2.

Strains were grown on a liquid minimal medium containing sodium glutamate (6 mM), sodium succinate (12 mM), and 1 ml/L of culture of a 1000x concentration vitamin mixture composed of biotin (500 µg/ml) and the Murashigare and Skoog preparation. When necessary, an inducer (1 µM apigenin) was added. These chemicals were purchased from Sigma. The media (1 L each) were inoculated with 2 ml precultures and incubated for 24 h at 28°C with shaking (160 rpm).

Total polysaccharide extraction
For the total polysaccharide study, the resulting growth medium, including the bacteria, was evaporated. The dry residue was redissolved in 200 ml deionized water and extracted by the hot phenol-water method (Carlson, 1978). To the water layer (200 ml) was added 100 ml phenol, and it was stirred 15 min at 65°C. The mixture was then centrifuged at 7500 x g, 5°C, for 20 min. The aqueous supernatant was dialyzed (dialysis tubing MWCO: 10 kDa) against two 6-L changes of deionized water to remove the phenol. It was then concentrated to 50 ml, made 10 mM in MgSO4 and 50 mM in Tris–HCl, and incubated with 1 mg RNase and 1 mg DNase overnight under stirring. One milligram of proteinase K (purchased from Sigma) was added, and the solution was stirred again at 37°C for 4 h. The sample was dialyzed for a second time and freeze-dried. This total polysaccharide crude extract obtained was then ready to be studied for further physicochemical analysis.

Cultured-cell PS isolation
Media were centrifuged at 12,000 x g for 20 min. The EPS-containing supernatant was removed, evaporated, freeze-dried, and treated with RNase, DNase, and proteinase it in the same way as the extract fraction. The harvested bacterial pellets (about 30 g wet weight) were washed twice with 200 ml 2% aqueous NaCl and centrifuged for 20 min at 12,000 x g. Supernatants were pooled, evaporated, and freeze-dried, constituting the second washing fraction presumed to be KPS-enriched. Then the washed bacteria were extracted by the hot phenol-water method and treated by RNase, DNase, and proteinase as described previously. The freeze-dried aqueous phase obtained consist in a crude extract, of which 20 mg was passed over a 1.6 x 23 cm polymixin column (Sigma) to separate the KPS from the LPS, as described by Forsberg and Carlson (1998)Go. After addition of the aqueous sample solution, the column was washed with 4% ammonium carbonate in aqueous solution to elute the KPS. LPSs were eluted with 2% DOC acid in H2O. The two KPS- and LPS-containing fractions were dialyzed, evaporated under vacuum, and freeze-dried. The LPSs were fractionated by a size-exclusion chromatography on Sephacryl S-300 (eluent: 0.1% deoxycholic acid, 292 mg/L ethylenediamine tetra-acetic acid, 0.2 M NaCl, 10 mM Tris). LPS-containing fractions were recognized by DOC-PAGE analysis and pooled for dialysis in 40% MeOH (2x 1 versus 30) essentially to remove the DOC acid present.

Bacteroidal LPS isolation
Eighteen grams of fresh nitrogen-fixing nodules were collected from Vigna leucocephaena roots in the Broughton lab. The LPSs were purified as previously described (Streeter et al., 1992Go) with some modifications. Nodules were ground in a mortar and pestle in 50% EtOH (2 volumes per nodule fresh weight), finally making a 36% EtOH concentration. The homogenate was centrifuged at 5°C, 7500 x g for 20 min. The pellets were resuspended in 36% EtOH in the mortar and pestle and then centrifuged. The two supernatant phases were pooled, and 95% fresh EtOH was added to reach a final concentration of 50% EtOH. The solution was stirred for 15 min and centrifuged. The supernatant was recuperated and concentrated to 10 ml. Fifty milliliters of 100% cold EtOH was added, and the solution was centrifuged. The precipitate was recovered and submitted to RNase, DNase, and proteinase treatments as described by An et al. (1995)Go. The solution was then dialyzed three times in water at 1 versus 60 and finally evaporated and freeze-dried. The sample was submitted to affinity chromatography on an agarose-beaded polymixin column as described. The resulting LPS enriched fraction was dialyzed in 50% MeOH three times at 1 versus 30. The dialyzed fraction was evaporated and submitted to PAGE and GC-MS analysis.

Polysaccharide identification and analysis
The PAGE protocol has been described previously (Reuhs et al., 1993Go). PAGE was performed on the phenol-water extracts, or purified polysaccharides using 18% polyacrylamide gels with DOC acid as detergent; the gels were either silver stained to detect LPS (Tsai and Frasch, 1982Go) or Alcian blue-silver stained to check for the presence of all polysaccharides (Corzo et al., 1991Go).

Polysaccharide monosaccharide analysis
Polysaccharide glycosyl compositions were determined by preparation of the alditol acetates or the peracetylated aldonitriles derivatives (York et al., 1985Go). The sample was dissolved in 200 µl 2 M TFA in a screw-cap Teflon vial, and heated for 2 h at 120°C for hydrolysis. The samples were evaporated at 60°C under a stream of filtered air. The addition of 3 x 200 µl isopropyl alcohol resulted in more complete removal of the TFA. The sugars were then reduced to give the corresponding alditols by the addition of a 1 M ammonia solution containing 10 mg/ml sodium borohydride (Sigma) (1 h at room temperature). The reaction was then quenched by the addition of two drops of pure acetic acid. The mixture was evaporated meanwhile 6 x 200 µl MeOH is added. For the aldonitrile method, the glycoses were reduced by hydroxylamine chlorhydrate (100 µg) in 100 µl pyridine at 80°C for 1 h. The alditols (or aldonitriles) were peracetylated by treatment with 100 µl pyridine and 100 µl acetic anhydride for 30 min at 80°C. The pyridine was evaporated, and the mixture was injected for GC in solution in chloroform. The products were identified and quantified by comparison with authentic standards in GC and GC-MS.

The GC chromatograms were performed on a flame-ionization detection Girdel series 30 gas chromatograph, using an OV-1 capillary column (50 m x 0.25 µm x 0.1 µm). The temperature program was 110°C (initial) up to 300°C (final) at 3°C · min–1. The vector gas was helium at 110 kPa.

GC-MS analysis was recorded on the same MicroMass Autospec 6F in the EI or CI mode (ionization energy: 70 eV). GC (HP 5890), split injector at 250°C, gas: He, 1 Bar. Temperature program: 110°C for 2 min, 110°C up to 310°C (3°C.min), 310°C for 10 min on an Optima MS capillary column (50 m x 0.25 µm x 01 µm; Macherey-Nagel, Hoert, France).

Infrared spectra
The infrared spectra were realized on a Perkin Elmer 1760X infrared spectrometer. The samples were deposited in the solid state directly on a diamante microcellule. The measurements were done by transmission with the help of a beam condenser fitted with ZnSe lenses.

Lipid A study
The lipid A moiety was isolated from the LPS using mild hydrolysis with 1% acetic acid for 1 h. Then the sample was submitted to centrifugation, and the precipitate was washed twice with water. Lipid A was subjected to acidic methanolysis, and the resulting fatty acids (monomethyl ester derivatives) were silylated with 100 µl bis-trimethyl sylil-trifluoroacetamide (Sigma) in pyridine (100 µl) at 70°C for a direct injection in GC and GC-MS analysis.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We wish to thank F. Debellé (INRA, Auzeville, France) for providing Sinorhizobium sp. NGR234 wild-type strains. We are grateful to W.J. Broughton (University of Geneva, Switzerland) for providing Vigna nodules infected with Sinorhizobium sp. NGR234. This work was supported by a grant of the French Ministère de la Recherche et de la Technologie (72-UPS-99).


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
ABC, ATP binding cassette; DOC, deoxycholic; EPS, exopolysaccharide; FAME, fatty-acid methyl ester; GC-MS, gas chromatography–mass spectrometry; Kdo, 2-keto-3-deoxy-octulosonic acid; KPS, capsular polysaccharide; NPS, nodular polysaccharide; ORF, open-reading frame; PAGE, polyacrylamide gel electrophoresis; rLPS, rough lipopolysaccharides; RLPS, rhamnose rich lipopolysaccharides; sLPS, smooth lipopolysaccharides; TFA, trifluoroacetic acid.


    Footnotes
 
1 To whom correspondence should be addressed; E-mail: poinsot@chimie.ups-tse.fr Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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