From the LBMPS, Université de Genève, 1 ch. de l'Impératrice, 1292 Chambésy/Genève,
Switzerland, ¶ Département de Chimie Organique,
Université de Genève, Sciences II, 30 Quai Ernest-Ansermet,
1211 Genève 4, Switzerland, and
Institut de Pharmacologie
et de Biologie Structurale, CNRS, 205 Route de Narbonne, 31077 Toulouse Cedex, France
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ABSTRACT |
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Loci unique to specific rhizobia direct the adjunction of special groups to the core lipo-oligosaccharide Nod factors. Host-specificity of nodulation (Hsn) genes are thus essential for interaction with certain legumes. Rhizobium sp. NGR234, which can nodulate >110 genera of legumes, possesses three hsn loci and secretes a large family of Nod factors carrying specific substituents. Among them are 3-O (or 4-O)- and 6-O-carbamoyl groups, an N-methyl group, and a 2-O-methylfucose residue which may bear either 3-O-sulfate or 4-O (and 3-O)-acetyl substituents. The hsnIII locus comprises a nod box promoter followed by the genes nodABCIJnolOnoeI. Complementation and mutation analyses show that the disruption of any one of nodIJ, nolO, or noeI has no effect on nodulation. Conjugation of nolO into Rhizobium fredii extends the host range of the recipient to the non-hosts Calopogonium caeruleum and Lablab purpureus, however. Chemical analyses of the Nod factors produced by the NodI, NolO, and NoeI mutants show that the nolO and noeI gene products are required for 3 (or 4)-O-carbamoylation of the nonreducing terminus and for 2-O-methylation of the fucosyl group, respectively. Confirmation that NolO is a carbamoyltransferase was obtained from analysis of the Nod factors produced by R. fredii containing nolO; all are carbamoylated at O-3 (or O-4) on the nonreducing terminus. Since mutation of both nolO and nodU fails to completely abolish production of monocarbamoylated NodNGR factors, it is clear that a third carbamoyltransferase must exist. Nevertheless, the specificities of the two known enzymes are clearly different. NodU is only able to transfer carbamate to O-6 while NolO is specific for O-3 (or O-4) of NodNGR factors.
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INTRODUCTION |
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Nodulation genes (nod, nol, and noe) of the symbiotic soil bacteria Azorhizobium, Bradyrhizobium, Mesorhizobium, and Rhizobium (collectively termed rhizobia), may be divided into two classes. One class comprises genes which, when mutated, completely abolish nodulation on all legumes. Since genes in this group share significant sequence homology and can be complemented between different rhizobial genera/species, they are often called "common." nodABCIJ and nodD are the best known examples. Genes of the second class are necessary for the interaction with certain, but not all, legumes. Expression of these genes permits nodulation of additional hosts, and for this reason, they have been termed host specificity of nodulation (= hsn) genes. By definition, they are unique to one or a few rhizobia.
In 1990, Lerouge et al. (1) showed that the products of the nod genes are N-acylated oligomers of N-acetyl-D-glucosamine. Numerous other investigators have confirmed these findings (for reviews see Refs. 2-5). Since these substances are the products of the nod genes, they are called Nod factors. Principal differences among the Nod factors of the various rhizobia concern the length of the core molecule as well as the substitutions to both the reducing and nonreducing residues. Presumably, the hsn genes are responsible for these substitutions.
With the discovery of Nod factors, it became possible to correlate nod gene expression with Nod factor structure. In their pioneering studies, Roche et al. (6) demonstrated that the hsn loci, nodH and nodPQ, are responsible for the 6-O-sulfation of the reducing N-acetyl-D-glucosamine of NodRm factors. Later work has shown that the first step in Nod factor assembly is performed by an N-acetylglucosaminyltransferase coded by nodC (7). Then, a deacetylase coded by nodB removes the N-acetyl moiety from the nonreducing end of the N-acetylglucosamine oligosaccharides (8). Finally, an acyltransferase coded by nodA, links the acyl chain to the NH2-free carbon C-2 of the nonreducing end of the oligosaccharide (9). NodI and NodJ are involved in the export of Nod factors (10, 11).
In Rhizobium sp. NGR234, three Hsn loci were discovered by transferring cosmids covering the symbiotic plasmid to heterologous rhizobia (i.e. rhizobia unable to nodulate NGR234 hosts), and asking if the transconjugants could form nodules on Vigna unguiculata (12, 13). In this way, we showed that HsnII, which is responsible for the host-specific nodulation of Leucaena species, contains nodSU (14), which are involved in N-methylation and 6-O-carbamoylation of NodNGR factors, respectively (15). HsnI contains five genes encoding a set of enzymes responsible for the synthesis GDP-fucose and its transfer by NodZ to NodNGR factors (16, 17).
Here we present a molecular analysis of the HsnIII locus (12, 13). In this, as in much of the work discussed above, we used the closely related R. fredii strain USDA257 because (a) it nodulates an exact subset of the NGR234 hosts; (b) many of the nod genes, and most of the essential, chromosomal genes, are extremely well conserved between the two strains (18-20); and (c) perhaps because of (b), transconjugants are more stable in the R. fredii background than in any other rhizobia. Combined, these properties facilitate extension of host-range studies in which NGR234 clones are introduced into USDA257 on broad host range, multiple copy plasmids. Since the nodulation requirements of the two bacteria are known, the initial screening for particular hosts can be performed on legumes that are nodulated by NGR234 but not by USDA257.
These analyses show that HsnIII is downstream of nodABC, and includes the nodIJnolOnoeI genes. NoeI is required for 2-O-methylation of the fucose and NolO for 3-O (or 4-O)-carbamoylation of the nonreducing terminus of NodNGR factors.
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EXPERIMENTAL PROCEDURES |
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Bacterial Genetics/Manipulation of DNA-- Bacterial strains and plasmids used in this study are listed in Table I. Rhizobia were grown in/on RMM3 (21). Microbiological techniques were performed as described in Lewin et al. (14). Recombinant DNA procedures were used as described previously (19, 22, 23). DNA sequencing was performed using chain-termination inhibitors (24).
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Construction of the Mutants NGRnolO and
NGR
nodI--
Plasmid p6.8HR was linearized with the
restriction enzymes BglII and EcoRI to liberate
nodI, and nolO respectively. Concomitantly, the
SpR Omega fragment was excised from pHP45 with
BamHI or EcoRI and ligated onto the linear
p6.8HR fragments. After transformation into
Escherichia coli XL.1 Blue, the plasmids were conjugated into a RifR derivative of NGR234 with the help of plasmid
pRK2013. RifRSpRTcS colonies were
checked for homologous recombination by Southern transfer. A 2.6-kb
EcoRI fragment of pNG77 carrying noeI was first cloned into the EcoRI site of pBluescript SK. The resulting
construct, pSK2.6E contains a unique StuI site that is
located 26-pb downstream of the start codon (ATG) of noeI.
Digestion of pSK2.6E with StuI permitted introduction of the
SpR Omega interposon (which was extracted from pHP45
by
digestion with SmaI) into the noeI gene (25).
Finally, the entire noeI-Omega cassette was cloned into the
XbaI and SalI sites of the suicide vector
pJQ200sk (26). Triparental matings (including the helper plasmid
pRK2013) were used to mobilize noeI-Omega into NGR234. Selection of NGR
noeI was performed as described
previously (27).
Construction of nodU/nolO Double Mutants--
The 2.2-kb
HindIII/BamHI fragment downstream of
nodABCIJ, which contains nolO, was cloned into
the HindIII and BamHI sites of pBluescript KS
(giving pKS2.2B/H). Digestion of pBluescript KS with BamHI
and HindIII led to deletion of the EcoRI site in the multiple-cloning site. Consequently, an EcoRI fragment
carrying the KmR Omega interposon (25) could be cloned into
the EcoRI site of nolO. The cassette containing
nolO:: was then cloned into XbaI and
XhoI sites of pJQ200sk. Finally, the recombinant plasmid was transferred to two distinct nodU mutants: NGR
26 and
NGR
1 that are a SpR insertion mutant (in
nodU) and a deletion mutant (nodSU) of NGR234, respectively (14). The two double mutants NGR
26
nolO
and NGR
1
nolO were verified as described in Hanin
et al. (27)
Nodulation Tests--
Seeds of Calopogonium caeruleum
(Benth.) Hemsl. were purchased from the Inland and Foreign Trading Co.,
Indus Road, Singapore; Gylcine soya Sieb. & Zucc. was a gift
from S. G. Pueppke, University of Missouri, Columbia, MO;
Lablab purpureus (L.) Sweet cv. Rongai was from Arthur Yates
Co., Rockhampton 4700, Australia; Macroptilium atropurpureum
Urb. cv. Siratro was purchased from Rawlings Seeds, Orpington, Kent,
UK; and Vigna unguiculata (L.) Walp. cv. Red Caloona was
bought from Rawlings Seeds. Nodulation capacity was assayed in modified
MagentaTM jars (28). Two replicate jars, each containing
four seedlings, were used per treatment. Inoculation with
107 colony-forming units was performed 3 d after
planting. All plants were grown at a daytime temperature of 30 °C, a
night temperature of 20 °C, and a light phase of 16 h
(including a 1-h stepped "sunrise" and a 1-h stepped "sunset";
maximum intensity of illumination was 350 µmol·m2·s
1 photosynthetically active
radiation).
Purification of Nod Factors--
Rhizobia strains were grown at
27 °C in 2-liter Erlenmeyer flasks containing 1 liter of RMM3 medium
with or without 106 M apigenin as the inducer
(21). Cells were grown to an A600 of 1. After
centrifugation (4,500 rpm, 30 min, 4 °C), extracellular Nod factors
were extracted from the supernatant as described previously (15, 21).
To label the Nod factors, RMM3 was supplemented with
D-[14C]glucosamine (54 mCi/mmol, Amersham
Pharmacia Biotech, Zürich).
Analytical Methods-- Separation of lipochitinoligosaccharides (LCOs)1 by HPLC, their analysis by liquid secondary ion mass spectrometry (LSI-MS), B/E linked scans (where B/E = fixed ratio of the magnetic (B) over the electric field (E) in daughter ion mass spectrometry), and one-dimensional NMR spectra were performed as described previously (15). 15N-1H-heteronuclear multiple quantum correlation NMR spectra of 15N-enriched NodNGR factors were recorded in Me2SO-d6 at 14 Tesla (600-MHz proton frequency) on an AMX-Z-600 spectrometer (Bruker GmbH, Karlsruhe, Germany). After alditol acetate derivatization, analyses of monosaccharides originating from the LCOs were performed by GC/MS (using an electron impact ion source) after separation on a 15-cm SupelcoTM SP fused silica column (Hewlett Packard, Palo Alto, CA). The synthesis and analysis of monoacetylated glucosamines by MS/MS was performed as described in Ferro et al.2
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RESULTS |
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Host Range Extension--
At the outset, large fragments (40
kb) of the symbiotic plasmid of Rhizobium sp. NGR234
(pNGR234a) were cloned into the nontransmissible cosmid
vector pJB8 (22, 30). Individual cosmids were mobilized into
heterologous rhizobia by introducing the cis-acting DNA
recognition site for conjugative DNA transfer (Mob site) of RP4 into
the clones. This was accomplished by conjugating the Tn5-Mob
vector pSUP5011 (31) into E. coli strains containing the
pJB8 cosmids. Matings with another RP4 derivative permitted
mobilization of the cosmids into various
Agrobacterium/Rhizobium strains including
Rhizobium loti strain NZP4010. R. loti (pWA54)
transconjugants nodulated V. unguiculata at low frequency,
and this locus was named HsnIII (12). Preliminary mapping showed that
pWA54 partially overlaps with pWA46, but that the latter does not
confer host-range extension on the transconjugants (13).
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Delimitation of the HsnIII Locus-- A series of subclones in pRK7813 were generated and mobilized into USDA257. The resulting transconjugants were used to inoculate Calopogonium, Lablab, and other legumes. Interestingly, the 7.6-kb EcoRI fragment of pNG77, which contains regions on either side of nodABC, was unable to extend the host range (Table II). On the other hand, clones containing the right most of two 6.8-kb HindIII fragments (p6.8HR) (Fig. 1), conferred the ability to nodulate both C. caeruleum and L. purpureus on the transconjugants. These data suggest that the HsnIII locus lies at the end of p6.8HR distal to nodABC.
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Mutational and Sequence Analysis of HsnIII-- As R. fredii transconjugants harboring p7.6E and p2.2B/H (Fig. 1) were unable to nodulate C. caeruleum while those containing p6.8HR were able to do so (Table II), the HsnIII locus is most probably located in the region of overlap between p7.6E and p2.2B/H. To delimit possible genes in this region, we sequenced the plasmids represented by p7.6E and p2.8HR. As confirmed by complete sequencing of pNGR234a (33), seven open reading frames are contained in this locus (Fig. 1). A noncoding sequence of 173 bp separates nodABC from the 1,005-bp nodI. After a gap of only 3 bp, this is followed by another ORF of 789 bp that is highly similar to nodJ. nolO is downstream of nodJ and is followed by part of noeI. Insertion of an Omega cassette into either nodI or nolO or noeI had no effect on the capacity of the mutant to form nodules (Table II).
RNA competition/hybridization experiments were used to see whether the loci contained on pNG77 and p6.8HR were inducible by flavonoids. One h after exposure to apigenin, all of the fragments contained on p6.8HR (e.g. the 1.7- and the 3.6-kb BamHI fragments) as well as the 7.6-kb EcoRI fragment of pNG77 hybridized, showing that the entire locus is inducible (data not shown). Although these data do not constitute proof, they suggest that nodABCIJnolOnoeI are transcribed as one operon.Involvement of HsnIII Genes in 2-O-Methylation of the
Fucose--
Thin layer chromatographic analyses of the supernatants
from [14C]glucosamine-fed cultures of
NGRnodI, NGR
nolO, and NGR
noeI did not reveal differences from the wild-type bacterium (data not
shown). We were thus able to exclude a role for these genes in
fucosylation or sulfation of NodNGR factors. Other possible roles of
the hsnIII genes were studied by subjecting partially purified (i.e. eluted from a C18-reverse-phase
column) Nod factors to acidic methanolysis. Hydrophilic compounds were
separated from the hydrophobic components by hexane extraction. GC/MS
analyses of the methyl-esterified hexane-fraction did not reveal any
changes in the N-linked acyl chain attached to the
nonreducing terminus. This way, we were able to rule out any affect of
the hsnIII genes on fatty acid synthesis.
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Nod Factors Produced by NGRnolO--
The LSI-MS spectra
(positive ionization mode), of RP-HPLC-purified compounds extracted
from the supernatants of NGR
nolO cultures revealed [M + H]+ molecular ions which correspond to sulfated or
acetylated molecules. Fragment ions from the nonreducing terminus
differ from analogous ions of Nod factors of the wild-type bacterium by
the absence of bis-carbamoylated products (absence of m/z
528, 526, and 498), which correspond to LCO species possessing two
carbamates, acylated with C18:0, C18:1, and
C16:1, respectively, and N-methylated. Molecules
in which the fucose is acetylated gave pseudomolecular ions that were
shifted down by 57 Da in comparison to ions from NodNGR factors (Fig.
2A). This 57-Da difference corresponds to the sum of the
mass of carbamoyl (43 Da) and methyl (14 Da) groups.
Nod Factors Produced by Various USDA257 Transconjugants-- As shown above, when p7.6E, which contains nodABCnodIJ (Fig. 1), was mobilized into USDA257, the transconjugants are unable to nodulate C. caeruleum. On the other hand, p6.8HR, which also contains nolO, was able to confer on R. fredii the capacity to nodulate C. caeruleum (Table II). This suggests that nolO (but not nodIJ) is necessary for nodulation of this plant. To determine the biochemical role of nolO, we studied Nod factors produced by USDA257(p6.8HR). LSI-MS spectra from the RP-HPLC peaks showed fragmentation sequences separated by 203 Da, which is characteristic of GlcNAc oligomers. The major peak, which corresponds to Nod factors containing five GlcNAc, N-C18:1, and methylfucose gave [M + H]+ ions at m/z 1459 instead of m/z 1416 from wild-type USDA257. This 43-Da augmentation in mass corresponds to a carbamoyl group. It is obvious that the carbamoyl group originates from the nonreducing terminus since all fragments were shifted up by 43 Da, particularly the B1 ion which arose at m/z 469 rather than m/z 426. Analyses of all HPLC fractions showed partial carbamoylation of tri-, tetra-, and pentameric Nod factors (Fig. 3).
Nod Factors Produced by nodU/nolO Double Mutants--
To establish
whether all carbamoylated NodNGR factors are the products of NodU and
NolO, double mutants in the two genes were made. Construction of the
double mutant was performed by inserting the SpR Omega
cassette into nolO of NGR1 in which a 9-kb fragment
containing the complete hsnII (nodSU) locus has
been deleted (see "Experimental Procedures"; Table I). Thus strain
NGR
1
nolO is deficient not only in nodSU but
because of the polar effects of Omega insertion in nolO and
noeI as well. RP-HPLC fractions of supernatants from cultures of NGR
1
nolO were analyzed by LSI-MS, while
the fatty acid and monosaccharide compositions were confirmed by GC/MS
using authentic standards. GC/MS analysis of alditol-acetate
derivatives of wild-type supernatants showed major peaks which
correspond to 2-O-methyfucose, GlcNAc, and MeGlcN (data not
shown). The chromatogram obtained from the crude extracts of the
supernatant of NGR
1 cultures lacked MeGlcN, while those from
NGR
1
nolO showed the disappearance of MeGlcN and
2-O-methyfucose as well as the presence of Fuc and GlcNAc
(data not shown). These results confirm the roles of NodS in
N-methylation and NoeI in O-methylation. All
LSI-MS spectra generated from supernatants of NGR
1 did not yield
fragment ions corresponding to N-methylated or
bis-carbamoylated Nod factors (Fig. 2B), but the
monocarbamoylated forms persisted (m/z 455, 469, and 471).
Unexpectedly, the B1 oxonium ions of mass spectra generated from
different fractions of the supernatants of NGR
1
nolO were similar to those from NGR
1, showing that mutation of
nodU and nolO were not sufficient to completely
abolish carbamoylation (data not shown). The 2-O-methyl
group was however absent in the supernatants of cultures from
NGR
nolO (Fig. 2A) and
NGR
1
nolO. Moreover the pseudomolecular ions from
NGR
1
nolO or NGR
1 revealed a short Nod factor
backbone (3 or 4 GlcNAcs) confirming the role of NodS in ensuring
pentameric NodNGR factors (15). In some cases, the molecular ions of
the tri- and tetrameric LCOs were similar to fragment ions from the
pentameric forms (see Fig. 2B, at 1299.7, 1256.7, 1096, and
1053 Da), but the relative abundances of the noncarbamoylated and
monocarbamoylated forms were reversed. MS and MS/MS analyses of pure
HPLC fractions of these short LCOs clearly proved these structures. The
monocarbamoylated forms were also observed in the LSI-MS spectra of
LCOs purified from NGR
nodU
nolO.
Specific Carbamoylation by NolO--
Further evidence that NolO
specifies carbamoylation on positions other than O-6 was obtained by
tandem mass spectrometry and by 15N or 13C NMR
spectroscopy. Using model compounds, we showed that the metastable ion
spectra of the oxonium ions from all isomers of mono-O-acetylated, N-acetylglucosamine were
different and that 3-O- and 6-O-carbamates
behaved as the corresponding O-acetates (4-O-acetate was not used as a reference
compound).2 In an attempt to locate their
O-carbamoyl substitutions, these experiments were extended
to NodNGR factors. Metastable decomposition of the B1 ion from Nod
factors isolated from NGRnodU showed the prominent loss
of carbamic acid (Fig. 4B).
This behavior was very similar to that found with
3-O-acetyl-N-acetylglucosamine in which the
3-O-substituent, acetic acid, was eliminated. It was
different from that found with the 6-O isomer however, which
was characterized by the loss of water. In contrast, the B1 ions from
Nod factors produced by the NGR
nolO mutant were
characterized by the prominent loss of water (Fig. 4C),
clearly locating the remaining carbamoyl group on C-6. This ion
spectrum was identical to that found with Nod factors from
Azorhizobium caulinodans (NodARc) in which the carbamate
group has been unambiguously assigned to O-6. Indeed, the metastable
ion spectra of the fragment ion at m/z 483, which corresponds to monocarbamoylated NodNGR factors, gave fragment ions at
m/z 465 and 422 (Fig. 4A) showing the loss of
carbamic acid or water. This suggests that the carbamoyl group could be at either of positions O-3 or O-6.
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DISCUSSION |
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Since mutation of noeI has no effect on nodulation while host-range extension (complementation) studies with nolO render USDA257 Nod+ on C. caeruleum, it seems likely that nolO is the principal host range determinant of the hsnIII locus. R. fredii transconjugants harboring p6.8HR (which contains the complete nolO gene but only part of noeI) or p6.8HRCRep (which contains a frameshift in noeI) allow USDA257 to nodulate C. caeruleum. On the other hand, transconjugants harboring p7.6E (in which nolO is truncated) or p6.8HRERep (having a frameshift in nolO) are unable to nodulate. As the fucosyl moiety of USDA257 Nod-factors is 2-O-methylated, an equivalent of the noeI gene must be active in this strain. Yet USDA257 does not nodulate C. caeruleum, confirming that adjunction of the 2-O-methyl group to the fucose is not essential for nodulation of this species. Mutation of nodI has a polar effect on the expression of the nodABCIJnolOnoeI operon and inhibits both the bis-carbamoylation and 2-O-methylation of NodNGR factors. Thus, through elimination it seems likely that NoeI is the 2-O-methyltransferase.
The predicted NolO protein is highly homologous to NodU of various
rhizobia as well as a carbamoyltransferase of Nocardia lactamdurans (Fig. 7). From the
present work, NolO seems to be specific to position O-3 or at least to
positions O-3 and O-4 of the nonreducing terminus (Fig. 6). In A. caulinodans and R. loti, a single carbamate group was
found at position O-6 (35) or position O-4 (36), respectively. Although
position O-6 had clearly been established by a set of convergent
methods, some doubt remained about positions O-3 and O-4 because of a
possible isomerisation occurring during purification. Despite this
reservation, it seems clear that at least two specific
carbamoytransferases exist in rhizobia. The present work, based on
LSI-MS, the absence of periodic cleavage, and 13C NMR
spectra confirm that NolO carbamoylates one of the secondary hydroxyl
groups of the terminal non-reducing GlcNAc. The continued production of
6-O-carbamoylated NodNGR factors in the double mutant NGRnodU
nolO, points to a third
carbamoyltransferase specific of the O-6 position. The physical
location of this third carbamoyltransferase remains to be determined,
since no other NodU/NolO homologues are present on pNGR234a
(33). Also, since 6-O-carbamoylated NodNGR factors cannot be
detected when nodU is mutated (15), the second
6-O-carbamytransferase must be less efficient than NodU (a
few percent of the 6-O-isomer cannot be ruled out by MS and
NMR data) or repressed when nolO is activated.
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Finally, since Leucaena sp. and
Calopogonium/Lablab react differently to NodNGR
factors modified by NodU and NolO respectively (Table II), it seems
likely that both groups of plants have specific requirements for the
position of the carbamoyl group. On the other hand, the absence of
clear phenotypes with the NGRnolO and
NGR
nodU
nolO mutants is probably due to the
third, carbamoyltransferase.
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ACKNOWLEDGEMENTS |
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We thank S. Reli and D. Gerber for
their assistance with many aspects of this work and G. Hardarson for
the gift of
(15NH4)2SO4.
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FOOTNOTES |
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* This work was supported by the Erna och Victor Hasselblads Stiftelse, the Fonds National Suisse de la Recherche Scientifique (Projects 31-36454.92 and 31-45921.95), and the Université de Genève.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ The first two authors contributed equally to this research.
** To whom correspondence should be addressed. Tel: 41 (22) 906 17 40; Fax: 41 (22) 906 17 41; E-mail: broughtw{at}sc2a.unige.ch.
1 The abbreviations used are: RP, reverse phase; LCO, lipochitinoligosaccharide; HPLC, high performance liquid chromatography; LSI, liquid secondary ion; MS, mass spectrometry; MS/MS, tandem MS; GC, gas chromatography; bp, base pair(s); kb, kilobase pair(s).
2 M. Ferro, M. Treilhou, S. Jabbouri, W. J. Broughton, C. Monteiro, and J.-C. Promé, manuscript submitted for publication.
3
B. Reli, unpublished results.
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REFERENCES |
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