From the Complex Carbohydrate Research Center, University of
Georgia, Athens, Georgia 30602
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INTRODUCTION |
Bacteria belonging to the family Rhizobiaceae are
Gram-negative and are able to form nitrogen-fixing symbiotic
relationships with legume plants. The surface polysaccharides,
including the lipopolysaccharides
(LPSs),1 are involved in the
normal infection process. Mutants that lack the O-chain
polysaccharide portion of their LPSs are symbiotically defective in
that they are unable to form normal infection threads (1, 2), and/or
they cannot invade the root nodule cells (3-5). In addition, it has
been shown that structural changes in the LPS occur during symbiotic
infection and that most of these changes appear to take place in the
O-chain polysaccharide portion of the LPS (6-14). These
structural adaptations in response to the environment of the host plant
are likely to be important in order for the symbiont bacterium to
induce a nitrogen-fixing nodule.
In addition to the importance of determining the symbiotic
"virulence" of these bacteria, rhizobial LPSs can have unique
structural features compared with the LPSs from enteric bacteria. The
most studied LPSs, the topic of this report, are those from
Rhizobium etli and Rhizobium leguminosarum. All
of the LPSs examined from wild-type or parent strains of these species
are devoid of phosphate. Their lipid A region has a trisaccharide
backbone consisting of one each of galacturonosyl (GalA), GlcN, and
2-aminogluconosyl (GlcN-onate) residues, the latter two residues being
O- and N-acylated with
-hydroxy fatty acids
and one very long chain fatty acid, 27-hydroxyoctacosanoic acid (15).
This lipid A also appears not to carry any acyloxylacyl substituents.
The core region has been found to consist of two oligosaccharides (16,
17) as shown below.
and
The core region of R. etli and R. leguminosarum LPSs does not contain heptose, a common glycosyl
residue found in the inner core region of enteric LPSs. Thus, although
the individual core components have been isolated and characterized,
the arrangement and proportions of these components in the intact LPS
from the various R. etli strains was unknown and is the
subject of this report.
The unique structural features of the R. etli LPS have also
prompted an investigation into the mechanism by which it is
synthesized. That work began by determining whether or not R. leguminosarum or R. etli contained any of the lipid A
biosynthetic enzymes found in Escherichia coli. It was found
that R. leguminosarum and R. etli contain all of
the enzymes necessary to make the E. coli lipid A precursor,
Kdo2lipid-IVa (a di-Kdo-sylated tetraacyl-1, 4
-bis-phosphorylated glucosamine disaccharide) (18).
Therefore, this component, or a close structural analog, is a common
precursor required for the synthesis of E. coli, R. etli, and R. leguminosarum lipids A. This result
implies that the biosynthetic steps leading to
Kdo2lipid-IVa are common to a very wide range of
Gram-negative bacteria. The fact that this precursor is essential for
cell viability in E. coli (19, 20) suggests that it, or a
similar rhizobial structural analog, may be required for the viability
of the R. etli and R. leguminosarum cells. The
presence of this common lipid A precursor also implied that these
rhizobial cells possess unique enzyme activities, which process their
Kdo2lipid-IVa analog into the mature rhizobial lipid A
structure. Several of these enzyme activities have been reported,
including a membrane-bound phosphatase that removes the 4
-phosphate
from Kdo2lipid-IVa and requires the presence of the Kdo
residues for maximum activity (21), a second phosphatase that removes
the 1-phosphate (22), and a unique acyl carrier protein required for
the transfer of the 27-hydroxyoctacosanoyl residue to the lipid A (23).
In addition, a rhizobial-specific mannosyl transferase activity has
been identified that transfers mannose from GDP-Man to
Kdo2lipid-IVa (24). It is apparent from these reports that
R. leguminosarum and R. etli contain novel
enzymes that can modify the enteric lipid A precursor, Kdo2lipid-IVa.
To fully understand how the unique R. etli and R. leguminosarum LPSs are biosynthesized and to understand their role
in determining symbiotic virulence, it is necessary to characterize
their structures. Thus, while the types of structures found in the core
region, e.g. oligosaccharides 1 and 2,
had been determined, as well as the lipid A structure, it was not known
how these structures were arranged in the intact LPS. In this report,
we describe the structures of the LPS from two R. etli
mutants, CE358 and CE359, which either lack the O-chain
polysaccharide entirely, or contain a truncated version of the
O-chain (17). A previous report showed that mild acid
hydrolysis of the LPS from CE359 produced core oligosaccharides
1 and 2 as well as monomeric Kdo and galacturonic
acid (17). The LPS from CE358 produced the same components except that
structure 1 lacked the galacturonosyl residue (17). In this
report, the complete structures of the lipid A-core region for the LPSs
from these two R. etli CE3 mutants are described.
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EXPERIMENTAL PROCEDURES |
Bacterial Strains--
R. etli CE3 (the parent
strain) and the R. etli mutants CE358 and CE359 were grown
in a tryptone/yeast extract supplemented with Ca2+ as
described previously (25). Bacteria were harvested by centrifugation at
late log/early stationary phase.
Lipopolysaccharide Purification--
The parent strain, CE3, and
the R. etli mutant strains CE358 and CE359, were extracted
using the hot phenol-water procedure as described previously (25, 26).
Polyacrylamide gel electrophoresis (PAGE) in the presence of
deoxycholate (DOC) (27, 28) of the water and phenol fractions indicated
that LPS was recovered predominantly in the water layer in the case of
strain CE3, while the two mutant strains yielded LPS in both the phenol
and water layers. Water and phenol layers containing LPS were dialyzed
and treated sequentially with ribonuclease, deoxyribonuclease, and
proteinase K and then dialyzed and subjected to further purification by
either gel filtration chromatography on Sepharose 4B (25, 29) or by
affinity chromatography on columns of polymyxin-agarose (Detoxi-Gel,
Pierce). For the latter procedure, the crude extracts were treated with
enzymes as described above and then dialyzed against 50 mM
NH4HCO3, pH 8.0. Portions of the dialyzed
extract (30 ml) were applied to a 10-ml bed volume column of
polymyxin-agarose equilibrated in 50 mM
NH4HCO3. The entire extract was recirculated
through the column overnight at a flow rate of 5.0 ml/h. Following
recirculation, the flow-through fraction was collected in a single
tube, and the column was eluted sequentially with solutions of
increasing ionic and/or chaotropic strength to elute the weakly bound
non-LPS components. The column was eluted first with 300 mM
triethylamine/acetic acid buffer (pH 6.4) containing 10% ethylene
glycol, followed by 2.0 M urea in 0.1 M
NH4HCO3, pH 8.0. The LPS was then eluted from
the column with 1% sodium DOC in 0.1 M
NH4HCO3, pH 8.0, or with 8.0 M
guanidine HCl. Fractions (3 ml) were monitored for LPS and other
components by DOC-PAGE analysis with silver staining (28).
Typically, when extracted using the traditional phenol-water
procedure, many of the R. etli mutants show relatively low
yields of LPS compared with that from the parent strain. An alternative extraction procedure was therefore developed and used for the CE358 and
CE359 mutant strains, consisting of extraction with a solution of 1.0 M TEA, 0.33 M EDTA containing 5% (v/v)
liquified phenol, pH 6.5-6.9. Typically, 80-100 g (dry weight) of
lyophilized cells were extracted with 500 ml of buffer solution with
stirring for 1 h at 60 °C. The resulting extract was
centrifuged (15,000 × g), yielding a pellet of cell
residue and an aqueous supernatant containing LPS, which was dialyzed
and treated with enzymes as described above. The aqueous extract was
then subjected to polymyxin-agarose affinity chromatography as
described above.
Purification of the Core Oligosaccharides--
Intact LPS was
subjected to mild hydrolysis by treating with 1% acetic acid for
1 h at 105 °C (30). The lipid A precipitate was removed by
centrifugation, and the supernatant, containing water-soluble core
fragments and an O-chain polysaccharide, was analyzed by
HPAEC on a Carbo Pac PA-1 column (Dionex) equipped with pulsed
amperometric detection. Separation was achieved using a gradient of
3-90% sodium acetate (1 M) in 100 mM NaOH
over 50 min at 1 ml/min (17). The oligosaccharide fractions were
collected, passed through Dionex OnGuard H cartridges to remove sodium,
and then lyophilized.
De-O-acylation of R. etli Mutant LPS--
Each purified LPS from
R. etli mutant strains CE358 and CE359 was
de-O-acylated with sodium methoxide (0.25 M) at
35 °C for 24 h (15). The reaction mixture was centrifuged
(3000 × g), and the supernatant was removed and
analyzed for released fatty acids. The precipitate, containing
de-O-acylated LPS, was dissolved in water, adjusted to pH
4.0 with dilute acetic acid, and washed two times with
hexane/chloroform 1:1 (v/v) to remove residual fatty acids. The
resulting de-O-acylated LPS was then desalted by sequential
dialysis (MWCO 1000) and treatment with Dowex 50 or Chelex (both in the
H+ form) and subjected to electrospray ionization mass
spectrometry analysis. In some cases, the de-O-acylated LPS
was converted to the ammonium form
(ROO
NH4+) by treating with
Chelex (NH4+) to assist in
identification of the charge state of ions during ESI-MS analysis.
Analysis of Glycosyl Residues--
Glycosyl compositions of
intact and de-O-acylated LPS were determined by preparation
of the trimethylsilyl methyl glycosides with GLC-MS (electron impact)
analysis (31). The purified LPS and de-O-acylated LPS were
subjected to methanolysis in methanolic 1 M HCl at 80 °C
for 18 h, N-acetylated, trimethylsilylated, and then
analyzed using a 30-m DB-5 fused silica capillary column (J & W
Scientific). Linkage analysis of neutral sugars was determined by
permethylation (Hakomori method), conversion to the partially methylated alditol acetates (PMAAs) as described previously (31), and
GLC-MS analysis using a 30-m SP2330 (Supelco) capillary column. The Kdo
and uronic acid linkages were identified by sequential permethylation,
reduction of the carboxymethyl groups with lithium triethylborodeuteride (Superdeuteride, Aldrich), mild hydrolysis (0.1 M trifluoroacetic acid, 100 °C, 30 min) to cleave
ketosidic linkages, reduction (NaBD4), normal hydrolysis (2 M trifluoroacetic acid, 121 °C, 2 h), and
conversion to the PMAAs and GLC-MS analysis (17). The identities of
trimethylsilyl methyl glycoside and PMAA derivatives were also
confirmed by chemical ionization, using a 30-m DB-1 column and ammonia
as the reactant gas.
Fatty Acid Analysis--
Ester-linked fatty acids, released from
the LPS by de-O-acylation as described above, were subjected
to methanolysis and trimethylsilylation and analyzed by GLC-MS using a
30-m DB-1 column (15). Total fatty acids were released by hydrolysis in
4 M HCl followed by methanolysis, trimethylsilylation, and
GLC-MS analysis as above. Amide-linked fatty acids were determined by
mild methanolysis, trimethylsilylation, and analysis of the resulting
N-acylglucosamine methyl glycosides by GLC-MS as described
(15, 32).
Mass Spectrometry--
Electrospray ionization mass spectrometry
(ESI-MS) was performed on a SCIEX API-III triple-quadrapole mass
analyzer (PE/SCIEX Thornhill, Ontario, Canada) operated in the positive
ion mode with an orifice potential of 35-50 V (15). Spectra are the
accumulation of 10-60 scans collected over the m/z range
200-2400 with a mass step of 0.2-1.0 atomic mass unit at 1 ms/step.
Samples were analyzed at a concentration of 2 µg/µl with a flow
rate of 3-5 µl/min using a solution of 15% (v/v) methanol in
deionized water containing 0.5% (v/v) acetic acid. Tandem mass
spectrometry (MS/MS) was performed on the SCIEX instrument by selecting
a parent ion for collision-induced dissociation using argon as the
collision gas. The predicted molecular weights of the various
de-O-acylated LPS were calculated using the following
average incremental mass values, based on the atomic weights of the
elements: hexose, 162.1424; hexuronic acid, 176.1259; Kdo, 220.1791;
N-acetylhexosamine, 203.1950; 2-amino-2-deoxyhexose, 161.1577; 6-deoxyhexose, 146.1430;
mono-O-methyl-6-deoxyhexose, 160.1699;
2-amino-2-deoxygluconic acid, 177.1571;
2-N-acetamido-2,6-dideoxyhexose, 187.1955;
-hydroxymyristate, 226.3592;
-hydroxypalmitate, 254.4130;
-hydroxystearate, 282.4667; free reducing end, 18.0153.
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RESULTS |
Purification of R. etli LPSs and Initial Characterization--
The
parent strain, CE3, was extracted with the hot phenol/water procedure,
and the LPSs were purified by the standard technique of size exclusion
chromatography on Sepharose 4B (25, 26, 29). DOC-PAGE analysis showed
the presence of typical high molecular weight LPS (LPS I, containing
O-chain polysaccharide) and low molecular weight LPS (LPS
II, which lacks the O-chain, and various LPSs that carry a
truncated O-chain) (12, 17). As described in previous
reports (12, 17), mutant CE358 produced only one type of LPS, having an
electrophoretic mobility identical to LPS II. Mutant CE359 also
produced an LPS II species, as well as an LPS of slightly higher
molecular weight, which carries a truncated O-chain
polysaccharide (17). The various forms of LPS that carry a truncated
O-chain have been designated LPS III, IV, and V and are
distinguishable based on their differential reactivity with monoclonal
antibodies (12). Mutant CE359 produces LPS V in addition to LPS II
(12).
In general, the two mutant strains showed substantially lower
yields of total LPS compared with the parent strain when extracted using the standard phenol/water procedure and its associated
purification scheme consisting of repeated enzyme treatments, dialysis,
and Sepharose 4B chromatography. The CE359 mutant yielded approximately 6 mg of LPS in each of the phenol and water layers from 50 g (dry weight) of lyophilized cells, compared with a typical yield of 700-800 mg of total LPS/50 g of lyophilized cells from the parent strain (CE3). DOC-PAGE analysis of the mutant LPS preparations obtained
by the Sepharose 4B procedure showed that they were highly contaminated
with capsular polysaccharides and other components that co-migrated
with the LPS during the chromatography step (data not shown). Due to
the need for reasonable amounts of high purity LPS from these strains,
an affinity chromatography approach utilizing polymyxin-agarose was
employed. DOC-PAGE analysis of polymyxin chromatography of the CE359
LPS extract is shown in Fig. 1. Using this procedure, the mutant LPSs could be conveniently isolated in a
single small volume fraction, free of contaminating polysaccharides. The CE358 LPS family, which consists only of the lower molecular weight
band (i.e. LPS II) was purified in a similar manner.

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Fig. 1.
DOC-PAGE analysis of polymyxin-agarose
affinity chromatography of the R. etli mutant CE359
LPS. The phenol layer obtained from phenol-water extraction was
dialyzed, treated with ribonuclease, and applied to a column of
polymyxin-agarose equilibrated in 0.1 M
NH4HCO3. Cell surface polysaccharides and other
non-LPS components were washed from the column with
NH4HCO3 (fractions 3-20),
TEA-acetic acid buffer (fractions 21-23), and 2.0 M urea (fractions 24-28) as described under
"Experimental Procedures." The phenol extract contained both LPS II
and LPS V, which were eluted in fraction 29 with 1% sodium
deoxycholate.
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To further improve the yields of mutant LPSs, several alternative
extraction procedures were examined that minimized or eliminated the
use of phenol. It was found that extraction with a solution of
TEA-EDTA-5% phenol (see "Experimental Procedures") produced relatively high yields of LPS, which could be directly applied to the
polymyxin column. Using this extraction procedure combined with
polymyxin-agarose chromatography, approximately 45 mg of LPS II could
be obtained from 50 g (dry weight) of CE358 cells, and 24 mg of
LPS II could be obtained from 50 g (dry weight) of CE359 cells. It
was further noted that extraction of the CE359 mutant with the TEA-EDTA
protocol yielded preparations highly enriched in LPS II, with
essentially no LPS V. In contrast, extraction by the standard
phenol/water procedure yielded mixtures of LPS II and V in the phenol
layer (as shown in Fig. 1) and preparations enriched in LPS V in the
water layer. The different extracts were purified separately by
polymyxin affinity chromatography and subjected to structural
analysis.
The glycosyl composition of the R. etli strains examined in
this study is listed in Table I. The
results show the presence of the O-chain glycosyl components
in the CE3 parent strain LPS and, to a lesser extent, in the CE359
mutant. The CE358 mutant has an LPS that is devoid of all
O-chain glycosyl components. The composition of the
O-chain repeating unit of the parent strain has been
reported (33, 34). The complete structure of the lipid A moiety of the
parent strain, including the fatty acid composition, has been published
previously (15), and composition analysis indicates that the mutant
LPSs contain a similar lipid A and fatty acid profile; i.e.
they contain galacturonic acid, glucosamine, and 2-aminogluconic acid
in the lipid A backbone as well as the previously reported 3-hydroxy
fatty acids (15). One notable difference in the mutant LPSs compared
with that from the parent is the significantly lower level of
2-aminogluconate. This may indicate that a significant portion of the
mutant LPSs contain molecules that lack the 2-aminogluconic acid
residue normally present in the lipid A.
Analysis of R. etli Mutant CE358 LPS--
The glycosyl composition
and linkages of the intact LPS from strain CE358 are reported in
Tables I and II, respectively. In
addition, a portion of the CE358 LPS was subjected to mild acid
hydrolysis, and the resulting oligosaccharides were analyzed by HPAEC
(Fig. 2A). The results verify
an earlier report (17) showing that the CE358 LPS core region consists
of two major oligosaccharide components: trisaccharide 2 (shown above) and trisaccharide 3.
In addition to trisaccharides 2 and 3,
monomeric Kdo and monomeric galacturonic acid were also released from the CE358 LPS during mild hydrolysis (Fig. 2A). These
results suggest that the CE358 LPS core region consists of the two
oligosaccharides (2 and 3) connected together in
an unknown arrangement with additional Kdo and galacturonosyl residues.
Alternatively, the individual oligosaccharide components could be
carried separately on individual LPS molecules, each having a similar
size and DOC-PAGE mobility.
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Table II
Glycosyl linkage analysis of R. etli LPS
Intact LPSs were subjected to linkage analysis for neutral sugars, Kdo,
and uronic acids as described under "Experimental Procedures," and
the total ion current peak areas from GLC-MS are reported. The lipid A
moiety contributes one residue of terminal GalA to each analysis, in
addition to glucosamine and 2-aminogluconate, which are not reported
here.
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Fig. 2.
Analysis by HPAEC of the core components
released by mild acid hydrolysis of the R. etli mutant
LPSs. A, R. etli CE358 LPS; B, CE359
LPS II; C, CE359 LPS V. The structures of tetrasaccharide 1 and trisaccharides 2 and 3 are
described in the Introduction and under "Results."
anhydTetra, the tetrasaccharide 1 containing an
anhydro-Kdo residue at the reducing end, a product of the acid
hydrolysis conditions (17). In panel A, the
-GalpA-(1 4)-Kdo disaccharide is a small component that
elutes at approximately 15 min. In panel C, the components
eluting between 2 and 5 min and at 13 min are oligosaccharides derived
from the truncated O-chain of the CE359 LPS V.
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Linkage analysis of the intact CE358 LPS, prior to mild acid
hydrolysis, yielded the following PMAA derivatives in a 1:1:2 ratio
(see Table II):
1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-mannitol (m/z 118, 162, 189, and 233),
1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-galactitol (m/z 118, 162, 189, and 233), and
1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-6,6-2H-galactitol
(m/z 118, 162, 191, and 235), derived from 6-linked mannose,
6-linked galactose, and terminal galacturonic acid, respectively. Methylation analysis of the oligosaccharides obtained after the mild
hydrolysis, shown in the HPAEC profile of Fig. 2A, gave the same results with the exception that all of the 6-linked galactose had
been replaced by terminal galactose (i.e.
1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-galactitol (m/z 118, 161, 162, and 205)). This result indicates that
the terminal galactosyl residue of trisaccharide 3 must
be substituted at O-6 in the intact LPS by a mild acid-labile residue, presumably Kdo or an oligosaccharide terminating in Kdo at the reducing
end. Methylation analysis of the intact CE358 LPS, according to a
procedure modified to detect Kdo linkages (see "Experimental Procedures") gave the PMAA derivatives of terminal-Kdo
(1,2,6-tri-O-acetyl-3-deoxy-4,5,7,8-tetra-O-methyl-1,1-2H-octitol
(m/z 89, 146, 205, 206, 250, and 366)), and 4,5-linked Kdo
(1,2,4,5,6-penta-O-acetyl-3-deoxy-7,8-di-O-methyl-1,1-2H-octitol
(m/z 89, 186, 228, 348, and 422)) (Table II and Fig. 3). These results indicate that a Kdo
residue must occupy a terminal (nonreducing) location on the majority
of the intact LPS molecules. In addition, the 5-linked Kdo residue
present in trisaccharide 3 must also be substituted at O-4
in the intact LPS, since a derivative arising from 5-linked Kdo,
detected during methylation of oligosaccharide 3, was not
observed during methylation of the intact LPS. In the intact LPS, this
5-linked Kdo residue of oligosaccharide 3 could be
substituted at O-4 by either the Kdo residue of oligosaccharide
2 (yielding 4) or by a single terminal Kdo
residue (yielding the alternative structure 5).
or
Other alternative structures would also fit these data,
particularly if the two core components, oligosaccharides 2 and 3, were carried separately on different LPS
molecules.

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Fig. 3.
GLC-MS analysis of the PMAA derivatives from
the Kdo linkages in the CE358 LPS. The profile shows selective ion
monitoring for m/z 89, which results from the C-7 to C-8
carbon fragment of all Kdo derivatives regardless of linkage position,
with the exception of Kdo residues linked at the C-7 or C-8 position.
The latter Kdo derivatives were not detected during total ion current analysis. Diastereomeric pairs arise due to nonstereospecific reduction
of the C-2 carbonyl with borodeuteride. The terminally linked GalA
(T-GalA) derivative yields m/z 89 as a result of
secondary fragmentation. T-Kdo, terminally linked Kdo.
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Methylation analysis also yielded several minor derivatives, detected
at nonstoichiometric levels, presumably arising from small populations
of molecules due to glycosyl heterogeneity. These minor derivatives
arise from 4-linked Kdo (i.e.
1,2,4,6-tetra-O-acetyl-3-deoxy-5,7,8-tri-O-methyl-1,1-2H-octitol
(m/z 89, 205, 278, 320, and 394)),
4,6-di-O-substituted mannose
(1,4,5,6-tetra-O-acetyl-2,3-di-O-methyl-mannitol
(m/z 118 and 261)), and terminal galactose
(1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-galactitol (m/z 118, 161, 162, and 205)). As discussed further below,
each of these minor derivatives corresponds to minor LPS species
identified by mass spectrometry. Thus, the derivatives obtained during
linkage analysis of the intact CE358 LPS are consistent with the
glycosyl compositions and core oligosaccharide structures
determined previously; however, the multiplicity of these derivatives
and the overall similarity of the core components detected during HPAEC
analysis precluded unambiguous structural assignments for the intact
LPS without the aid of molecular mass information.
To distinguish between the alternative structures, a portion of the
intact CE358 LPS was subjected to de-O-acylation and ESI-MS analysis. The positive ion ESI-MS spectrum is shown in Fig.
4, and the major molecular ions are
listed along with their proposed formulas in Table
III. Clearly, the most prominent species
is the single ion at m/z 960.2. This ion and all others in
the spectrum are doubly charged protonated molecular ions of the
general formula (M + 2H)2+. The charge state and type of
cationization were readily determined by examination of the incremental
differences between various ions in the spectrum and by observing the
alterations in m/z values produced during controlled
cationization with the addition of ammonium or sodium to the sample.
The molecular mass of this species (1918.4 atomic mass units) was then
arrived at by multiplying the m/z value by the number of
charges (z) and subtracting an equivalent number of protons.
This molecular species does not include the 2-aminogluconosyl residue
of the lipid A, indicating that it is either absent in a significant
portion of the LPS, as indicated from the composition results (Table I)
discussed above, or is somewhat labile to the alkaline
de-O-acylation conditions. The lability of this residue was
indicated in the previously reported fast atom bombardment mass
spectrum of the de-O-acylated lipid A from the R. etli parent strain (15), in which the major ion observed was that
of the molecule without 2-aminogluconate. The major
de-O-acylated CE358 LPS carries the 14-carbon chain length fatty acid (
-hydroxymyristic acid) in amide linkage to the
glucosamine residue of the lipid A backbone. This is in agreement
with fatty acid analysis of the CE358 LPS, which shows that the
amide-linked fatty acids consist of
-hydroxymyristate,
-hydroxypalmitate, and
-hydroxystearate in a 7.6:1.0:1.2 ratio. A
similar ratio, in which
-hydroxymyristic acid was the predominant
amide-linked fatty acid, was observed for the parent strain lipid A
(15, 35). A family of minor ions in the spectrum also arises from this
same molecule, due to lactone formation [(M + 2H)
H2O]2+ (m/z 951.2), from residual
cationization with ammonia (m/z 968.5), and from the
amide-linked fatty acid heterogeneity (m/z 974.2 and 988.5)
due to the 16 and 18 carbon fatty acids, respectively (Table III, Fig.
4, inset).

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Fig. 4.
Positive ion electrospray mass spectrum of
the R. etli CE358 de-O-acylated LPS II.
The LPS preparation yielded doubly charged protonated molecular ions
for the major LPS species (m/z 960.2, calculated mass 1918)
and for minor LPS species present in the mixture. The inset
shows the family of ions arising from amide-linked fatty acid
heterogeneity and lactone formation of the major molecular species. All
ions and their proposed formulas are listed in Table III.
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The major molecular ion at m/z 960.2 (Mr 1918) and its associated ions clearly
indicate that the major form of the CE358 de-O-acylated LPS
carries both core oligosaccharides 2 and 3 together on the same molecule. Molecular ions for individual LPS
molecules carrying either trisaccharide 2 or trisaccharide
3 alone were not observed, and such molecules therefore do
not contribute to the glycosyl heterogeneity of the CE358 LPS
preparation. To distinguish between the resulting structural
alternatives (i.e. structures 4 or 5 above), the major molecular ion (m/z 960.2) was selected as
parent ion for collision-induced dissociation using the electrospray
instrument and argon as collision gas. The resulting fragment ions
(Fig. 5) consisted of both Y-type sequence ions (36) resulting from glycosidic bond cleavages, with
charge retention on the reducing end moiety, and the complementary B-type sequence ions. The Y-type ions demonstrate the sequential loss
of terminal Kdo (yielding m/z 850.2, 1698.4 atomic mass
units), followed by loss of hexose (yielding m/z 769.2, 1536.4 atomic mass units), indicating that Kdo indeed occupies a
terminal (nonreducing) position and that a hexosyl residue occupies the
penultimate position. These results, in combination with the
methylation data, clearly support the core arrangement shown in
4, and indicate that the major de-O-acylated LPS
from CE358 has the structure indicated in Fig. 5.

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Fig. 5.
Positive ion ESI-MS/MS spectrum of the major
CE358 LPS species shown in Fig. 4. The doubly charged ion
m/z 960.2 was selected as parent ion for collision-induced
dissociation-MS/MS analysis. Consecutive loss of the first three
glycosyl residues yielded doubly charged Y-type fragment ions (36) with
charge retention at the reducing end, m/z 850, 769, and 688. Singly charged Y-fragment ions were also observed at m/z
1536 and 1373 and become intense at m/z 802 and 582. The
spectrum also contains a series of complementary B-type sequence ions
resulting from oxonium ion formation with charge retention on the
nonreducing end sequences: Kdo-Hex-Hex (m/z 545), Kdo-Hex
(m/z 383), Kdo (m/z 221),
[GalA]2Kdo (m/z 573), and GalA-Kdo
(m/z 397).
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The ESI-MS spectrum of the de-O-acylated CE358 LPS shows, in
addition to the m/z 960.2 ion family, several doubly charged molecular ions of minor intensity at m/z 1048.2 (Mr 2094.4), 872.2 (Mr
1742.4), 850.2 (Mr 1698.2), and 674.0 (Mr 1346.0) (see Table III). These minor ions
show that there is glycosyl heterogeneity in the CE358 LPS. The
m/z 1048.2 ion is consistent with an additional galacturonosyl residue. The presence of this additional residue is
supported by the HPAEC analysis of the mild acid released
oligosaccharides (Fig. 2A), which shows the presence of a
small amount of tetrasaccharide 1, containing galacturonic
acid linked to O-4 of the mannosyl residue. As discussed above,
methylation analysis (Table II) shows small amounts of 4,6-linked
mannose, which also supports the presence of this structure. The
m/z 872.2 ion arises from a structure that lacks one
galacturonosyl residue. Again, such a structure is supported by the
presence of a small amount of 4-linked Kdo (Table II and Fig. 3) and
the HPAEC analysis (Fig. 2A), which indicates a small amount
of an oligosaccharide with a retention time corresponding to a
disaccharide of
-GalpA-(1
4)-Kdo. Mild acid hydrolysis
of R. etli LPS has been shown previously to release a small
amount of this
-GalpA-(1
4)-Kdo disaccharide (17). The
minor ion m/z 850.2 indicates the presence of LPS molecules
lacking one Kdo residue. Methylation analysis (Table II) again shows
the presence of minor amounts of terminally linked galactose,
indicating that a small percentage of LPS is present that lacks the Kdo
residue that is terminally linked to O-6 of this galactosyl residue.
Finally, the minor ion at m/z 674.0 is consistent with the
absence of two galacturonosyl and one Kdo residues. This minor species
could lack the trisaccharide 2 unit; however, since
methylation analysis of this LPS does not reveal any 5-linked Kdo, a
more likely possibility for the arrangement of this minor species
is as follows.
Thus, both methylation analysis of the CE358 LPS and HPAEC
analysis of the core components support the identification of minor
molecular species having the mass assignments indicated by ESI-MS
analysis (Fig. 4 and Table III). That these minor molecular ions result
from true glycosyl heterogeneity in the LPS rather than ion fragments
formed during electrospray is further supported by the fact that
fragmentation of individual maltooligosaccharide standards was not
observed under identical ionization conditions. The structures of the
minor de-O-acylated CE358 LPS species are shown in Fig.
4.
Analysis of the R. etli Mutant CE359 LPS--
As described above,
mutant CE359 produces two forms of LPS, i.e. LPS II and LPS
V, as shown in PAGE analysis (Fig. 1). However, the relative proportion
of these two bands varies considerably depending on the extraction
procedure as well as on the particular cell preparation. Fig. 1 shows
the LPS pattern typically observed from the phenol layer of
phenol/water extracts. The water layer of such extracts is often
enriched in LPS V, while LPS II-enriched preparations are readily
obtained by extraction using the TEA-EDTA protocol. Glycosyl
composition analysis (Table I) shows that in addition to the core
region glycosyl components found in CE358, the CE359 LPS contains small
amounts of glycosyl residues that are typical of the parental
O-chain polysaccharide (i.e. fucose, 3-O-methylrhamnose, glucuronic acid,
N-acetylquinovosamine, and additional mannose). This result,
together with the increased electrophoretic mobility compared with the
higher molecular weight LPS I of the parent strain, shows that CE359
LPS V contains a truncated version of the O-chain and is
consistent with previous reports showing that LPS V binds monoclonal
antibodies that are specific for the O-chain (12).
A portion of the CE359 LPS preparation containing both LPS II and V
(Fig. 1) was de-O-acylated, converted to the ammonium form
(ROO
NH4+), and analyzed by
ESI-MS. The spectrum, Fig. 6,
demonstrated the presence of two major families of ions consistent with
the presence of the two LPS forms, LPS II and LPS V, as revealed by DOC-PAGE (see Fig. 1). The doubly charged molecular ions are of the
general formula (M + 2NH4)2+. The ion at
m/z 1065.0 thus indicates an (experimental) mass of 2093.9 atomic mass units for the LPS II, and the ion m/z 1554.0 yields an experimental mass of 3071.9 atomic mass units for LPS V. Minor molecular species were also detected, and those results are
described below.

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Fig. 6.
Complete positive ion ESI-MS spectrum of the
de-O-acylated R. etli CE359 LPS II and LPS
V mixture, obtained from polymyxin chromatography of the intact LPSs
shown in Fig. 1. The de-O-acylated LPSs were purified
and converted to the ammonium form as described under "Experimental
Procedures," yielding major doubly charged ammoniated molecular ions
at m/z 1065.0 and m/z 1554.0 as labeled above.
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Mild acid hydrolysis and HPAEC analysis of the CE359 LPS II
obtained by TEA-EDTA extraction yielded five core components with identical retention times to those released from the CE358 core although in different proportions (Fig. 2B). The CE359 LPS
II contained relatively equal amounts of oligosaccharides 1 and 2 and only small amounts of trisaccharide 3, a major component in CE358 LPS. As with CE358 LPS, monomeric Kdo and
galacturonic acid were also products of mild acid hydrolysis of CE359
LPS II. These CE359 LPS oligosaccharides are consistent with those
previously reported for this mutant (17).
The molecular mass and core oligosaccharide arrangement of the CE359
LPS II was determined by ESI-MS analysis of the
de-O-acylated LPS II sample (Fig.
7 and Table
IV). The major LPS species yielded a
doubly charged protonated molecular ion, [M + 2H]2+, at
m/z 1048, indicating a mass of 2094.8 atomic mass units. This species is 176 mass units greater than the major CE358 LPS, indicating the presence of an additional galacturonosyl residue on the
CE359 LPS II. This is consistent with HPAEC analysis (Fig. 2), which
shows that tetrasaccharide 1, carrying the GalA residue, is
a major core component of the CE359 LPS II in place of trisaccharide
3, which lacks this GalA residue and is found in the CE358
LPS II. Methylation analysis of CE359 LPS confirmed these results in
that the major mannosyl residue was 4,6-linked mannose instead of the
6-linked mannosyl residue found in CE358 LPS. MS/MS analysis of the
m/z 1048 ion (data not shown) supported the same glycosyl
sequence as that observed for CE358 LPS with the exception that CE359
LPS II contains the additional galacturonosyl residue linked to the O-4
of mannose.

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Fig. 7.
Positive ion ESI-MS spectrum of the
de-O-acyl R. etli CE359 LPS II. Intact LPS
II was obtained by TEA-EDTA extraction of the CE359 mutant as described
under "Experimental Procedures"; LPS V is not extracted from the
cells by this TEA-EDTA procedure. The inset shows minor
molecular ions arising from glycosyl and fatty acid heterogeneity and
lactone formation in the LPS family; all ions are doubly charged and
are listed in Table IV.
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Glycosyl heterogeneity in the CE359 LPS II family was evident from
several minor ions in the ESI-MS spectrum (Fig. 7, Table IV), at
m/z 960 (Mr 1918), m/z 938 (Mr 1874), and m/z 762 (Mr 1522). The Mr 1918 LPS is a minor species that lacks a galacturonosyl residue and is
likely to have the same structure as the major LPS species described
above for CE358; i.e. the core region lacks the GalA residue
attached to O-4 of mannose. This is supported by methylation analysis
of the CE359 LPS, which, in addition to 4,6-linked mannose, shows small
amounts of 6-linked mannose (Table II), and by HPAEC analysis (Fig.
2B), which shows the presence of a small amount of
trisaccharide 3. The Mr 1874 LPS
species is 220 mass units less than the major CE359 LPS II species and therefore lacks a Kdo residue. Methylation analysis of the CE359 LPS
yielded a minor amount of terminally linked galactose, supporting the
presence of a minor LPS species lacking the Kdo that is terminally linked to O-6 of galactose in the major form of CE359 LPS II. The minor
Mr 1522 LPS species (ion m/z 762) in
CE359 LPS II is consistent with an LPS that is missing two
galacturonosyl residues and one Kdo residue, compared with the major
LPS species at Mr 2094. However, as with the
CE358 LPS, 5-linked Kdo was not observed during methylation analysis of
CE359 LPS, and therefore the absence of trisaccharide 2 would not appear likely. The minor m/z 762 ion may be a
fragment ion or, alternatively, could arise from additional core region
heterogeneity as follows.
The types of de-O-acylated structures obtained from the
CE359 LPS II fraction are summarized in Fig. 7.
The CE359 LPS V-enriched fraction, obtained from water layers of
phenol/water extracts, was also further characterized. Mild acid
hydrolysis of the LPS V and HPAEC analysis yielded the profile in Fig.
2C. The results show that tetrasaccharide 1,
trisaccharide 2, and monomeric galacturonic acid are again
major core components as with the CE359 LPS II. However, a decrease in
monomeric Kdo was observed in comparison with that released from CE359
LPS II or that from CE358 LPS. In addition, several new components, not
found in CE359 LPS II, were observed during HPAEC analysis (Fig.
2C). These new components have mobilities identical to those of O-chain oligosaccharides derived from the LPS of the
parent strain after mild acid hydrolysis (not shown). Composition
analysis of these oligosaccharides showed that they contain glycosyl
residues characteristic of the O-chain polysaccharide
(i.e. mannose, N-acetylquinovosamine, fucose,
3-O-methylrhamnose, glucuronic acid, and Kdo). Methylation analysis of the intact CE359 LPS (Table II) also showed the presence of
O-chain glycosyl linkages, namely terminal rhamnose,
3,4-linked fucose, 4-linked glucuronic acid, 3-linked mannose, 3-linked
N-acetylquinovosamine, and 4-linked Kdo. These derivatives
were also observed during methylation analysis of the intact parent
strain LPS (Table II) and of the intact purified O-chain,
recovered after mild hydrolysis of the parent strain LPS (results not
shown).
ESI-MS analysis of the de-O-acylated CE359 LPS V produced
the positive ion spectrum shown in Fig.
8, and the molecular ions and proposed
compositions are listed in Table V. The
sample yields doubly charged protonated molecular ions ([M + 2H]2+) with the major ion at m/z 1537.0, (calculated mass 3072.8 atomic mass units). The mass difference between
LPS V and LPS II (3072.8
2094.8) corresponds to the addition of
two fucosyl residues, one glucuronosyl, one methylrhamnosyl, one
mannosyl, and one N-acetylquinovosaminosyl residue, which
are the glycosyl residues characteristic of the O-chain and
the O-chain attachment region.

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Fig. 8.
Positive ion ESI-MS spectrum of the
de-O-acyl R. etli CE359 LPS V. The intact
LPS V was obtained in the water layers from phenol-water extraction of
the CE359 mutant. The LPS V exhibits heterogeneity at the nonreducing
end due to the attachment of variable lengths of the O-chain
glycosyl residues. The major LPS species (m/z 1537.0, calculated mass 3072) contains a single repeat of the
O-chain repeating unit; minor molecular species give rise to
the less intense doubly charged ions as shown above and listed in Table
V.
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Minor molecular species in the CE359 LPS V preparation that lack one or
more residues of the truncated O-chain are also evident in
the ESI-MS spectrum (Fig. 8 and Table V). The ion at m/z
1456.8 (calculated mass of 2912.6 atomic mass units) is due to an LPS species that is 160.2 mass units less than the major LPS species and
indicates the loss of a 3-O-methylrhamnosyl residue. The ion m/z 1296 (Mr 2590) corresponds to an
LPS that lacks fucose, 3-O-methylrhamnose, and glucuronic
acid, the residues that comprise the single repeating unit of the
truncated O-chain. That these ions reflect true glycosyl heterogeneity in the LPS V preparation is also indicated by the results
of methylation analysis, which show that the O-chain sugars are present in a nonstoichiometric ratio; e.g. small amounts
of 3-linked fucose and a higher proportion of terminal fucose than terminal 3-O-methylrhamnose were detected (Table II).
These minor molecular species, together with the glycosyl linkages of
these O-chain residues, support the glycosyl sequence of the
truncated O-chain indicated in Fig. 8. The complete core
region structure of the various R. etli mutant LPSs is
depicted in Fig. 9. The exact location of
the 3-linked N-acetylquinovosaminosyl residue is not known
with certainty; however, since methylation analysis of the LPS from
CE359 gives only 4- and 4,5-linked Kdo, it is proposed that this
N-acetylquinovosaminosyl residue may be linked to O-4 of Kdo
as shown in Fig. 8. Furthermore, isolation of the intact
O-chain released by mild acid hydrolysis from the parent strain LPS shows that the single 3-linked
N-acetylquinovosaminosyl residue is an O-chain
component, located distally to the reducing end Kdo residue (25).
Further work is in progress to determine if this proposed location is
correct.

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Fig. 9.
The complete glycosyl sequence of the
R. etli LPS core region, showing a single repeating unit of
the truncated O-chain, the outer and inner core regions,
and the linkage to lipid A. The structure of the R. etli lipid A moiety and the position of core attachment to lipid A
were determined previously (15). Fatty acyl chains are ester-linked
(R1, 3-OH-C14:0, -C16:0,
-C18:0, and -C15:0) and amide-linked
(R2, 3-OH-C14:0, -C16:0, and
-C18:0). The 27-OH-C28:0 acyl chain
(R3) is ester-linked; however, the location at the C-5 of
GlcN-onate is hypothetical. The single QuiNAc residue is 3-linked in
both the parent strain LPS and the CE359 LPS V. As described under
"Discussion," the anomeric configurations were assigned previously
(15, 16, 37), with the exception of the Kdo residues, proposed to be in
the -configuration. The positions of glycosyl truncation for LPS
from the various R. etli mutants are indicated by
bars.
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DISCUSSION |
The complete core region structure of the R. etli LPS
is shown in Fig. 9, which shows the structural arrangement of the core oligosaccharides isolated previously, together with the proposed structure for the truncated O-chain. Fig. 9 also indicates
the structural defects found in the LPSs from various R. etli mutants based on the results reported here and on previous
reports (16, 17, 37).
The results described in the present study clearly show that the
previously reported core oligosaccharides from R. etli CE3 (16, 17), i.e. structures 1 and 2, are
carried together on a single LPS molecule and are arranged as shown in Fig. 9. The data do not support the idea that there are two separate LPS species, one with structure 1 as the core region, and a
second with structure 2, in which the former contains the
O-chain polysaccharide. The results also show that the
mutant LPSs examined in this report contain a number of minor LPS
species due to heterogeneity in both the core region, particularly in the case of CE358 LPS, and in the truncated O-chain of CE359
LPS V. In addition, it was previously shown that mild acid hydrolysis of the LPSs from R. leguminosarum biovars trifolii and
viciae strains yields core oligosaccharides identical to those obtained from R. etli CE3 LPS (16, 17, 38-40), strongly suggesting
that the R. leguminosarum LPS core region is identical to
that shown in Fig. 9 for R. etli. The identity of the
R. leguminosarum LPS core with that of R. etli
CE3 is also supported by the fact that monoclonal antibodies to the
core region of an R. leguminosarum bv. viciae LPS
cross-react with equal affinity to the LPSs from numerous strains of
R. leguminosarum bv. trifolii, bv. viciae, and R. etli CE3 (41).2 Thus, it
is proposed that this core structure is common to the LPSs from strains
of R. leguminosarum and R. etli.
The core region of R. etli LPS differs in several respects
from the typical cores of enterobacterial LPS, specifically in the
absence of both heptose and phosphate, and notably in the location of a
Kdo residue in the O-chain attachment region, removed four
residues distally from the lipid A moiety. This Kdo residue serves as
the linking residue between the O-chain and the core region.
Mild acid hydrolysis of R. etli LPS and isolation of the resulting O-chain by size exclusion chromatography indicate
that this 4-linked Kdo residue occupies the reducing end of the
polysaccharide and is readily reduced with borodeuteride to a stable
4-linked Kdo alditol. As the structure of the complete R. etli LPS carbohydrate backbone becomes more clear, it appears that
both an inner and outer core region can be distinguished, analogous to
the enterobacterial LPS. The R. etli and R. leguminosarum common inner core contains Kdo, mannose, galactose,
and an abundance of galacturonic acid, the latter probably serving as a
substitute for phosphate found in the enteric LPS core regions. The
resulting inner core would have a high negative charge density,
allowing the LPS to stabilize in the membrane in the presence of
Ca2+ and other divalent cations, such as proposed for the
enteric LPS inner cores (42, 43). It is well documented that pectic polysaccharides rich in GalA exhibit a high degree of cross-linking in
the presence of Ca2+ (44). The outer core region of the
R. etli LPS is the O-chain attachment region and
consists of one residue each of 3-linked mannosyl, 3-linked
N-acetylquinovosaminosyl, and 4-linked Kdo residues. This
region contains considerably less hexose than the hexose-rich outer
cores found in many enteric LPS. We suggest that the
N-acetylquinovosaminosyl residue may be a signal for proper
O-chain attachment or elongation, analogous to the role proposed for fructose and sedoheptulose in Vibrio cholerae
LPS (45, 46) or for the core modifications proposed for
Salmonella and Shigella (47, 48). Other
structural features of the core region are undoubtedly required for
O-chain attachment in R. etli and R. leguminosarum and are currently under study in other R. etli mutants.
Fig. 9 also shows the proposed structure of the truncated
O-chain in the LPS from mutant CE359. This structure is
based on the ESI-MS data and linkage analysis of the
de-O-acylated LPS described in this report as well as on
unpublished data for the O-chain repeating unit from the
parent strain.3 The single
residues of 3-linked mannose and 3-linked
N-acetylquinovosamine are not part of the repeating unit
and, as discussed above, are defined as an outer core region. Both
residues are carried in the polysaccharide portion released by mild
acid hydrolysis of the parent strain LPS; the relative order of these
residues with respect to the reducing end 4-linked Kdo residue will be
determined by MS/MS analysis of the O-chain polysaccharide.
While the order of these residues is not known with certainty,
preliminary results indicate that a portion of the reducing Kdo
residues are destroyed during mild acid hydrolysis and that subsequent
reduction of the O-chain polysaccharide with borodeuteride
yields the deuterated alditols of Kdo and, to a lesser extent, QuiNAc,
suggesting a penultimate location for this residue as indicated in Fig.
9.
Previous 1H NMR analysis (16, 37) of the core
oligosaccharides 1 and 2 has shown that all of
the glycosyl residues, with the exception of the Kdo residues, are
-linked as shown in Fig. 9. The anomeric configurations of the Kdo
residues have not yet been determined in the intact LPS; however, they are shown as
-anomers, analogous to the Kdo of LPS from enteric bacteria. The configurations of the lipid A glycosyl residues were
previously determined by 1H NMR (15). The anomeric
configurations of the O-chain glycosyl residues are based on
preliminary results obtained from chromium trioxide oxidation and will
be described in more detain in a forthcoming report on this
polysaccharide.
An interesting feature of these R. etli LPSs is the fact
that mild acid hydrolysis releases monomeric galacturonic acid. In a
previous report (17), it was suggested that this may be due to an
additional galacturonosyl residue (i.e. additional to those found in structures 1 and 2) that is attached in some acid-labile manner to the core region. However, the ESI-MS results
reported here clearly show that the de-O-acylated LPSs from
CE358 and CE359 do not contain any additional galacturonosyl residues.
Furthermore, the alkaline conditions of the de-O-acylation experiment do not cause the release of galacturonic acid from the LPS
(data not shown). Thus, it is concluded that one or more of the
galacturonosyl residues shown in the structure of Fig. 9 is labile to
the mild hydrolytic conditions. Such a possibility is not without
precedence, since model compound studies have demonstrated mild
acid-catalyzed elimination of O-4 substituents
(e.g., phosphate) from Kdo, resulting in unsaturated Kdo
residues (49, 50).
The R. etli CE3 lipid A-core structure shown in Fig. 9
serves as the foundation for the attachment of the O-chain
polysaccharide. In order for symbiosis to be successful, it is
necessary that the O-chain be polymerized (i.e.
it cannot be a truncated version) and that the polymerized
O-chain be present in sufficient amounts (i.e. a
sufficient percentage of the LPS molecules must contain the
O-chain polysaccharide). In addition, it is known that
modification to the O-chain occurs during symbiotic
infection. Knowledge of this LPS structure will greatly facilitate our
further characterization of the LPS modifications required for
symbiosis.