From the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602
Received for publication, October 13, 2002, and in revised form, November 15, 2002
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ABSTRACT |
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A broad-host-range endosymbiont,
Sinorhizobium sp. NGR234 is a component of several
legume-symbiont model systems; however, there is little structural
information on the cell surface glycoconjugates. NGR234 cells in
free-living culture produce a major rough lipopolysaccharide (LPS,
lacking O-chain) and a minor smooth LPS (containing O-chain), and the
structure of the lipid A components was investigated by chemical
analyses, mass spectrometry, and NMR spectroscopy of the underivatized
lipids A. The lipid A from rough LPS is heterogeneous and consists of
six major bisphosphorylated species that differ in acylation.
Pentaacyl species (52%) are acylated at positions 2, 3, 2', and 3',
and tetraacyl species (46%) lack an acyl group at C-3 of the proximal
glucosamine. In contrast to Rhizobium etli and
Rhizobium leguminosarum, the NGR234 lipid A contains a
bisphosphorylated The family Rhizobiaceae includes the
Rhizobium and Sinorhizobium, Gram-negative
bacteria able to form nitrogen-fixing symbioses with legumes in a
host-specific manner. Sinorhizobium sp. NGR234 is a fast
growing, broad-host-range symbiont able to colonize a diverse range of
commercially important legumes (1, 2), including both indeterminate and
determinate nodule-forming hosts. Partly because of its agricultural
role, the molecular genetics of NGR234 are of interest, and the
symbiotic plasmid was recently sequenced (3). However, there is little
complementary structural information on the cell surface
macromolecules or the alterations that occur in these
molecules during symbiotic infection and bacteroid differentiation.
Lipopolysaccharides (LPS)1
are the major structural and antigenic components of the rhizobial
outer membrane (4-8) and are proposed to contribute to the biochemical
processes that result in symbiotic infection (8-16). Rhizobial LPS
structural mutants typically yield phenotypes with underdeveloped
nodules (Ndv The lipid A moieties of rhizobial LPS are of interest, because of their
highly unusual structures and, by analogy to enterobacterial lipid A,
because of their essential role in maintaining cell viability and
membrane integrity. The structure of the Rhizobium
etli-Rhizobium leguminosarum common lipid A-core region
was recently elucidated from laboratory cultured cells (24-27) and was
found to have an entirely different structure from the typical lipid
A-core of enterobacterial LPS. The lipids A of R. etli-R. leguminosarum lack phosphate and instead have
trisaccharide backbones containing a distal galacturonic acid residue
in In contrast to R. etli-R. leguminosarum, there is no
detailed information on the structures of any of the
Sinorhizobium LPS. Compositional studies have indicated that
sinorhizobial LPS core regions are substantially different from those
of R. etli (8, 12, 32, 33) or from enteric bacteria and that
the O-chains are often expressed in trace amounts or consist of simple
homopolymers (8, 12), at least on vegetative cells. A single study on the lipid A moiety from the Sinorhizobium meliloti mutant
10406 indicated the presence of a bisphosphorylated glucosamine
disaccharide backbone; however, detailed structures, component
heterogeneity, and the locations of some fatty acids, including the
LCFA, were not characterized (34). Interestingly, acyloxyacyl
residues were not detected in the S. meliloti mutant lipid
A, possibly because effective solvents for NMR analysis of
underivatized lipids A were unavailable at the time.
In this report we describe structural analysis of the lipid A moiety
from the LPS produced by Sinorhizobium sp. NGR234 grown in
vegetative culture. Our results demonstrate several structural features
that are shared with Rhizobium etli-Rhizobium
leguminosarum lipid A, which may be important in establishing or
maintaining symbiosis. These features include a remarkable similarity
in the type and location of acyloxyacyl residues containing the LCFA, when compared with R. etli- R. leguminosarum (24). In
marked contrast to R. etli lipid A, the NGR234 lipid A also
shares certain features with the enterobacterial lipids A, constituting
a "hybrid" structure that may prove useful in endotoxin-elicitor
studies. Structural information on the NGR234 lipid A will provide
additional insight on the biosynthesis of rhizobial and sinorhizobial
lipids A and facilitate comparisons with bacteroid-derived and other structurally altered lipids A.
Growth of Bacteria--
Agar slants of Sinorhizobium
sp. strain NGR234 were provided by Dr. Peter van Berkum at the
National Rhizobium Germplasm Collection Center in Beltsville, MD. The
bacteria were transferred to liquid media (tryptone/yeast extract
supplemented with Ca2+) and grown in fermentor culture at
28 °C as described previously for related species (8, 35). The cells
were harvested by centrifugation at late log phase
(A600 = 2.70) yielding 500 g (wet weight)
of cells/100 liters of culture.
Isolation of LPS and Purification of Lipid A--
LPSs were
extracted by the hot phenol/water procedure (5, 36) and analyzed by
deoxycholate-PAGE (37-39). Water layer extracts containing LPS were
dialyzed and treated sequentially with ribonuclease, deoxyribonuclease,
and proteinase K (5) and then redialyzed and subjected to size
exclusion chromatography under dissociative conditions (0.25% sodium
deoxycholate, 0.2 M NaCl, 1.0 mM EDTA, 10 mM Tris, pH 9.2) on a column of Sephadex G-150 (2.2 × 100 cm). This procedure separates the R-LPS from the S-LPS. 60 mg of
water layer extract yielded ~23 mg of R-LPS and 1 mg of S-LPS per
run. Portions (2-3 mg) of the G-150 purified R-LPS were subjected to further purification on Superose-12 HR 10/30 (Amersham Biosciences) under associative conditions (50 mM ammonium formate) at
0.3 ml/min.
The R-LPS was hydrolyzed in 10 mM sodium acetate buffer, pH
4.5, containing 1% SDS, following a published procedure (40), to yield
the free lipid A (R-lipid ASDS). For comparison, lipid A
was also isolated by hydrolyzing R-LPS in 1.0% acetic acid (pH 2.8, 105 °C, 21/2 h), and the precipitated lipid A (R-lipid
AHOAc) was recovered by ultracentrifugation at 160,000 × g for 1 h (16). The resulting lipids A were analyzed
by TLC, MALDI-TOF mass spectrometry, and GC-MS analysis of derivatives
as described below. Selected lipid A species were isolated by
preparative TLC (described below) and analyzed by two-dimensional NMR
spectroscopy, MALDI-TOF-MS, and GC-MS of derivatives.
Glycosyl Composition Analysis--
Glycosyl compositions of LPS
and the intact and de-O-acylated lipid A samples were
determined by GC-MS analysis (electron impact) of the TMS methyl
glycosides (41) using a 30-m DB-5 fused silica capillary column (J & W
Scientific). Neutral and amino sugars were also determined by GC-MS
analysis of the alditol acetates using a 30-m SP2330 capillary column
(Supelco) (42).
Fatty Acid and N-Acylglucosamine Analysis--
Total fatty acids
were released by hydrolysis of lipid A in 4 M HCl at
100 °C for 4 h (43). The liberated fatty acids were converted
to methyl esters using methanolic 1 M HCl at 80 °C for 2 h, followed by trimethylsilylation and analysis by GC-MS using the DB-5 column. Ester-linked fatty acids were selectively released by
de-O-acylation of intact lipid A with sodium methoxide (0.5 M) at 35 °C for 16 h (26); liberated fatty acids
were then methyl-esterified, trimethylsilylated, and analyzed by GC-MS.
Amide-linked fatty acids were analyzed by hydrolyzing the
de-O-acylated lipid A in 4 M HCl; the liberated
fatty acids were then analyzed as the TMS methyl esters by GC-MS.
Amide-linked fatty acids were also determined by mild methanolysis of
the intact lipid A, followed by preparation and GC-MS analysis of the
N-acylglucosamine TMS methylglycoside derivatives (44).
Chemical analysis of amide-linked acyloxyacyl groups was performed on
lipid ASDS by methylation with silver oxide/methyl iodide
(Kraska methylation), following the procedures of Wollenweber and
Rietschel (43).
Thin Layer Chromatography--
TLC of lipid A was performed on
Silica Gel 60 high performance TLC plates (Merck) using two solvent
systems as follows: A, chloroform/methanol/water/triethylamine
(30:12:2:0.1, v/v) (45); and B, chloroform, pyridine, 88% formic acid,
methanol, water (60:35:10:5:2, v/v) (27). Lipids A were visualized by
spraying with 15% ethanolic sulfuric acid and charring, and by a
molybdenum blue spray reagent (Sigma). Preparative TLC was performed in
the same manner except bands were localized by misting the plate
lightly with water and holding the plate under UV light, under which
conditions the hydrophobic lipid A bands were readily visible.
Individual bands containing lipid A were removed by scraping, and the
lipid A was solubilized in chloroform/isopropyl alcohol/water
(5:3:0.25 v/v). Silica gel and particulates were removed by passage
through a small column (5 × 8 mm) of Sephadex LH-20 equilibrated
in the same solvent.
NMR Spectroscopy--
1H spectra and all
two-dimensional homonuclear and heteronuclear spectra of the intact
lipid ASDS were recorded at 30 °C on a Varian Inova 500 NMR spectrometer using standard Varian software. The lipid
ASDS was dissolved and analyzed in the ternary solvent CDCl3/CD3OD/D2O (2:3:1 v/v) (24,
46), yielding clear solutions at ~3 mg/ml; spectra were referenced to
internal tetramethylsilane (0.00 ppm). COSY data were recorded in the
absolute value mode with a 5.0-kHz spectral width, collecting 512 increments at 16 scans per increment. 1H-1H
TOCSY was recorded with a mixing time of 80 ms and 2 sets of 512 time
increments at 24 scans per increment. Phase-sensitive NOESY was
recorded with a 500-ms mixing time using 2 arrays of 256 increments at
80 scans per increment. 1H-13C HSQC was
performed with an acquisition time of 0.199 s by collecting 2 arrays of
200 increments at 80 scans per increment.
1H-31P HMBC correlation spectra were recorded
without 31P decoupling on the R-lipid ASDS in
the ternary solvent system. Direct observed 1H-decoupled
31P spectra of intact lipid ASDS and lipid
AHOAc were obtained by dissolving the lipid A in
D2O containing 2% sodium deoxycholate and 5 mM
EDTA; pH adjustments were made with triethylamine. Spectra were
recorded on a Varian 300 instrument at 121.51 MHz, and 31P
chemical shifts were recorded relative to an external standard of 85%
phosphoric acid at 0.00 ppm.
Mass Spectrometry--
MALDI-TOF mass spectrometry was performed
on a Kratos Analytical Kompact SEQ instrument using delayed extraction,
in both positive and negative ion modes. Samples were desorbed with a nitrogen laser ( Analytical Procedures--
Total phosphorus was determined by a
colorimetric procedure (47). Column eluants were monitored by
colorimetric assays for neutral carbohydrate (41) and Kdo (48) and by
refractive index detection.
Purification of LPS--
The major LPS produced by vegetative
cultures of Sinorhizobium sp. NGR234 is a rough LPS (R-LPS),
which lacks the polysaccharide O-chain (8, 49). This is typical of many
Sinorhizobium strains examined to date (8, 9, 14, 32, 35)
and contrasts with R. leguminosarum and R. etli,
which synthesize a major smooth LPS (S-LPS, containing O-chain) when
grown as free-living cultures (14, 16). In the case of strain NGR234,
the majority of the total LPS (consisting of the major R-LPS and a
minor S-LPS) was recovered in the water layer during hot phenol/water
extraction. In Fig. 1, fractionation of
the water layer by size exclusion chromatography under dissociative
conditions afforded separation of the R-LPS and S-LPS, as well as other
cell surface components, the capsular polysaccharides (K-antigens),
extracellular polysaccharides (EPS), cyclic glucans, and derived
fragments. The recovery of total LPS (R-LPS + S-LPS) was 340 mg per
40 g of dry cell weight (compare R. etli strain CE3,
which yields ~600 mg of LPS from the same amount of cells (25)). Of
the total LPS, over 96% consisted of R-LPS. Final purification of
R-LPS was achieved by size exclusion chromatography under associative
conditions on Superose-12 (Fig. 1). This step removed a low molecular
weight population of K-antigen/EPS fragments which co-migrated with the
R-LPS during dissociative chromatography.
Isolation and Initial Characterization of Sinorhizobium sp. NGR234
Lipid A--
In previous studies on the lipid A from R. etli and the closely related R. leguminosarum, mild
hydrolysis in 1% acetic acid was routinely used to cleave the
core-lipid A ketosidic linkage, and the free lipid A was isolated
without extensive degradation (26, 30). This procedure was applied to
NGR234 R-LPS, and the resulting R-lipid AHOAc was analyzed
by TLC (see figures in the Supplemental Material). The mobility of the
major components suggested the presence of monophosphorylated species,
which typically migrate near the solvent front in this solvent.
Composition analysis of lipid AHOAc (Table
I), indicated a total
phosphate/glucosamine ratio of ~1:2, also consistent with
monophosphorylated species.
The presence of trace components having low Rf
values suggested that some bisphosphorylated or other more highly phosphorylated lipid A species might be present. This suggested that
the 1% HOAc treatment may have caused substantial cleavage of labile
glycosidic phosphate or other polar head groups and that more highly
phosphorylated lipid A species may be the true biosynthetic products.
Additional lipid A was therefore isolated using the mild hydrolytic
procedure of Caroff et al. (40) (pH 4.5, 1% SDS), and the
products (R-lipid ASDS) were analyzed and compared (Table I
and see figures in the Supplemental Material). This procedure yielded a
markedly different lipid A profile in which the most abundant
components had lower Rf values, typical of those
reported for various bisphosphorylated lipids A (45, 50). Analysis for
phosphate (Table I) indicated a molar ratio of ~1:1 relative to
glucosamine, also suggesting that R-lipid ASDS contained
bisphosphorylated species. In addition to elevated levels of phosphate,
GC-MS analysis indicated that R-lipid ASDS had elevated
levels of several fatty acids (3-OH-14:0, 27-OH-28:0, and 29-OH-30:0)
compared with R-lipid AHOAc (Table I).
Analysis of lipid AHOAc and lipid ASDS for
total fatty acids indicated a high degree of acyl group heterogeneity.
As with R. etli, the Sinorhizobium sp.
NGR234 R-lipid A contained the unusual LCFA 27-OH-28:0, in addition to
the common series of 3-hydroxy-14- to 18-carbon fatty acids. The NGR334
lipid A also contained substantial amounts of 29-OH-30:0, and several
unsaturated hydroxy fatty acids, not found in R. etli/R. leguminosarum lipids A. In contrast to the
lipid A of R. etli/ R. leguminosarum,
glucosamine was the only carbohydrate detected by GC-MS analysis of the
alditol acetates and TMS-methyl glycosides; 2-amino-gluconate and
galacturonic acid, both components of the R. etli lipid A
(26), were not detected. Mild hydrolysis of R-lipid ASDS in
0.2 M HCl, conditions which allow recovery of
4-amioarabinose, indicated that this glycosyl residue was not a
component of NGR234 lipid A.
Analysis of Phosphate Residues--
The number and type of
phosphate residues in lipid AHOAc and lipid
ASDS from R-LPS was investigated by 31P NMR of
the intact lipids A. In Fig.
2A, R-lipid AHOAc
yielded two sharp signals when analyzed at pH 10.6. The chemical shifts of both signals are characteristic of ester-linked, unsubstituted monophosphates. The signal at
The location of phosphate in lipid ASDS was unequivocally
determined by 1H-31P HMBC (Fig. 2D),
using a ternary solvent system (46). The lipid ASDS,
determined by chemical analysis and mass spectrometry to consist mainly
of bisphosphorylated species (see below), revealed 3-bond couplings
indicating two sites of phosphate attachment, at C-1 of the proximal
GlcN residue (glycosidic attachment) and at C-4' of the distal GlcN
residue. In addition, a 4-bond coupling between the glycosidic
phosphorous and H-2 of the proximal GlcN residue was observed. The
identities of protons H-1 ( Determination of Ester- and Amide-linked Fatty
Acids--
Ester-linked fatty acids were released from R-lipid
AHOAc and analyzed by GC-MS (Table
II). The 3-OH-14:0 fatty acid was
exclusively ester-linked, accounting for 54% of the base-labile fatty
acids, and the two LCFAs accounted for 40%. Excluding LCFA, 3-OH-14:0 composed 88% of the ester-linked fatty acids. Analysis of the de-O-acylated lipid A precipitate (DOA-lipid
AHOAc) indicated that 3-OH-18:0 composed 46% of the
amide-linked fatty acids and that unsaturated 3-hydroxy fatty acids
composed 26%.
The N-acyl heterogeneity was also assessed by GC-MS analysis
of the N-acylglucosamine derivatives, prepared by mild
methanolysis (44) of R-lipid AHOAc. Four different
N-fatty acyl derivatives of glucosamine were detected
(ratios from GC-MS ion current peak areas): GN-3-OH-16:0 (1.00);
GN-3-OH-17:0 (0.18); GN-3-OH-18:0 (2.52); and GN-3-OH-18:1 (0.08).
Other derivatives were not detected; however, these ratios are in
reasonably good agreement with the amide-linked fatty acid ratios
determined by total acid hydrolysis of the DOA-lipid AHOAc
(Table II).
Amide-linked acyloxyacyl residues were investigated by the Kraska
methylation procedure as described (43). Two acyloxyacyl derivatives were detected, consistent with those of
27-O-( Characterization of NGR234 Lipid A Species by MALDI-TOF Mass
Spectrometry--
Analysis of R-lipid AHOAc by negative
ion MALDI-TOF MS revealed a complex pattern, which was found to consist
of 6 major species, all having molecular ions corresponding to
monophosphorylated lipids A (AMONO
The major mono- and bisphosphorylated components were isolated from
lipid AHOAc and lipid ASDS by preparative TLC,
and the components were analyzed by negative ion MALDI-TOF MS (Fig.
4). The major upper band from lipid
AHOAc was enriched in species EMON and
FMON, and the major lower band from lipid ASDS
was enriched in species EBIS and FBIS. The
results confirm the degree of phosphorylation for both the high and low
mobility TLC species.
Localization of the Long Chain Fatty Acyl Substituent--
In
previous studies (27) of R. etli lipid A, positive ion
MALDI-TOF MS was an effective method for localizing fatty acyl substituents to the distal or proximal glucosamine residues. At high
laser power settings, cleavage of the GlcN-
The two B1+ ion families originate
from glycosidic cleavage of the four parent species, C-F. The
B1+ ion family at
m/z 1173 originates from parent species C and E, and the B1+ family at
m/z 1273 originates from parent species D and F. Additional ±14 mass unit heterogeneity arises from partial endogenous
O-methylation of some of the
Additional information on the nature of the LCFA linkage was obtained
by further examination of the B1+
ions. In order to accommodate the mass of a LCFA component in any of
the B1+ ions, the LCFA would need to
be esterified as the secondary component of an acyloxyacyl residue, the
most likely location being the (distal) amide-linked fatty acid. This
is suggested by the following rationale. 1) Both 3-OH-14:0 and LCFA are
totally released from lipid A by sodium methoxide or mild hydrazine
treatments (Table II), indicating they are both ester-linked. 2)
3-Methoxy fatty acid derivatives were not detected during
sodium methoxide-catalyzed de-O-acylation of lipid A,
indicating that ester-linked acyloxyacyl residues were not
present (43); thus, the LCFA could not be attached as a secondary acyl
group to an ester-linked 3-OH-14:0 residue. 3) The methyl ester
derivative of 3-(3-hydroxytetradecanoyloxy)-octadecanoic acid, which if
present would be released from its amide-linkage during Kraska
methylation of lipid ASDS, was not observed. This indicates
that 3-OH-14:0 was not esterified as a secondary fatty acid to an
amide-linked 3-OH-18:0 residue. 4) The majority of the LCFA is
esterified at the Localization of Amide-linked Fatty Acids by MALDI-TOF Analysis of
the De-O-acylated Lipid A--
To assess further N-acyl
heterogeneity, the de-O-acylated lipid AHOAc was
analyzed by MALDI-TOF mass spectrometry (Fig.
6). The DOA-lipid AHOAc
yielded a family of molecular ions (M + Na)+1 having
m/z values and peak ratios in close agreement
with the types of species predicted on the basis of amide-linked fatty acid composition (Table II). The most abundant ions and proposed species (Table IV) were at
m/z 1021.0, P-GN2[3-18:0 + 3-19:0]; m/z 1019.7, P-GN2[3-18:0 + 3-19:1];
and m/z 1007.6, P-GN2[3-18:0 + 3-18:0], where P-GN2 designates the 4'-monophosphoryl
glucosamine disaccharide. At high laser power,
B1+ fragment ions were again
observed, representing the distal 4'-phosphoglucosamine residue and its
attached N-fatty acyl chain. Major
B1+ ions (Table IV) were
m/z 565, P-GN[3-18:0], and
m/z 537, P-GN[3-16:0].
Two-dimensional NMR Analysis of the Intact NGR234 Lipid
ASDS--
The intact R-lipid ASDS was enriched
for species EBIS and FBIS by preparative TLC
(Fig. 4B) and analyzed by homo- and heteronuclear NMR
spectroscopy. The lipid A was analyzed without derivatization, using a
ternary solvent system reported to give well resolved spectra of intact
and underivatized lipids A (24, 53). 1H-1H COSY
(not shown) and TOCSY (Fig. 7) revealed
two glycosyl ring systems, as well as five major spin systems arising
from the hydroxylated fatty acyl chains. The H-1 proton of the proximal
GlcN residue was assigned to a doublet-doublet resonance downfield at
The
1H-1H NOESY analysis of the R-lipid
ASDS showed numerous intra-residue and a single
inter-residue NOE (see figures in the Supplemental Material). A strong
inter-residue NOE between H-1' (
Carbon chemical shifts (Table V) were determined from
1H-13C HSQC experiments. H-1 was found to
correlate to a carbon resonance at Analysis of Lipid ASDS Derived from the S-LPS--
Due
to the limiting amounts of S-LPS (12 mg), extensive analysis of the
S-lipid A was not conducted. Analysis of S-lipid ASDS by
TLC (see figures in the Supplemental Material) revealed a pattern suggesting mainly bisphosphorylated species, although the
Rf values appeared to be somewhat different from
those of the rough lipid A. MALDI-TOF analysis suggested that most of
the components (86%) were indeed bisphosphorylated; however, three new
triacyl species, presumably lacking O-acyl chains, were
detected. The proposed structures of these unique S-lipid
ASDS components are shown in Fig.
8, and the observed molecular ions and
calculated masses are summarized in Table III. The relatively low
proportion of 3-OH-14:0 in the S-lipid A preparation, relative to the
R-lipid A, was also confirmed by GC-MS analysis (not shown).
In free-living culture, the major LPS synthesized by
Sinorhizobium sp. NGR234 is a rough-LPS, which yields a
heterogeneous lipid A consisting of six major components that differ in
acylation (Fig. 8). As estimated from the relative intensities of
molecular ions observed during mass spectrometry, 52% of the lipid A
components are pentaacylated (components E and F) and 46% are
tetraacylated (components B-D). Over 90% of these R-lipid A molecules
contain a single copy of an LCFA, carried as the secondary fatty acid of an acyloxyacyl substituent on the distal glucosamine residue. The
absence of the labile It is generally recognized that all Rhizobiaceae examined to
date contain LCFA in their lipid A (14, 29, 30), at least when the LPS
are extracted from free-living, vegetative cells. However, because of
the lack of detailed structural studies on the lipids A from these
organisms, and because of difficulties associated with analyzing LCFA,
the location, stoichiometry, and type of attachment of these
substituents have not been reported. It is also not clear if LCFA is
restricted to a specific location among all rhizobial lipids A or is
variable in location, perhaps characteristic of different strains or
genera. Although this latter point is still not answered, the present
study shows that there is a striking similarity in the overall
acylation pattern, including the specifics of LCFA attachment, for the
lipids A of Sinorhizobium sp. NGR234 and R. etli-R.
leguminosarum (24, 26, 27). Features of lipid A acylation that are
common to both but that are not characteristics of E. coli or other enteric lipids A include the following: 1) a high
degree of acyl chain length heterogeneity of the amide-linked fatty
acids, primarily localized to the proximal carbohydrate residue; 2)
variability in the occurrence of an ester-linked fatty acid at C-3 of
the proximal residue; 3) the presence of a single amide-linked
acyloxyacyl substituent, on the distal glucosamine, in which the
LCFA is attached as the secondary fatty acid; 4) the absence of any
ester-linked acyloxyacyl residues, with the exception of 5) the
occurrence, in at least 50% of the molecules, of a These common structural features have implications for both the
biological function and biosynthetic pathway of rhizobial and
sinorhizobial lipids A. Considering the structure of the carbohydrate backbone of Sinorhizobium sp. NGR234 lipid A, the early
stage enzymes required for the initial synthesis of the
Kdo2-lipid IVA bisphosphorylated precursor are
most likely present (in homologous forms), as they are in
Rhizobium sp. and E. coli (55). The structures described here further suggest that several of the enzymes involved in
lipid A acylation, unique to R. etli-R. leguminosarum
(i.e. not found in enteric bacteria), may also exist in
Sinorhizobium sp. NGR234. These would include a
3-O-deacylase, which in R. etli selectively
removes ester-linked 3-OH-14:0 from the proximal glucosamine (54), and
a homologous system for delivering and attaching the LCFA to the 2'
amide-linked fatty acid, forming the distal acyloxyacyl residue of the
completed lipid A. The sinorhizobial LCFA transfer system would include
a special long chain acyltransferase (LpxXL) and a dedicated acyl
carrier protein (AcpXL), homologous to those identified in R. etli (56). Compositional analyses indicate that in NGR234, the
putative LpxXL and AcpXL proteins would have slightly altered chain
length specificities compared with those of R. etli, because
~30% of the LCFA in NGR234 lipid A is C30, whereas C28 is the sole
component in R. etli, under the described growth conditions
(26, 30, 54). The structures reported here also show that prior removal
of the 4'- and 1-phosphates from the Kdo2-lipid
IVA precursor is not required for the transfer of LCFA to
substrate, in the case of NGR234 lipid A. It was previously reported
that the 3-O-deacylase synthesized by R. etli/R. leguminosarum was not detectable in S. meliloti Rm1021 (54). The possible existence of a
3-O-deacylase in NGR234 could represent one of the variables
in lipid A structure, among the many strains of S. meliloti and the related Sinorhizobium fredii. The fine
structural differences in lipid A acylation among these bacteria have
not been characterized; however, it is noteworthy that the profile of
lipid A components derived from S. fredii USDA257 is
considerably different from the profile reported here for
Sinorhizobium sp. NGR234.2 Alternatively, the
3-O-deacylated species (C and D) could arise from
degradation of lipid A during isolation. However, all of these
components quantitatively retained the very labile C-1 glycosidic phosphate, and based on the observed
B1+ ions (Fig. 5),
3'-O-deacylated species were not observed in the rough lipid
A, suggesting that loss of 3-O-acyl groups occurred specifically. Finally, in NGR234, distinct acyltransferase/acyl carrier
proteins may be required for attachment of the unsaturated N-linked fatty acids, which are not components of the
R. etli lipid A (26, 27). These unsaturated fatty acids are
primarily localized to the proximal residue, as evidenced from
MALDI-TOF analysis of the de-O-acylated lipid A (Table IV
and Fig. 6).
The fact that key features (i.e. the occurrence, type, and
location of acyloxyacyl residues), which distinguish the R. etli acylation pattern from that of E. coli, are
retained almost identically in NGR234 further strengthens the
supposition that these features are in some way essential to successful
symbiotic infection. During R. leguminosarum colonization of
Pisum sativum, Kannenberg and Carlson (31) observed an
increase in the mol % of LCFA in bacteroid-derived LPS, compared with
LPS from free-living cells. The LCFA was proposed to stabilize the
bacteroid membrane, which is closely surrounded by the plant-derived
symbiosome membrane during endocytosis, symbiosome formation, and
symbiosome/bacterial cell division. Recently, an acpXL
mutant of R. leguminosarum did not incorporate C28 LCFA into
its lipid A and showed delayed nodulation in pea
plants.3 This lipid A mutant
also demonstrated less competitive growth in vegetative culture.
The NGR234 lipid A isolated from smooth LPS appeared to have a
different acylation profile than the R-lipid A components. In addition
to molecular species C-F, several unusual triacylated lipid A
components were observed, which appeared to lack all ester-linked fatty
acids (Fig. 8). The absence of ester substituents may have occurred
through degradation; however, it is interesting that all of these
species retained the other labile substituents, i.e. phosphorylation at C-1, and the LCFA-acyloxyacyl residue. Recent studies with Sinorhizobium sp. NGR234 (49, 57) have
indicated that the proportion of S-LPS is increased relative to R-LPS,
when vegetative cells are cultured in the presence of apigenin or other plant flavonoids (inducers of bacterial nod and
nol genes). These "flavonoid-induced LPS" appear to have
an altered lipid A and core region structure compared with normal
(uninduced) LPS (49). Smooth LPS may also be the dominant form of LPS
on the bacteroid surface in the case of Sinorhizobium sp.
NGR2344 and in
R. leguminosarum 3841 (31). The biological significance of
these observations requires further structural analysis of both the
vegetative and bacteroid-derived forms.
-(1'
6)-glucosamine disaccharide, typical of
enterobacterial lipid A. However, NGR234 lipid A retains the unusual
acylation pattern of R. etli lipid A, including the
presence of a distal, amide-linked acyloxyacyl residue containing a
long chain fatty acid (LCFA) (e.g.
29-hydroxytriacontanoate) attached as the secondary fatty acid. As in
R. etli, a 4-carbon fatty acid,
-hydroxybutyrate, is
esterified to (
1) of the LCFA forming an acyloxyacyl
residue at that location. The NGR234 lipid A lacks all other
ester-linked acyloxyacyl residues and shows extensive heterogeneity
of the amide-linked fatty acids. The N-acyl heterogeneity,
including unsaturation, is localized mainly to the proximal
glucosamine. The lipid A from smooth LPS contains unique triacyl
species (20%) that lack ester-linked fatty acids but retain
bisphosphorylation and the LCFA-acyloxyacyl moiety. The unusual
structural features shared with R. etli/R.
leguminosarum lipid A may be essential for symbiosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phenotype) in which nitrogen fixation is
absent or diminished (Fix
) (5, 9, 15, 17-23).
1,4-linkage to glucosamine, linked
1,6 to a proximal
2-aminogluconic acid residue (26). The latter is generated from normal
glucosamine by a membrane-associated dehydrogenase (24). The acylation
pattern of R. etli-R. leguminosarum lipid A also differs in
a number of key respects from that of enteric bacteria (24, 26, 27).
These features include extensive heterogeneity of the ester and
amide-linked fatty acids, particularly on the proximal 2-aminogluconate
residue, the absence of ester-linked acyloxyacyl groups, and the
presence of a single amide-linked acyloxyacyl residue, containing a
unique long chain fatty acid (LCFA, e.g.
27-hydroxyoctacosanoic acid), carried as the secondary acyl group.
These unique structures can be rationalized by considering their
possible role in bacteroid survival and symbiotic infection. It has
been proposed, for example, that replacement of phosphate with less
acidic, negatively charged residues could help attenuate stimulation of
the plant defense response to the invading rhizobia (27, 28). The
occurrence of the C28 LCFA is particularly interesting, because its
presence (but not specific location) has been demonstrated in a variety
of bacteria that survive within intracellular host membrane-derived
compartments, including all of the Rhizobiaceae studied to date, the
plant pathogen Agrobacterium, and the phylogenetically related intracellular animal pathogen Brucella (29, 30). The C28 LCFA is long enough to span the entire lipid bilayer and has been
suggested to promote membrane stability during the critical stages of
symbiotic infection, when the bacterial and plant membranes appear to
be in close proximity (i.e. during endocytosis-symbiosome formation and during the synchronized cell division of the
bacterium-symbiosome (31)).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
= 337 nm) and extraction voltage of 20 kV.
Lipid A samples were dissolved in CHCl3/isopropyl
alcohol/water (5:3:0.25 v/v) and desodiated by treating with OnGuard-H
cation exchange resin (H+) (Dionex) for 20 min with
shaking. The resin was removed by centrifugation (12,000 × g), and the supernatant containing lipid A was dried, adjusted to a concentration of 2 µg/µl in fresh solvent, and mixed in a 1:1 ratio with matrix solution (2,4,6-trihydroxyaceto-phenone, 0.5 M in methanol). 1 µl was applied to the MALDI stage. Mass calibration was performed with malto-oligosaccharides (positive ion
mode) and with R. etli CE3 lipid A (negative ion mode). The predicted molecular weights of the various lipid A species were calculated from the average incremental masses of subunits, based on
the atomic weights of the elements: 2-amino-2-deoxyhexose, 161.1577;
phosphate, 79.9799;
-hydroxybutyrate, 86.0904; 3-OH-14:0, 226.3592;
3-OH-16:0, 254.4130; 3-OH-17:0, 268.4399; 3-OH-18:0, 282.4667;
3-OH-18:1, 280.4509; 3-OH-18:2, 278.4351; 3-OH-19:0, 296.4935;
3-OH-19:1, 294.4777; 27-OH-28:0, 422.7355; 29-OH-30:0, 450.7891; and
free reducing end, 18.0153.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Isolation of Sinorhizobium
sp. NGR234 rough and smooth LPS by sequential
chromatography. A, size exclusion chromatography on
Sephadex G-150 in detergent (dissociative conditions) allows separation
of the smooth and rough LPS. Most of the high molecular weight K
antigens (Kdo-containing polysaccharides) were removed from the LPS
during this step. B, chromatography of the R-LPS fraction
(from step 1) on Superose-12 under associative conditions allows the
R-LPS to reaggregate, migrating at the void volume. Contaminants that
co-eluted with the LPS during step 1, including lower molecular weight
K antigen and EPS fragments, do not reaggregate. Inset,
deoxycholate-PAGE analysis of the purified components: lane
1, K antigens; lane 2, S-LPS; lane 3,
R-LPS.
Chemical composition of Sinorhizobium sp. NGR234 lipid A, prepared by
different methods
4.804 was attributed to ester-linked phosphate on molecules that are lacking some of their
O-linked fatty acids; loss of ester-linked fatty acids has
been shown to produce 31P signal heterogeneity of this type
(34). The majority of partially de-O-acylated species was
probably generated during 1% HOAc hydrolysis and is a major component
of R-lipid AHOAc, as shown below by MALDI-TOF and chemical
analyses. The signal at
4.090 arises from fully (or more highly)
O-acylated species. The chemical shifts of both signals are
in close agreement with the two signals observed for the
4'-monophosphate ester of R. meliloti 10406 lipid A (34). In Fig. 2B, R-lipid ASDS retained these two
signals, in addition to a new signal at
2.352, characteristic of a
glycosidically linked phosphomonoester (34, 51). As shown below, lipid
ASDS contains a higher proportion of fully
O-acylated species than does lipid AHOAc,
accounting for the decreased intensity of the
4.804 signal relative
to the
4.121 signal. In Fig. 2C, all signals showed a
pH-sensitive chemical shift, typical of unsubstituted monophosphate
residues, both ester- and glycosidically linked.
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Fig. 2.
31P NMR analysis of
Sinorhizobium sp. NGR234 lipids A from R-LPS.
Lipid A samples were dissolved in D2O containing 2% sodium
deoxycholate, 5 mM EDTA, and pH adjustments were made with
triethylamine. A, lipid AHOAc released from
R-LPS by 1% acetic acid hydrolysis contains a single ester-linked
monophosphate, yielding two signals ( 4.804 and 4.090) recorded at
pH 10.6. The signal at
4.804 originates from partially
de-O-acylated species; the signal at
4.090 arises from
fully O-acylated species. B, lipid
ASDS released by mild hydrolysis in sodium acetate
buffer/SDS produced these two signals, along with a new signal (
2.352) characteristic of a glycosidically linked monophosphate,
indicating that lipid ASDS consists primarily of
bisphosphorylated species. C, all signals show a
pH-sensitive chemical shift, typical of unsubstituted monophosphate
residues. D, 1H-31P HMBC spectrum of
the NGR234 R-lipid ASDS. The sample was dissolved in
CDCl3/CD3OD/D2O (2:3:1 v/v) (24)
and analyzed at 30 °C. 3-Bond couplings revealed two sites of
phosphate attachment, at C-1 of the proximal GlcN residue (glycosidic
attachment) and at C-4' of the distal GlcN residue (ester-linked
PO4). A 4-bond coupling between the glycosidic phosphate
and H-2 was also observed. Heterogeneity in the H-4'-P coupling was
observed, due to partial loss of O-linked fatty acids at
various positions, including the C28 LCFA.
5.44), H-2 (
4.12), and H-4' (
4.17 and
4.15) were determined from 1H-1H
COSY and TOCSY experiments (described below). The C-1 phosphorus signal
was located at
2.21, and the C-4' phosphorus was found at
0.65 (major) and
0.22p (minor). Heterogeneity of the H-4'-P
coupling was again attributed to partial de-O-acylation of
lipid ASDS, which appeared to occur to a minor extent in
the solvent during the analysis. The downfield location of the C-4' phosphorus signal relative to C-1 phosphorus is in agreement with previous studies (46) on enterobacterial lipid A using this ternary
solvent; however, the absolute values reported here for NGR234 lipid
ASDS are somewhat different, probably due to differences in
temperature, pH, solvent ratios, and acylation.
Relative recovery of ester- and amide-linked fatty acids during
de-O-acylation of lipid AHOAc from Sinorhizobium sp. NGR234
rough-LPS
-hydroxybutoxy)-C28:0 and
29-O-(
-hydroxybutoxy)-C30:0. The EI spectrum of the
former product was identical to that identified previously in R. etli lipid A (26); the spectrum of the latter showed an increase of 28 mass units for ions containing the LCFA moiety, i.e.
m/z 437
465, and m/z
453
481. The detection of these products indicated that the
1
hydroxyl groups of the LCFA are esterified with a
-OH-butyrate
residue; as shown below, this substitution is not stoichiometric. No
other derivatives were detected, indicating that the base-labile fatty
acids (with the exception of LCFA) were not secondary components of
amide-linked acyloxyacyl residues. As a positive control, E. coli lipid A yielded the expected methyl ester derivatives of
[14:0(3-O(14:0))] and [14:0(3-O(12:0))], during the Kraska procedure. The failure of the LCFA to yield an
acyloxyacyl derivative during Kraska methylation was also observed during the original analysis of the R. etli lipid A
(26).
FMONO,
Fig. 3A). Analysis of R-lipid
ASDS also revealed 6 major components (Fig. 3B),
each 80 mass units higher than the corresponding lipid
AHOAc species, confirming their identification as
bisphosphorylated species (ABIS
FBIS). Table
III summarizes the observed molecular
ions, proposed composition, and calculated mass of the different lipid
ASDS species. Comparison of lipid AHOAc and
lipid ASDS (Fig. 3, A and B) also
showed that lipid AHOAc is enriched in partially
de-O-acylated species (species A-D) in comparison to lipid
ASDS, which has a higher proportion of fully acylated
species (E and F). Species A and B each lack a single copy of a LCFA
(either 27-OH-28:0 or 29-OH-30:0), resulting in a decrement of 423 (or
451) mass units compared with species C and E, respectively. Species A,
C, and D each lack a single 3-OH-14:0 fatty acid (
mass 226) in
comparison to species B, E and F, respectively. This mass decrement
represents the absence of an ester-linked 3-OH-14:0 residue (as shown
below, in lipid ASDS the loss of this residue occurs
specifically at O-3 of the proximal residue, rather than at O-3').
Another incremental difference of 86 mass units exists between species
E and F, and between species C and D. This mass difference is
attributed to partial substitution by
-hydroxybutyrate, esterified
to some of the LCFA acyl chains at the (
1) hydroxyl group. Based
on the relative intensities of the MALDI-TOF molecular ions, it was
estimated that 96% of the R-lipid ASDS components were
bisphosphorylated, and ~4% were monophosphorylated. The lipid
ASDS from R-LPS consisted of pentaacylated species (52%),
tetraacylated species (46%), and triacylated species (2%), and 93%
of the R-lipid ASDS components contained a single LCFA
substituent.
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Fig. 3.
Negative ion MALDI-TOF mass spectrometry of
NGR234 lipid A isolated from the R-LPS. A, lipid
AHOAc yields six ion families, arising from
monophosphorylated components (A-FM). B,
lipid ASDS consists of the same 6 species, which are
bisphosphorylated (A-FB). The 6 major lipid A
families, designated A-F, differ from each other by their acylation
pattern; species A and B lack the 27-OH-28-carbon fatty acid, and
species A, C, and D lack a single 3-OH-14:0 fatty acid. Species D and F
contain a 4-carbon hydroxy fatty acid ( -hydroxybutyrate,
mass 86 units) as an acyloxyacyl substituent on the 27-OH-28-carbon fatty acid.
The observed molecular ions [M
H]
1, and
proposed formulas are summarized in Table III. Individual ions within
each family differ by ±14 mass units, due to differences in acyl chain
length. Additional heterogeneity within each ion family was revealed by
MALDI-TOF analysis of the de-O-acylated lipid
AHOAc (Fig. 6).
Summary of the major lipid ASDS species derived from
Sinorhizobium sp. NGR234 rough-LPS and smooth-LPS
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Fig. 4.
Preparative TLC and MALDI-TOF analysis of
NGR234 R-lipid A species. A, inset, lane
1, the upper band was isolated by preparative TLC
and then rechromatographed as shown. A, this upper band is
enriched in 2 monophosphorylated species (EMON and
FMON), as shown by the negative ion MALDI-TOF-MS
spectrum. B, inset, lane 2, the
lower band was isolated then rechromatographed as shown.
B, the negative ion spectrum of lane 2 indicates
that the major lower TLC band is enriched in two bisphosphorylated
species (EBIS and FBIS).
(1-6)-GlcN glycosidic
linkage occurred readily and specifically, yielding prompt fragment
ions of the B+ type (52) carrying the respective
(distal) acyl substituents. When applied to NGR234 R-lipid
ASDS, two distinct families of B1+ ions were observed, along with
an attenuation of the parent molecular ions, as shown in Fig.
5. All
B1+ ions had masses corresponding to
LCFA attachment, indicating that the LCFA must be located on the distal
GlcN residue. The two B1+ ion
families differed by 86 mass units, consistent with the presence or
absence of a
-hydroxybutyrate moiety, which occurs in acyloxyacyl linkage to either of the two LCFAs (27-OH-28:0 and 29-OH-30:0) as
discussed above. Molecular ions (M + Na)+1 for the intact
lipid ASDS species were also observed (Fig. 5), each 24 mass units higher than the corresponding (M
H)
1
ions shown in Fig. 3B, thus confirming the mass assignments
for these species.
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Fig. 5.
Localization of the 27-OH-28:0 fatty acid to
the distal glucosamine residue of the NGR234 R-lipid ASDS
by positive ion MALDI-TOF MS. At high laser energy settings,
prompt fragmentation of the GlcN- (1-6)-GlcN glycosidic linkage
occurs, and the resulting B1+
fragment ions carry the distal fatty acyl substituents, allowing the
distal and proximal fatty acids to be distinguished (27). The two
B1+ ion families differ by 86 mass
units due to the variable content of
-hydroxybutyrate, an
acyloxyacyl substituent carried on some of the 27-OH-28-carbon fatty
acids. The parent molecular ions are [M + Na]+1.
-hydroxybutyrate residues
(sometimes found as 3-methoxybutyrate by GC-MS). In Fig. 5, it is also
noted that there are no B1+ ions
having a decrement of 226 mass units compared with the observed B1+ ions, i.e. ions at
m/z 1047 and 947 were not observed. This
indicates that there are no B1+
fragments (distal fragments) lacking a 3-OH-14:0 residue, even though
parent molecular species are clearly present which lack this fatty acid
(species C and D in Figs. 3B or 5). This in turn indicates
that the absence of the 3-OH-14:0 fatty acid in species C and D occurs
at a specific location, on the proximal residue (presumably C-3), in
the case of lipid ASDS. At lower power settings, the
B1+ cleavage products were not
observed, and the (M + Na)+ ions displayed increased
intensity (not shown).
1 hydroxyl with a
-hydroxy-butyryl residue, precluding attachment of a 14 carbon fatty acid (or any other
fatty acid) at this location. These points also rule out the
possibility that LCFA could be esterified directly to the 3'-position.
All of these points indicate that, on the distal glucosamine residue,
the LCFA is in acyloxyacyl linkage to amide-linked 3-OH-18:0 and that
3-OH-14:0 is esterified to the 3'-position. NMR analysis of the intact
R-lipid ASDS (described below) also indicated the existence
of a single 3-acyloxyacyl residue; however, its location (distal or
proximal) could not be unambiguously assigned.
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Fig. 6.
Analysis of de-O-acylated
lipid AHOAc by positive ion MALDI-TOF mass
spectrometry. All parent ions (M + Na)+1, and the
B1+ cleavage products, are readily
accounted for by the amide-linked fatty acyl composition (Table II).
The cluster of ions around m/z 565 are the
B1+ cleavage products, consisting of
the distal 4'-P-GlcN with the attached N-linked fatty acid.
The inset is a blow up of the de-O-acyl lipid A
parent ion region; the unsaturated N-linked fatty acids give
rise to species having a decrement of 2 mass units. Ions having this
± 2 mass unit difference were observed mainly in the parent
ion region, and much less so in the
B1+ region (representing the distal
end), indicating that the unsaturated fatty acids are localized mainly
to the proximal residue. The more abundant species also show weak
disodiated ions (M
H + Na)Na+ (e.g.
m/z 1041.9, 1029.7, and 599.0). Ions and proposed formulas
are listed in Table IV.
Selected ions from MALDI TOF mass spectrometry of the de-O-acylated
lipid AHOAc from the rough-LPS of Sinorhizobium sp.
NGR234
5.45. This proton also showed a strong connectivity with the
(glycosidic) phosphate during 1H-31P HMBC (Fig.
2). The chemical shift of this proton is consistent with glycosidic
phosphate substitution and the
-anomeric configuration for the
proximal GlcN residue. H-1 showed a strong COSY cross-peak with a
proton at
4.12 (H-2), which showed coupling to H-3 (
5.20). The
remaining glycosyl protons of the proximal residue were readily
assigned from TOCSY connectivities, and the complete proton assignments
are listed in Table V. Analysis of the
distal GlcN residue was also initiated at the anomeric proton. A COSY doublet at
4.60 was assigned to H-1', indicating the
-anomeric configuration for the distal GlcN. H-1' showed strong COSY cross-peaks with H-2' (
3.93) and a proton subsequently identified as H-5' (
3.48). H-4' (
4.17) was identified from TOCSY cross-peaks with H-2'
(
3.93), H-3' (
5.20), and H-5' (
3.48). The downfield shift
of H-4' is consistent with coupling to an ester-linked phosphate, observed during 1H-31P HMQC (Fig. 2). H-5'
showed cross-peaks with H-4' (
4.17), H-6a' (
3.94), and H-6b'
(
3.79); strong cross-peaks with the remaining (axial) ring protons
were also observed, typical of the gluco-configuration. A
distinct feature of these spectra is that both H-3 and H-3' are located
downfield (coincident at
5.20) indicating that both the
proximal (C-3) and distal (C-3') positions are acylated. The proton
chemical shifts for both glycosyl ring systems were found to be quite
similar to those reported for the intact, bisphosphorylated lipid A
from Escherichia coli, using the same solvent system
(53).
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Fig. 7.
500-MHz 1H-1H TOCSY
spectrum of the intact lipid ASDS from NGR234 R-LPS.
The two glycosyl-ring systems are identified, with H-1 of the proximal
residue resonating downfield at 5.45 due to substitution by
-linked PO4. H-1' (
4.60) of the distal residue
indicates the
-anomeric configuration for that residue. Both H-3 and
H-3' are found downfield (coincident at
5.20) indicating that both
the proximal (C-3) and distal (C-3') positions are acylated. A single,
prominent acyloxyacyl moiety was indicated by a downfield
-oxymethine proton (
5.13) having cross-peaks with the
-methylene protons (
2.57,
2.45) and the
-methylene
protons (
1.58,
1.31) of a 3-O-acylated fatty acyl
residue.
1H and 13C chemical shifts of NGR234 lipid ASDS
components
H ppm) were
assigned from COSY and TOCSY experiments; carbon chemical shifts (
C
ppm) were assigned from HSQC experiments; "BHB" =
-OH-butyrate.
-hydroxy fatty acyl protons of the NGR234 lipid ASDS
showed strong TOCSY connectivities, characterized by 4 partially overlapping sets of
/
/
-proton cross-peaks (Fig. 7). In these sets, the
H values of the
-oxymethine protons are consistent with
unsubstituted
-hydroxy fatty acids, both ester- and amide-linked (i.e. 3-hydroxy fatty acids that are not involved in
acyloxyacyl linkage). The
-oxymethine protons of these fatty acyls
had chemical shifts ranging from
3.93 to 4.00 (Table V) similar to
those of glycosyl ring protons having unsubstituted hydroxyl groups. A
fifth set of
/
/
-proton cross-peaks was distinct, due to the downfield chemical shift of its
-oxymethine proton (
5.13) having cross-peaks with the two
-methylene protons (
2.57,
2.45) and
the
-methylene protons (
1.58,
1.31) (Table V and Fig. 7).
This set of cross-peaks was assigned to a single acyloxyacyl residue;
the downfield shift of the
-proton is due to acyl substitution at
the 3-hydroxy group. The chemical shifts of the
/
and
/
cross-peak sets reported here for NGR234 lipid A are remarkably similar
to those reported recently for R. etli lipid A (24). Although their carbohydrate backbones are totally different, both lipids A contain a single acyloxyacyl residue. The cross-peak at (
5.35,
2.03) was assigned to the vinylic and allylic proton couplings of the unsaturated 3-hydroxy fatty acids; further TOCSY connectivities were traced to upfield secondary and terminal alkyl protons (not shown).
4.60) and both H-6a and H-6b of the
proximal residue provided convincing evidence for the
(1-6)-glycosidic linkage in the disaccharide backbone. Strong
intra-residue NOEs were observed between H-3/H-5, H-3'/H-5', H-2/H-4,
and H-2'/H-4' providing evidence for the
4C1 chair conformation for both GlcN
residues. All NOEs were found to be negative (cross-peaks phased with
the same polarity as the diagonal signal, i.e. negatively),
indicating that the lipid A existed as small aggregates during the
analysis, as observed previously for E. coli lipid A
(53).
96.2 (C-1), confirming the
-pyranosidic form for the proximal residue. H-2 was coupled to a
carbon signal at
54.5 (C-2); the upfield shift was indicative of
substitution by the amino group. H-3 was coupled to a carbon resonance
at
76.3 (C-3), which is slightly downfield from that expected for
an unsubstituted C-3 hydroxyl, reflecting acyl substitution at that
position. Carbon resonances originating from the distal residue showed
similar downfield/upfield shifts, confirming acyl substitution at C-3' and aminoacyl substitution at C-2'. C-4' (
73.3) was found downfield in comparison to C-4 (
69.9), reflecting phosphorus substitution at
C-4'.
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Fig. 8.
Structural diagrams of the R-lipid
ASDS and S-lipid ASDS components isolated from
Sinorhizobium sp. NGR234 R-LPS and S-LPS, by mild
hydrolysis in pH 4.5 buffer containing SDS. The triacylated C and
D species were detected only in the S-lipid A. The proposed formulas
and relative abundance of individual species are summarized in Table
III.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxybutyrate moiety (components C and E)
could arise from degradation or from endogenous heterogeneity in
acylation. As discussed below, the specific absence of a
3-O-acyl substituent (components C and D) may reflect a
biosynthetic origin, as reported for R. leguminosarum (54).
At the present time, detailed structures are known for three rhizobial
lipids A, the Rhizobium etli-R. leguminosarum common lipid A
(24-27) and that of Sinorhizobium sp. NGR234.
-hydroxybutyrate
moiety, attached in ester linkage to the (
1) hydroxyl of
the LCFA, thus forming an acyloxyacyl moiety at that location (Fig. 8,
species D and F).
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ACKNOWLEDGEMENTS |
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We thank Dr. John Glushka for advice and assistance with NMR and Dr. Brad Reuhs for discussions. The Complex Carbohydrate Research Center was supported in part by Department of Energy Grant DE-FG09-93ER20097.
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FOOTNOTES |
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* This work was supported by National Research Initiative/United States Department of Agriculture CSREES Grant 99-35305-8143 (to L. S. F.).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 on-line version of this article (available at
http://www.jbc.org) contains additional figures.
Present address: Emory University School of Medicine, Atlanta, GA 30303.
§ To whom correspondence should be addressed. Tel.: 706-542-4401; Fax: 706-542-4412; E-mail: SFORSB@ccrc.uga.edu.
Published, JBC Papers in Press, November 26, 2002, DOI 10.1074/jbc.M210491200
2 L. S. Forsberg and S. Gudlavalleti, unpublished MALDI-TOF and TLC results.
3 V. Vedam, E. L. Kannenberg, J. G. Haynes, D. J. Sherrier, A. Datta, and R. W. Carlson, submitted for publication.
4 B. L. Reuhs, B. Relic, C. Marie, S. Jabbouri, T. Ojanen-Reuhs, S. Stevens, L. S. Forsberg, C.-H. Wong, and W. J. Broughton, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are: LPS, lipopolysaccharide; R-LPS, rough LPS; S-LPS, smooth LPS; Kdo, 3-deoxy-D-manno-2-octulosonic acid; LCFA, long chain fatty acid; 27-OH-28:0, 27-hydroxyoctacosanoic acid; GC-MS, gas-liquid chromatography-mass spectrometry; TMS, trimethylsilyl; MALDI-TOF, matrix assisted laser desorption ionization-time of flight; COSY, 1H-1H correlation spectroscopy; TOCSY, total correlation spectroscopy; HMBC, heteronuclear multiple bond coherence spectroscopy; NOE, nuclear Overhauser effect; EPS, extracellular polysaccharides; NOESY, NOE spectroscopy; HSQC, heteronuclear single quantum coherence spectroscopy.
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