For the first time,
the complete structure of a lipopolysaccharide (LPS) core region from
Salmonella enterica has been identified that is different
from the Ra core type generally thought to be present in all
Salmonella LPS. The LPSs from two rough mutants and the
smooth form of S. enterica sv. Arizonae IIIa O62, which all
failed to react with an Ra core type-specific monoclonal antibody and
were resistant to phage FO1, were analyzed after chemical modification
using monosaccharide analysis, mass spectrometry, and NMR spectroscopy.
In the novel core type, the terminal D-GlcNAc residue
present in the Ra core type, is replaced by a D-Glc
residue. The O-specific polysaccharide is
1
4-linked to the second
distal Glc residue of the core. Furthermore, phosphoryl substituents attached to O-4 of
L-glycero-D-manno-heptose
(Hep) I and II were identified as 2-aminoethyl diphosphate (on Hep I)
and phosphate (Hep
II).
 |
INTRODUCTION |
Lipopolysaccharides
(LPSs),1 major components of
the outer membrane of Gram-negative bacteria, are composed of
covalently linked structural domains that are differentiated by their
structure, genetics, biosynthesis, and biological properties. They
consist of the O-specific polysaccharide, the inner and outer core
oligosaccharides, and the lipid A (1). Smooth form bacteria synthesize
LPSs that are built up of all three structural regions, while LPSs from rough type bacteria comprise only the lipid A and the core region, due
to genetic defects in the rfb locus, which encodes
O-specific polysaccharide biosynthesis. Furthermore, mutations in the
rfa locus encoding core biosynthesis may lead to truncated
rough type LPSs (2, 3). Such mutants were first identified in
Salmonella enterica sv. Minnesota, and the analysis of their
LPSs led to the elucidation of the structure of the Ra type core region
(Fig. 1) (2). In contrast with the many diverse structures of
O-specific polysaccharides identified in LPS from S. enterica (4), to date only one core type (the Ra core) has been
identified in LPSs from S. enterica.
The current procedure for detecting Salmonella in food or
other specimens involves the isolation and identification of the organisms using biochemical and serological methods that are
time-consuming, labor-intensive, and also expensive. The application of
immunological methods for detecting Salmonella either
directly in food or in selective media could help to circumvent these
problems. The success of an immunological approach depends on the
availability of a specific antibody, and the outer core region was
thought to be common to the LPSs of all S. enterica serovars
and was thus suggested to represent a genus-specific antigen against
which such an antibody could be raised. Two murine monoclonal
antibodies (mAbs) T6 (5) and 105 (6) were obtained, the epitopes for
which were identified in the distal part of the outer core region of
S. enterica LPS. However, some serotypes, including S. enterica sv. Arizonae IIIa O62, failed to react with these mAbs
(6-9), suggesting the presence of a modified core oligosaccharide
structure in some serotypes. mAb T6 is known to be specific for the T6
epitope, which comprises the
1
2-linked GlcNAc residue in the Ra
type core. To investigate the possibility of developing mAb T6 as a
polyvalent Salmonella serological reagent, and to gain
information on the molecular diversity of the Salmonella LPS
outer core structure, Tsang et al. (7, 8) tested a broad
variety of S. enterica strains for the presence of the T6
epitope. Ten serotypes of subspecies IIIa were identified that did not
react with mAb T6. In early structural investigations of LPSs from
S. enterica sv. IV and sv. Djakarta (9), the results of
monosaccharide and methylation analyses led to the proposal of the
existence of two novel LPS core structures in the LPS of S. enterica. Here, we describe the results of detailed structural
analysis of the core region of LPSs from S. enterica sv.
Arizonae IIIa O62, and of the linkage position of the O-specific
polysaccharide. This report represents the first complete
characterization of a novel LPS core type from S. enterica
and allows the lack of reactivity with mAb T6 to be rationalized in
terms of chemical structures.
 |
EXPERIMENTAL PROCEDURES |
Bacteria and Bacterial LPS--
S. enterica sv.
Arizonae IIIa O62 (smooth form), its rough mutants R4 and R5, and
S. enterica sv. Minnesota R60 were obtained from the strain
collection of the Max-Planck-Institut für Immunbiologie, Freiburg, Germany. Bacteria were grown at 37 °C in a fermentor using
media and growth conditions as described (10); washed successively with
ethanol, acetone (twice), and ether; and then dried. From smooth
bacteria and R4 and R5 mutants, the LPSs were isolated using the hot
phenol/water method (11), and from the R4 mutant, LPSs were extracted
in addition with phenol/chloroform/light petroleum (12) for purposes of
comparison, as were LPSs from S. enterica sv. Minnesota R60
(Ra chemotype). The two differently extracted LPSs from mutant R4 are
in the following designated as R4a (from phenol/water extraction) and
R4b (from phenol/chloroform/light petroleum extraction).
Isolation of Oligosaccharides--
The LPSs from the rough
mutants of S. enterica sv. Arizonae (R4a, 125.9 mg; R4b,
73.6 mg; R5, 161.2 mg) were de-O-acylated by mild acid
hydrolysis as described (13). After lyophilization (yields: R4a, 67.6 mg (53.7% of the LPS); R4b, 28.6 mg (38.9%); R5, 99.2 mg (61.5%)),
dephosphorylation was carried out in 5 ml of 48% aqueous hydrogen
fluoride (HF, 48 h, 4 °C) with constant stirring, followed by
neutralization with 8 M potassium hydroxide (KOH) at
10 °C. The suspensions were dialyzed against water (10 liters,
three times, 20 °C) and reduced with sodium borohydride (NaBH4, 1 h, 20 °C). Sodium was removed with an
Amberlite IR 120H+ cation exchanger, followed by dialysis
of the samples against water (10 l, 8 h, 20 °C).
De-N-acylation of LPS was performed with 4 M KOH
(20 mg of LPS ml
1) as described (14), and samples were
desalted using gel-permeation chromatography on Sephadex G50 (yields:
R4a, 41.3 mg (32.8% of the LPS); R4b, 18.1 mg (24.6%); R5, 52.3 mg
(32.4%)).
The core oligosaccharides of the de-O-acylated and
dephosphorylated LPS from mutant R5 (3 mg) were isolated after
hydrolysis with 1% acetic acid (0.5 ml, 2 h, 100 °C),
centrifugation (3000 × g, 30 min), and lyophilization of
the supernatant.
Smooth form LPSs (202.1 mg) were hydrolyzed with 1% acetic acid (100 min, 100 °C), and the polysaccharide fraction (105.8 mg, 52.4% of
the LPS) was isolated after centrifugation (100,000 × g, 30 min) and lyophilization of the water phase and was separated using
gel-permeation chromatography on Sephadex G50. The second fraction
(15.0 mg, 7.4%) was further purified by semipreparative high
performance anion-exchange chromatography (HPAEC) at 4 ml min
1 as described (14, 15) with the modification that the
column was eluted using a gradient program of water to 120 mM aqueous sodium acetate (pH 6.0) over 25 min, followed by
isocratic elution at 120 mM sodium acetate over 35 min. The
purity of the fractions obtained was analyzed by analytical HPAEC (15),
and the second fraction (fraction SR) was desalted on Sephadex G50,
lyophilized (2.7 mg, 1.3% of the LPS), and used for further
analyses.
Mild base treatment of 100 µg of the smooth form LPS was carried out
for MS analysis in 250 µl of methanol and 250 µl of ammonium hydroxide solution (25% NH3 in water) overnight at ambient
temperature followed by lyophilization.
The LPSs from S. enterica sv. Minnesota R60 (100 mg) were
first dephosphorylated with 48% aqueous HF (5 ml, 48 h, 4 °C),
then reduced and de-O-acylated (13). The sample (50 mg, 50%
of the LPS) was de-N-acylated (10 mg ml
1) with
4 M KOH and separated using gel-permeation chromatography on Sephadex G10. The fractions obtained were lyophilized, and their
purity and composition were investigated by 1H NMR
spectroscopy. The third fraction comprising the complete carbohydrate
chain of the core-lipid A region was used for NMR analyses (yield: 2.3 mg (2.3% of the LPS)).
General and Analytical Methods--
The conditions for gas
chromatography (GC), gas chromatography-mass spectrometry (GC-MS),
gel-permeation chromatography, and determination of the absolute
configurations of monosaccharide residues were as reported (16), as
well as the analytical methods for the determination of phosphates,
GlcN, Kdo, and neutral monosaccharide residues (17). Acetylation and
methylation together with the remaining steps of linkage analysis
(hydrolysis, reduction, and acetylation) were carried out as described
(18).
For methylation analysis of the Kdo region (19), the LPS from S. enterica sv. Arizonae mutant R5 was methylated according to Ref.
20, followed by evaporation of methyl iodide and then dialysis. This
procedure was repeated, followed by carboxyl reduction (NaBH4), dialysis, and a third methylation. The sample was
hydrolyzed with 0.1 M trifluoroacetic acid (1 h,
100 °C), evaporated, carbonyl-reduced (NaB2H4), and divided into two halves. One half
was acetylated and analyzed by GC-MS. The other half was methylated
with C2H3I, then hydrolyzed with 1 M trifluoroacetic acid (1 h, 120 °C), carbonyl-reduced
(NaB2H4), acetylated, and analyzed by GC-MS.
For methylation analysis of the complete core region, S-form LPS was
dephosphorylated, then reduced, and de-O-acylated. Three mg
of this sample were methylated according to (20), after which methyl
iodide was evaporated and the sample was dialyzed, followed by
repetition of this procedure. The sample was divided into two halves,
one of which was successively hydrolyzed in 90% aqueous formic acid (1 ml, 3 h, 100 °C) and 0.25 N sulfuric acid
(H2SO4, 1 ml, 16 h, 100 °C), and the
other in 4 M trifluoroacetic acid (1 ml, 4 h,
100 °C). After neutralization, the samples were
N-acetylated (21), reduced (NaB2H4),
acetylated, and analyzed by GC-MS. For methylation analysis of the core
region of mutant R5, its de-O-acylated LPS (4 mg) was
methylated according to Ref. 20, followed by evaporation of methyl
iodide and dialysis, and repetition of this procedure. The sample was
hydrolyzed in 4 M trifluoroacetic acid (1 ml, 4 h,
100 °C), evaporated, reduced (NaB2H4),
acetylated, and analyzed by GC-MS.
Mass Spectrometry--
Positive and negative ion mode
fast-atom-bombardment mass spectrometry (FAB-MS) was carried out using
the first two sectors of a Jeol JMS-SX/SX102A tandem mass spectrometer
operating at 7 kV (mass range m/z 200-3500), 8 kV (mass
range m/z 200-3000), or 10 kV (mass range m/z
200-2400) accelerating voltage. The FAB gun was operated at an
emission current of 10 mA with xenon as the bombarding gas. Spectra
were scanned at a speed of 30 s for the full mass range specified
by the accelerating voltage used, and were recorded and processed on a
Hewlett-Packard HP 9000 data system. Tandem mass spectra (FAB-MS-MS)
were obtained following collision-induced dissociation (CID) in the
third field free region using the same instrument under similar
conditions with helium as collision gas at a pressure sufficient to
reduce the parent ion to one half of its original intensity. In all
experiments, the matrix used was thioglycerol (2-3 µl) acidified by
adding 1 µl of concentrated trifluoroacetic acid in water (for
underivatized samples) or 1 µl of methanolic hydrochloric acid (for
derivatized samples). Underivatized samples were dissolved in water,
and derivatized samples were dissolved in methanol. The probe was
loaded with 1 µl of sample solution.
GC-MS was performed on a Fisons MD 800 mass spectrometer fitted with an
Interscience 8000 gas chromatograph using an on-column injector with
helium as the carrier gas. The monosaccharide derivatives were
separated on a DB-5MS column (0.25 mm × 30 m, J&W
Scientific) using the following temperature program: 50 °C for 2 min, a gradient of 40 °C min
1 to 130 °C, 130 °C
for 2 min, a gradient of 4 °C min
1 to 230 °C, and
finally 230 °C for 2 min. Mass spectra were obtained under
conditions of electron impact in the positive ion mode and were
recorded by linear scanning of the mass range m/z 55-400 at
an ionization potential of 70 eV.
Fraction SR (2.7 mg) was dissolved in 2.7 ml of water so that a final
concentration of about 1 µg µl
1 was obtained.
Negative mode electrospray ionization (ESI) mass spectra were obtained
on a VG Platform II single quadrupole mass spectrometer. Aliquots of 5 µl of the sample were infused into a mobile phase of
acetonitrile/water (50:50, v/v) and introduced into the electrospray
source at a flow rate of 5 µl min
1. Spectra were
scanned at a speed of 10 s for m/z 800-2200, with a
cone voltage of
80 V or
60 V and recorded and processed using the
MassLynx software, version 2.0. Mass calibration was performed by
multiple-ion monitoring of horse-heart myoglobin signals.
NMR Spectroscopy--
Structural assignments were made from
sample solutions in 2H2O (about 3.8 mg
ml
1) at p2H 7.1. Spectra were recorded in
5-mm tubes at 500.13 and 600.13 MHz for 1H and 125.77 or
150.90 MHz for 13C with a Varian Unity INOVA 500 or a
Bruker AMX 600 spectrometer, and chemical shifts reported relative to
internal acetone (2.23 ppm, 2HOH at 4.67 ppm at 37 °C).
Coupling constants (±0.5 Hz) were determined on a first-order basis.
The 13C resonances are reported relative to internal
dioxane (67.4 ppm). A one-dimensional 1H NMR spectrum was
recorded on the Bruker AMX 600 spectrometer using 16,000 data points
and a sweep width of 6250 Hz.
The phase-sensitive correlated spectroscopy (COSY) experiments were
performed using double-quantum filtering (22, 23) with the Bruker
COSYPHDQ microprogram. In the F2 dimension, 4000 data
points were collected giving an acquisition time of 0.492 s. The data
matrix was zero-filled in the F1 dimension to give a matrix
of 4000 × 2000 points and was resolution-enhanced in both
dimensions by a shifted sine-bell function before Fourier transformation. The total correlation spectroscopy (TOCSY) (24) and the
nuclear Overhauser enhancement spectroscopy (NOESY) (25) experiments
were carried out in the phase-sensitive mode according to the method of
States et al. (26). A decoupling in the presence of scalar
interactions (27) spin lock pulse sequence was used in the TOCSY
experiment with a mixing time of 125 ms and a spin lock power of 7300 Hz. The NOESY experiments were performed with a mixing time of 200 ms.
The intensities of NOESY cross-peaks were classified as either strong,
medium, or weak using cross-peaks from intra-ring proton-proton
contacts for calibration.
The 1H,13C correlation experiment was performed
in the inverse mode, as a heteronuclear multiple quantum coherence
(HMQC) experiment (28). This uses the bilinear rotation decoupling (29)
pulse sequence to suppress protons attached to 12C with a
pulse delay of 400 ms (30). 13C couplings were decoupled
during acquisition using the globally optimized alternating phase
rectangular pulse sequence (31) with a transmitter power corresponding
to a 90° pulse of 106 ms. The experiment was performed in the
phase-sensitive mode by the States time-proportional phase
incrementation phase-cycling method (32) acquiring a total of 4000 points over a sweep width of 4545 Hz in F2 and 1000 in
F1 over 13,500 Hz. Processing was performed using standard
Bruker software after zero-filling to 2000 in F1.
Broad band 1H-decoupled 31P NMR spectra were
recorded at 101.25 Hz on a Bruker DRX 250 at ambient temperature.
Spectra were acquired using a sweep width of 10,100 Hz and 8000 data
points. Pulses of 90° were applied for 9 ms with a pulse repetition
time of 0.4 s and a pre-acquisition delay of 2 s. Chemical
shifts were given in ppm relative to an external reference of 85%
phosphoric acid. A proton-detected one-dimensional
1H,31P HMQC spectrum was recorded on the same
instrument at ambient temperature with an evolution delay of 60 ms
using a sweep width of 2000 Hz and 4000 data points. The
two-dimensional 1H,31P HMQC experiment was
carried out under the same conditions using a sweep width of 624 Hz. In
the F2 dimension, 1000 data points were collected giving an
acquisition time of 0.82 s. The data matrix was zero-filled twice
in the F1 dimension to give a matrix of 1000 × 256 points using linear prediction.
The COSY, TOCSY, NOESY, and HMQC spectra were assigned using the
computer program PRONTO (33), which allows the simultaneous display of
different two-dimensional spectra and the individual labeling of
cross-peaks. The HSEA/Monte Carlo calculation was performed as reported
(34).
 |
RESULTS AND DISCUSSION |
Compositional Analysis of LPS--
The compositions and structures
of the core region of S. enterica sv. Minnesota R60
(reviewed in Refs. 2 and 35) and of the O-specific polysaccharide of
S. enterica sv. Arizonae O62 (36) have been reported.
Monosaccharide and phosphate analyses of LPSs from S. enterica sv. Arizonae O62 rough mutants R4 and R5 (Table
I) reveals the presence of Glc, Gal,
L-glycero-D-manno-heptose (Hep), 3-deoxy-D-manno-oct-2-ulopyranosonic acid
(Kdo), GlcN, organic phosphate, and small amounts of Rha (indicating a
smooth LPS fraction) in LPSs from the rough mutants. GC analysis of the acetylated (R)-butyl glycosides showed that Glc, Gal, and
GlcN residues possess the D-configuration. The
L-configuration of Rha was determined by others (36). The
fatty acids identified in the LPS preparations are
3-hydroxytetradecanoic acid, tetradecanoic acid, hexadecanoic acid, and
dodecanoic acid.
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Table I
Monosaccharide and phosphate compositions of LPSs from strains R4 and
R5 of S. enterica sv. Arizonae IIIa O62
Molar ratios are based on Gal = 2.0 (absolute amounts in nmol/mg
LPS). Kdo was determined after hydrolysis with 0.1 M sodium acetate buffer (pH 4.4, 1 h, 100 °C).
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The low relative molar ratios of GlcN (Table I) determined for the
rough mutant LPS samples are noteworthy. The values suggest that one of
the three expected GlcN residues is absent; since the lipid A moiety
should possess two N-acylated GlcN residues, we assume that
the GlcNAc residue in the outer core region (compare Fig.
1) is the one that is absent. In
addition, the data indicate the presence of only one Glc residue. An
additional quantification of Kdo after hydrolysis with 0.1 M sodium acetate buffer (pH 4.4, 100 °C, data not shown)
(37) revealed one terminal Kdo residue in each LPS.

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Fig. 1.
Structure of the Ra chemotype core
oligosaccharide of Salmonella LPS (2, 35). A third Kdo
residue -2 4-linked to the second one is in most LPSs present in
non-stoichiometric amounts.
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The success and fidelity of chemical manipulations were checked by
monosaccharide analysis. These data (not shown) show no significant
differences from those for the unmodified samples presented in Table
I.
Mass Spectrometry of Rough Type LPS--
Mass spectrometry was
used to determine the details of the core structures present in the
LPSs from mutants R4a, R4b, and R5. The dephosphorylated, reduced, and
deacylated LPS preparations were desalted on Sephadex G50 and then
examined using positive ion FAB-MS. Analysis of the early eluting G50
fraction obtained from the LPS from strain R5 (Fig.
2) yields a spectrum containing a series
of intense [M+H]+ pseudo-molecular ions in the region
m/z 1300-2050. The most intense ion at m/z 1845 corresponds to a species with a monosaccharide composition of
Hex3Hep3Kdo2(HexNH2)2,
representing a species having a monosaccharide composition similar to
that of the Salmonella Ra core with the lipid A but lacking
one HexNH2 and Hex residue. A related but very minor ion is
present at m/z 2007, which would correspond to a species
with the same composition as that giving rise to the ion at
m/z 1845, but bearing an extra Hex residue. In addition, the
ion at m/z 1683 corresponds to a species again related to
the Ra core but this time lacking one HexNH2 and two Hex
residues. The remaining ions in the spectrum correspond to species
originating from smaller related components that, in addition to
lacking one HexNH2 and one or two Hex residues, also lack
one Kdo and/or a second HexNH2 residue. Partial cleavage of
monosaccharide residues during the chemical modifications is assumed to
account for this heterogeneity. The acid-labile Kdo glycosidic linkage is partially cleaved on HF treatment, giving rise to the ions at
m/z 1625 and 1463, representing species containing only one Kdo residue. The ions at m/z 1684 and 1464 also arise as
artifacts of HF treatment2
and represent species in which the reducing terminal HexNH2
of the lipid A is cleaved prior to reduction. These species thus possess only one lipid A HexNH2 residue and this became
reduced. Similarly, the ions at m/z 1464 and 1302 correspond
to species lacking both the Kdo and the lipid A HexNH2
residue. The less intense ion observed at m/z 1359 probably
arises by
-cleavage, and corresponds to a component with the
monosaccharide composition Hep3Kdo2(HexNH2)2.
Apart from small differences in relative ion intensities, the FAB-mass
spectra of the two later eluting G50 fractions from R5, as well as
those from the G50 fractions from R4a and R4b resemble that of the
early eluting G50 fraction of R5. This indicates that the difference in
extraction methods used to obtain R4a and R4b LPSs does not influence
the types of LPS molecules isolated, but only their relative
amounts.

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Fig. 2.
Positive ion mode FAB-mass spectrum of the
dephosporylated, reduced, and deacylated LPS from S. enterica
sv. Arizonae rough mutant R5. Mass numbers are given as
nominal monoisotopic masses.
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The de-O-acylated, dephosphorylated LPSs from strain R5 were
further modified to reduce the heterogeneity of the preparation to
obtain more detailed information on the arrangement of the hexoses in
the outer core. Mild hydrolysis in 1% aqueous acetic acid was carried
out to cleave the linkage between the inner core and lipid A, and to
remove the remaining terminal Kdo residue. The positive ion FAB-mass
spectrum (not shown) of the product reveals a reduction in
heterogeneity following acid hydrolysis to yield a mixture of only two
components. The major ion is observed at m/z 1301 for the
protonated component Hex3Hep3Kdo. The minor ion
at m/z 1161 reflects the sodium-cationized
Hex2Hep3Kdo component. Both ions are
accompanied by signals 18 mass units lower, probably corresponding
to the presence of Kdo artifacts (38).
To further improve sensitivity and to direct mass spectrometric
fragmentation, allowing the sequence and branching patterns of the
monosaccharide residues to be determined, the acid-hydrolyzed, de-O-acylated, dephosphorylated R5 LPS sample was
per-O-acetylated. The positive ion mode FAB-mass spectrum
(not shown) reveals a series of oxonium (or B-) ions (43, 44). The two
most intense of these are at m/z 1627 and 1987 and
correspond to Hex3Hep2+ and
Hex3Hep3+, respectively,
deriving from the Hex3-containing major component. The
presence of these ions, together with the absence of an oxonium ion for
Hex3Hep1+, indicates that
the third Hep residue in the core is terminal and linked to the Hep
residue which is attached to the Glc residue (see Fig. 1). The most
abundant Hex3Hep2+ oxonium
ion (m/z 1627) was used as the parent ion for a CID tandem mass spectrometric experiment (Fig. 3)
designed to allow determination of the arrangement of the Hex residues
in the outer core. The most intense fragment ions at m/z 331 and 403 represent oxonium ions for terminal Hex and terminal Hep,
respectively. The major ions formed from an acetylated derivative are
known to originate from single glycosidic bond cleavages to form
oxonium ions. Thus, if the Hex3 outer core is linear,
oxonium ions at m/z 331 (Hex1+), m/z 619 (Hex2+), and m/z 907 (Hex3+) may be expected to be formed,
while a branched structure can only yield ions at m/z 331 (Hex1+) and m/z 907 (Hex3+) with no ion at m/z
619 for Hex2+. The
Hex3+ oxonium ion is present in the
spectrum, while the ion for Hex2+ is
notably absent. Thus, from our data, we conclude that the major
component having a composition of
Hex3Hep3Kdo2(HexNH2)2, contains a branched hexose structure, as well as a branched heptose arrangement.

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Fig. 3.
CID tandem mass spectrum of the
dephosphorylated and acetylated core oligosaccharide preparation
obtained after hydrolysis with 1% acetic acid (2 h, 100 °C) from
de-O-acylated LPS from S. enterica sv. Arizonae
rough mutant R5. Parent ion m/z 1627. Fragment ion
m/z 1267 represents the loss of a Hep residue to form a
fully acetylated Hex3Hep+ ion. Formation of
this ion involves the elimination of an internal heptose residue in a
rearrangement process (39-41) or the transfer of an acetyl group
instead of a hydrogen atom during a -cleavage reaction (42).
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Methylation Analysis--
Methylation analysis of the LPS from the
R5 mutant (19) was performed to determine the linkages in the Kdo
region. After hydrolysis of the methylated sample with 0.1 M trifluoroacetic acid, the derivative for a terminal
(B in Fig. 4) Kdo was identified, although the derivative corresponding to a 4-substituted Kdo residue indicating a Kdo2
4Kdo branch was not observed. After additional strong acid hydrolysis with 1 M trifluoroacetic
acid and further derivatization, a 4,5-disubstituted Kdo residue was detected. This product originates from the first Kdo residue
(A) that links the core region to lipid A and is substituted
on O-5 by the first Hep (C) residue and on O-4 by the second
Kdo residue (B, product obtained after mild acid
hydrolysis). On additional methylation analysis to determine the
substitution pattern of hexoses and heptoses, derivatives corresponding
to 3,7-disubstituted Hep, 3,6-disubstituted Hex, 3-substituted Hep, 6-substituted Hex, and terminal Hep and Hex residues were identified, indicating the presence of a Glc residue that is substituted on O-3 and
O-6 by Gal. Derivatives corresponding to either 2- or 3-substituted Hex
were not detected. These data confirm the presence of a branched
Hex3 outer core containing a 3,6-disubstituted Hex residue.
In the outer core region of the minor component (see MS results above),
the 3-linked Gal residue is absent (Rb3 chemotype), which
we assume is due to incomplete biosynthesis. The presence of such a
minor Rb3 LPS fraction (2) is consistent with the results
of immunoblot analyses of various rough type LPSs from S. enterica (45), which indicated the presence of different terminal
structures in each of these LPSs.

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Fig. 4.
Structure of the repeating unit of the
O-specific polysaccharide of serotype O62 (36) (A) and the
novel core oligosaccharide identified in LPS from S. enterica
sv. Arizonae IIIa O62 (B) . Monosaccharide
residues are labeled A-Q as used in Tables II-V and
throughout the text. Corresponding minor residues are labeled a-q. PPEA, 2-aminoethyl diphosphate;
P, phosphate.
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In summary, the core oligosaccharides from the LPSs of the rough
mutants of S. enterica sv. Arizonae IIIa lack the distal GlcNAc and Glc residues found in the common Salmonella Ra
core. This indicates that, in the rough mutants, biosynthesis or
transfer of the distal two monosaccharide residues is absent. A minor
component was identified that also lacks the 3-linked Gal residue
(i.e. Rb3 chemotype LPS), probably as a result
of incomplete core biosynthesis. Thus, the major LPS fractions from the
rough type strains investigated are of the Rb2
chemotype (2).
Analysis of the Core Region from Smooth Form LPS--
To determine
whether the changes to the Ra core observed in the rough mutant LPS
preparations are due to mutations leading to the Rb2
chemotype or are indeed indicative of the occurrence of a novel core
structure in Salmonella, the structure of the core region of
smooth from LPS isolated from the parent strain (S. enterica
sv. Arizonae IIIa O62) was analyzed. In addition, the position of the
linkage between the core region and the O-specific polysaccharide was
investigated. Monosaccharide analysis of smooth form LPS revealed the
presence of Rha, GalANAc (constituents of the O-specific
polysaccharide) Glc, Gal, Hep, GlcN, and Kdo residues. The structure of
the repeating unit of the O-specific polysaccharide of this LPS has
been reported (Fig. 4A) (36). After hydrolysis of the smooth
form LPS with 1% acetic acid and centrifugation, the resulting mixture
of poly- and oligosaccharides in the supernatant was separated by
gel-permeation chromatography on Sephadex G50. Three fractions were
obtained, the first of which eluted with the void volume contained the
O-specific polysaccharide attached to the core region, and the third
eluted in the region where mono- and disaccharides elute. Both
fractions were not further investigated. The second fraction was
expected to contain oligosaccharides of the SR-type, i.e.
the complete core region substituted by one or few repeating units of
the O-specific polysaccharide, and was therefore further separated
using HPAEC. Five fractions were obtained which were desalted and
investigated by 1H NMR spectroscopy indicating that the
second fraction contained only one compound that possessed the complete
core and probably one repeating unit. This fraction was called
"fraction SR" and used for structural analysis.
Mass Spectrometry of Fraction SR--
FAB-MS and ESI-MS of
fraction SR were carried out to determine the structure of the
novel Salmonella core when linked to the O-specific
polysaccharide. The positive ion mode FAB-mass spectrum (Fig.
5A) contains an intense series
of poorly resolved pseudomolecular ion clusters in the m/z
3000 region. The ions in the cluster are separated by 22 mass unit
increments and are centered at m/z 2957, 2979, 3001, 3021, 3043, and 3064. The increments of 22 mass units indicate that the LPS
molecule bears several sodium adducts, consistent with the presence of
several strongly acidic moieties. The most intense pseudomolecular ion
cluster centered at m/z 3021 for a species bearing five
sodium atoms and having a monosaccharide composition of
HexANAc(DeoxyHex)4HexNAcHex5Hep3Kdo
corresponds to one repeating unit of the O-specific polysaccharide and
the full Ra core in which a HexNAc is replaced by a Hex residue (Fig. 4B) and includes three phosphates and an ethanolamine group.
The other pseudomolecular ions represent the same species possessing from two to seven sodium atoms. Three additional ion clusters (labeled
III, IV, and V) centered at
m/z 3037, 3058, and 3080 derive from potassium-cationized
pseudomolecular ions representing the same species. Minor
pseudomolecular ions representing a species lacking one phosphate group
(P, 80 mass units) are present centered at m/z
2898 ([M-P-2H+3Na]+) and m/z 2920 (labeled
II, [M-P-3H+4Na]+). In addition, very minor
ions that correspond to the species lacking the 2-aminoethyl phosphate
group (PEA, 123 mass units) are observed at m/z
2856 ([M-PEA-2H+3Na]+) and m/z 2877 (labeled
I, [M-PEA-3H+4Na]+).

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Fig. 5.
Mass spectra obtained from fraction SR of LPS
from S. enterica sv. Arizonae IIIa O62. A,
positive ion mode FAB-MS; B, negative ion mode ESI-MS,
doubly charged species; inset, triply charged
pseudomolecular ion region. Peak assignments for minor ions:
I, m/z 2877 [M-PEA-3H+4Na]+;
II, m/z 2920 [M-P-3H+4Na]+;
III, m/z 3037 [M-4H+4Na+K]+;
IV, m/z 3058 [M-5H+5Na+K]+;
V, m/z 3080, [M-6H+6Na+K]+;
VI, 1405.4 [M-PEA-3H+Na]2 ; VII,
m/z 1425.2 [M-P-3H+Na]2 ; VIII,
m/z 1435.2 [M-P-4H+2Na]2 .
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Highly phosphorylated LPS molecules may be expected to form negative
ions readily, therefore, to reduce the number of salt adducts, negative
ion mode ESI-MS was performed (Fig. 5B). Indeed, a doubly
charged ion ([M-2H]2
) is observed at m/z
1455.3 for the complete oligosaccharide structure (HexANAc(DeoxyHex)4HexNAcHex5Hep3Kdo)
including ethanolamine and three phosphate groups but without any
sodium atoms. The remaining ions in the cluster at m/z
1466.5, 1476.3, 1487.5, 1498.2, and 1509.1 correspond to species
containing one to five sodium atoms. A similar ion cluster is present
at m/z 970.4, 978.2, 984.4, and 991.9 corresponding to
triply deprotonated ions. The minor ions observed in the FAB-mass
spectrum and ascribed to the main species lacking either a phosphate or
an ethanolamine phosphate group, are also present in the ESI-mass
spectrum, both as doubly and triply deprotonated ions.
Since both of the mass spectrometric experiments were carried out at
resolutions of about 1000, this high mass LPS sample gives broad
signals. To obtain well resolved signals and thus better mass
assignments a higher resolution (resolving power of 3000) was used for
a negative ion mode FAB-MS experiment. Prior to this, the sample was
treated with ammonia in methanol to reduce the number of different ions
formed by both reducing the amount of salt and excluding the possible
presence of artifacts arising from acid treatment of the Kdo residue
(38). Two weak but resolved signals were observed in the negative ion
mode FAB-mass spectrum having their 12C isotope peaks at
m/z 2910.8 and 2932.8 (data not shown). These correspond
precisely to the expected m/z values for the monoisotopic [M-H]
and [M-2H+Na]
ions for the
oligosaccharide
HexANAc(deoxyHex)4HexNAcHex5Hep3Kdo including 2-aminoethanol and three phosphate groups. The mass spectrometric data are thus consistent with the presence of an LPS
species bearing one GalANAcRha4GlcNAc O-specific
polysaccharide repeating unit attached to a novel Salmonella
core composed of five Hex residues in the outer core and lacking the
terminal GlcNAc epitope. It is reasonable to postulate that the GlcNAc
residue present in the Ra core is replaced in the core region of
S. enterica sv. Arizonae O62 by a Glc residue.
NMR Spectroscopy of Fraction SR--
Fraction SR was further
subjected to NMR analyses to confirm the presence of a novel
Salmonella core structure, to identify the linkage positions
of the monosaccharide residues, to determine the manner of attachment
of the O-specific polysaccharide to the core, and to define the nature
and position of the phosphate substituents. The 1H NMR (600 MHz) chemical shifts are given in Table
II. The assignments are based on
two-dimensional phase-sensitive COSY and TOCSY experiments. Anomeric
configurations are assigned on the basis of the chemical shifts
observed and J1,2 values measured from the COSY
experiment (Table III). Ring
configurations are assigned on the basis of coupling constants as
follows: J2,3 ~ 9-10 Hz and
J3,4 ~ 9-10 Hz for
-gluco, J2,3 ~ 9-10 Hz and
J3,4 ~ 9-10 Hz for
-gluco,
J2,3 ~ 9-10 Hz and
J3,4 ~ 3-4 Hz for
-galacto, and
J2,3 ~ 3-4 Hz and J3,4 ~ 8-10
Hz for
-manno (in Hep, Rha). The NOESY data in Table
IV confirm the assignments using
intra-residue nuclear Overhauser effect (NOE) signals and allow the
unambiguous determination of the monosaccharide sequence. Assignments
of chemical shifts from 13C NMR spectroscopy (Table
V) are based on the HMQC experiment (500 MHz) together with the proton signal assignments. For comparison, the
core oligosaccharide of S. enterica sv. Minnesota R60
(chemotype Ra) was also analyzed using the same methods as described
above and the results are presented in parantheses in Tables II and V.
The coupling constants and NOE data obtained for this sample are
completely consistent with the published structure (2, 35) and very
similar to those obtained for fraction SR.
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Table II
1H NMR chemical shifts (in ppm) for fraction SR from LPS of S. enterica sv. Arizonae IIIa O62 and from S. enterica sv. Minnesota R60 (values given in parentheses)
Spectra were recorded from a solution in 2H2O
(p2H 7.1) at 500.13 and 600.13 MHz at 37 °C. Chemical shifts
are relative to internal acetone (2.23 ppm). Assignments were made from
COSY and TOCSY experiments. Monosaccharide units A-Q (major
fraction with -Kdo as reducing terminus) and a-m (minor
fraction with -Kdo as reducing terminus) are as shown in Fig. 4.
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Table III
JH,H and JH,P coupling constants (in Hz) observed for
fraction SR from LPS of S. enterica sv. Arizonae IIIa O62
Values are given ±0.5 Hz. Monosaccharide units A-Q (major
fraction with -Kdo as reducing terminus) and c-m (minor
fraction with -Kdo as reducing terminus) are as shown in Fig. 4.
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Table IV
NOE signals observed in the NOESY spectrum of fraction SR from LPS of
S. enterica sv. Arizonae IIIa O62
Monosaccharide units C-Q (major fraction with -Kdo as
reducing terminus) and c-m (minor fraction with -Kdo as
reducing terminus) are as shown in Fig. 4. s, strong NOE; m, medium NOE; w, weak NOE.
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Table V
13C NMR chemical shifts (in ppm) for fraction SR from LPS of S. enterica sv. Arizonae IIIa O62 and from S. enterica sv. Minnesota R60 (values given in parentheses)
Spectra were recorded from a solution in 2H2O
(p2H 7.1) at 125.77 or 150.90 MHz at 37 °C. Chemical shifts
are relative to internal dioxane (67.4 ppm). Assignments were made from
an HMQC experiment. Monosaccharide units A-Q (major
fraction with -Kdo as reducing terminus) and a-m (minor
fraction with -Kdo as reducing terminus) are as shown in Fig. 4.
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In the low field region of the 1H NMR spectrum (4.6-5.9
ppm) of fraction SR, signals for 14 anomeric protons, integrating each as a single proton, are present, together with some additional minor
proton signals (Fig. 6). The major
monosaccharide residues are identified as residues from the O-specific
polysaccharides and the core oligosaccharide and are consistent with
the results of monosaccharide analysis and mass spectrometry.
Characteristic signals for H-3 of the major Kdo residue are present at
1.84 ppm (axial) and 2.17 ppm (equatorial). Except for the
-GlcNAc
residue, all other residues are assigned as
-linked. The
-configuration of the GlcNAc residue is confirmed by NOE signals
(Table IV) observed between protons L-1 and L-3
and L-5 (see Fig. 4 for labeling of residues), which are not
observed for
-anomers. Therefore, this GlcNAc residue corresponds to
the O-specific polysaccharide (36).

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Fig. 6.
1H NMR spectrum of fraction SR
from S. enterica sv. Arizonae IIIa O62. The spectrum
was recorded from a solution in 2H2O
(p2H 7.1) at 500.13 or 600.13 MHz at 37 °C. Assignments were
made from COSY and TOCSY experiments. See Fig. 4 for labeling of the residues.
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All Rha, Hex, HexNH2, and Hep residues give NOE signals
between their H-1 and the proton on the carbon atom to which they are
glycosidically linked, from which the linkage positions were assigned.
Interestingly, the NOE signals observed between protons L-1
and I-4 (see Fig. 7) identify
the linkage of the
-GlcNAc L of the O-specific
polysaccharide to O-4 of
-Glc I of the core
oligosaccharide; thus, the O-specific polysaccharide is linked to the
core via GlcNAc L rather than Rha M, as might be
expected from the published structure of the repeating unit (36). The
presence of the terminal
-Glc residue K 1
2-linked to
-Glc I is established by NOE signals and confirms the
structure of a novel core oligosaccharide for Salmonella
(see Fig. 7). Interestingly, an NOE signal is observed between protons
K-1 and F-4 that may indicate an unusual
conformation of the outer core of the smooth form LPS in the region
that might be responsible for recognition by specific antibodies.
Consequently, an HSEA/Monte Carlo calculation for the tetrasaccharide
Glc
1
2Glc
1
2Gal
1
3Glc
was carried out to simulate the
conformation of the outer core of the smooth form LPS. It nicely shows
the short distance between H-1 of Glc K and H-4 of Glc
F. In the minimum energy conformation, the distance is 2.25 Å and the average distance from the simulation is 2.70 Å. Both
results fit very well with the NOE signal observed and thus give an
indication for the conformation of the outer core of the smooth form
LPS.

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Fig. 7.
Sections of two-dimensional spectra (COSY,
TOCSY, and NOESY) of fraction SR from S. enterica sv.
Arizonae IIIa O62 identifying the glycosidic linkage of Glc I to Gal H,
and its substitution by Glc K and GlcNAc L from the NOE
signals.
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Fraction SR contains one repeating unit of the O-specific
polysaccharide (see MS data); its terminal residue,
-GalANAc
Q gave only NOE signals to
-Rha P. The
chemical shifts corresponding to protons of the O-specific
polysaccharide are similar to those reported for serotype O62 by
Vinogradov et al. (36) identifying the structure of the
repeating unit present to be essentially that published, except that
-GlcNAc L is now identified as the residue linking the
O-specific polysaccharide to the core.
The presence of a major and a minor structure in fraction SR was
confirmed by a 1H,13C-HMQC spectrum. The minor
residues observed are
-Gal h,
-Glc i,
-Hep c,
-Hep d,
-Gal g,
-Rha m,
-Hep e,
-GlcNAc l,
and Kdo a. Their intensities are about one third of those
corresponding to the major residues. Comparison of the 1H
chemical shifts of the Kdo residues A and a with those for the two reference monosaccharides in Ref. 46 (allyl
-Kdo
and allyl
-Kdo) shows that the mixture has probably arisen due to
/
anomerization of the reducing terminal (unreduced) Kdo residue.
The chemical shifts for H-3eq (2.07 ppm) and H-4 (4.10 ppm)
of allyl
-Kdo differ significantly from those for allyl
-Kdo
(2.38 ppm and 3.71 ppm, respectively). Comparable differences are
observed between the shifts for Kdo A and a,
indicating that the presence of two Kdo residues is explained by
/
anomerization. From the chemical shifts, Kdo A
corresponds to the
-anomer and Kdo a corresponds to the
-anomer. Comparison of the carbon shifts of A-4 and
a-4 with those for the reference monosaccharides confirm this assignment.
Evidence for the presence of two components in fraction SR originating
from
/
anomerization is also found in the NOESY spectrum. Most
NOE signals observed are either between monosaccharide residues of the
major portion (
-Kdo-containing) or between those of the minor
(
-Kdo-containing). Interresidual NOE signals between major and minor
residues are only found when minor counterparts of the major residues
are absent (see Fig. 7). NOE signals for H-1 of
-Glc K
are observed between H-4 of both
-Glc residues I and
i, suggesting that
-Glc i has not arisen due
to the partial absence of
-Glc K. This interpretation is
consistent with the results obtained on MS analysis. The residues for
which no minor version is observed probably have the same chemical
shifts as the corresponding residue in the minor component.
Core Substituents in Fraction SR--
31P NMR spectra
were recorded to determine the composition and position of the
phosphate groups substituted to the LPS. The proton-decoupled
31P NMR spectrum of fraction SR (Fig.
8) contains equally intense resonances at
10.99 and
10.24 ppm, indicative of a diphosphodiester. In addition,
a signal at 4.25 ppm is observed in the chemical shift range expected
for phosphomonoesters. The two-dimensional 1H,31P-HMQC spectrum reveals that the phosphate
signal at 4.25 ppm is coupled to a proton with a chemical shift of 4.34 ppm which is assigned to H-4 of Hep D. This result is
consistent with the coupling pattern of the COSY cross-peaks of
D-4 showing a coupling constant JH,P
of 9 Hz. The signal at
10.99 ppm correlates with a broad signal in
the two-dimensional spectrum around 4.6 ppm identified as H-4 of major
Hep C and minor Hep c. Further evidence is found
in the coupling pattern of the cross-peaks in the COSY spectrum of
both, Hep C and c, that show an additional, large
coupling JH, P 8 Hz. Furthermore, correlation of the second
signal at
10.24 ppm to a proton with a chemical shift of 4.21 ppm is
observed, consistent with a diphosphodiester. In the proton-detected
one-dimensional HMQC spectrum, this proton appears together with the
H-4 protons of Hep residues C and c. An
additional, weak signal is observed in the spectrum at 3.3 ppm. In the
COSY spectrum, this proton rise to only one cross-peak, that to the
proton at 4.21 ppm. Compared with the 1H chemical shifts of
authentic 2-aminoethyl phosphate (4.08 ppm, CH2-P, 3.25 ppm, CH2-N3), these two signals can
thus be assigned to the protons of a 2-aminoethyl group attached to
diphosphate which in turn is attached to O-4 of Hep C and
c. Evidence for the presence of a 2-aminoethyl group is also
supported by the results of calculations of the proton and carbon
chemical shifts for a 2-aminoethyl phosphodiester using the simulation
programs ACD/HNMR and ACD/CNMR (47), respectively, which give results
consistent with the experimental data. In general, charged substituents
are present in non-stoichiometric proportions so that structural
determination of phosphate substituents in the core region is
difficult. In this study, however, purification of one component from a
hydrolytic mixture using HPAEC has allowed the presence and position of
phosphate substituents to be established. The 2-aminoethyl diphosphate
is thus linked to O-4 of Hep C and c, and the
monophosphate group is attached to O-4 of Hep D (compare
Fig. 4B).

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Fig. 8.
31P NMR spectrum with broad band
1H decoupling of fraction SR of S. enterica sv.
Arizonae IIIa O62. The spectrum was recorded at 101.25 MHz at
ambient temperature. Peak assignment: 10.99 and 10.24 ppm,
diphosphodiester; 4.25 ppm, phosphomonoester.
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Methylation Analysis--
Methylation analysis of
dephosphorylated, carbonyl-reduced, and de-O-acylated smooth
form LPS identified (from the O-specific polysaccharide) derivatives
for 2,3-disubstituted Rha, 2- and 3-substituted Rha, and 3-substituted
GlcN, and, after hydrolysis with formic acid and
H2SO4, derivatives for terminal GalNA, and from
the core region, 3,7-disubstituted Hep, 2,4-disubstituted Hex
(indicating the attachment of the O-specific polysaccharide to O-4 of
I, compare Fig. 4), 3,6-disubstituted hexose, 3-substituted
Hep, the two 6-substituted GlcN residues from lipid A
(1,6-di-O-acetyl-2-deoxy-2-methylacetamido-3,4,5-tri-O-methyl-glucose from the reducing GlcN and
1,5,6-tri-O-acetyl-2-deoxy-2-methylacetamido-3,4-di-O-methyl-glucose), 4-substituted Hex (attachment of the O-specific polysaccharide, but
indicating the presence of an smooth form fraction lacking the terminal
Glc residue K in the core region, compare Fig. 4), two
different 2-substituted Hex residues (one in minor amounts, probably
indicating the presence of a rough type fraction, compare Fig. 4), and
terminal Hep, Glc, and Gal derivatives. These results are consistent
with the structure (Fig. 4) deduced from MS and NMR experiments.
In conclusion, we have successfully identified the chemical structure
of a novel core type of LPS from S. enterica. Its presence had been proposed based on serological and early chemical analyses (6-9). The analysis of an SR-type oligosaccharide isolated from smooth
form LPS from S. enterica sv. Arizonae O62 demonstrates unambiguously that the terminal
-D-GlcNAc residue which
is present in the core regions of LPS from S. enterica sv.
Minnesota or sv. Typhimurium is replaced by
-D-Glc.
Furthermore, the site attachment of the O-specific polysaccharide to
the core region was determined as the O-4 of the penultimate
-D-Glc residue (see Fig. 4), a site that is identical to
that in LPS of S. enterica sv. Typhimurium (2). The chemical
structure of the repeating unit of the O-specific polysaccharide from
serotype O62 has already been published (36); however, we now show that
this polysaccharide is linked to the core region via
-D-GlcNAc L and, thus, establish the
monosaccharide sequence of the "biological repeating unit" in the
LPS (see Fig. 4). Finally, the sites of phosphoryl substitution in the
core region were identified as O-4 of Hep residues C
(substituted by PPEA, also proposed for the core region of S. enterica sv. Minnesota and sv. Typhimurium (2)) and D
(substituted by a phosphate residue, also proposed for the core region
of S. enterica sv. Minnesota and sv. Typhimurium, albeit at
an unknown position (2)).
Methylation analysis of S-form LPSs of S. enterica sv.
Arizonae O62 reveals the presence of a 4-substituted Hex residue. Since the linkage of the O-specific polysaccharide to the core region (
1
4 to Glc I) is the only linkage to an O-4 of a
monosaccharide residue in this LPS, this derivative as well as that
corresponding to a 2,4-disubstituted Hex residue must originate from
Glc I, indicating that one fraction of the smooth form LPSs
comprises an Ra analogue (complete core, Fig. 4) and another fraction
an Rb1-type core region that lacks Glc K. A
possible partial substitution of the core region by the terminal GlcNAc
residue in various S. enterica serovars has been discussed
(9), but no data have been published. Tsang et al. (9)
proposed the presence of the Rb1-type core structure in LPS
of S. enterica sv. Djakarta and suggested it as a third new
Salmonella LPS core type. However, from our findings and
from the discussion in Ref. 9, this core type may generally be present
in a fraction of S. enterica smooth form LPSs, either as
minor or even as a major component. This core type was observed mass
spectrometrically ([M+H]+ at m/z 2007) as a
minor component in the LPS from one of the rough-type mutants from
S. enterica sv. Arizonae IIIa. In S. enterica, the O-specific polysaccharides are first synthesized as
undecaprenol-linked intermediates and then transferred as a whole to
the core region (2, 3). On the basis of the presence of the
Rb1-type core region one may speculate that in the
biosynthesis of S-form LPS of S. enterica sv. Arizonae O62,
both the Ra analogue and the Rb1-type (which lacks Glc
K) core regions may be used as acceptor molecules for this
transfer. If this is so, then the terminal Glc (in S. enterica sv. Arizonae IIIa O62) and possibly also the GlcNAc
(e.g. in S. enterica sv. Typhimurium) residues do
not represent structural parameters important for this biosynthetic step. Alternatively, the terminal GlcNAc or Glc residues could be
removed in a late step of smooth form LPS biosynthesis (9). These
contrasting hypotheses await further investigation.
In addition to S. enterica sv. Arizonae IIIa O62, there
exist other serovars of subspecies IIIa, which do not react with mAb T6
and are resistant to phage FO1 (7). Further investigations will show
whether the LPSs from these serovars also possess the novel core
structure as in S. enterica sv. Arizonae IIIa O62.
We thank F.H. Garbers, B. Pers, and R. Engel
for expert technical assistance and H. Moll for help with GC-MS. We are
grateful to J. Duus for carrying out the HSEA/Monte Carlo
calculation and to U. Zähringer for the 1H NMR
data of 2-aminoethyl phosphate. We thank the Carlsberg Laboratory for
providing temporary accommodation and facilities for M. M. A. O.