Tài liệu Báo cáo khoa học: A novel type of highly negatively charged lipooligosaccharide from Pseudomonas stutzeri OX1 possessing two 4,6-O-(1-carboxy)-ethylidene residues in the outer core region ppt

A novel type of highly negatively charged lipooligosaccharide
from
Pseudomonas stutzeri
OX1 possessing two
4,6-
O
-(1-carboxy)-ethylidene residues in the outer core region
Serena Leone
1
, Viviana Izzo
2
, Alba Silipo
1
, Luisa Sturiale
3
, Domenico Garozzo
3
, Rosa Lanzetta
1
,
Michelangelo Parrilli
1
, Antonio Molinaro
1
and Alberto Di Donato
2
1
Dipartimento di Chimica Organica e Biochimica and
2
Dipartimento di Chimica Biologica, Universita
`

degli Studi di Napoli Federico II,
Napoli, Italy;
3
Istituto per la Chimica e la Tecnologia dei Materiali Polimerici – ICTMP – CNR, Catania, Italy
Pseudomonas stutzeri OXI is a Gram-negative microorgan-
ism able to grow in media containing aromatic hydrocar-
bons. A novel lipo-oligosaccharide from P. stutzeri OX1
was isolated and characterized. For the first time, the pres-
ence of two moieties of 4,6-O-(1-carboxy)-ethylidene resi-
dues (pyruvic acid) was identified in a core region; these two
residues were found to possess different absolute configur-
ation. The structure of the oligosaccharide backbone was
determined using either alkaline or acid hydrolysis. Alkaline
treatment, aimed at recovering the complete carbohydrate
backbone, was carried out by mild hydrazinolysis (de-O-
acylation) followed by de-N-acylation using hot KOH. The
lipo-oligosaccharide was also analyzed after acid treatment,
attained by mild hydrolysis with acetic acid, to obtain
information on the nature of the phosphate and acyl groups.
The two resulting oligosaccharides were isolated by gel
permeation chromatography, and investigated by composi-
tional and methylation analyses, by MALDI mass spectro-
metry, and by
1
H-,
31
P- and
13
C-NMR spectroscopy. These
experiments led to the identification of the major oligosac-

charide structure representative of core region-lipid A. All
sugars are
D
-pyranoses and a-linked, if not stated otherwise.
Based on the structure found, the hypothesis can be ad-
vanced that pyruvate residues are used to block elongation
of the oligosaccharide chain. This would lead to a less
hydrophilic cellular surface, indicating an adaptive response
of P. sutzeri OX1 to a hydrocarbon-containing environment.
Keywords: Pseudomonas stutzeri OXI; lipopolysaccharide;
NMR spectroscopy; mass spectrometry; pyruvic acid.
Environmental pollution is recognized worldwide as an
emergency for its negative effects on the biosphere and on
human health. Bioremediation strategies have recently been
devised, based on microbial biotransformations, given the
metabolic potential of selected microorganisms, in partic-
ular by Gram-negative bacteria, and their adaptability to
many different pollutants [1].
Pseudomonas stutzeri OX1 is a Gram-negative bacterium
isolated from the activated sludge of a wastewater treatment
plant, and endowed with unusual metabolic capabilities for
the degradation of aromatic hydrocarbons [2]. In fact, in
contrast with other Pseudomonas strains, this microrganism
is able to grow on a large spectrum of aromatic compounds
including phenol, cresol and dimethylphenol, and on
nonhydroxylated molecules such as toluene and o-xylene,
the most recalcitrant isomer of xylene. Moreover, it is able to
metabolize tetrachloroethylene (PCE), one of the ground-
water pollutants commonly resistant to degradation [3].
Degradation of aromatic hydrocarbons by aerobic bac-

teria comprises an upper pathway, which produces dihy-
droxylated aromatic intermediates by the action of
monooxygenases, and a lower pathway, which processes
these intermediates to molecules that enter the citric acid
cycle [4]. We have recently cloned, expressed and charac-
terized three different enzymatic systems from P.stutzeri
OX1: (a) toluene-o-xylene monooxygenase (ToMO) [5],
endowed with a broad substrate specificity [6] and (b)
phenol hydroxylase (PH) [7], both belonging to the upper
pathway; and (c) catechol 2,3 dioxygenase (C2,3O) (A. di
Donato, unpublished observations), the ÔgatewayÕ enzyme
to the lower pathway. However, chemical toxicity of wastes
can hamper the use of this and other microorganisms in
bioremediation strategies, especially when organic solvents
are present at high concentrations.
Several mechanisms have been described that contribute
to solvent resistance in Gram-negative bacteria, all based on
structural changes in outer and inner membranes [8].
Different short- and long-term responses have been
observed including modifications of the fatty acid and
phospholipid composition of the membrane, extrusion
mechanisms using vesicles, and energy-dependent active
efflux pumps that export toxic organic solvents outside the
cytoplasm [9].
Correspondence to A. Molinaro, Dipartimento di Chimica Organica e
Biochimica, Universita
`
di Napoli Federico II, Complesso
Universitario Monte S. Angelo, via Cintia 4, 80126 Napoli, Italy.
Fax: + 39 081 674393, Tel.: + 39 081 674123,

E-mail:
Abbreviations: DEPT, distorsionless enhancement by polarization
transfer; GlcN, 2-amino-2-deoxy-glucose; Hep,
L
-glycero-
D
-
manno-heptose; Kdo, 3-deoxy-
D
-manno-oct-2-ulosonic acid;
LOS, lipooligosaccharide; LPS, lipopolysaccharide.
(Received 1 March 2004, revised 23 April 2004,
accepted 30 April 2004)
Eur. J. Biochem. 271, 2691–2704 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04197.x
Even though lipopolysaccharides (LPSs) are major
components of the outer membrane of Gram-negative
bacteria, little is known about their role and their chemical
modifications under environmental stress [1,9]. It is certain,
however, that LPSs are unique and vital components of
these microorganisms and that they play an important role
in their survival and their interaction with the environment
[10,11]. Smooth-form lipopolysaccharides (S-LPSs) include
three regions, the O-specific polysaccharide (or O-antigen),
the oligosaccharide region (core region) and the lipid part
(lipid A). Conversely, rough (R) form LPSs do not possess
an O-specific polysaccharide and are frequently named lipo-
oligosaccharides (LOSs). LOSs have been found either in
wild-type strains and in mutant strains harboring mutations
in the genes encoding enzymes of the biosynthesis and/or
the transfer of the O-specific polysaccharide [12,13].

The core region from both smooth and rough forms of
enteric bacteria generally includes oligosaccharides built of
up to 11 units [12,13], and consists of two distinct domains:
an inner core, characterized by the presence of 3-deoxy-
D
-
manno-oct-2-ulosonic acid (Kdo) and
L
-glycero-
D
-manno-
heptose (Hep), and an outer core, which contains common
sugars. It is worth noting that the core oligosaccharide of
LOSs has been reported to play a role in the interaction of
the microorganism with the environment [12,13].
In this paper, the structural characterization of the
carbohydrate backbone of the rough form LPS of P. stutzeri
OX1 is reported, obtained by chemical analyses, MALDI-
TOF mass spectrometry and two-dimensional NMR
spectroscopy. This novel oligosaccharide chain was found
to possess unusual structural features, which might be
biologically relevant. Among these is a GalN residue
substituted by two gluco-configured residues, which are
blocked at position O-4 and O-6 by a pyruvate ketal linkage,
a structure peculiar and new to lipopolysaccharide core
regions.
Based on this finding, the hypothesis can be advanced
that the insertion of pyruvate residues at the end of the
oligosaccharide chain blocks its elongation, thereby leading
to a shorter LOS and hence to a less hydrophilic cellular

surface. Moreover, as it has already been proposed [1,9],
these residues may also contribute to the rigidity and
stability of the Gram-negative cell wall by binding cations.
Experimental procedures
Bacterial growth and LPS extraction
Cells were routinely grown on M9-agar plates supplemented
with 10 m
M
malic acid as the sole carbon source, at 27 °C.
For growth in liquid medium, 1 mL was inoculated with a
single colony from a fresh plate, and grown for 18 h at 27 °C
with constant shaking. This saturated culture was used to
inoculate 100 mL of the same medium and grown at 27 °C
until D
600
 1. Final growth was started by inoculating the
appropriate volume of the latter culture into 1 L of fresh
medium, to D
600
¼ 0.02. Cells were grown at 27 °C, until
D
600
¼ 1 was reached and then recovered by centrifugation
at 3000 g for 15 min at 4 °C, washed with an isotonic buffer
and lyophilized. Growth was carried out in M9 salt medium
supplemented with 4 m
M
phenol as the sole carbon and
energy source. Dried cell yield was 0.13 gÆL
)1

.
Dried cells were extracted three times with a mixture of
aqueous 90% phenol/chloroform/petroleum ether (50 mL,
2 : 5 : 8 v/v/v) as described previously [14]. After removal of
the organic solvents under vacuum, the LOS fraction was
precipitated from phenol with water, washed first with
aqueous 80% (v/v) phenol, and then three times with cold
acetone, each time centrifuged as above, and lyophilized
(the yield was 90 mg of LOS, about 4.3% of the dry mass).
Sodium dodecyl sulfate polyacrylamide gel electrophor-
esis (SDS/PAGE) was performed as described previously
[15]. For detection of LPS and LOS, gels were stained with
silver nitrate [15].
Isolation of oligosaccharides
An aliquot of LOS (40 mg) was dissolved in anhydrous
hydrazine (2 mL), stirred at 37 °C for 90 min, cooled,
poured into ice-cold acetone (20 mL), and allowed to
precipitate. The precipitate was then centrifuged (3000 g,
30 min, 4 °C), washed twice with ice-cold acetone, dried,
dissolved in water and lyophilized (32 mg, 80% of the
LOS). This material was de-N-acylated with 4
M
KOH as
described [16]. Salts were removed using a Sephadex G-10
(Pharmacia) column (50 · 1.5 cm). The resulting oligosac-
charide 1 constitutes the complete carbohydrate backbone
of the lipid A-core region (16 mg, 40% of the LOS).
Another aliquot of LOS (40 mg) was hydrolyzed in 1%
(v/v) acetic acid (100 °C, 2 h) and the precipitate (lipid A)
was removed by centrifugation (8000 g,30min).The

supernatant was separated by gel-permeation chromatog-
raphy on a P-2 column (85 · 1.5 cm). Two fractions were
obtained, the first contained oligosaccharide 2 (28 mg, 70%
of the LOS), whereas the second fraction contained a
mixture of reducing pyranose, furanose, anhydro and
lactone forms of 3-deoxy-
D
-manno-oct-2-ulosonic acid
(3 mg, 7.5% of the LPSs).
General and analytical methods
Determination of Kdo, neutral sugars, carbamoyl analysis,
including the determination of the absolute configuration of
the heptose residues, organic bound phosphate, absolute
configuration of the hexoses, fatty acids and their absolute
configuration, GLC and GLC-MS were all carried out as
described previously [17–21]. For methylation analysis of
Kdo region, LOS was carboxy-methylated with methanolic
HCl (0.1
M
,5min)andthenwithdiazomethanetoimprove
its solubility in dimethyl sulfoxide. Methylation was carried
out as described [22,23]. LOS was hydrolyzed with 2
M
trifluoroacetic acid (100 °C, 1 h), carbonyl-reduced with
NaBD
4
, carboxy-methylated as described above, carboxyl-
reduced with NaBD
4
(4 °C, 18 h), acetylated and analyzed

by GLC-MS.
Methylation of the complete core region was carried out
as described previously [22–24]. The sample was hydrolyzed
with 4
M
trifluoroacetic acid (100 °C, 4 h), carbonyl-reduced
with NaBD
4
, acetylated and analyzed by GLC-MS.
NMR spectroscopy
For structural assignments of oligosaccharides 1 and 2,1D
and 2D
1
H-NMR spectra were recorded on a solution of
2692 S. Leone et al.(Eur. J. Biochem. 271) Ó FEBS 2004
5mgin0.6mLofD
2
O, at 55 °Corat30°C, at pD 14 and
7 (uncorrected values), respectively.
1
H- and
13
C-NMR
experiments were carried out using a Varian Inova 500 or a
Varian Inova 600 instrument, whereas for
31
P-NMR spectra
a Bruker DRX-400 spectrometer was used. Spectra were
calibrated with internal acetone [d
H

2.225, d
C
31.45].
Aqueous 85% phosphoric acid was used as external
reference (0.00 p.p.m.) for
31
P-NMR spectroscopy.
Nuclear Overhauser enhancement spectroscopy
(NOESY) and rotating frame Overhauser enhancement
spectroscopy (ROESY) were measured using data sets
(t
1
· t
2
)of4096· 1024 points, and 16 scans were acquired.
A mixing time of 200 ms was used. Double quantum-filtered
phase-sensitive COSY experiments were performed with
0.258 s acquisition time, using data sets of 4096 · 1024
points, and 64 scans were acquired. Total correlation
spectroscopy experiments (TOCSY) were performed with a
spinlock time of 80 ms, using data sets (t
1
· t
2
)of
4096 · 1024 points, and 16 scans were acquired. In all
homonuclear experiments the data matrix was zero-filled in
the F1 dimension to give a matrix of 4096 · 2048 points and
was resolution enhanced in both dimensions by a shifted
sine-bell function before Fourier transformation. Coupling

constants were determined on a first-order basis from 2D
phase sensitive double quantum filtered correlation spectro-
scopy (DQF-COSY) [25,26]. Intensities of NOE signals were
classified as strong, medium and weak using cross-peaks
from intraring proton-proton contacts for calibration.
Heteronuclear single quantum coherence (HSQC) and
heteronuclear multiple bond correlation (HMBC) experi-
ments were measured in the
1
H-detected mode via single
quantum coherence with proton decoupling in the
13
C
domain, using data sets of 2048 · 512 points, and 64 scans
were acquired for each t
1
value. Experiments were carried
out in the phase-sensitive mode according to the method of
States et al. [27]. A 60 ms delay was used for the evolution
of long-range connectivities in the HMBC experiment. In all
heteronuclear experiments the data matrix was extended to
2048 · 1024 points using forward linear prediction extra-
polation [28,29].
MALDI-TOF analysis
MALDI mass spectra were carried out in the negative
polarity in linear or in reflector mode on a Voyager STR
instrument (Applied Biosystems, Framingham, MA, USA)
equipped with a nitrogen laser (k ¼ 337 nm) and provided
with delayed extraction technology. Ions formed by the
pulsed laser beam were accelerated through 24 kV. Each

spectrum is the result of approximately 200 laser shots. A
saturated solution of 2,4,6-trihydroxyacetophenone was
used as the matrix.
Results
Isolation and characterization of the LOS fraction
The LOS fraction was isolated from dried cells by extraction
with phenol/chloroform/petroleum ether, and further puri-
fied with gel permeation chromatography. SDS/PAGE
showed, after silver nitrate gel staining, the presence of fast
migrating species in agreement with their oligosaccharide
nature. Compositional monosaccharide analysis of the LOS
fraction led to the identification of
L
,
D
-Hep,
D
-GalN,
D
-GlcN,
D
-Glc, Kdo (2 : 1.0 : 3.2 : 1.1 : 1.8) and trace
amounts of
L
-Rha. 7-O-Carbamoyl-
L
,
D
-Hep was present in
a stoichiometric ratio with

L
,
D
-Hep. Methylation analysis
showed the presence of terminal Kdo, 6-substituted-HexN,
3-substituted-Hep, 4,5-disubstituted-Kdo, 3,4-disubstituted-
HexN, 4,6-disubstituted-Glc, 4,6-disubstituted-HexN and,
in small amounts, terminal-Rha and 6-substituted-Glc.
In addition, the disaccharide 7-O-carbamoyl-Hep-(1fi3)-
Hep was found.
Fatty acid analysis revealed the presence of typical fatty
acids of pseudomonads LPS [30], i.e. (R)-3-hydroxydodec-
anoic acid [C12:0 (3-OH)], present exclusively in amide
linkage and (R)-3-hydroxydecanoic [C10:0 (3-OH)] (S)-2-
hydroxydecanoic [C12:0 (2-OH)] and dodecanoic acid
(C12:0), present in ester linkage. Moreover, phosphate
colorimetric assays gave positive results.
The LOS fraction was then subjected to both alkaline and
acid degradations and complete structural characterization.
NMR spectroscopy and MALDI-TOF MS spectrometry
of oligosaccharide 1
Oligosaccharide 1 was isolated by gel permeation chroma-
tography after complete deacylation of the LOS of
P. stutzeri OX1. The complete structure of fully deacylated
oligosaccharide 1 (Fig. 1) was determined by
1
H-,
31
P- and
13

C-NMR spectroscopy. Chemical shifts were assigned
using DQF-COSY, TOCSY, NOESY, ROESY,
1
H,
13
C-
DEPT-HSQC,
1
H,
31
P-HSQC,
1
H,
13
C-HMBC and
1
H,
13
C-
HSQC-TOCSY experiments. Anomeric configurations
were assigned on the basis of
1
Hand
13
C chemical shifts,
of
3
J
H1,H2
values determined from the DQF-COSY experi-

ment (Table 1), and of
1
J
C1,H1
values derived by
1
H,
13
C-
HSQC spectrum recorded without decoupling during
acquisition.
All sugars were present as pyranose rings, as indicated by
1
H- and
13
C-NMR chemical shifts and by the HMBC
spectrum that showed for all residues intraresidual scalar
connectivity between H-1/C-1 and C-5/H-5 atoms (for Kdo
units, between C-2 and H-6). The anomeric region of the
1
H-NMR spectrum (Fig. 2) showed seven major signals in
the region between 5.46 and 4.47 p.p.m. relative to seven
different spin systems (A–G, in order of decreasing chemical
shift), and in addition two AB methylene resonances at high
fields, typical of Kdo residues (I–L). Each spin system was
completely assigned by COSY and TOCSY starting from
anomeric resonances. For Kdo residues I and L the starting
point was the H-3 diastereotopic methylene resonance.
Both spin systems A and D (5.46 and 5.27 p.p.m.) were
characterized by low

3
J
H1, H2
and
3
J
H2, H3
values, indicative
of two a-manno-configured residues. Moreover, all other
cross peaks within each spin system were assigned in the
TOCSY spectrum from H-2 proton signals, leading to their
identification as two heptoses. Residue B was identified as
a-gluco-configured hexosamine on the basis of chemical
shifts and
3
J
H,H
values. Moreover, based on its anomeric
signal at 5.42 p.p.m. present as a double doublet (
3
J
H1,H2
¼
2.9 Hz and
3
J
H1,P
¼ 8.3 Hz), with one of the couplings due
to a phosphate signal as shown below, it was identified as
GlcN I of the lipid A skeleton. The spin system at

Ó FEBS 2004 LPS from Pseudomonas stutzeri OX1 (Eur. J. Biochem. 271) 2693
Table 1.
1
H,
13
Cand
31
P NMR chemical shifts (p.p.m) of deacylated core-lipid A backbone (oligosaccharide 1) of LOS from P. stutzeri OX1.
Chemical shifts are relative to acetone and external aq. 85% (v/v) phosphoric acid (
1
H, 2.225 p.p.m.;
13
C, 31.45 p.p.m.;
31
P, 0.00 p.p.m. at 55 °C).
Residue Nucleus 1 2 3
ax/
45678
A
1
H 5.46 4.39 4.09 4.46 4.39 4.20 3.86/4.08
Hep
13
C 97.8 73.8 76.7 72.0 73.7 69.8 63.9
31
P 1.9 4.0
B
1
H 5.42 2.73 3.64 3.45 4.09 4.30/3.74
GlcN

13
C 95.1 55.9 73.7 70.8 72.0 70.1
31
P 3.0
C
1
H 5.32 3.22 4.06 4.27 4.02 3.71
GalN
13
C 101.3 51.5 78.3 77.5 71.5 61.7
D
1
H 5.27 4.43 4.14 4.05 4.16 4.45 3.75/4.09
Hep
13
C 102.7 69.9 78.2 67.4 71.7 74.2 63.7
31
P 4.3
E
1
H 4.73 3.44 3.71 3.43 3.66 3.93/3.65
Glc
13
C 105.8 75.3 72.9 75.8 67.7 64.6
F
1
H 4.67 2.70 3.54 3.71 3.36 4.04/3.93
GlcN
13
C 104.7 58.2 76.6 77.7 67.2 64.7

G
1
H 4.47 2.67 3.66 3.82 3.47 3.71/3.45
GlcN
13
C 103.5 56.9 73.5 73.4 76.9 63.7
31
P 3.9
I
1
H 1.81/2.07 4.27 4.08 3.69 4.03 3.86/3.70
Kdo
13
C 175.0 101.7 36.1 65.9 67.7 73.0 70.1 63.7
L
1
H 2.00/2.34 4.12 4.24 3.67 3.93 3.86/3.69
Kdo
13
C 175.0 100.9 35.0 71.8 69.5 73.6 70.6 63.9
S-Pyr
1
H 1.48
13
C 175.5 101.9 25.2
R-Pyr
1
H 1.62
13
C 175.8 99.5 17.2

Fig. 1. The structure of oligosaccharide 1 obtained by alkaline hydrolysis of the core region of the LPS of P. stutzeri OX1.
2694 S. Leone et al.(Eur. J. Biochem. 271) Ó FEBS 2004
5.32 p.p.m. (C;
3
J
H1, H2
¼ 3.6 Hz) was identified as a-GalN
by its J
H,H
values for H-3/H-4 and H-4/H-5, diagnostic of a
galacto configuration (3.4 Hz and less than 1 Hz, respect-
ively). Three spin systems E, F and G (doublets;
3
J
H1, H2
¼
8.6, 7.8 and 7.7 Hz, respectively) were identified as b-gluco-
configured monosaccharides given their large
3
J
H,H-
values.
A further indication of their b configuration was the
observation of NOE contacts in the ROESY spectrum
among H-1, H-3 and H-5, for all E, F, G residues.
TheH-3methylenesignalsoftwoa-Kdo residues were
present at 1.82 p.p.m. (H-3ax) and 2.07 p.p.m. (H-3eq)
(residue I), and 2.00 p.p.m. (H-3ax) and 2.34 p.p.m.
(H-3eq) (residue L), respectively. Their a configuration
was established on the basis of the chemical shift of their

H-3eq protons and by measurement of the
3
J
H7,H8a
and
3
J
H7,H8b
coupling constants [31,32]. Two methyl singlet
signals were present at higher fields, at 1.48 and 1.62 p.p.m.,
respectively. Each methyl signal was in a 3 : 1 ratio with the
anomeric signals, i.e. in a stoichiometric ratio.
The
13
C-NMR chemical shifts could be assigned by a
DEPT-HSQC experiment, using the assigned
1
H-NMR
spectrum. Seven anomeric carbon resonances were identi-
fied (Table 1), numerous carbon ring signals and four
nitrogen-bearing carbon signals assigned to C-2 of B, C, F
and G spin systems. Considering the
13
Cchemicalshiftsof
nonsubstituted residues [33], several low-field shifted signals
indicated substitutions at O-3 of residue A, O-6 of residue B,
O-3 and O-4 of residue C, O-3 of residue D,O-4andO-6of
residues E and F, O-6 of residue G, O-5 and O-4 of residue
L,whereasI was a terminal residue. In the high field region
of the spectrum two cross peaks at 1.48/25.2 and 1.62/

17.2 p.p.m. were present.
Phosphate substitution was established on the basis of
31
P-NMR spectroscopy. The
31
P-NMR spectrum showed
the presence of five monophosphate monoester signals
(Table 1). The site of substitution was inferred by a
1
H,
31
P-HSQC spectrum that showed correlations of
31
P
signals with H-1 B (GlcN), H-4 A and H-2 A (Hep I), H-4 G
(GlcN) and H-6 D (Hep II).
The sequence of the monosaccharide residues was
determined using NOE effects of the ROESY (Fig. 3) and
NOESY spectra, and by
1
H,
13
C-HMBC correlations. The
typical lipid A carbohydrate backbone was eventually
assigned on the basis of the NOE signal between H-1 G
and H-6
a,b
B. In the case of Kdo units, which lack the
anomeric proton, the sequence was inferred by NOE
contacts between the methylene-proton H-3

eq
of Kdo L
and H-6 of Kdo I,whereasKdoL was substituted by
heptose A as indicated by the NOE effect found between
H-1 A and H-5 L, and, in addition, between H-5 A and
H-3
ax
L. All of these NOE contacts were characteristic of
the sequence a-
L
-glycero-
D
-manno-heptose-(1fi5)-[a-D-
Kdo-(2fi4)]-a-D-Kdo [34,35].
Heptose A was, in turn, substituted at the O-3 position
by heptose D, as demonstrated by the NOE cross peak
between H-1 D and H-3 A. A disaccharide 7-O-carbamoyl-
Fig. 2.
1
H-NMR spectrum of oligosaccharide 1. The spectrum was recorded under the following conditions: 5 mg of oligosaccharide 1 in 0.6 mL
D
2
O, pD 14 at 30 °C.
Ó FEBS 2004 LPS from Pseudomonas stutzeri OX1 (Eur. J. Biochem. 271) 2695
Hep-(1fi3)-Hep was also identified by methylation analysis
of the intact LOS, thus, the carbamoyl group should be
attached to O-7 of the heptose moiety D.TheGalNC was
attached to the O-3 position of this last heptose as shown by
the NOE effect between the anomeric proton of GalN and
H-3 of D.

GalN is the branching point of the chain and, conse-
quently, it should carry two sugar residues at O-3 and O-4.
Indeed, the anomeric proton of b-glucose E gave a NOE
effect with H-3 of GalN, whereas the anomeric proton of
b-glucosamine F gave a NOE effect with H-4 of GalN.
In determining the L Kdo location, its linkage to unit G
was deduced by exclusion. In particular, the linkage to O-6
of G was inferred by taking into account the downfield shift
of the carbon signal C-6 (63.7 p.p.m., Table 1) indicating its
involvement in a glycosydic linkage.
The HMBC spectrum confirmed the sequence proposed
for oligosaccharide 1, as it contained the significant long-
range correlations required for the determination of the
sequence and of the attachment points. In fact, together
with intraresidual long-range cross-peaks, interresidual
long-range connectivity was found between H-5/C-5 L
and C-1/H-1 A, H-3/C-3 A and C-1/H-1 D,H3/C-3D and
C-1/H-1 C, H-1/C-1 E and C-3/H-3 C, H-1/C-1 F and C-4/
H-4 C, H-1/C-1 G and C-6/H-6 B.
The HMBC experiment was also crucial for the
identification and localization of the two methyl
groups belonging to noncarbohydrate constituents. Plain
long-range correlations (Fig. 4A) were found in the
spectrum for each methyl signal. The signal at
1.48 p.p.m. correlated to two different carbon signals at
101.9 and 175.5 p.p.m., whereas the signal at 1.62 p.p.m.
correlated to two other signals at 99.5 and 175.8 p.p.m.
None of these four carbon signals was present in the
HSQC spectrum. These data pointed to two cyclic ketals
of pyruvic acid present on two distinct residues, namely E

and F, whose C-4 and C-6 signals experienced a downfield
displacement. This was confirmed by the HMBC spec-
trum where each ketal carbon signal of pyruvate residues
correlatedtoH-4andH-6ofE and F residues (b-
D
-Glc
and b-
D
-GlcN, respectively). It should be noted that the
signal discrepancy in the
1
Hand
13
C chemical shifts of the
two pyruvate moieties is due to the different absolute
configuration at C-2. In fact, the methyl signal occurring
at 1.48 and 25.2 p.p.m. is assigned to the S-pyruvate
group, whereas the one occurring at 1.62 and 17.2 p.p.m.
is assigned to an R-pyruvate group, as already described
[36]. Moreover, the ROESY spectrum (Fig. 4B) was in
complete agreement with the assignment above. In fact,
the methyl signal of the R-pyruvate residue at 1.62 p.p.m.
gave a strong NOE effect with H-4 and H-6
a
of residue F.
This is in agreement with an axial orientation of the
methyl group on a 1,3-dioxane ring in a chair-like
conformation in which H-4 and H-6
a
are sin diaxial with

respect to it. The methyl signal of S-pyruvate, being in
equatorial orientation, only gave NOE effect with the
Fig. 3. Section of the ROESY spectrum of oligosaccharide 1. Monosaccharide labels are as indicated in Fig. 1. NOE cross-peaks are in black, in
antiphase with diagonal (grey lines). Spectrum was recorded at pD 14, 55 °C.
2696 S. Leone et al.(Eur. J. Biochem. 271) Ó FEBS 2004
adjacent H-6 of residue E. Thus, all main spin systems
were assigned in the NMR spectra, and all chemical data
found a rational explanation.
The presence of a minor spin system (10%) belonging to
rhamnose (anomeric signal at 4.89 p.p.m) and 6-substituted
glucose (overlapped with terminal glucose) might be
explained by the presence of a second outer core glycoform
in which rhamnose is attached at O-6 of the glucose residue,
which obviously lacks the pyruvate group.
The MALDI mass spectrum confirmed the proposed
structure. In fact, an ion peak at m/z 2188.4 (Fig. 5A) was
present, corresponding to the complete carbohydrate back-
bone bearing five phosphate goups and two pyruvic acid
acetal residues. Moreover, at higher laser intensity (Fig. 5B)
various ion peaks related to fragments were found, all fitting
with the structure shown in Fig. 1.
In conclusion, the data above allowed the identification
of the carbohydrate backbone from alkaline degradation of
the rough form LPS from P. stutzeri OX1.
Isolation, NMR and MS analyses of oligosaccharide 2
from acetic acid hydrolysis
Further information on alkaline labile groups that could
be present in the core region (i.e. acyl groups) was obtained
by treating the LOS with acetic acid to split the Kdo
linkage. An oligosaccharide mixture was isolated after gel

permeation chromatography, which was purified further
and the resulting oligosaccharide 2 (Fig. 6) analyzed by
compositional/methylation analyses, 2D NMR and mass
spectrometry.
Compositional and methylation analyses led to the
identification of 3-substituted-
L
,
D
-Hep, 7-O-carbamoyl-
3-substituted-
L
,
D
-Hep 3,4-disubstituted-
D
-GalN, terminal
D
-glucose and terminal
D
-GlcN. Traces of 5-substituted
Kdo, 6-substituted-
D
-Glc and terminal
L
-rhamnose were
also found.
The
1
H-NMR spectrum revealed the absence of anomeric

signals from GlcN I and GlcN II of Lipid A, the lack of
pyruvate methyl groups, as a consequence of the cleavage of
the ketal group under acid treatment, and the presence
of singlet signals at 2.00 p.p.m. Methylene signals of Kdo
were spread because of its presence as reducing end unit, i.e.
pyranose, furanose, anhydro and lactone forms present at
same time. The anomeric region of the spectrum consisted
of six main signals (Fig. 6), five of which belonging to the
main oligosaccharide backbone, named U–Z. All resonanc-
es of the monosaccharides (Table 2) were obtained from 2D
NMR spectroscopy (DQF-COSY, TOCSY, NOESY,
ROESY,
1
H,
13
C-DEPT-HSQC
1
H,
31
P-HSQC,
1
H,
13
C-
HMBC and
1
H,
13
C-HSQC-TOCSY). Evaluation of
chemical shifts and of

3
J
H,H
coupling constants led to
identification of residues Hep (U), 7-O-carbamoyl-Hep (V),
GalN (X), GlcN (W), Glc (Z).
Fig. 4. Sections of the high field region of the (A) ROESY and (B) HMBC spectra. Correlations of pyruvate methyl groups are shown. (A) The 4,6
Pyr-GlcN residue is drawn in the middle of the figure with arrows indicating the relevant NOE contacts between methyl protons of the R-pyruvate
group and H-4 and H-6 of GlcN residue. Spectra were recorded at pD 14, 55 °C.
Ó FEBS 2004 LPS from Pseudomonas stutzeri OX1 (Eur. J. Biochem. 271) 2697
Low-field shifted signals were present in the HSQC
spectrum indicating substitutions at O-3 (U, V and X)and
O-4 (X), whereas residues W and Z were not substituted.
Cross-peaks were also detected only for two nitrogen atoms
bearing carbon signals, at 4.26/49.8 p.p.m. (H-2/C-2 X)and
3.67/56.1 p.p.m. (H-2/C-2 W), in agreement with the
absence of Lipid A disaccharide and with the presence of
a a-galacto and a b-gluco configured 2-amino-2-deoxy
hexoses. Moreover, given the downfield H-2 chemical shifts
of the X and W residues, the amino groups should have
been present as acylamido.
ThehighfieldprotonregionoftheHMBCgavecluesfor
the identification of the nature of acyl groups. Three
different singlet signals were present in this region (Fig. 6),
two with a smaller area that probably account for the same
methyl group that experienced oligosaccharide heterogen-
eity. All signals in the region of 2.0 p.p.m. showed long-
range correlations with a carbonyl signal at 174.6 p.p.m.
which in turn correlated to protons at 3.67 p.p.m. (H-2 W)
and 4.26 p.p.m. (H-2 X). These correlations indicated the

presence of two acetamido groups at the C-2 of GlcN W
and GalN X. Thus, the two smaller methyl signals are both
due to GalN X and are consequences of oligosaccharide
heterogeneity, possibly due either to the adjacent heptose V
bearing heterogeneous phosphate substitution (see below)
or to a reducing Kdo residue.
In addition, all diagnostic interresidue NOE effects were
found in the ROESY spectrum. This confirmed the
oligosaccharide sequence as determined in the previous
paragraph.
Other information on noncarbohydrate substituents
(phosphate and carbamoyl groups) was gained by the
observation of the downfield displaced heptose signals,
namely H-2/C-2 and H-4/C-4 of heptose U and H-6/C-6
and H-7
a,b
of heptose V.
The H-7
a,b
downfield shift was clearly due to the presence
of a carbamoyl group that did not undergo hydrolysis in
mild acid conditions, and that has already been located at
position O-7 of the second heptose residue on the basis of
methylation analysis. In agreement with this assignment, a
Fig. 5. Negative ion MALDI-TOF mass spectra of oligosaccharide 1 obtained in linear mode at normal (A) and higher laser intensity (B). Assignments
ofmainionpeaksareshown.P,phosphate;Pyr,pyruvicacid.
2698 S. Leone et al.(Eur. J. Biochem. 271) Ó FEBS 2004
signal at 160.0 p.p.m. in the HMBC spectrum correlated
with both protons H-7 of V.
The degree of phosphorylation and localization of

phosphate substituents was established by 1D and 2D
31
P-NMR spectroscopy (Fig. 7, Table 2). Several signals
were found in the
31
P-NMR spectrum, whose chemical
shift clearly indicated that they derived from phosphate
groups present in different magnetic/chemical environ-
ments. In fact, in addition to a number of phosphate
monoester signals in the region of 1.4–3.2 p.p.m., two
peaks of lower intensity were present at )5.5 and
)9.7 p.p.m. These last two signals derived from a
diphosphate monoester bond, i.e. a pyrophosphate group.
In particular, the signal at )5.5 p.p.m. could be identified
as the distal phosphate group, while the phosphate at
)9.7 p.p.m. was identified as the proximal phosphate
group. The
1
H,
31
P-HSQC spectrum showed correlation
between H-2 and H-4 of heptose U, with typical signals of
a phosphate monoester group. The H-6 V resonance,
present as two different signals, showed two different
cross-peaks, one with a phosphate monoester group at
4.46/3.2 p.p.m. and the other at 4.63/)9.7 p.p.m., with the
proximal phosphate of a diphosphate monoester residue.
Thus, heptose U is substituted at O-2 and O-4 by a
phosphate group, whereas heptose V carries at O-6 a
phosphate group or alternatively, a pyrophosphate group.

The MALDI-TOF mass spectrum (Fig. 8) of oligosac-
charide 2 confirmed all the assignments, as all ion peaks
corresponding to the structures above were present. In fact,
ion peaks characteristic of an oligosaccharide were found,
composed of two HexNAc, one Hex, two Hep, one Kdo,
one carbamoyl group and two, three, four and five
phosphate groups. Moreover, additional peaks at Dm/z
146 accounted for the presence of a second core glycoform,
which differs from the most abundant one by an additional
rhamnose residue that must be linked at O-6 of glucose. Ion
peaks derived from the loss of water from molecular ions,
probably Kdo lactone or anhydro-Kdo forms, were also
present. Furthermore, the MALDI-TOF mass spectrum
also accounted for the presence of a very small amount of
pentaphosphorylated species, which was not detected by
NMR. Because no different phosphate substitution was
visible in 1D and 2D
31
P-NMR, we propose that the fifth
phosphate group is present as pyrophosphate on heptose U.
In conclusion, information derived from both acid and
alkaline hydrolysis leads to the proposal of the following
structure of the major oligosaccharide from the LOS of
P. stutzeri OX1.
Fig. 6.
1
H-NMR spectrum of oligosaccharide 2 obtained by acetic acid hydrolysis. The spectrum was recorded under the following conditions: 5 mg
of oligosaccharide 2 in 0.6 mL D
2
O, pD 7. Monosaccharides are as shown; rhamnose residue anomeric signal is not labeled as it belongs to the

minor oligosaccharide. Dotted bonds indicate a nonstoichiometric linkage. Chemical shifts are shown in Table 2.
Ó FEBS 2004 LPS from Pseudomonas stutzeri OX1 (Eur. J. Biochem. 271) 2699
Table 2.
1
H,
13
Cand
31
P NMR chemical shifts (p.p.m.) of the oligosaccharide product deriving from acetic acid treatment of the LOS from P. stutzeri
OX1. O-6 V resonances are given in parentheses when this position is monophosphorylated. P/P refers to both resonances of pyrophosphate.
Because Kdo signals are spread due to its multiple forms, resonances are not given. Resonances of the minor fragment Rha-C1fi6)-Glc are also
shown at the bottom. Chemical shifts are relative to acetone and external aq. 85% (v/v) phosphoric acid (
1
H, 2.225 p.p.m.;
13
C, 31.45 p.p.m.;
31
P, 0.00 p.p.m. at 30 °C).
Residue 1 2 3456 7
1
H/
13
C/
31
P
U 5.23 4.56 4.21 4.68 4.23 4.07 3.77
Hep 99.2 73.8 76.8 73.7 74.5 70.9 64.1
1.4 1.8
V 5.14 4.39 4.02 4.11 4.15 4.63 (4.46) 4.59/3.96
Hep 102.7 69.17 78.1 67.9 71.6 71.2 (70.2)

)9.7/)5.5 (3.2)
62.4
X 5.14 4.26 4.12 4.39 4.12 3.72
GalN 99.8 49.8 77.3 75.0 73.5 60.1
W 4.91 3.67 3.67 3.61 3.41 3.612/3.88
GlcN 101.2 56.1 72.6 73.6 70.0 61.2
Z 4.53 3.29 3.49 3.48 3.40 3.74/3.89
Glc 104.4 73.5 75.6 70.1 76.9 60.7
Ac 2.0–2.1
174.6–175.0 22.3
Cm 160.0
4.53 3.30 3.52 3.79 3.80 3.93
Glc 104.4 73.5 76.5 70.9 75.1 69.7
4.86 3.96 3.73 3.42 4.01 1.26
Rha 101.7 70.5 70.2 72.9 69.2 17.0
Fig. 7. Section of the
1
H,
31
P-HSQC spectrum of oligosaccharide 2. The spectrum shows cross peaks relevant for the localization of the phosphate
groups.
2700 S. Leone et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Discussion
Several core oligosaccharides from Pseudomonas strains
have already been isolated and characterized [12,13], mainly
from Pseudomonas aeruginosa strains [21,37–43]. The carbo-
hydrate backbone of the so-called inner core region of
Pseudomonas LPS determined so far has always been found
to be identical in different strains [12,13,21,37–43], which
indicates a strict biosynthetic control. It contains two residues

of 3-deoxy-
D
-manno-oct-2-ulosonic acid (Kdo), two residues
of
L
-glycero-
D
-manno-heptose (Hep), one O-carbamoyl
group (Cm), which links the O-7 of a Hep residue, and a
2-amino-2-deoxy galactose (GalN), which is the branching
point of the oligosaccharide. The amino function of the GalN
residue is frequently acylated with alanine, but in a few cases
an acetyl group has been found [38]. The inner core region is
always characterized by the presence of a large number of
negative charges, usually carried by phosphate groups, which
are linked to heptose residues, in addition to the key
phosphate residues attached to the lipid A backbone. The
outer core is more variable than the inner part, usually
resulting in two outer core glycoforms. However, the
architecture of the outer core is common, with a GalN
residue linked by two glucose moieties, one of which could
carry a rhamnose residue, the key residue substituent for the
O-polysaccharide transfer [40–42].
The structure of the inner core region of the LOS from
P. stutzeri OX1, as determined in this study, was found to
agree closely with the general structure described above. It
includes the characteristic monosaccharide residues, a large
number of phosphate groups at the proper location, a
carbamoyl moiety, and an acetyl group.
It is worth noting that the nature and the localization of

phosphate substituents on heptose residues in core oligo-
saccharides of Pseudomonas have recently been resolved,
mainly by MS techniques following alkaline and acid
degradations [37–43]. Also in this case, the comparative
analysis by NMR and MS of products obtained by either
Fig. 8. Negative ion MALDI-TOF-MS spectrum of oligosaccharide 2 recorded in reflector mode. Assignments of main ion peaks are shown. Dm/z 18
is due to Kdo present in reducing or lactone form. P, phosphate; Cm,7-O-carbamoyl; Ac, acetyl.
Ó FEBS 2004 LPS from Pseudomonas stutzeri OX1 (Eur. J. Biochem. 271) 2701
alkaline and acid degradation allowed the complete iden-
tification and localization of the labile groups, i.e. pyro-
phosphate groups, which are commonly lost in alkaline
treatment.
As for the outer core structure of the LOS from P. stutzeri
OX1, the structure we have determined was found to be of
special interest, both for its novelty with respect to the
structures already determined [12,13,21,37–43], and for its
biosynthetic implications. A GalNAc residue substituted by
two gluco-configured residues, at O-3 by glucose and at O-4
by GlcNAc, was found. To our knowledge, a GlcNAc
residue directly linked to GalN has never been found in the
core region structure of Pseudomonas. Moreover, both of
the two gluco-configured residues are blocked at position
O-4andO-6byapyruvateketallinkage.
The presence of pyruvate residues in the core region of
lipopolysaccharides is also new. Although pyruvate resi-
dues are frequently found in bacterial exopolysaccharides
[44,45], and in O-polysaccharides [46] as a postpolymer-
ization decoration, they have never been found as core
constituents.
In our opinion, this finding could be relevant to the

understanding of adaptive chemical alterations of the
outer membrane of P. stutzeri OX1 as a consequence of
its exposure to solvents, which has already been docu-
mented in other cases [1,8,9]. Most of the modifications
described essentially involve phospholipid head groups
and fatty acid composition, whereas little is known about
LPS alterations. On the basis of this novel structure, we
can advance the hypothesis that the presence of pyruvate
residues in lipopolysaccharides is a new, structurally
mediated, biochemical response to a harsh and odd
environment, such as that in which P. stutzeri OX1 was
selected.
Our hypothesis is based on the consideration that
insertion of two bulky pyruvate groups at the end of the
oligosaccharide chain blocks its elongation, by creating a
structural hindrance to the action of glycosyl transferases.
This leads to a shorter LOS, and hence to a less hydrophilic
cellular surface. Thus, the presence of pyruvate moieties
might represent a chemical camouflage of the glycosyl
transferase substrate, obtained by linking a key molecule
from the primary metabolism in a simple and mild chemical
bond. This chemical protection is very similar to the
isopropylidene group, one of the most widespread protect-
ing groups in coupling reactions of oligosaccharide synthe-
ses. It should be added that this hypothesis is also supported
by the finding that an alternative minor glycoform of the
LOS has been found, with the typical oligosaccharide
structure of a potential substrate for chain elongation and
O-polysaccharide attachment [40–42]. In this latter chain a
rhamnose residue is present at O-6 of the glucose residue

substituting the pyruvate moiety. This finding would
confirm that pyruvate residues are used to block chain
elongationintheLOSofP. stutzeri OX1.
Thus, the structure of the R-type LPS (LOS) that we have
found in Pseudomonas stutzeri OX1 would represent an
adaptive response of the microorganism to a hydrocarbon-
containing environment, because the presence of a long
hydrophilic O-polysaccharide chain could have hindered its
suitability to an external medium characterized by the
presence of aromatic hydrocarbons.
Moreover, the peculiar presence of bulky pyruvate
residues as blocking groups might offer an additional
advantage to P. stutzeri OX1, as these residues could help
prevent the massive entrance of external organic com-
pounds, which could be detrimental to its catabolism. In
fact, their presence increases the total negative charges of
the LOS, thus altering the physical properties of the
external membrane. It is already known [1,8,9,47,48] that
polyanionic LOS molecules electrostatically bind divalent
cations; thus, an increased capability to bind cations
might favor better packing of membrane molecules and
constitute a selective barrier to the entrance of organic
molecules.
Acknowledgements
This work was supported by grants (to A.D.D. and M.P.) from the
Ministry of University and Research (PRIN/2000 and PRIN/2002).
A.M. thanks Hermann Moll (Research Center Borstel) for carbamoyl
and methylation analyses.
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