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
`

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-

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].

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

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

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

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

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-

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

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

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

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

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

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

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

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

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

)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

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

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.
References
1. Ramos, J.L., Duque, E., Gallegos, M.T., Godoy, P., Ramos-

8. Ramos, J.L., Duque, E., Rodrı
`
guez-Herva, J.J., Godoy, P., Hai-
dour, A., Reyes, F. & Fernandez- Barrero, A. (1997) Mechanisms
for solvent tolerance in bacteria. J. Biol. Chem. 272, 3887–3890.
9. Ramos, J.L., Gallegos, M.T., Marque
´
s, S., Ramos-Gonza
`
lez,
M.I., Espinosa-Urgel, M. & Segura, A. (2001) Responses of
Gram-negative bacteria to certain environmental stressors. Curr.
Opin. Microbiol. 4, 166–171.
10. Alexander, C. & Rietschel, E.Th. (2001) Bacterial lipopoly-
saccharides and innate immunity. J. Endotoxin Res. 7, 167–202.
11. Mamat, U., Seydel, U., Grimmecke, D., Holst, O. & Rietschel,
E.Th. (1998) Comprehensive Natural Products Chemistry (Barton,
D., Nakanishi, K., Meth-Cohn, O. & Pinto, B.M., eds), Vol. 3,
pp. 179–239. Elsevier, Oxford.
2702 S. Leone et al.(Eur. J. Biochem. 271) Ó FEBS 2004
12. Holst, O. (1999) Endotoxin in Health and Disease (Brade, H.,
Morrsion, D.C., Opal, S. & Vogel, S., eds), pp. 115–154. Marcel
Dekker Inc, New York.
13. Holst, O. (2002) Chemical structure of the core region of lipo-
polysaccharides - an update. Trends Glycosci. Glyctechnol. 14,87–
103.
14. Galanos, C., Lu
¨
deritz, O. & Westphal, O. (1969) A new method
for the extraction of R. lipopolysaccharides. Eur J. Biochem. 9,

703–710.
20. Su
¨
sskind, M., Brade, L., Brade, H. & Holst, O. (1998) Identifi-
cation of a novel heptoglycan of a1fi2-linked
D
-glycero-
D
-manno-
heptopyranose. Chemical and antigenic structure of lipopolysac-
charides from Klebsiella pneumoniae ssp. pneumoniae rough strain
R20 (O1
)
:K20
)
). J. Biol. Chem. 273, 7006–7017.
21. Beckmann, F., Moll, H., Jager, K E. & Za
¨
hringer, U. (1995)
Preliminary communication 7-O-carbamoyl-
L
-glycero-
D
-manno-
heptose: a new core constituent in the lipopolysaccharide of
Pseudomonas aeruginosa. Carbohydr. Res. 267, C3–C7.
22. Ciucanu, I. & Kerek, F. (1984) A simple and rapid method for the
permethylation of carbohydrates. Carbohydr. Res. 131, 209–217.
23. Hakomori, S. (1964) A rapid permethylation of glycolipid, and
polysaccharide catalyzed by methylsulfinyl carbanion in dimethyl

S., eds), pp. 93–114. Marcel Dekker Inc, New York.
31. Birnbaum, G.I., Roy, R., Brisson, J.R. & Jennings, H. (1987)
Conformations of ammonium 3-deoxy-
D
-manno-2-octulosonate
(Kdo) and a-andb-ketopyranosides of Kdo: X-ray structure and
1
H NMR analyses. J. Carbohydr. Chem. 6, 17–39.
32. Holst, O., Thomas-Oates, J.E. & Brade, H. (1994) Preparation
and structural analysis of oligosaccharide monophosphates
obtained from the lipopolysaccharide of recombinant strains of
Salmonella minnesota and Escherichia coli expressing the genus-
specific epitope of Chlamydia lipopolysaccharide. Eur. J. Biochem.
222, 183–194.
33. Lipkind, G.M., Shashkov, A.S., Knirel, Y.A., Vinogradov, E.V. &
Kochetkov, N.K. (1988) A computer-assisted structural analysis
of regular polysaccharides on the basis of 13C-n.m.r. data.
Carbohydr. Res. 175, 59–75.
34. Holst, O., Bock, K., Brade, L. & Brade, H. (1995) The structures
of oligosaccharide bisphosphates isolated from the lipopoly-
saccharide of a recombinant Escherichia coli strain expressing the
gene gseA [3-deoxy-
D
-manno-octulopyranosonic acid (Kdo)
transferase] of Chlamydia psittaci 6BC.Eur. J. Biochem. 229,194–
200.
35. Bock, K., Vinogradov, E.V., Holst, O. & Brade, H. (1994) Isola-
tion and structural analysis of oligosaccharide phosphates con-
taining the complete carbohydrate chain of the lipopolysaccharide
from Vibrio cholerae strain H11 (non-O1). Eur. J.Biochem. 225,

269, 2194–2203.
41. Bystrova, O.V., Shashkov, A.S., Kocharova, N.A., Knirel, Y.A.,
Za
¨
hringer, U. & Pier, G. (2003) Elucidation of the structure of the
lipopolysaccharide core and the linkage between the core and the
O-antigen in Pseudomonas aeruginosa immunotype 5 using strong
alkaline degradation of the lipopolysaccharide. Biochemistry
(Moscow) 68, 918–925.
42. Bystrova, O.V., Lindner, B., Moll, H., Kocharova, N.A., Knirel,
Y.A., Za
¨
hringer, U. & Pier, G. (2003) Structure of the lipopoly-
saccharide of Pseudomonas aeruginosa O-12 with a randomly
O-acetylated core region. Carbohydr. Res. 338, 1895–1905.
43. Kooistra, O., Bedoux, G., Brecker, L., Lindner, B., SancheZ.
Carballo, P., Haras, D. & Za
¨
hringer, U. (2003) Structure of a
highly phosphorylated lipopolysaccharide core in the Delta algC
mutants derived from Pseudomonas aeruginosa wild-type strains
Ó FEBS 2004 LPS from Pseudomonas stutzeri OX1 (Eur. J. Biochem. 271) 2703
PAO1 (serogroup O5) and PAC1R (serogroup O3). Carbohydr.
Res. 338, 2667–2677.
44. Cescutti,P.,Toffanin,R.,Polesello,P.&Sutherland,I.W.(1999)
Structural determination of the acidic exopolysaccharide pro-
duced by a Pseudomonas sp. strain 1.15. Carbohydr. Res. 315, 159–
168.
45. Cescutti, P., Osman, S.F., Fett, W.P. & Weisleder, D. (1995) The
structure of the acidic exopolysaccharide produced by