Structural studies on the core and the O-polysaccharide repeating
unit of
Pseudomonas aeruginosa
immunotype 1 lipopolysaccharide
Olga V. Bystrova
1
, Aleksander S. Shashkov
1
, Nina A. Kocharova
1
, Yuriy A Knirel
1
, Buko Lindner
2
,
Ulrich Za¨ hringer
2
and Gerald B. Pier
3
1
N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia;
2
Research Center Borstel,
Center for Medicine and Biosciences, Borstel, Germany;
3
Channing Laboratory, Department of Medicine,
Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA
The structure of the lipopolysaccharide (LPS) of Pseudo-
monas aeruginosa immunotype 1 was studied after mild acid
and strong alkaline degradations by MS and NMR spec-
troscopy. Three types of LPS molecules were found, inclu-

region at unknown position.
The site and the configuration of the linkage between the
O-polysaccharide and the core and the structure of the O-
polysaccharide repeating unit were defined in P. aeruginosa
immunotype 1. The QuiNAc residue linked to the Rha
residue of the core was found to have the b configuration,
whereas in the interior repeating units of the O-polysac-
charide this residue is in the a-configuration. The data
obtained are in accordance with the initiation of biosynthesis
of the O-polysaccharide of P. aeruginosa O6, which is closely
related to immunotype 1, by transfer of
D
-QuiNAc-1-P to
undecaprenyl phosphate followed by synthesis of the
repeating O-antigen tetrasaccharide.
Keywords: lipopolysaccharide; core oligosaccharide struc-
ture; repeating unit; O-polysaccharide; Pseudomonas
aeruginosa.
Correspondence to Y. A. Knirel, N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 47, 119991
Moscow, GSP-1, Russia. Fax: + 7095 1355328. E-mail: [email protected]
Abbreviations: aKdo, anhydro form of 3-deoxy-
D
-manno-octulosonic acid; Cm, carbamoyl; FT-ICR, Fourier transform ion cyclotron resonance;
Fo, formyl; Kdo, 3-deoxy-
D
-manno-oct-2-ulosonic acid; LPS, lipopolysaccharide; OS, oligosaccharide; Hep,
L
-glycero-
D
-manno-heptose; HexN,

biological repeating unit was defined only in P. aeruginosa
serogroup O5 [4]. Structures of the core [5–7] and lipid A
[8,9] of P. aeruginosa LPS have also been investigated. The
inner core region is composed of two residues of 3-deoxy-
D
-
manno-oct-2-ulosonic acid (Kdo) and two residues of
L
-glycero-
D
-manno-heptose (Hep), one of which is specific-
ally 7-O-carbamoylated. The inner core region is character-
ized by a high degree of phosphorylation but data on the
location of the phosphate groups are contradictory [5–7].
The outer core region contains up to four
D
-glucose
residues, one
L
-rhamnose residue, and one residue of
N-(
L
-alanyl)- or N-acetyl-
D
-galactosamine; it may include
also O-acetyl groups. Recently, it has been reported that
strain P. aeruginosa PAO1 and a cystic fibrosis isolate
P. aeruginosa 2192 produce two different glycoforms of the
LPS outer core [4,5].
Strains belonging to P. aeruginosa immunotype 1 (sero-

tant was dialyzed against distilled water, nucleic acids were
precipitated using Cetavlon [13] and removed by centrifu-
gation (5000 g, 90 min). The supernatant was dialyzed
against distilled water and lyophilized.
Mild acid degradation of the lipopolysaccharide
LPS (200 mg) was dissolved in 0.1
M
sodium acetate buffer
pH 4.2 and heated for 13 h at 100 °C. The precipitate was
removed by centrifugation (12 000 g, 10 min), the superna-
tant fractionated by gel-permeation chromatography on a
column (80 · 2.5 cm) of Sephadex G-50 (Pharmacia-
Upjohn, Uppsala, Sweden) in pyridinium acetate buffer
pH 4.5 (4 mL pyridine and 10 mL HOAc in 1 L water) at
30 mLÆh
)1
, monitoring with a Knauer differential refrac-
tometer, and 5-mL fraction volume. Fractions 24–34 were
pooled to give a polysaccharide and fractions 41–54 an
oligosaccharide mixture (19.8 and 9.1% of the LPS mass,
respectively), the latter containing the core and the core with
one O-antigen repeating unit attached.
Alkaline degradation of the lipopolysaccharide
LPS (200 mg) was treated with anhydrous hydrazine
(4 mL) for 1 h at 37 °C, then 16 h at 20 °C, hydrazine
was flushed out in a stream of air at 30–33 °C, the residue
washed with cold acetone and dried in vacuum. The
O-deacylated LPS was dissolved in 4
M
NaOH (8 mL), the

2-propanol, water, and triethylamine at a concentration of
 20 ngÆlL
)1
and sprayed with a flow rate of 2 lLÆmin
)1
.
NMR spectroscopy
The NMR spectra were obtained on a Bruker DRX-500
spectrometer at 30 °C in 99.96% D
2
O. Prior to the
measurements, the samples were lyophilized twice from
D
2
O. Chemical shifts are referenced to internal acetone (d
H
2.225, d
C
31.45) or external aqueous 85% H
3
PO
4
(d
P
0.0).
Bruker software
XWINNMR
1.2 was used to acquire and
process the data. A mixing time of 200 or 100 ms was used
in 2D TOCSY and ROESY experiments, respectively.

Ac
0)1
with a Kdo
residue in an anhydro form [14] and a variable number of
phosphate (P
0-2
) and O-acetyl (Ac
0)1
) groups (Fig. 1A).
The major ion peak at m/z 1590.41 corresponded to the
monophosphorylated non-O-acetylated derivative (the cal-
culated molecular mass 1591.48 Da). A similar series was
observed in the ESI mass spectrum of the core oligosac-
charide from the rough (R)-type LPS of P. aeruginosa 2192
[5]. In addition, a less intense series of [M-H]
–
pseudo-
molecular ions was present for the core with one O-poly-
saccharide repeating unit attached (Fig. 1B). Again,
heterogeneity of the oligosaccharides was associated with
nonstoichiometric phosphorylation (P
0-2
) and O-acetyla-
tion (Ac
0)2
) as well as with incomplete amidation of
GalNAcA or/and GalNAcFo residues resulting in a mass
difference of 1 or 2 Da. The major ion peak at m/z
2383.62 corresponded to the monophosphorylated bisam-
idated derivative containing one O-acetyl group (mostly

N-deacylated by strong alkaline hydrolysis [15]. The alkaline
degradation was accompanied by depolymerization of the
O-polysaccharide by b-elimination in 4-substituted GalNA
residues, which were converted into the corresponding hex-
4-enuronic acid (DHexNA) (Fig. 2). The negative ion mode
ESI FT-ICR mass spectrum of the product (Fig. 3) showed
the presence of the core-lipid A backbone oligosaccharide
6dHexHex
3
(HexN)
3
Hep
2
Kdo
2
P
5
and that with a DHexNA-
QuiN disaccharide remainder of the repeating unit of the O-
polysaccharide attached (the determined and calculated
molecular masses 2357.54 and 2357.51 Da for the former
and 2659.64 and 2659.61 Da for the latter compound,
respectively). In addition to the major pentakisphosphoryl-
ated compounds (P
5
), there were minor compounds con-
taining six (P
6
) and four (P
4

H- and
13
C-NMR spectra of the alkaline degrada-
tion product were assigned using 2D shift-correlated NMR
experiments (COSY, TOCSY, ROESY, and H-detected
1
H,
13
C HMQC) (Tables 1 and 2). The monosaccharide spin
systems were assigned based on the coupling constant values
and those for amino sugars by correlation of the protons at
the nitrogen-bearing carbons to the corresponding carbons.
The configurations of the glycosidic linkages of the gluco
and galacto sugar residues (Glc, GlcN, GalN, QuiN, and
DHexNA) followed from the J
1,2
coupling constant values
and those of Rha, Hep
I
,Hep
II
,Kdo
I
,andKdo
II
from
typical
1
H NMR chemical shifts (compare published data
[7,16]). Two series of NMR signals were present for most

degradation products of the LPS. Shown are
regions of [M-H]
–
pseudomolecular ions for
the core oligosaccharide [M, 6dHex-
Hex
3
(HexNAla)Hep(HepCm)aKdo] (A),
the core oligosaccharide with one O-polysac-
charide repeating unit [M
I
,6dHexNAc
(HexNAcAN)(HexNFoAN)(6dHex)
2
Hex
3
(HexNAla)Hep(HepCm) aKdo] (B), and
[M-3H]
3–
pseudomolecular ions and fragment
ions from the reducing end (C), which repre-
sent all essentials ion peaks found in the
complete spectrum, except for peaks for
doubly charged pseudomolecular ions. An
explanation of the fragments is shown at the
top of the region C. M
P1
,M
P1Ac1
, etc., refer to

the H1 signals of GlcN
I
,H4ofGlcN
II
,H2andH4of
Hep
I
, and H6 of Hep
II
at d )1.80/5.74, 0.18/3.85, )0.1/
4.53, 1.30/4.50, and 1.33/4.54, respectively. The assignment
of the Hep
I
P2/H2 and Hep
II
P6/H6 cross-peaks was
confirmed by Hep
I
P2/H3 and Hep
II
P6/H5 four-bond
correlations.
Therefore, the alkaline degradation products have struc-
tures shown in Fig. 5. Taking all of the data together, the
structures of the core and core with one O-polysaccharide
repeating unit in the LPS of P. aeruginosa immunotype 1
were established (Fig. 7).
DISCUSSION
Previously, the structure of the O-polysaccharide of
P. aeruginosa immunotype 1 LPS was elucidated [3,10–

Hep
2
Kdo
2
P
5
, respectively.
Fig. 2. Alkaline degradation of the LPS. The
glycosidic linkage of QuiNAc is a between the
O-polysaccharide repeating units and b
between the O-polysaccharide and the core.
2198 O. V. Bystrova et al. (Eur. J. Biochem. 269) Ó FEBS 2002
approaches. One was mild acid degradation, which,
together with a long-chain polysaccharide, resulted in an
oligosaccharide mixture containing a core oligosaccharide
and that with one O-polysaccharide repeating unit at-
tached. The other was strong alkaline degradation, which
caused b-elimination in GalNA residues present in the O-
polysaccharide to give a core oligosaccharide with a
truncated single O-polysaccharide repeating unit from the
LPS species with both long-chain O-polysaccharide and
one repeating unit.
Analysis of the products with one O-polysaccharide
repeating unit or its remainder by ESI MS and NMR
spectroscopy enabled determination of not only the biolo-
gical repeating unit but also of the mode and the site of the
linkage between the O-polysaccharide and the core. It was
found that the QuiNAc residue, which occupies the
reducing end of the biological repeating unit, has the b con-
figuration when linking the O-polysaccharide to the core but

Inner core-lipid A backbone
fi 6)-a-
D
-GlcpN
I
-1-P 5.74 3.47 3.92 3.63 4.11 4.28 3.82
fi 6)-b-
D
-GlcpN
II
-(1 fi 4.84 3.14 3.89 3.85 3.76 3.73 3.51
fi 4,5)-a-Kdop
I
-(2 fi 2.07 2.24 4.11 4.29 3.73 3.85 3.89 3.60
a-Kdop
II
-(2 fi 1.85 2.09 4.13 4.08 3.73 4.11 3.96 3.69
fi 3)-a-Hepp
I
2,4P-(1 fi 5.36 4.53 4.18 4.50 4.28 3.98 3.94 3.80
fi 3)-a-Hepp
II
6P-(1 fi 5.14 4.41 4.22 3.84 4.01 4.54 3.80 3.73
Outer core, glycoform 1
fi 3,4)-a-
D
-GalpN-(1 fi 5.58 3.84 4.45 4.40 4.20 3.88 3.88
fi 6)-b-
D
-Glcp

D
-Glcp
III
-(1 fi 5.00 3.56 3.70 3.40 3.67 3.86 3.73
fi 3)-a-
L
-Rhap-(1 fi 5.16 4.25 4.01 3.65 4.03 1.26
Remainder of the O-polysaccharide repeating unit
fi 3)-b-
D
-QuipN-(1 fi 5.06 3.32 4.20 3.51 3.62 1.34
b-
L
-DHexpNA-(1 fi 5.65 3.73 4.52 5.98
Ó FEBS 2002 LPS of Pseudomonas aeruginosa immunotype 1 (Eur. J. Biochem. 269) 2199
accordance with the biosynthesis pathway of O-polysac-
charides, which involves multiple enzymes that mediate the
formation of the QuiNAc glycosidic linkage. One of them,
glycosyltransferase WbpL, transfers
D
-QuiNAc-1-P from
UDP-
D
-QuiNAc to Und-P to initiate the O-polysaccharide
repeating unit biosynthesis in P. aeruginosa O6 [18], which is
closely related to P. aeruginosa immunotype 1. Another
enzyme, O-antigen polymerase Wzy, mediates polymeriza-
tion of the preassembled oligosaccharide attached to Und-
PP to form a long-chain O-polysaccharide. Finally, ligase
WaaL ligates the preassembled oligosaccharide or the long-

C-NMR data (d, p.p.m.).
Sugar residue C1 C2 C3 C4 C5 C6 C7 C8
Inner core-lipid A backbone
fi 6)-a-
D
-GlcpN
I
-1-P 92.9 55.2 70.5 70.7 73.8 70.5
fi 6)-b-
D
-GlcpN
II
-(1 fi 100.2 56.7 72.8 75.5 75.0 63.7
fi 4,5)-a-Kdop
I
-(2 fi 174.2
a
100.1 35.3 72.2 69.5 73.1 70.3 64.9
a-Kdop
II
-(2 fi 174.2
a
102.4 36.0 66.8 67.7 73.2 70.3 64.3
fi 3)-a-Hepp
I
2,4P-(1 fi 98.6 75.6 75.3 74.2 73.3 71.8 64.4
fi 3)-a-Hepp
II
6P-(1 fi 103.2 70.5 78.9 67.7 72.6 71.8 62.7
Outer core, glycoform 1

a-
D
-Glcp
II
-(1 fi 100.5 73.0 73.8 70.3 72.6 61.5
a-
D
-Glcp
III
-(1 fi 99.3 72.4 74.4 70.8 73.5 62.1
fi 3)-a-
L
-Rhap-(1 fi 101.7 71.7 80.7 72.3 70.1 17.6
Remainder of the O-polysaccharide repeating unit
fi 3)-b-
D
-QuipN-(1 fi 101.1 55.9 80.1 76.1 73.5 17.7
b-
L
-DHexpNA-(1 fi 97.4 53.3 63.3 107.3 147.1 174.7
a
a
Assignment could be interchanged.
Fig. 5. Structures of the major alkaline degra-
dation products of the LPS. Glycoform 1 core
is unsubstituted (A) and glycoform 2 core
substitued with a remainder of the O-poly-
saccharide repeating unit (B). All sugars are in
the pyranose form and have the
D

the core oligosaccharide is the site of the attachment of the
O-polysaccharide. This residue only occurs in the core
glycoform 2, whereas the terminal 1 fi 6-linked Rha
residue in the other, isomeric glycoform 1 cannot accept
the O-polysaccharide. No unsubstituted core glycoform 2
was detected, nor a core glycoform with two Rha residues.
It could be thus suggested that the attachment of the 1 fi
6-linked Rha blocks the attachment of the 1 fi 3-linked
Rha, which is the acceptor of the O-polysaccharide. A
competition of the corresponding rhamnosyl transferases
may provide a mechanism for regulation of the content of
long-chain (S-type) and short-chain (R-type) LPS species on
the cell surface by an enhanced synthesis of the appropriate
glycoform. Like the O-polysaccharide chain length, the
content of the LPS species containing the core with one
O-polysaccharide repeating unit attached (SR-type LPS) is
controlled by the O-antigen chain length regulator Wzz [18],
which influences the functioning of O-antigen polymerase
and ligase by a mechanism that is not clearly understood.
As in P. aeruginosa strains studied previously [5–7,19,20],
the core of the LPS of P. aeruginosa immunotype 1 is
distinguished by a high degree of phosphorylation. Three
major phosphorylation sites were determined in the core,
two of which are at positions 2 and 4 of Hep
I
and one at
position 6 of Hep
II
. This finding is in agreement with the
phosphorylation pattern in P. aeruginosa strains H4 [6] and

1
H,
31
P HMQC spectrum of alka-
line degradation products. Three-bond and
four-bond correlations are shown by positive
and negative levels, respectively. Other four-
bondcorrelationsweretooweaktobedis-
tinguished from noise signals.
Ó FEBS 2002 LPS of Pseudomonas aeruginosa immunotype 1 (Eur. J. Biochem. 269) 2201
which produces an R-type LPS [5]. The outer core of this
strain has at least four O-acetylation sites, and the major
LPS species is mono-O-acetylated. A similar O-acetylation
pattern with up to five O-acetylation sites has been found in
thecoreofP. aeruginosa immunotype 5, O3a,3b,3c, and
O12 (O. V. Bystrova, A. S. Shashkov, N. A. Kocharova,
Y. A. Knirel, B. Lindner, U. Za
¨
hringer & G. B. Pier,
unpublished data). In immunotype 1, the outer core has at
least one O-acetylation site and the O-acetyl group is present
in the core of a minority of the LPS molecules. The position
of the O-acetyl groups in the core of P. aeruginosa LPS, as
well as their biological significance, remains to be deter-
mined.
ACKNOWLEDGEMENTS
This work was supported by the Civilian Research and Development
Foundation (CRDF, USA) grant RB1-2042 (to Y. A. K. and
G. B. P.), the Sonderforschungsbereich (SFB, Germany) 470 (project
B4) (to U. Z.), the Deutsche Forschungsgemeinschaft grant LI-448/1-1

tetrasaccharide trisphosphate from the lipopolysaccharide of
Pseudomonas aeruginosa mutant H4. Eur. J. Biochem. 261,500–
508.
7. Sadovskaya, I., Brisson, J R., Lam, J.S., Richards, J.C. &
Altman, E. (1998) Structural elucidation of the lipopolysaccharide
core regions of the wild-type strain PAO1 and O-chain-deficient
mutant strains AK1401 and AK1012 from Pseudomonas aerugi-
nosa serotype O5. Eur. J. Biochem. 255, 673–684.
8. Bhat, U.R., Marx, A., Galanos, C. & Conrad, R.S. (1990)
Structural studies of lipid A from Pseudomonas aeruginosa PAO1:
occurrence of 4-amino-4-deoxyarabinose. J. Bacteriol. 172, 6631–
6636.
9. Kulshin, V.A., Za
¨
hringer, U., Lindner, B., Ja
¨
ger, K.E., Dmitriev,
B.A. & Rietschel, E.T. (1991) Structural characterization of the
lipid A component of Pseudomonas aeruginosa wild-type and
rough mutant lipopolysaccharides. Eur. J. Biochem. 198, 697–704.
10. Vinogradov, E.V., Knirel, Y.A., Shashkov, A.S. & Kochetkov,
N.K. (1987) Determination of the degree of amidation of 2-deoxy-
2-formamido-
D
-galacturonic acid in O-specific polysaccharides of
Pseudomonas aeruginosa O4 and related strains. Carbohydr. Res.
170, C1–C4.
11. Knirel, Y.A., Shashkov, A.S., Dmitriev, B.A. & Kochetkov, N.K.
(1984) Structural studies of the Pseudomonas aeruginosa
immunotype 1 antigen, containing the new sugar constituents:

15. Holst, O. (2000) Deacylation of lipopolysaccharides and isolation
of oligosaccharide phosphates. In Bacterial Toxins. Methods and
Protocols (Holst, O., ed.), pp. 345–353. Humana Press, Totowa,
New Jersey.
16. Knirel, Y.A., Grosskurth, H., Helbig, J.H. & Za
¨
hringer, U. (1995)
Structures of decasaccharide and tridecasaccharide tetraphos-
phates isolated by strong alkaline degradation of O-deacylated
lipopolysaccharide of Pseudomonas fluorescens strain ATCC
49271. Carbohydr. Res. 279, 215–226.
17. Bock, K. & Pedersen, C. (1983) Carbon-13 nuclear magnetic
resonance spectroscopy of monosaccharides. Adv. Carbohydr.
Chem. Biochem. 41, 27–66.
18. Rocchetta, H.L., Burrows, L.L. & Lam, J.S. (1999) Genetics of
O-antigen biosynthesis in Pseudomonas aeruginosa. Microbiol.
Mol. Biol. Rev. 63, 523–553.
19. Masoud, H., Altman, E., Richards, J.C. & Lam, J.S. (1994)
General strategy for structural analysis of the oligosaccharide
region of lipooligosaccharides. Structure of the oligosaccharide
component of Pseudomonas aeruginosa IATS serotype O6
mutant R5 rough-type lipopolysaccharide. Biochemistry 33,
10568–10578.
20. Masoud, H., Sadovskaya, I., De Kievit, T., Altman, E., Richards,
J.C. & Lam, J.S. (1995) Structural elucidation of the lipopoly-
saccharide core region of the O-chain-deficient mutant strain A28
from Pseudomonas aeruginosa serotype 06 (International Anti-
genic Typing Scheme). J. Bacteriol. 177, 6718–6726.
21. Beckmann, F., Moll, H., Ja
¨