Tài liệu Báo cáo khoa học: The structure of the carbohydrate backbone of the lipopolysaccharide from Acinetobacter baumannii strain ATCC 19606 docx

The structure of the carbohydrate backbone of the lipopolysaccharide
from
Acinetobacter baumannii
strain ATCC 19606
Evgeny V. Vinogradov
1,
*, Jens é. Duus
1
, Helmut Brade
2
and Otto Holst
2
1
Department of Chemistry, Carlsberg Laboratory, Valby, Copenhagen, Denmark;
2
Division of Medical and Biochemical Microbiology,
Research Center Borstel, Borstel, Germany
The c hemical structure of the phosphorylated carbohydrate
backbone of the lipopolysacchar ide ( LPS) from Acineto-
bacter baumannii strain ATCC 19606 was investigated by
chemical analysis and NMR spectroscopy of oligosaccha-
rides obtained a fter deacylation or mild acid hydrolysis.
From the c ombined i nformation the following carbohydrate
backbones c an be deduced:
-GalpNR
1
-GlcpN -Kdo
1 1 2

4 7 4
R

2
3)- -GlcpNAcA-(1 4)- -Kdo-(2 5)- -Kdo-(2 6)- -GlcpN4P-(1 6)- -GlcpN1P
where R
1
 HandR
2
 a-Glcp-(1 ® 2) -b-Glcp-(1 ® 4)-b-
Glcp-(1 ® 4)-b-Glcp-(1 as major and R
1
 Ac and R
2
 H
as minor products.
All monosaccharides are
D
-con®gured. Also, smaller oli-
gosaccharide phosphates were identi®ed that are thought to
represent d egradation products of the above structures.
Keywords: Acinetobacter baumannii; lipopolysaccharide;
core region; structural analysis; NMR spectroscopy.
The Gram-negative bacterium Acinetobacter (Moraxella-
ceae) i s isolated from soil and water w hich represent its
natural habitats. However, the genus has gained increasing
importance as a causative agent of nosocomial infections
(e.g. bacteremia, secondary meningitis) in recent years [1].
As in other G ram-negative bacteria, Acinetobacter pos-
sesses lipopolysaccharides (LPS) in t he outer membrane of
the cell wall t hat have been shown to represent useful
chemotaxonomic and a ntigenic markers for its identi®ca-
tion and differentiation. The o ccurrence of S-form L PS in

Acinetobacter has unequivocally b een proven recently, and
the s tructures of a number of O-speci®c polysaccharides
and t heir antigenic characterization have b een published
[2±9]. T he core region of Acinetobacter LPS possesses
particular structural features which clearly distinguish it
from other core structures [10]. First, it belongs to the
group of heptose-de®cient core regions. Second, it may
contain
D
-glycero-
D
-talo-oct-2-ulopyranosonic acid (Ko)
which can replace the 3-deoxy-
D
-manno-oct-2-ulopyrano-
sonic a cid (Kdo) residue linking the core region to lipid A
[11±15].
Another core type h as been identi®ed in A. baumannii
strain NCTC 10303 [16] that is devoid of Ko. It comprises
the tetrasaccharide a-Kdo-(2 ® 5)-[a-Kdo-(2 ® 4)-]-a-
Kdo-( 2 ® 5)-a-Kdo-(2 ® [a-Kdo IV-(a-Kdo III)-a-Kdo
II-a-Kdo I], of which Kdo IV is substituted at O-8 by a
short-chain rhamnan and Kdo III at O-4 by the disaccha-
ride a-
D
-GlcpNA c-(1 ® 4)-a-D-GlcpNA (GlcpNA, 2-ami-
no-2-deoxy-glucopyranosuronic acid). Kdo I links the core
region to the lipid A. This Kdo tetrasaccharide is unique in
nature; however, a second Kdo tetrasaccharide of different
structure has been identi®ed in LPS of Chlamydophila

psittaci 6BC [17]. The latter has been shown to be assembled
by one Kdo t ransferase, which is therefore multifunctional
[18]. However, the biosynthesis of th e Kdo-tetrasaccharide
from LPS of A. baumannii strain NCTC 10303 has not yet
been elucidated. For A. baumannii strain ATCC 15303,
which possesses i n its LPS c ore r egion the trisaccharide
a-Kdo- (2 ® 5)-[a-Kdo-(2 ® 4)-]-a-Kdo-(2 ® , it has been
shown that the Kdo t ransferase is able to transfer the ®rst
two a-(2 ® 4)-linked Kdo residues [19]. The mechanism of
the transfer of the third Kdo residue has not yet been
identi®ed.
In addition to the work on the determination of the
chemical and antigenic structures of O-speci®c polysaccha-
rides from LPS of Acinetobacter in order to establish an
O-serotyping scheme, t here exists considerable interest in
investigating the LPS core regions from this genus which
obviously allows novel insights in structure, genetics and
biosynthesis of LPS. Here, the structures of the carbohy-
drate backbones of the LPS from A. baumannii strain
ATCC 19606 are reported.
Correspondence to O. Holst, Research Center Borstel, Parkallee 22,
D-23845 Borstel, Germany. Fax: + 49 4537 188419,
Tel.: + 49 4537 188472, E-mail:
Abbreviations: LPS, lipopolysaccharide; GlcpNAcA, 2-acetamido-2-
deoxy-glucopyranosyluronic acid; DGlcpNA, 2 -amino-2,4-dideoxy-b-
L
-threo-hex-4-enopyranosyluronic acid; HMBC, heteronuclear mul-
tiple bond correlation; HMQC, heteronuclear multiple quantum co-
herence; HPAEC, high-performance anion-exchange
chromatography; Kdo, 3-deoxy-

D
-manno-oct-2-ulopyranosonic acid.
Dedication: this article is dedicated to Prof Dr Joachim Thiem,
Institute of Organic Chemistry, University of Hamburg, Germany on
the occasion of his 60th birthday.
*Present address: Institute for Biological Sciences, National Research
Council Canada, Ottawa, Ontario K1A 0R6, Canada.
(Received 23 July 2001, revised 29 October 2001, accepted 31 October
2001)
Eur. J. Biochem. 269, 422±430 (2002) Ó FEBS 2002
MATERIALS AND METHODS
Bacteria and Bacterial LPS
A. ba umannii strain ATCC 19606 was cultivated and the
LPS was obtained as described previously [11].
Preparation of oligosaccharides
The LPS (80 mg) was hydrolysed in 5% acetic acid (100 °C,
5 h ), the precipitate was removed by ultracentrifugation
(100 000 g, 4 h), and the supernatant was lyophilized (yield:
50 mg, 62.5% of the LPS). The latter sample was reduced
with NaBH
4
, and, after working up, was d esalted by
gel-permeation chromatography. High-performance anion-
exchange chromatography (HPAEC) of this f raction yield-
ed oligosaccharides 1 (4 mg, 5% of the LPS) and 2 (10 mg ,
12.5% of the LP S), and two stereoisomers of 1 and 2 in
minor amounts that differed b y the con®guration at C2 o f
Kdo-ol.
Another portion of the LPS (350 mg) was de-O-acylated
as described previously [20] (yield: 236 mg, 67.4% of the

LPS), and 150 mg of this de-O-acylated LPS was then
de-N-acylated [20], and, after working up, desalted by
gel-permeation chromatography. H PAEC of this sample
yielded oligosaccharides 3 and 4 (10 and 25 mg, 2.9 and
7.1% of the LPS, respectively).
General and analytical methods
Gel-permeation chromatography was carried out on a
column (3.5 ´ 40 cm) of TSK HW40 (S) gel (Merck) in
water, and runs were monitored with a differential
refractometer (Knauer). Preparative HPAEC was per-
formed on a c olumn (9 ´ 250 mm) of CarboPac PA1
(Dionex Corp.) as described previously [20] usi ng linear
gradient programs of 3 to 40% 1
M
sodium acetate in
0.1
M
NaOH over 80 min (for separation of oligosaccha-
rides 1 and 2, F ig. 1) and 30 t o 70% o ver 8 0 min (for
separation of oligosaccharides 3 and 4, F ig. 1). Samples
Fig. 1. Structures of oligosaccharides 1±5.
DGlcpNA, 2-amino-2,4-dideoxy-b-
L
-threo-
hex-4-enopyranosyluronic acid (the product of
the 4,5-b-eliminat ion of a-glucosaminuronic
acid).
Ó FEBS 2002 LPS from Acinetobacter baumannii (Eur. J. Biochem. 269) 423
Table 1 .
1

H NMR data o f oligosaccharides 1±4. Sp ectra were recorded o f solutions in
2
H
2
O relative to internal acetone (2.225 p.p.m.). Oligo,
oligosaccharide.
Chemical shift (in p.p.m.) and J
H,H
coupling constants (in Hz) of proton
123(ax)3eq 4 5 6(a) 6b
78a8b
Residue Oligo J
1,2
J
2,3
J
3,4
,J
3ax,4
J
3eq,4,
J
3ax,3eq
J
4,5
J
5,6
J
6a,6b
J

5,6b
A, a-GlcpN 3 5.62
a
3.38 3.85 3.62 4.10 3.72 4.24
3.5 12 1.9
4 5.63
b
3.39 3.86 3.63 4.12 3.73 4.26
3.5 10 10 9 9 12 1
B, b-GlcpN 3 4.83 3.03 3.82 3.74 3.72 3.38 3.65
4
4.85 3.03 3.83 3.75 3.73 3.39 3.68
8.5 10.2 10
C, a-Kdo
3 2.31
12.1
1.90
4
12.2
3.93 4.47 3.66 3.85 3.59 3.85
4 2.33
12.5
1.92
3.5
12
3.95
<1
4.48 3.67
9
c

3.86 3.60 3.86
D, a-Kdo
3 1.69
12.3
2.06
2.4
12.8
3.99
<1
3.95
<1
3.68 3.94 3.69 3.90
4 1.70
12.7
2.07
4.6
12.3
4.01
<1
3.96
<1
3.69
9
c
3.96 3.70 3.89
E, aKdo-ol/a-Kdo
1 4.06
4
d
1.93

6
e
2.08
6
3.99
6
f
4.10
6
3.73
4
6
c
v
3.87 3.69 3.83
2 4.16 2.00 2.17 4.02 4.17 3.78 3.89 3.76 3.88
3 2.29
12
2.90
2.7
12.0
4.84
<1
4.43
<1
4.27
9
c
4.12 3.91 3.91
4 2.30

12
2.91
4
12
4.87
<1
4.43
<1
4.29
9
d
4.13 3.92 3.92
F, a-GlcpAN 1
4.99 3.92 3.86 3.71 4.12
3.5 10 10 10
2
4.97 4.20 4.29 3.94 4.25
3.5 10 9 10
3
5.45 3.54 4.45 5.88
2 4.2 4.2
4
5.470 3.762 4.658 5.982
1.6 4.1 4.1
G, a-GalpN
g
1
5.36 4.09 3.82 3.93 3.90 3.63 3.70
4 10 2.5
2

5.74 3.26 3.87 3.96 3.92 3.68 3.68
3.8 9.7 2
Y, a-Glc 2 5.34 3.51 3.73 3.43 4.04 3.75 3.78
4
5.34 3.51 3.72 3.42 4.04 3.75 3.77
3.8 10 3.5 13
R, b-Glc 2
4.63 3.49 3.57 3.41 3.43 3.69 3.90
7.8 10
4
4.63 3.49 3.56 3.41 3.44 3.70 3.89
7.8 9.4
T, b-Glc 2 4.50 3.34 3.64 3.75 3.63 3.85 3.96
4
4.50 3.33 3.64 3.74 3.63 3.85 3.97
8.0 9.5 9 9 4 12
S, b-Glc 2 4.61 3.32 3.60 3.68 3.52 3.85 3.96
4 4.72 3.33 3.64 3.64 3.63 3.81 3.97
8.0 9.5 4 12
Z, b-GlcN 1 4.77 2.98 3.54 3.40 3.43 3.67 3.85
2
4.54 2.68 3.37 3.36 3.43 3.69 3.90
8.2 9.5 9.5
3
4.96 3.09 3.65 3.42 3.50 3.69 3.90
8.4 10 9 9.9 2.3 6.2
h
4 4.97 3.12 3.66 3.45 3.52 3.72 3.92
8.3 10.6
a

J
1,P
6.5 Hz;
b
J
1,P
6.6;
c
J
6,7;
d
J
2,3;
e
J
2,3¢
;
f
J
3¢,4
;
g
additional signal in oligosaccharide 1:CH
3
CO, 2.033 p.p.m;
h
J
5,6a
.
424 E. V. Vinogradov et al. (Eur. J. Biochem. 269) Ó FEBS 2002

were desalted using a Dowex 50 ´ 4(H
+
) cation exchanger
in water, and amino-group containing compounds were
then eluted with 5% aqueous ammonia. GLC-MS , mono-
saccharide analysis, and the determination of the absolute
con®guration of monosaccharides were performed as
described p reviously [14,15,21]. Conformational analysis
was carried out as described previously [16].
NMR spectroscopy
For structural assignments, 1D and 2D
1
Hand
13
CNMR
spectra were recorded on solutions of oligosaccharides 1±4
in
2
H
2
O(500lL) with a Bruker AMX-600 spectrometer at
27 °C using standard Bruker software. Chemical shifts are
given relative to internal acetone (CH
3
-, 2.225 p .p.m. for
1
H, 34.5 p.p.m. for
13
C). The assignment of the proton
chemical shifts was a chieved by c orrelation spectroscopy

(COSY), total correlation spectrosco py (TOCSY), and
nuclear Overhauser enhancement s pectroscopy (NOESY),
and the assignment of the carbon chemical shifts was
performed b y heteronuclear multiple quantum coherence
(HMQC) and heteronuclear multiple bond correlation
(HMBC) experiments. Broad band
1
H-decoupled
31
P NMR spect ra w ere r ecorded at 10 1.25 MHz with a
Bruker DRX 250 spectrometer at 2 7 °C, and chemical shifts
are given relative to an external reference of 85% phospho-
ric acid (0.0 p.p.m.).
Mass spectrometry
The electrospray mass spectra (ES-MS) were acquired in
the negative mode on a VG Quattro triple quadropole
mass spectrometer (VG Biotec h, Altrincham, Cheshire,
UK) with 50% aqueous CH
3
CN as the mobile phase at a
¯ow rate of 8 lLámin
)1
. Ten microlitres of a 20 l
M
aqueous solution of the samples were injected into the
electrospray source.
RESULTS AND DISCUSSION
Sugar analyses of the LPS
Monosaccharide a nalyses of the LPS f rom A. baumannii
strain ATCC 19606 revealed the presence of

D
-Glc,
D
-GlcN,
D
-GalN, and Kdo. The
D
-con®guration of Kdo was
determined on the basis of the optical rotation value
[a]
D
 +56 ° (C 1, in water) of Kdo methyl ester a-methyl
glycoside isolated after methanolysis of the LPS [14].
Table 2.
13
C NMR data of oligosaccharides 1±4. Spectra were r ecorde d of solutions in
2
H
2
O relative to internal acetone (34.5 p .p.m.). Oligo,
oligosaccharide.
Residue Oligo
Chemical shift of carbon (in p.p.m.)
12345678
A, a-GlcpN 3 91.7
a
55.4
b
70.4 70.3 73.4 70.7
4 91.6 55.4 70.4 70.3 73.4 70.4

B, b-GlcpN 3 100.2 56.4 72.7 75.2
c
74.6
d
63.2
4 100.4 56.4 72.8 75.1 74.7 63.2
C, a-Kdo 3 175.9 100.8 35.2 73.4 70.0 73.3 70.2 64.6
4 176.1 100.8 35.1 73.4 70.0 73.3 70.3 64.6
D, a-Kdo 3 176.0 103.4 35.1 66.7 67.7 72.8 71.8 63.7
4 176.0 103.4 35.2 66.7 67.6 72.8 71.6 63.7
E, Kdo-ol/a-Kdo 1 182.4 71.2 36.6 76.8 70.5 70.0 80.5 61.9
2 182.0 70.7 37.0 76.5 70.4 69.2 80.1 61.2
3 176.0 101.9 30.0 71.8 63.1 71.0 77.3 60.6
4 176.0 101.8 30.0 71.8 63.1 71.0 77.1 60.6
F, a-GlcpAN 1 97.0 55.3 73.4 77.2 74.4 177.3
2 97.6 54.0 77.7 74.0 74.2 175.0
3 91.8 53.4 63.3 105.8 147.7 169.0
4 92.2 51.3 71.6 104.4 148.4 168.7
G, a-GalN 1 98.3 50.9 68.7 69.4 71.8 61.9
2 96.7 51.5 68.5 68.7 71.7 61.3 23.1 176.3
R, b-Glc 2 102.2 77.1 75.3 70.7 76.7 61.8
4 102.2 77.1 75.3 70.4 76.7 61.4
T, b-Glc 2 103.2 73.5 74.5 76.6 75.7 60.8
4 103.1 73.9 74.8 76.6 75.7 60.8
S, b-Glc 2 103.5 73.5 74.7 79.1 75.4 59.7
4 102.2 73.5 74.5 79.1 75.7 60.6
Y, a-Glc 2 98.2 72.1 73.5 70.1 72.4 61.1
4 98.2 72.1 73.5 70.1 72.4 61.1
Z, b-GlcN 1 100.4 57.3 74.1 71.1 77.3 61.7
2 103.3 57.4 76.4 70.7 76.7 61.4

3 98.2 56.6 72.9 71.8 76.7 61.4
4 98.1 56.6 72.9 70.7 76.7 61.5
a
J
1,P
0.5 Hz;
b
J
2,P
7.5 Hz;
c
J
4,P
3.5 Hz;
d
J
5,P
6.5 Hz.
Ó FEBS 2002 LPS from Acinetobacter baumannii (Eur. J. Biochem. 269) 425
Isolation and structural analysis
of oligosaccharides 1±4
Acetic acid degradation of the LPS and reduction yielded as
main products two stereoisomers each of the two oligosac-
charides resulting from r eduction of the carbonyl group of
the reducing Kdo residue. Oligosaccharides 1 and 2 as the
major isomers were isolated by HPAEC and were examined
by NMR spectroscopy (data summarized in Tables 1±3).
All proton and carbon resonances could be a ssigned. Most
relative con®guration s and ring sizes of the monosac-
charides were determined on the basis of vicinal proton±

proton coupling constants. The ano meric con®gu rations of
hexoses followed from J
H1,H2
coupling constants (3.5±4 Hz
for a-linked and 7.8±8.4 Hz for b-linked residues). All
residues were present in the pyrano se form, as deduced from
the absence of low-®eld signals i n t he
13
C-NMR spectra that
are characteristic for furanoses, and from the vicinal proton
coupling constan ts. The NOE data (Table 3) allowed the
unambiguous determination of the sequence of monosac-
charide residues.
In the low-®eld region of t he
1
H-NMR spectra of
oligosaccharides 1 and 2 (4.4±5.4 p.p.m.), signals for t hree
and seven anomeric protons were present, respectively. The
two oligosaccharides differed from each other by the
tetrasaccharide fragment Y-R-T-S- (see Fig. 1). In both
oligosaccharides, the Kdo residue was present as an octitol
derivative (H2 at 4.061 and 4.159 p.p.m., respectively). For
each oligosa ccharide, all aldose residues gave NOE signals
between the H1 protons and the proton on the carbon atom
to which they w ere glycosidically linked (Table 3). Corre-
lations between the anomeric protons and the transglycosi-
dic carbon atom could also be detected in HMBC spectra,
con®rming the monosaccharide sequence and attachment
sites, the latter being assigned from
13

C-NMR spectroscopic
data using HMQC experiments (Table 2). The structures of
oligosaccharides 1 and 2 were con®rmed by e lectrospray
mass spectrometry, the spectra of which gave for o ligosac-
charide 1 an ion at m/z 822.4 ([M + H]
+
,calculated
molecular mass 821.9 Da) and for oligosaccharide 2 ions at
m/z 1428.6 (calculated molecular mass 1428.4 Da) and
715.2, representing [M + H]
+
and [M + 2H]/2
+
,respec-
tively.
From the resulting mixture of deacylated LPS, the two
major oligosaccharides 3 and 4,aswellasthetetrasac-
charide a-Kdo- (2 ® 4)-a-Kdo-(2 ® 6)-b-GlcpN4P-( 1
®6) -a-GlcpN1P, the trisaccharide a-Kdo- (2 ® 6)-b-
GlcpN4P-(1 ® 6)-a-GlcpN1P, and several minor com-
pounds were isolated by HPAEC. The tetrasaccharide and
the trisaccharide were readily identi®ed on the basis of
published NMR data [24,25], and the minor compounds
were not further investigated. The structures of oligosac-
charides 3 and 4 were fully characterized by NMR
spectroscopy (Tables 1±3). They differed from each other
by the tetrasaccharide fragment Y-R-T-S- (Fig. 1), as
observed also for oligosaccharides 1 and 2. T he substituent
at O4 of GlcpNA F, Gal pN G present i n 1 and 2,waslostby
b-elimination owing to the alkaline c onditions that also

converted the GlcpNA into 2-amino-2,4-dideoxy-b-
L
-threo-
hex-4-enopyranosyluronic a cid. Oligosaccharides 3 and 4
were similar to the oligosaccharide 5,isolatedfromLPSof
A. baumannii strain NCTC10303 [16], d iffering by the
presence of b-GlcN Z and (in 4) b y t he tetrasaccharide
sequence attached to O-3 of DGlcpNA F.
Both oligosaccharides 3 and 4 contained three Kdo
residues. All monosaccharide residues were present in the
pyranose form, as deduced from the absence of low-®eld
signals in the
13
C-NMR spectra that are characteristic for
furanoses, and from the vicinal proton coupling c onstants
(Tables 1 and 2). NOE contacts between H-1 of DGlcpNA
FandH-3ax,H-3eq, H-4 and H-5 of one of the Kdo
residues identi®ed the latter as residue E (Fig. 3), which was
present as an octitol residue i n compounds 1 and 2.The
Kdo residue E w as substituted by b-GlcN Z, as was
apparent from the observation of an NOE between protons
Z1 (b-GlcpN) and E7 ( Fig. 2, Table 3). The NOE contacts
between protons C5 and E3ax,E3eq , E4, and E6 (Fig. 3)
suggested t he attachment of E to C5. The sequence of the
sugar residues C, D, E , and F was ®n ally proven by long-
range H -C correlations that were present in t he HMBC
spectrum of heptasacch aride 3, namely between proton C4
and carbon D2, C5 and E2, and F1 and E4. The signals of
the anomeric carbons in Kdo residues were assigned on the
basis of the intraresidual correlations between H-3eq and

C-2.
The a-con®guration of the Kdo residues C and D
followed from the observed NOE between protons C3eq
and D6 (Fig. 3), characteristic for the fragment a-Kdo-
(2 ® 4)-a-Kdo [25], which were also consistent with the
sequence D-(2 ® 4)-C. The a-con®guration of the Kdo
residues C and D in 3 and 4 each was con®rmed by the
chemical shifts of their H-3ax, signals (1.7±2.3 p .p.m.)
[22,23]; however, the chemical shifts of H-3ax,ofCwere
interchanged. The chemical shifts of protons H-3, H-4, H-5,
and H-6 of Kdo E occurred at unusual low ®eld positions,
Table 3. Interresidual NOE contacts observed in oligosaccharides 1±4.
s, Stro ng NOE; m, medium NOE; w, weak NOE. Labeling i s a s f or
Fig. 1.
Oligosaccharides
Observed NOE contact
From proton To protons
3, 4 B-1 A-6a m, A-6b m
3, 4 C-3ax D-6 s, E-6 m
3, 4 C-3eq D-6 s, D-7 m
3, 4 D-3eq C-5 w, E-5 m, E-6 m
3, 4 E-3ax C-5 m, F-1 w
3, 4 E-3eq C-5 s, D-3eq m, F-1 m
3 E-4 C-5 s, F-1 s
4 E-4 C-5 s, C-7 w, D-4 w, D-7 w, F-1 s
3, 4 E-6 C-3 axw, C-5 s, C-7 s, D-3 eqm
1, 2 F-1 E-4 s, E-5 m, E-6 m
3 F-1 E-3 eqw, E-3 axw, E-4 s, E-5 s
4 F-1 E-3 eqw, E-3 axw, E-4 s, E-5 s,
E-6 w, D-4 w

1, 2 G-1 F-2 w, F-3 w, F-4 s
2, 4 R-1 T-3 m, T-4 s, T-6 am, T-6 bm
2 S-1 F-3 s
4 S-1 F-2 s, F-3 s
2, 4 T-1 S-4 s, S-6 am, S-6b m
2, 4 Y-1 R-1 w, R-2 s, T-1 w, T-5 m, T-6 m
1, 2 Z-1 E-7 s
3, 4 Z-1 E-5 w, E-7 s, E-8 s
426 E. V. Vinogradov et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Fig. 2. Part of the NOESY spectrum of oligosaccharide 4.
Fig. 3. Some important observed interresidual
NOE connectivities in the structural element
C-D-E-F of oligosaccharide 4. The depicted
structure represents one minim um energy
conformation.
Ó FEBS 2002 LPS from Acinetobacter baumannii (Eur. J. Biochem. 269) 427
nevertheless, on the basis of conformational analysis (see
below) and comparison o f the data with those of structure 5,
this residue is proposed to possess th e a-con®guration.
Oligosaccharide 3 possessed a structure similar t o oligo-
saccharide 5 (Fig. 1), which had been isolated earlier f rom
the LPS of A. baumannii strain NCTC10303 [16]. The only
difference was the presence of a-GlcpNZin3. However,
signi®cant differences of up to 0.4 p .p.m. w ere observed
between some proton signals of 3 and 5, i.e. C3 ax,E3ax,
E3eq, E4 and E5. In o rder to exclude any in¯uence o f
varying experimental conditions,
1
H-NMR a nd COS Y
spectra of a mixture of oligosaccharides 3 and 5 were

recorded, resulting in much closer chemical shifts: the
signals of C3ax,E3ax,E3eq, E4 and E5 of 5 were observed
at 2.174, 2.125, 2.607, 4.632, and 4.251 p.p.m., respectively,
and of 3 at 2.190, 2 .184, 2.764, 4.709 , and 4.270 p.p.m,
respectively. Other signals (except those from p rotons close
to the attachment point of GlcpNZ)overlappedinboth
structures. The differences i n chemical shifts for 3 and 5
obtained for the isolated samples must therefore be due to
small differences in sample conditions, e.g. concentration,
pH or ionic strength. Also, most of the NOE contacts
(Fig. 2 ) were similar. Thus, oligosaccharides 3 and 5 possess
the common hexasaccharide fragment F ® E ® (D ® )
C ® B ® A, and Kdo E is proposed to be present in
oligosaccharides 3 and 4 in a-pyranosidic form, a s shown
earlier for the o ligosaccharide 5 isolated from the LPS of
A. baumannii strain NCTC10303 [16]. The conformation o f
the fragment F-C was similar in 3, 4 and 5, but not identical.
An NOE contact between protons E3eq and D3eq was only
observed in the oligosaccharides from strain A TCC 1960 6
(compounds 3 and 4). In contrast, an NOE contact between
E3eq and F5 was only observed in the p roducts from strain
NCTC 10303 (e.g. compound 5). Conformational analyses
showed that a close proximity of protons E3eq to D3eq and
of E3eq and E4 to C5 could not simultaneously occur in the
same low energy conformation (assuming the chair confor-
mation of Kdo E). Thus, the observed NOE contacts
originate probably from different conformational minima
or from a d istorted ring conformation of Kdo E . T he last
possibility was supported by the observation of the intra-
residual NOE contact between E3 eq and E6, which is n ot

possible in the
5
C
2
con®guration. The existence of residue E
in the equilibrium of several conformers agrees with the
observation of its broadened and unresolved H-3 signals,
whereas all other signals in the spectra of 3 and 4 were sharp
and well resolved.
31
P NMR sp ectra o f oligosaccharides 3 and 4 showed two
signals of equal intensity at 2.2 and 4.5 p.p.m., which in
1
H,
31
P-HMQC spectrum correlated with protons A1 and
B4, respectively, thus proving phosphorylation at positions
O-1 of A and O -4 of B.
In summary, the structures of oligosaccharides 1±4 were
elucidated as depicted in Fig. 1. The structures possess a
common branched tetrasaccharide fragment G-F-[Z-]-E,
which is substituted in compound 2 at O- 3 of GlcpNA F
with the tetraglucosyl sequ ence Y-R-T-S Oligosaccharides
1 and 2 differ also in the acetylation o f the amino group of
GalN G. In the octasaccharide, this amino g roup is not
acetylated and H-2 of the residue G resonates at
3.264 p.p.m., whereas in the tetrasaccharide this amino-
group is acetylated, thus, this signal is shifted to
4.092 p.p.m. Structures 1±4 can b e combined to a complete
structure of the carbohydrate backbone of the LPS from

A. baumannii strain ATCC 19606 as depicted in Fig. 4.
The structures o f four core regions from LPS of d ifferent
Acinetobacter strains have been characterized during this
work. Two of these core regions (i.e. o f LPS from
A. haemolyticus strain ATCC 17906/NCTC10305 and from
Acinetobacter strain A TCC 1 7905) have i n c ommon t he
presence of Ko, which replaces in part the K do residue that
links the core oligosaccharide to the lipid A [14,15]. In bo th
cases, this ®rst Ko or Kdo residue was substituted at O-5 by
the t risaccharide a-
D
-Glcp-(1 ® 6)-b-
D
-Glcp-( 1 ® 4)-a-
D
-
Glcp (which in the core region of strain ATCC 17906/
NCTC10305 is phosphorylated at O-6 o f the 4-substituted
a-Glcp). The other two core regions were identi®ed in L PS
of A. baumannii, i.e. in s trains A TCC 17904/NCTC 10303
[16] and ATCC 19606 (this w ork). Their common feature is
the p rese nce o f t he trisaccharide a-Kdo-(2 ® 5)-[a-Kdo-
(2 ® 4)-]-a-Kdo-(2 ® , which has also been identi®ed in
the core r eg ion of LPS from A. baumannii strain ATCC
15303 [19]. Thus, it is possible t hat this t risaccharide
represents a partial structure o f the core region which i s
speci®c for LPS of A. b aumannii.
The oligosaccharides identi®ed in the core region of LPS
from A. baumannii strain ATCC 19606 suggests how a part
of its b iosynthesis may occur. Approximately one-third of

the core region is terminated by a
D
-GalpNAc residue (G in
Fig. 1), as indicated b y the yield of oligosaccharide 1. Here,
the
D
-GlcpNAcA residue F is not substituted at O -3. The
other two-thirds of the core comprised an oligosaccharide in
which G is a
D
-GalpN residue and F bears at O-3 the
tetrasaccharide a-Glcp-(1 ® 2)-b-Glcp-(1 ® 4)-b-Glcp-
(1 ® 4)-b-Glcp-(1 ® . Thus, it is r easonable to assume
that the tetrasaccharide is introduced only after enzymatic
de-N-acetylation of GalpNAc and that 1 represents a
precursor o f core region biosynthesis. Additionally, the
isolation of the tetrasaccharide a-Kdo-(2 ® 4)-a-Kdo-
(2 ® 6)-b-GlcpN4P-(1 ® 6)-a-GlcpN1P and the trisaccha-
ride a-Kdo-(2 ® 6)-b-GlcpN4P-(1 ® 6)-a-GlcpN1P from
deacylated LPS suggests that the a-( 2 ® 4)-linked Kdo
disaccharide is furnished ®rst in biosynthesis of t he core
region, as found e.g. i n LPS biosynthesis of Escherichia coli
Fig. 4. The complete chemical structure of the carbohydrate backbone of the LPS from Acinetobacter baumannii strain ATCC 19606.
428 E. V. Vinogradov et al. (Eur. J. Biochem. 269) Ó FEBS 2002
[26,27]. Then, the biosynthesis of the core region of LPS
from A. baumannii strain ATCC 19606 should proceed with
the introduction of a third Kdo residue to O-5 of the second
Kdo. This represents a remarkable difference to the
biosynthesis of LPS in E. coli (and in other bacteria), in
which the second Kdo is substituted at O-5 by one residue of

L
-glycero-
D
-manno-heptopyranose [10,26,27].
The K do transf erase o f strain A TCC 1 5303 has b een
cloned and characterized in vitro whereby it was shown that
it is able to transfer two Kdo residues in a-(2 ® 4)-linkage
to a lipid A precursor [ 19]. No evidence was obtained for a
second Kdo t ransferase for which we searched by Southern
blots. Therefore, the a-( 2 ® 5)-linked Kdo is either trans-
ferred by a second Kdo transferase that does not share
enough similarity with the one that was cloned or the
speci®city of t he enzyme is different under in vitro and in vivo
conditions.
ACKNOWLEDGEMENTS
We thank Veronika Susott for technical assistanc e and Klaus Bock fo r
valuable discussions and critical r eading of the manuscript.
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