The structure of the O-chain of the lipopolysaccharide of a prototypal
diarrheagenic strain of
Hafnia alvei
that has characteristics of a new
species under the genus
Escherichia
Reine Eserstam
1
, Thushari P. Rajaguru
1,2
, Per-Erik Jansson
1
, Andrej Weintraub
3
and M. John Albert
4
1
Clinical Research Center, Analytical unit, Karolinska Institute, Huddinge Hospital, Huddinge, Sweden;
2
Department of Chemistry,
University of Peradeniya, Peradeniya, Sri Lanka;
3
Karolinska Institute, Department of Microbiology, Pathology and Immunology,
Division of Clinical Bacteriology, Huddinge University Hospital, Sweden;
4
Department of Microbiology, Faculty of Medicine, Kuwait
University, Safat, Kuwait
The structure of the O-polysaccharide of the lipopolysac-
charide from a diarrheal strain isolated in Bangladesh
was studied with sugar, and methylation analysis, NMR
spectroscopy, mass spectrometry and partial acid hydrolysis.

the family Enterobacteriaceae. There are reports of associ-
ation of H. alvei with diarrhoea in Canada [1] and Finland
[2], but the mechanism of diarrhoea caused by this organism
in these locations remains unknown [3]. However, some
isolates of a bacterium typed as H. alvei from patients with
diarrhoea in Bangladesh produced diarrhoea in rabbits by
attaching and effacing (AE) lesions in the intestinal mucosa
that are characteristic of the lesions produced by entero-
pathogenic Escherichia coli [4]. Like enteropathogenic
E. coli,theseH. alvei isolates possess a homologous patho-
genicity island in the chromosome locus for enterocyte
effacement (LEE), which is responsible for producing
attaching and effacing lesions [5]. LEE encodes a type III
secretory system [6]. Secretion of the virulence factors leads
to effacement of the microvillus structure and reorganiza-
tion of the actin cytoskeleton to form a pedestal-like
structure, the attaching and effacing lesion [7]. AE lesion
formation is critical in mediating diarrhoea production in
the host, but its exact role in disease is not known. Recent
results from conventional biochemical analyses, testing of
susceptibility to cephalothin, lysis by a Hafnia-specific
phage, and amplification of the outer membrane protein
gene phoE with species-specific primers support the identi-
fication of these isolates as members of the genus Escheri-
chia rather than Hafnia alvei [8]. We studied the structure of
the O-chain of the lipopolysaccharide of one them.
MATERIALS AND METHODS
Bacterium, cultivation and isolation
of lipopolysaccharide and O-specific polysaccharide
The Hafnia alvei, strain number 10457, was from the culture

Hydrolysis was performed with 2
M
trifluoroacetic acid
(120 °C, 2 h), and monosaccharides identified by GLC as
their alditol acetates. Sugars were analysed on a Hewlett–
Packard 5880 GLC instrument on a DB-5 fused-silica
capillary column and a temperature gradient of 160 °C
(1 min) to 250 °Cat3°CÆmin
)1
. The absolute configura-
tions were determined by GLC of acetylated glycosides
with (+)-2-butanol, as described previously, but with the
modification that acetates were used [9–11]. Neuraminic
acid methyl glycoside methyl ester was analyzed as the
trimethylsilyl (TMS)-derivative and authentic reference.
A colorimetric test for Kdo using thiobarbituric acid was
also made [17].
Methylation analysis
Methylation was carried out with methyl iodide in dimethyl
sulfoxide in the presence of sodium methylsulfinylmethanide
[18]. The methylated polysaccharide was purified using Sep–
Pak C18 cartridges. Hydrolysis was performed as described
for sugar analysis; partially methylated monosaccharides
were converted into alditol acetates and analyzed by GLC
and GLC-MS on a Hewlett Packard 5890 chromatograph
equipped with a NERMAG R10–10 L mass spectrometer,
using the above conditions. Identification was made using
reference data.
NMR spectroscopy
1

and freeze dried. Ultracentrifugation of the lipopolysaccha-
ride gave a pellet and an upper phase, the latter containing
most of the material. SDS/PAGE of the two materials
(Fig. 1) in the upper phase and the pellet showed identical
patterns and it was therefore concluded that the same
polysaccharide was present. A hydrolysate of the upper
phase, analyzed as alditol acetates, revealed as
D
-glucose,
D
-galactose,
D
-galactosamine,
L
-glycero-
D
-manno-heptose,
and
D
-glucosamine in the proportions 6 : 65 : 19 : 6 : 3.
The relatively high proportion of heptose may be the result
of short chains. It is not a component in the polysaccharide
as demonstrated in the MS analysis (see below). The
absolute configurations of the sugars were established by
GLC analysis of the acetylated (+)-2-butyl glycosides
[9–11]. Methanolysis of the sample and analysis by GLC-
MS gave, in addition to the sugars mentioned, neuraminic
acid. The pellet showed essentially the same compounds.
To verify that the material was an O-polysaccharide or
exclude the possibility, the content of Kdo was checked with

ultracentrifugation of the Hafnia alvei lipopolysaccharide. For com-
parison lipopolysaccharide from a smooth (Shigella flexneri,3)anda
rough bacterium (Salmonella typhimurium Ra, 4) was run simulta-
neously.
3290 R. Eserstam et al. (Eur. J. Biochem. 269) Ó FEBS 2002
mine were detected, thus indicating a linear polysaccharide
consisting of repeats with three sugar residues. This was
corroborated by the
1
H-NMR spectrum, which showed
signals for anomeric protons at d 5.11 (J small), 4.76
(J 7.7 Hz) and 4.46 (J 7.7 Hz), for ring protons, and for an
N-acetyl methyl group at d 2.05. The first chemical shift
should belong to a furanoside as these normally have small
J-values, the second and third signal have typical J-values
for b-linked sugars with galacto-pyranose configuration.
The absence of NeuAc was evident as no methylene-
deoxyresonances could be detected. The
13
C-NMR spec-
trum showed signals inter alia for anomeric carbons at d
109.9, 104.1 and 103.6. The first value is very high and
characteristic of a b-furanosidic sugar. A distorsionless
enhanced polarization transfer (DEPT) spectrum revealed
that the substituted hydroxymethyl group, C-6 of the
galactofuranose, is located at d 71.8, thus among those of
secondary carbons. The
1
H- and
13

alvei lipopolysaccharide in D
2
O.
Table 1.
1
H- and
13
C-NMR chemical shifts (d, p.p.m.) for different H. alvei polysaccharides. The N-acetyl group in the GalNAC residue appears at d
2.05/22.7/176.1 in the O-polysaccharide and in the GalNAc and NeuAc residues at d 2.05/22.8–22.9/175.8 in the lipopolysaccharide.
Sugar residue 1 2 3 4 5 6 7 8 9
H. alvei O-polysaccharide
fi 6)-b-
D
-Galf-(1 fi (A) 5.11 4.09 4.06 4.06 4.02 3.75, 4.04
109.9 82.1 77.6 83.7 70.2 71.8
fi 3)-b-
D
-GalpNAc-(1 fi (B) 4.76 4.05 3.82 4.02 3.71  3.77
103.6 52.2 78.7 70.2 75.6 61.6
fi 3)-b-
D
-Galp-(1 fi (C) 4.46 3.62 3.76 4.16 3.71  3.77
104.1 70.7 82.6 69.3 75.6 61.6
Native H. alvei lipopolysaccharide
fi 6)-b-
D
-Galf-(1 fi (A) 5.11 4.09 4.05 4.04 4.00 3.89, 3.89
109.9 82.8 77.8 83.8 70.6 72.1
fi 3)-b-
D

-galactose. In addition, smaller
peaks corresponding to the minor component polysaccha-
ride were observed. The repeating unit of the polysaccharide
thus contains a terminal NeuAc, and the above mentioned
residues. A comparison to the methylation analysis data on
the O-polysaccharide, indicates that the terminal NeuAc
should be substituting the galactose residue in the
6-position.
For the full characterization of the lipopolysaccharide,
with NeuAc still present, an NMR sample was prepared
from the native lipopolysaccharide. The spectrum had
broadened lines but were surprisingly good with resolved
couplings (Fig. 2). The
1
H-NMR spectrum of the lipopoly-
saccharide showed signals for three anomeric protons at
d 5.11 (J small), 4.73 (J 7.7 Hz), 4.43 (J 7.7 Hz), thus close
to those observed for the O-polysaccharide. In the high field
region signals for a methylene group, assigned to CH
2
in
NeuAc were observed at d 2.75 and 1.68, the large difference
establishing the presence of an axial carboxyl group and an
a-linkage in the NeuAc residue. Signals for N-acetyl groups
deriving from NeuAc and GalNAc were present at d 2.05. In
the
13
C-NMR spectrum, the corresponding signals were
present inter alia at d 109.9, 104.2, 103.7, and 101.2 for
anomeric carbons and at d 41.0 and 22.8–22.9 for methylene

and B–C. From the NOE spectrum the following inter-
residue correlations between H-1 and protons on linkage
carbons were observed: A H-1/B H-3 (5.11/3.82), demon-
strating the element A–B. Correlations 4.73/3.72 and 4.43/
3.90 are in accord with elements B–C and C–A but
ambiguous due to signal overlap. From the combined data,
however, the following structure can be postulated for the
repeating unit
→6)-β-
D
-Galf-(1→3)-β-
D
-GalpNAc-(1→3)-β-
D
-Galp-(1→
α-NeuAc
↑
6
2
A
B
C
D
Fig. 3. The COSY spectrum the of the Hafnia
alvei lipopolysaccharide showing the anomeric
and the ring proton region.
3292 R. Eserstam et al. (Eur. J. Biochem. 269) Ó FEBS 2002
MALDI-MS of the O-polysaccharide
The O-polysaccharide, i.e. the desialylated polysaccharide
chain, was also characterized by MALDI-MS of the

n
and the sequence is
further established.
The initial phenotypic characterization of the strain 10457
with a commercial identification system, API-20 E identified
the strain as Hafnia alvei [4]. Additional phenotypic
characterization and partial 16S rRNA sequencing of a
set of isolates identified them not as typical Hafnia alvei, but
Fig. 5. HSQC spectrum of the Hafnia alvei
lipopolysaccharide showing the anomeric and
the ring proton/carbon region and including
high resolution
1
H- and
13
C-NMR spectra.
Fig. 4. TOCSY spectra of the Hafnia alvei
lipopolysaccharide showing the correlations
deriving from the anomeric protons. Mixing
times were from 30 to 160 ms.
Ó FEBS 2002 New species under the genus Escherichia (Eur. J. Biochem. 269) 3293
unique isolates [12]. Further phenotypic characterization
suggested that these isolates are neither Hafnia alvei nor
Escherichia coli, but closely related to the genus Escherichia
[8]. DNA hybridization studies suggested that these isolates
deserve a new species name under the genus Escherichia
(J. Albert, Kuwait University, Safat, Kuwait, personal
communication). The unique structure of the O-chain of
one of these isolates further confirms this conclusion.
The lipopolysaccharide structures of both H. alvei and

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